Relevance to Disorders of Propionyl-Coa Metabolism

Home , Aminoaciduria, Glutamine synthetase

INTERRELATIONS BETWEEN PROPIONATE,

3-HYDROXYPROPIONATE, AND

β-ALANINE METABOLISM:

RELEVANCE TO DISORDERS OF PROPIONYL-COA METABOLISM

By KIRKLAND A. WILSON

Submitted in fulfillment of the requirements for the Degree of Doctor of Philosophy

Thesis Advisor: Henri Brunengraber, M.D., Ph.D.

Department of Nutrition School of Medicine CASE WESTERN RESERVE UNIVERSITY

January, 2018

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Kirkland A Wilson

candidate for the degree of Doctor of Philosophy.

Committee Chair

Danny Manor

Committee Member

Michelle Puchowicz

Committee Member

George Dubyak

Committee Member

Gregory Tochtrop

Committee Member

Shawn McCandless

Committee Member

Douglas Kerr

Date of Defense

06/21/17

*We also certify that written approval has been obtained

for any proprietary material contained therein.

Dedication

I dedicate this work to my parents, family, and loved ones, all of whom have provided continued love and support throughout my life. They have encouraged me to set lofty goals and have given me the strength to reach for them. Their love has helped me succeed in my work.

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Table of Contents List of Tables ...... viii List of Schemes and Figures ...... ix Acknowledgements ...... xii List of Abbreviations ...... xv Abstract ...... xvi

Chapter 1: Disorders of Propionyl-CoA Metabolism 1.1 Introduction ...... 1 1.2 Propionic Acidemia ...... 2 1.2.1 Overview ...... 2 1.2.1.1 Propionyl-Coenzyme A Carboxylase ...... 4 1.2.2 Signs and Symptoms ...... 5 1.2.2.1 Neurological Complications ...... 6 1.2.2.2 Cardiac Abnormalites ...... 8 1.2.2.3 Additional Complications ...... 11 1.2.3 Biochemical Parameters ...... 12 1.2.4 Diagnosis and Treatment ...... 14 1.2.5 Patient Outcomes Post Newborn Screening ...... 19 1.3 Methylmalonic Acidemia ...... 21 1.3.1 Overview ...... 21 1.3.1.1 Methylmalonyl-CoA Epimerase and Methylmalonyl-CoA Mutase ...... 22 1.3.1.1.1 Methylmalonyl-CoA Epimerase ...... 23 1.3.1.1.2 Methylmalonyl-CoA Mutase ...... 24 1.3.2 Signs and Symptoms ...... 24 1.3.2.1 Renal Complications ...... 25 1.3.2.2 Neurological Complications ...... 26 1.3.2.3 Additional Complications ...... 27 1.3.3 Biochemical Parameters ...... 28 1.3.4 Diagnosis and Treatment ...... 29

1.3.5 Patient Outcomes Post Newborn Screening ...... 31

Chapter 2: Metabolism of Propionate 2.1 Normal Metabolism of Propionate ...... 33 2.2 Metabolism of Propionate and Metabolic Toxicity in Disorders of Propionyl-CoA Metabolism ...... 34

Chapter 3: Metabolism of Methylmalonate 3.1 Normal Metabolism of Methylmalonate ...... 41 3.2 Metabolism of Methylmalonate and Metabolic Toxicity in Disorders of Propionyl- CoA Metabolism ...... 42

Chapter 4: Metabolism of 3-Hydroxypropionate 4.1 Normal Metabolism of 3-Hydroxypropionate ...... 44 4.2 Metabolism of 3-Hydroxypropionate and Metabolic Toxicity in Disorders of Propionyl-CoA Metabolism ...... 46

Chapter 5: Hyperammonemia in Disorders of Propionyl-CoA Metabolism 5.1 Ammonia Homeostasis ...... 49 5.2 ATP Utilization in Ureagenesis ...... 51 5.3 Dyregulation of the Urea Cycle in Disorders of Propionyl-CoA Metabolism ...... 51

Chapter 6: Metabolism of β-alanine 6.1 Normal Metabolism of β-alanine ...... 55 5.2 Metabolism of β-alanine and Metabolic Toxicity in Disorders of Propionyl-CoA Metabolism ...... 57

Chapter 7: Research Plan

7.1 Overview ...... 59 7.2 Metabolism of 3HP and Propionate in Perfused Rat Livers: Summary of Strategy and Methods ...... 60 7.2.1 Characterizing the Metabolism of 3HP ...... 60 7.2.2 Investigating the Metabolism of 3HP and/or Propionate ...... 62 7.2.3 Health Relevance ...... 64 7.2.4 Strategy ...... 64 7.2.4.1 Perfused Liver Experiments ...... 65 7.2.4.2 Analytical Procedures ...... 65 7.2.4.2.1 Ultraviolet-Visible Absorption Spectroscopy ...... 66 7.2.4.2.2 Nuclear Magnetic Resonance Spectroscopy ...... 66 7.3 Metabolism of β-alanine: Summary of Strategy and Methods ...... 66 7.3.1 Characterizing and Investigating the Metabolism of β-alanine ...... 66 7.3.2 Public Health Relevance ...... 67 7.3.3 Strategy ...... 68 7.3.3.1 Perfused Liver Experiments ...... 68 7.3.3.2 In vivo Experiments ...... 69 7.3.3.3 Analytical Procedures ...... 69

Chapter 8: Publications 8.1 Overview ...... 70 8.1.1 Wilson, K.A., Han, Y., Zhang, M., Hess, J., Chapman, K.A., Cline, G.W., Tochtrop, G.P., Brunengraber, H., Zhang, G-F. Interrelations between 3- hydroxypropionate and propionate metabolism in rat liver: Relevance to disorders of propionyl-CoA metabolism. Am. J. Physiol. Endocrinol. Metab. (In Press): 2017...... 71 8.1.2 Wilson, K.A., Bedoyan, J., Zhang, G-F., Venditti, C.P., Brunengraber, H. Maleate, a nephrotoxic plasma biomarker of propionic and methylmalonic acidemias in humans and mice. (To be submitted to Mol Genet Metab.): 2017...... 125 8.1.3 Wilson, K.A., Hess, J., Zhang, M., Chapman, K.A., Derave W., Zhang, G-F,

Brunengraber, H., Tochtrop, G.P. Metabolism of β-alanine in the perfused rat liver and live rats: Stimulation of CoA synthesis and carboxylation to 2- (aminomethyl)-malonate. (To be submitted to Am. J. Physiol. Endocrinol. Metab.): 2017 ...... 143

Chapter 9: Discussion, Implications, and Future Directions 9.1 CoA Trapping in Disorders of Propionyl-CoA Metabolism ...... 179 9.1.1 Discussion and Conclusions ...... 179 9.1.2 Future Directions ...... 180 9.2 Modulation of the CAC in Disorders of Propionyl-CoA Metabolism ...... 181 9.2.1 Discussion and Conclusions ...... 181 9.2.2 Future Directions ...... 183 9.3 Possible Fates of the Carbon of 3HP ...... 185 9.3.1 Discussion and Conclusions ...... 185 9.3.2 Future Directions ...... 186 9.4 Production of Maleate from Propionate and from 3HP ...... 186 9.4.1 Discussion and Conclusions ...... 186 9.4.2 Future Directions ...... 187 9.5 Novel Metabolism of β-alanine ...... 188 9.5.1 Discussion and Conclusions ...... 188 9.5.2 Future Directions ...... 189 9.6 Altered CoA Metabolism from High β-alanine ...... 190 9.6.1 Discussion and Conclusions ...... 190 9.6.2 Future Directions ...... 191 9.7 Effects of High β-alanine on Neurotransmitter Balance ...... 191 9.4.1 Discussion and Conclusions ...... 191 9.4.2 Future Directions ...... 192 9.8 Summary and Implications ...... 193

Literature Cited ...... 195

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List of Tables Chapter 8

Table 8.1 Rates of uptake and release of substrates ...... 111 Table 8.2 Metabolite concentrations (nmol/g dry weight) ...... 112 Table 8.3 Release of amino acids and urea from perfused rat livers, expressed as (microequivalents of N)·(g dry weight)-1·hr-1 ...... 113 Table 8.4 Metabolite concentrations in plasma samples from humans (µM) ...... 137 Table 8.5 Plasma samples from single patient taken as patient metabolic status changed ...... 138 Table 8.6 Metabolite concentrations in plasma samples from mice (µM) ...... 139 Table 8.7 Release of amino acids and urea from livers, in nitrogen equivalents (nanoequivalent of N·g dry weight-1·hr-1) ...... 164 Table 8.8 Metabolite concentrations (nmol/g dry weight) ...... 165

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List of Schemes and Figures Chapter 1 Figure 1.1 Mechanism of Propionyl-CoA anaplerosis ...... 5 Figure 1.2 β-oxidation of Odd-Chain Fatty Acids ...... 20

Chapter 2 Figure 2.1 Summary of Major Propionyl-CoA Metabolic Pathway in the Liver ...... 34 Figure 2.2 Sites of Effect by Methylcitrate ...... 37

Chapter 3 Figure 3.1 Presence of a Methylmalonate Shunt in the Propionyl-CoA Metabolic Pathway in the Liver ...... 42

Chapter 4 Figure 4.1 Mechanism of 3HP Metabolism ...... 45

Chapter 5 Figure 5.1 Overview of the Urea Cycle ...... 50

Chapter 6 Figure 6.1 Overview of β-alanine Metabolism ...... 56

Chapter 8 Scheme 8.1 Pathways of propionate and 3-hydroxypropionate metabolism in rat liver .114 Scheme 8.2 Transfer of C-2 and C-3 of 3-hydroxypropionate to C-4 and C-5 of glutamate via acetyl-CoA ...... 115 Figure 8.1 Crossover analyses of liver metabolite concentrations ...... 116 Figure 8.2 Mass isotopomer distribution of 3HP released in perfusate ...... 117 Figure 8.3 Mass isotopomer distributions of CoA esters ...... 118 Figure 8.4 NMR spectra of glutamate in livers perfused with 2 mM [2-13C]3HP, 13 13 [3- C]3HP or [ C3]3HP ...... 119

Figure 8.5 Mass isotopomer distributions of non-CoA metabolites ...... 120 Figure 8.6 Mass isotopomer distributions of glucose ...... 121 Figure 8.7 Comparison of M2 enrichments of citrate, acetyl-CoA and C-1+2 of acetoacetate ...... 122 Figure 8.8 Mass isotopomer distributions (MID) of fumarate and maleate released by perfused livers ...... 123 Figure 8.9 Relative concentrations of maleate released by perfused livers ...... 124 Figure 8.10 Calibration Curve of Maleate Concentration ...... 140 Figure 8.11 Correlation of Maleate to Metabolites Related to Disorders of Propionyl- CoA Metabolism in Humans ...... 141 Figure 8.12 Correlation of Maleate to Metabolites Related to Disorders of Propionyl- CoA Metabolism in Mice ...... 142 Scheme 8.3 Relationship of β-alanine metabolism to pathways of propionate and 3- hydroxypropionate metabolism in rat liver ...... 166 Scheme 8.4 Protocol for in vivo rat experiments ...... 167 Figure 8.13 Concentration and mass isotopomer distribution of select nitrogenous compounds released by the perfused liver ...... 168 Figure 8.14 Mass isotopomer distributions of metabolites in livers perfused with 2 mM 15 13 β-[ N, C3]alanine ...... 169 Figure 8.15 Electron ionization mass spectra of TBDMS derivatives ...... 170 Figure 8.16 Evidence that AMM is formed by carboxylation of β-alanine ...... 171 Scheme 8.5 2-aminomethylmalonate (AMM), an isomer of aspartate formed by addition

of CO2 onto β-alanine ...... 172 Figure 8.17 Metabolite concentrations in PLASMA of rats injected with AOA or saline and subsequently gavaged with β-alanine or saline ...... 173 Figure 8.18 Metabolite concentrations in LIVER of rats injected with AOA or saline and subsequently gavaged with β-alanine or saline ...... 174 Figure 8.19 Metabolite concentrations in LIVER of rats injected with AOA or saline and subsequently gavaged with β-alanine or saline...... 175 Figure 8.20 Metabolite concentrations in rats injected with AOA or saline and subsequently gavaged with β-alanine or saline...... 176

Figure 8.21 Metabolite concentrations in BRAIN of rats injected with AOA or saline and subsequently gavaged with β-alanine or saline ...... 177 Figure 8.11 Metabolite concentrations in BRAIN of rats injected with AOA or saline and subsequently gavaged with β-alanine or saline ...... 178

Acknowledgements

Completing my PhD degree would not have been possible without the continued support of my my family, friends, and colleagues for their support. I must also thank my teachers and other faculty who provided advice and help. First and foremost, I must thank my advisor, Dr. Henri Brunengraber, for his invaluable guidance and education. Dr.

Brunengraber further fueled my love of research and was an amazing advisor. His patience, enthusiasm, teaching, and leadership abilities made learning the complexities of isotope and metabolic research possible. The research environment he fostered was perfect for my academic and scientific growth and allowed for the development of my independence. The research I conducted was perfect for both my interests and my development as a medical scientist.

I must also thank Drs. GuoFang Zhang and Michelle Puchowicz, who aided me with their

scientific expertise, patience, and guidance. Dr. Puchowicz was a vital source of help

throughout my research in overcoming the difficulties of metabolomic work, and I was

extremely fortunate to work side-by-side with Dr. Zhang as he guided me in my learning

of the various techniques and problems associated with GC-MS and LC-MS/MS technology. Their help and advice allowed me to grasp the intricacies of metabolic research and isotope technology.

I would also like to thank Dr. Gregory Tochtrop who provided many of the compounds used to complete my work. Additionally, he gave time and input that allowed me to overcome many technical hurdles that would have been insurmountable otherwise.

Without Dr. Tochtrop and his lab, progress would have been severely hindered.

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I want to thank Dr. Douglass Kerr for his insights into pyruvate carboxylase. Without his

help I would not have been prepared to explore this unexpected avenue of research. His advice on the subject allowed for rigorous investigation and completion of my dissertation research.

I must also thank Drs. George Dubyak and Danny Manor, for their continued help and support throughout my pursuit of a combined MD/PhD degree. Their advice allowed me to best utilize my time and the resources available to me and I am appreciative of all the

time and effort they gave. With the help of Dr. Dubyak and Dr. Manor I have been able

to navigate the difficulties of the program and reach this point.

I also must thank Dr. Shawn McCandless, who has provided input on my project, given

guidance on the clinical aspects of my career path, and has given his time to provide me with exposure and advice concerning medical genetics. Through his efforts and guidance

I have a solidified my ideal future career path.

I would furthermore like to thank my entire dissertation committee: Dr. Michelle

Puchowicz, Dr. Colleen Croniger, Dr. Danny Manor, and Dr. Gregory Tochtrop. They have continually provided advice, promoted my intellectual development, and taught me

how to think critically about my research. They have also provided consistent

encouragement and irreplaceable advice.

Last but not least, I want to thank all of my colleagues in the Brunengraber Lab, the

Mouse Metabolomic Phenotyping Center, the Department of Nutrition and the

Department of Chemistry. John Koshy, Sophie Roussel-Kochheiser, Sharon Zhang,

Lindsay Kaydo, and Cindy Wang also contributed to my research. They provided

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assistance, advice, and technical support that allowed me to complete the research

presented.

It was a pleasure and privilege to work with all of these individuals, and I am indebted to them for their help and guidance. I have learned much in this process and will cherish all that I have been taught as I continue on in my future endeavors

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List of Abbreviations

CAC Citric Acid Cycle

CASTOR CoA Sequestration, Toxicity, and Redistribution

CNS Central Nervous System

CoA Coenzyme A

CPSI Carbamoyl Phosphate Synthetase I

CSF Cerebral Spinal Fluid

GC-MS Gas Chromatography-Mass Spectrometry

LC-MS Liquid Chromatography-Mass Spectrometry

MCEE Methylmalonyl-CoA Epimerase

MMA Methylmalonic Acidemia

MUT Methylmalonyl-CoA Mutase

NAG N-acetylglutamate

NAGS N-acetylglutamate synthetase

NBS Newborn Screening

OLT Orthotopic Liver Transplant

PA Propionic Acidemia

PCC Propionyl-coenzyme A carboxylase

3HP 3-Hydroxypropionate

GABA Gamma-aminobutyrate

Interrelations Between 3-Hydroxypropionate, Propionate,

And β-Alanine Metabolism:

Relevance To Propionic Acidemia

Abstract

KIRKLAND A. WILSON

Propionic acidemia (PA) and methylmalonic acidemia (MMA), both disorders of propionyl-CoA metabolism, are both diseases of toxic compound accumulation.

Propionate, 3-hydroxypropionate (3HP), methylcitrate, methylmalonate, related compounds, and ammonium accumulate in body fluids of patients. Although liver transplant alleviates hyperammonemia, high concentrations of toxic compounds persist in body fluids. We hypothesized that metabolic perturbations result from the simultaneous presence of propionate and 3HP in body fluids. We then confirmed this hypothesis and detailed our findings. First, in the perfused rat liver, propionate and 3HP result in major overload of the citric acid cycle (CAC) and large perturbations in CAC metabolite ratios.

Second, energy homeostasis is greatly perturbed; [ATP]/[ADP] and [ATP]/AMP] ratios were decreased while total adenine nucleotide pool was increased, thus the overloaded

CAC doesn’t supply enough reducing equivalents to maintain adenine nucleotide homeostasis. Third, there is major CoA trapping in propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA and tripling of total CoA concentration.

Further, we identified new fates of propionate and 3HP metabolism in perfused rat livers, plasma samples from heterozygous and conditional knockout mice of PA and MMA, and

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plasma samples from MMA and PA patients. Propionate and 3HP form nephrotoxic

maleate via independent processes not involving the CAC. Propionate conversion to

maleate likely occurs by propionyl-CoA metabolism to cytosolic methylmalonyl-CoA then further conversion to maleate by unknown processes. This has clinical relevance to the high occurrence of renal failure in MMA and the increased rate of renal failure in PA post-orthotopic liver transplant.

Lastly, we identified new metabolic fates of the 3HP metabolite, β-alanine. We showed that β-alanine (i) stimulates CoA synthesis but does not directly contribute to the β- alanine moiety of CoA, (ii) reversibly forms 3HP and forms the novel metabolite 2-

(aminomethyl)-malonate by carboxylation via pyruvate carboxylase, and (iii) perturbs neurotransmitter balance. These discoveries were made in perfused rat livers and in live rats using a combination of metabolomics and stable isotope techniques.

Overall, research into disorders of propionyl-CoA metabolism is a complex area of investigation. We explored propionyl-CoA metabolism in the setting of these disorders and report findings that have implications for the clinical management of these patients.

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Chapter 1

Disorders of Propionyl-CoA Metabolism

1.1 Introduction

Metabolism of propionyl-CoA to succinate is a vital mechanism for the catabolism of fats

and proteins. As such, abnormalities involving the enzymes and coenzymes that

participate in this pathway lead to profound illness and death in the absence of adequate

treatment. There are numerous inherited abnormalities in the pathway of propionyl-CoA metabolism, differentiated by the specific disease-causing mutation. This work focuses

on mutations resulting in propionic acidemias and methylmalonic acidemias that alter the

enzymes directly associated with the propionyl-CoA to succinate pathway, namely,

propionyl-CoA carboxylase, methylmalonyl-CoA epimerase, and methylmalonyl-CoA

mutase. These enzymes allow for the refilling of the CAC by propionyl-CoA in a process

termed anabolism, and when defective, lead to disease phenotypes that are least

confounded by deficiencies not directly related to the anabolism of propionyl-CoA.

Specifically, the enzymes involved in propionyl-CoA metabolism also require use of coenzymes. While it is recognized that abnormalities in either the efficacy of (i) biotin, the coenzyme for propionyl-CoA carboxylase, or (ii) cobalamin, the coenzyme for methylmalonyl-CoA mutase, result in disease, these will not be discussed to any great

extent. This decision was made due to the broad scope of the resulting disease, effects on

other pathways which involve these coenzymes, and the resulting overlapping and

confounded biochemical profiles.

1.2 Propionic Acidemia

1.2.1 Overview

Propionic Acidemia (PA) was first described in 1961 as a neonatal disease characterized

by hyperglycinemia and hyperglycinuria. A defect of glycine metabolism was quickly ruled out as the cause of illness; instead, it was discovered that a deficiency in propionate metabolism was the underlying defect. The first described patient, a 3 year old male, in addition to hyperglycinemia and hyperglycinuria, presented with developmental retardation, episodic vomiting, lethargy, dehydration, and ketosis. His history showed repeated infections, hypogammaglobulinemia, neutropenia, periodic purpura, and thrombocytopenia (38). His first signs began approximately 18 hours after birth. PA has never been associated with clinically relevant intrauterine complications and PA patients appear normal at birth (38, 100, 218).

This initial phenotype was then expanded upon and the deficiency was characterized as a block in propionate metabolism. The altered glycine metabolism originally described has yet to be elucidated, though it is suspected that either propionyl-CoA, or a metabolite related to PA, inhibits an aspect of the glycine cleavage system (76, 102, 218). In 1971 the underlying cause of illness was described as a deficiency of propionyl-coenzyme A

(CoA) carboxylase (PCC, EC 6.4.1.3) (82). Genetically, PA shows an autosomal recessive inheritance pattern, with the majority of patients presenting as compound heterozygotes (65, 82, 156). The incidence of PA has been estimated at approximately

1:50,000, making it one of the most frequently diagnosed organic acidemias (111). If left untreated, patients can quickly deteriorate, and succumb to a terminal hyperammonemic coma. Symptoms of the late-onset form, may not develop for several years after birth, but

2 are typically similar to, though milder than, those seen in the neonatal onset form of disease (195, 218). All PA patients, however, are susceptible to metabolic crises (68).

Given the similar but milder phenotype in the late onset form, this review will focus on the more common neonatal onset form of PA.

The first case study of PA, conducted by Childs [1961, 522-538] involved a series of amino acid tolerance tests whereby the patient was administered various amino acids and followed for signs of metabolic decompensation and/or changes in plasma and urine glycine concentrations. Their results showed that, of the amino acids administered, only isoleucine, threonine, valine, and leucine resulted in the now classic presentation of PA.

All other amino acids were well tolerated. Isoleucine, threonine, and valine are all directly related to propionate metabolism, explaining the adverse effects seen upon administration. Leucine, though not directly related to propionate metabolism was also poorly tolerated, likely due to its ketogenic metabolism as the patient had a much greater than normal ketotic response. Whole protein also elicited the same symptoms in the patient as observed previously, as did prior reported infections (38). It is now known that there is a spectrum of tolerance in patients; the health of the patient prior to the onset of symptoms is an indication of subsequent severity. Overall, as a result of altered metabolism, PA results in a buildup of various toxic metabolites, such as 3- hydroxypropionate (3HP), methylcitrate, ammonium, and propionylcarnitine. Thus PA can be classified as a disease of toxic compound accumulation (9).

1.2.1.1 Propionyl-Coenzyme A Carboxylase

PCC catalyzes the conversion of propionyl-CoA to S-methylmalonyl-CoA, as seen in

Figure 1.1. It is a dodecamer holoenzyme composed of six α-subunits, each transcribed from the PCCA gene, and 6 β-subunits, each transcribed from the PCCB gene, and has

highly conserved amino acid sequences. The quaternary structure has the six β subunits at

its core, surrounded by the six alpha subunits. Additionally, the biotin carboxylase

domain and biotin carboxyl carrier protein domains in the alpha subunit are homologous

to equivalent regions in acetyl-CoA carboxylase and pyruvate carboxylase. The β

subunit, which confers carboxyltransferase activity is furthermore homologous to the

equivalent domain of acetyl-CoA carboxylase (83). PCC enzyme activity is present in most tissues, including the liver, heart, brain, kidney, skin, and various types of blood cell

(218). Most of the mutations that lead to PA affect the active and/or binding sites of the subunits, with most occurring in the β subunit. These mutations likely affect substrate binding and/or catalysis, while very few mutations causing PA have been located to the interface between alpha and β subunits. Disease causing mutations in the alpha subunit often have a variable presentation with regard to both age of onset and symptoms (83,

156). Notably, neither the defined mutation nor the residual activity of PCC in a patient have been shown to correlate well to severity of disease nor is the specific mutation a good predictor of patient outcome (12, 65, 173).

Though PCC is a biotin dependent enzyme, PCC deficiency resulting in PA is clinically unresponsive to biotin supplementation. Interestingly, Wolf [1980, 964-966] showed that biotin supplementation to PA patient fibroblasts resulted in a 122% increase in leukocyte

PCC activity and a 76% increase in fibroblast PCC activity. However, this resulted in a

4 rescue of only 15-20% of PCC effect clinically, compared to the normal range, furthermore rescue of activity only occurred in a subset of patients (82, 217).

Methylmalonyl – CoA Epimerase

Figure 1.1 Mechanism of Propionyl-CoA anaplerosis The enzyme mechanism outlined in red is deficient in PA. McCarthy, A. et al, Structure. 9: 637-646, 2001.

1.2.2 Signs and Symptoms

If treatment is not initiated during the neonatal period, approximately 74% of patients with early-onset PA present with clinical symptoms within the first 8 days of life. The remaining 26% typically present between days 11 and 90. The most prominent findings include muscular hypotonia, refusal to feed, and lethargy. Hyperammonemia is also a frequent finding with one study suggesting that it is found in at least 84% of untreated newborns. PA patients as they age may trend toward a short stature with decreased head

circumference, but preserved body mass index. Without treatment, death usually occurs

within the first year of life (173). Symptoms may be exacerbated by catabolic stressors

such as increased protein intake, infection, and/or constipation. Overall, a large amount of clinical heterogeneity is present when assessing the phenotype of PA patients (65, 218).

Late-onset PA patients usually present clinically by 16 months and with a highly variable presentation. Most have few to no metabolic crises documented. Neurological development may be normal in this patient population, with few cases of developmental retardation (173).

No specific physical abnormalities have been directly associated with PA. Any decreases in body length seen in a PA patient is instead most likely the result of recurrent illness and/or the restrictive diet. Clearly abnormal neurological development was noted by cerebral magnetic resonance imaging in a subset of PA patients studied, 2 out of 17.

Neurological complications of PA are further discussed in the subsequent section (218).

1.2.2.1 Neurological Complications

Characteristic neurologic findings of disease include ataxia and basal ganglia necrosis,

hypotonia, lethargy, and seizures. The seizures are typically generalized or myoclonic in

early childhood, but are mild generalized or absence seizures in later childhood.

Metabolic stroke-like episodes, defined as the presence of vegetative symptoms, such as bradycardia, low body temperature, and drowsiness, are also common. It is still unknown

whether the acute neurologic events in PA, namely metabolic stroke-like episodes and

seizures, are the direct result of the systemic metabolic acidosis seen in PA patients or are

due to some other process. Furthermore, it is unknown whether prenatal neurological

development is affected by PA metabolites. It is well documented that even in the

presence of optimal therapy, long term chronic neurological complications occur, such as

spastic quadriplegia and athetosis. Furthermore, neurologic symptoms may develop

independent of a documented metabolic crisis. While an overview of neurologic sequela

will be given, a comprehensive review of the neurologic findings associated with PA can

be found in (175, 177, 218).

A survey conducted through the Propionic Acidemia Foundation showed that

approximately three-quarters of the total responders had delays in gross motor, fine

motor, and/or language development. Additionally, more than half of the responders that attended or had attended school required an individualized learning plan (156). Overall,

there is a significant negative relationship between the number of metabolic crises and

patient neurological development and IQ (68).

While many of the underlying causal mechanisms of damage are unknown, much of the neuropathophysiology is known in PA. In a normal setting it was shown that cerebral arterioles are responsive to pH changes in extracellular fluid. Specifically, dilation resulting in cerebral edema occurs in normal cerebral arterioles at low pH, a known

occurrence in PA. Sustained dilation would lead to hypoperfusion, causing damage

similar to what is seen in urea cycle disorders. In addition to decreased cerebrovascular

resistance, edema may be the direct result of astrocyte vulnerability (43). Damage to this

cell type would occur from inhibition of the urea cycle, causing hyperammonemia,

another known event in PA. Furthermore, autopsy of PA patient brains show alterations

similar to what is seen in patients suffering from hyperammonemia and urea cycle

enzyme defects (40, 218). The basal ganglia seem to be especially vulnerable and are often damaged during metabolic attacks; brain metabolite concentrations are more severely altered during these metabolic crises (43).

While circulating propionate can enter the brain, locally synthesized toxic metabolites,

such as methylcitrate, cannot easily escape the blood brain barrier and thus likely

accumulate to neurotoxic levels. Autopsies of PA patient brains, who died during an

acute metabolic crisis, show hypoxic-ischemic changes, possibly due to altered energy

metabolism, perturbations in the CAC, metabolite trapping, CoA trapping, and/or a

deficient response to oxidative stress (101, 142, 177). Additionally, it has been shown

under various conditions that concentrations of metabolites in plasma are not reflective of

metabolite concentrations in the CSF. It is likely that due to the inability of many

substances to cross the blood brain barrier there is an accumulation of cerebrally

produced toxic metabolites, such as 3HP and methylcitrate. Thus, the concentration of

PA metabolites in the brain may be very different than what is measured in plasma or

other organs (101, 175). Increases in CSF metabolites related to energy metabolism, such

as lactate, even when patients are metabolically stable, suggest that oxidative

phosphorylation is disturbed, as seen in mitochondria (43, 175, 179).

1.2.2.2 Cardiac Abnormalities

There are numerous cardiac complications associated with PA, and only a minority of PA

patients present with cardiac abnormalities during a metabolic crisis. The absence of

correlation between cardiac abnormalities and metabolic decomposition, suggests

chronic, rather than acute, damage to the heart. Long-term altered energy balance in the

heart is possible as propionate, along with pyruvate, are the major anaplerotic substrates

for this organ (61). In one patient, cardiac hypertrophy developed prior to the classic

onset of PA symptoms (126). A retrospective study showed that 23% of patients

developed dilated cardiomyopathy with an average age of onset of seven years (169). All

patients in the retrospective study were under similar treatment strategies and had

comparable ages of diagnosis. Interestingly, this study showed normal L-carnitine plasma

levels, suggesting that cardiomyopathy may develop in the setting of apparent whole

body carnitine sufficiency (169). Due to the high rate of β-oxidation in the heart, carnitine

transport of fatty acids is a major determinant of mitochondrial function, and increased

carnitine requirement may be present. Decreased carnitine is a documented cause of cardiomyopathy (122, 212). It was noted on autopsy that cardiac tissue from PA patients

may present with decreased levels of carnitine in the setting of normal circulating plasma

carnitine levels and normal peripheral muscle carnitine concentrations. This, however, needs to be confirmed in a larger study. Currently, there is no mechanism to explain the suspected decreased carnitine uptake by the heart. Additionally, in the study, both cardiac and skeletal muscle acylcarnitine was low, despite skeletal muscle having normal free carnitine concentrations. If true, this would suggest gross anomalies in carnitine uptake and utilization, but these findings also need to be confirmed in a larger study (126, 169).

Currently no clear mechanism for cardiomyopathy in PA has been identified. Decreased oxidative phosphorylation is the suspected cause, though there is continued debate as to the role that toxic accumulation of metabolites plays in the development of cardiomyopathy (169).

Prolonged QTc interval, defined as greater than 440 ms, is also a frequent cardiac finding

in PA (11, 89). This is alarming as prolonged QTc interval is an independent risk factor for sudden cardiac death due to fatal arrhythmia. Both the risk of developing QTc prolongation and the length of the interval seem to increase with age. This would suggest that the underlying cause is an ongoing progressive process, possibly due to toxic metabolite accumulation and resulting damage, rather than a direct genetic effect due to mutation(s) in PCCA and/or PCCB. Repolarization abnormalities, palpitations, and supraventricular tachycardia have also been noted (126, 169). Numerous potential explanations exist such as a direct toxic metabolite effect and/or intracardiac depletion of propionyl-CoA anaplerotic pathway metabolites, namely methylmalonyl-CoA and succinyl-CoA, secondarily leading to depletions in CAC intermediate metabolites, resulting in an overall energy deficit (11). These hypotheses have yet to be fully explored.

Acidosis is known to decrease cardiac contractility and predispose patients to arrhythmias. Rat models of severe metabolic acidosis have shown reduced cardiac output which may also result in decreased organ perfusion and hypoxia (40).

There has been no established relationship between the severity of disease, including the

presence of neurologic and cardiac abnormalities, and clinical measures of PA, such as

genotype, the resulting enzymatic activity, clinical history, and/or biochemical

measurements. Whereas there are known discrepancies in CSF concentrations and

plasma/urinary metabolite concentrations due to the blood brain barrier, urinary

measurements are considered accurate measurements of cardiac tissue concentrations. It

is therefore assumed that plasma concentrations of metabolites are reflective of cardiac

concentrations of metabolites. To date, the metabolites produced as a result of PA do not

correlate with either the presence or severity of the patient’s PA phenotype (126, 169).

1.2.2.3 Additional Complications

Pancreatitis has been described in PA patients with and without symptoms of metabolic

decompensation. Though clinical presentation of pancreatitis is variable, amylase and

lipase levels are elevated in most PA patients. The mechanism underlying pancreatitis in this patient population is unclear but elevated triglycerides have been reported, and hypertriglyceridemia is a known risk factor for pancreatitis. Free fatty acids and odd chain fatty acids are not part of the standard metabolite panel in PA, so it is unknown whether elevations in free fatty acids is a common finding in PA (26, 156). As a buildup

of C15 and C17 straight chain fatty acids has been reported in the liver of PA patients, a similar accumulation in the pancreas of PA patients is possible (79). An alternate explanation for the increased susceptibility to pancreatitis in PA, is direct damage to acinar cells from the buildup of toxic metabolites (26, 156). Pathologic protease sensitization and ligand-induced activity within pancreatic acinar cells have been shown to occur under acidotic conditions, resulting in pancreatitis (87).

Immunodeficiency is suspected in PA, which may worsen the condition. Illness acts as a catabolic stressor, increasing metabolism and resulting in increased concentrations of toxic metabolites. Müller [1980, 551-552] described a patient with PA who presented with T and B cell depletion on autopsy. The patient history included multiple infections,

repeated episodes of leucopenia, lympopenia, granulopenia, and thrombopenia, as well as

low serum IgM. Additionally, the patient’s serum prevented normal lymphocyte activity,

suggesting a toxic metabolite present in serum. It is known that even chain fatty acids can

inhibit lymphocyte response, and it is possible that odd-chain fatty acids act in a similar

manner (139). Stork [1986, 783-788] described a stable PA patient who presented with altered bone marrow morphology, which resolved with dietary management. They

showed an association between propionate and hematologic abnormalities (193). There

was also a report of degeneration of macrophages in both the bone marrow and spleen in

a PA patient on autopsy (79). Recently, it has been suggested that 30-65% of all PA

patients have some sort of immunodeficiency (131). If immunodeficiency is a real

complication of PA, it would result in an increased risk of infection, and increased

catabolic stress. Instead, however, an altered immune state may be due to altered

nutrition, poor protein management, and/or increased protein breakdown, resulting in

hypogammaglobulinemia (218). A definitive link between PA and immunodeficiency has

yet to be confirmed or refuted. If present, more study would be needed to discover the

underlying cause.

PA has been shown to cause myopathy and ultrastructural changes of muscle

mitochondria. PA patient muscle tissue shows severely reduced ATP and

phosphocreatine concentrations, as well as increased lactate (179).

1.2.3 Biochemical Parameters

PA is characterized by hyperglycinemia and hyperglycinuria of unconfirmed etiology,

severe metabolic ketoacidosis due to the accumulation of organic acid metabolites, and

hyperammonemia due to urea cycle inhibition. Organic acids that are known to

accumulate in PA include, propionate, β-hydroxybutyrate, methylcitrate,

propionylglycine, tiglylglycine, and 3HP. Some report PA patients emitting an acetone

odor. Additionally, lactate is typically elevated along with an elevated anion gap. Urinary

propionate may be only slightly elevated or normal depending on the metabolic state of the patient when urine is collected. Serum propionate, however, is consistently elevated

(12, 218). Lehnert [1994, S68-S80] give a comprehensive overview of the various

compounds produced and the biochemical perturbations that occur in PA (112).

Hypoglycemia is a common finding during metabolic decompensation but hyperglycemia has also been described, and patients may develop insulin resistance (156). The dysregulation of glucose metabolism and presence of insulin resistance may be due to decreased responsiveness of insulin-mediated glucose metabolism in the setting of acidosis. Furthermore, in multiple organ systems glycolysis is inhibited in acidosis, due to pH regulation of phosphofructokinase. The suspected changes in energy balance have raised the question as to whether the increased lactate would stimulate gluconeogenesis, or whether the increased acidosis would inhibit gluconeogenesis (40). The carbon from proteolysis, which would occur as the result of increased protein ingestion or increased catabolism, is an additional source of glycogen which may exacerbate glucose metabolism dysregulation (36). Metabolite toxicity and enzymatic inhibition by methylcitrate, discussed in subsequent chapters, is another possible mechanism

underlying altered glucose metabolism. It is likely that a confluence of these various

contradicting factors result in the variability of glucose metabolism seen in PA patients.

Measurement of metabolites characteristic of PA, such as propionylcarnitine, 3HP, and methylcitrate, do not correlate well with the metabolic state of the patient. Instead acid-

base balance, plasma ammonia, and other indirect measures of PA are better predictive

indicators. Such measures also show better correlation with the severity of metabolic

decompensation episodes (222).

1.2.4 Diagnosis and Treatment

PA is a part of the recommended uniform newborn screening panel. Early detection, in

the neonatal period, may allow for initiation of treatment in clinically asymptomatic individuals, especially those with late onset PA (29, 47, 177). Depending on the US state, the NBS calls for measurement of primary and secondary tier metabolites such as

propionylcarnitine and methionine (29). Alternatively, enzymatic activity can be

measured in amniotic fluid cells allowing for prenatal diagnosis (218). More recently,

fetal DNA testing has been used in combination with measurements of metabolites in

amniotic fluid or amniocyte enzymatic assays to achieve a diagnosis. Often, a diagnosis

of PA in the neonate is confirmed by characterizing the underlying defect in skin

fibroblasts or by genetic analysis (12). However, even with early diagnosis and optimal

treatment neurologic impairment is still present to some degree (177). Additionally,

cardiomyopathy can develop even in the setting of aggressive metabolic control (169).

Current treatment strategies of PA involve: a strict low-protein diet with a supplemented

caloric intake and nocturnal feeding to prevent fasting, supplementation with L-carnitine,

and periodic treatment with antibiotics to minimize propionate producing intestinal microflora, a major source of free propionate (169, 200). Additionally, regular nutritional assessments are needed to prevent protein malnutrition and/or micronutritional

deficiencies (12). Diet restriction, however, is not completely effective because of an obligate amino acid catabolism requirement. Also, antibiotic usage, while effective at reducing propionate is not consistently effective, likely due to variations in gut

microflora. Metronidazole, either alone or in combination with colistin is currently the

standard of care for antibiotic treatment in PA (67). It must be noted however, that metronidazole alone is only moderately effective in reducing the production of propionate from the gut (200). More effective antibiotic regimens may exist.

The frequency of metabolic decompensations is typically highest in the first year of life, likely due to poor ability by the infant to tolerate even small periods of fasting.

Decompensated PA patients should immediately be supplied with fluids to prevent dehydration, as even a mild dehydration can worsen the symptoms due to the resulting gross metabolic changes. Long term neurological outcomes are related to both the duration of the metabolic decompensation episode and blood ammonia levels. Therefore, treatment should be initiated based on presenting symptoms, rather than waiting for metabolic testing (12, 67, 218). Unfortunately, the mainstay of PA treatment, limitation of intake of propiogenic amino acids to the minimum daily requirement, has shown only limited effectiveness in preventing the neurologic sequelae common in PA (67).

Chapman [2012, 16-25] detail complete recommendations for the management of PA patients in an acute metabolic decompensation episode (33). Briefly, recommendations involve (i) stabilization of the patient using standard life support techniques, (ii) reversal of catabolism through the cessation of all sources of protein, for no more than 36 hours, and (iii) supplementation with non-protein calories, such as intravenous glucose or oral

polysaccharides, to maintain normal caloric intake. Insulin has been used sporadically to

further drive glucose into cells and reverse catabolism. Additional measures include,

carnitine supplementation which should be started or increased, initiation of an antibiotic

protocol, initiation of hemodialysis if necessary/appropriate to detoxify blood, and/or N-

carbamylglutamate, Carbaglu, or sodium benzoate/sodium phenylacetate treatment to

correct clinical hyperammonemia. Phenylacetate conjugates with glutamine in the liver; the resulting phenylacetylglutamine is excreted in urine. Ammonia concentration should be checked every three hours, since as many as 95% of PA patients presenting with an acute decompensation episode have hyperammonemia on admission (12, 34, 67, 164,

218). Al-Hassnan [2003,89-91] have raised concern about the use of sodium phenylacetate due to possibly altered nitrogen metabolism in this patient population.

Sodium phenylacetate may also decrease CoA levels in PA patients (1).

Treatment with sodium bicarbonate was quickly proven to be ineffective because the

acidosis seen in PA is due to an accumulation of propionate and other metabolites as the

result of the inability to fix carbon dioxide on Propionyl-CoA (79).

Biotin supplementation has resulted in only subclinical or anecdotal improvements in

patient quality of life and outcomes (216, 218).

Orthotopic liver transplant (OLT) has been shown in some patients to reverse or at least halt further organ damage and cardiomyopathy with the possibility of prolonging

survival, likely in part due to reduced toxic metabolite plasma concentrations. It must be

noted that transplantation likely has little direct effect on extrahepatic production of toxic

metabolites (33, 169). While liver transplant may decrease or eliminate the metabolic

acidosis suffered by PA patients, neither urinary methylcitrate, nor serum and CSF

propionylcarnitine, were significantly changed after liver transplant in the PA patients

studied, though a larger cohort is needed (91). Furthermore, PA patients are

recommended to maintain the low protein diet and carnitine supplementation post-

transplant. Even if the diet is relaxed, meat must still be avoided (33, 91). Short-term

outcome studies suggest that donor livers from PCC haploinsufficient living relatives

give similar outcomes to PA patients receiving transplants of livers with full PCC activity

(196). Liver transplant is a high risk procedure in this patient population. Seven of 23

children died from various post-liver transplant complications (30% mortality rate) (9,

195). Additionally, as stated in Charbit-Henrion [2015, 786-791], centers report a 5 year

graft survival rate of only 60% in this patient population. Interestingly, renal failure

seems to increase in this patient population post-OLT, suggesting a possible correlation

between rescue of propionate metabolism and aberrations in renal function, in the setting

of PA (35). An explanation for this association is explored in subsequent chapters.

Because of the limited clinical data associated with OLT in PA patients, as well as recent

changes in post-transplant patient management, periodic re-evaluation of liver transplant safety and organ efficacy are needed. Firm conclusions as to efficacy of OLT cannot be

made for PA. However, liver transplant is unlikely to be a “curative” measure.

As stated previously, neurologic damage is likely due to local CNS metabolism and metabolite trapping rather than systemic metabolism. The effects of OLT are unknown in regards to neurological damage, though de Baulny [2005,415-423] summarized numerous reports citing that OLT either only delayed neurological deterioration or shifted the presentation from acute to a chronic insidious progression (9). The brain has a low capacity for carnitine conjugation, thus carnitine supplementation in this patient population is not expected to significantly impact the neurologic complications noted in

PA (101). It is possible that CNS metabolite concentrations differ from plasma levels, thus magnetic resonance imaging in this patient population may be prudent (206). Some even recommend that a detailed neurological examination should be a part of every visit to a metabolic clinic prior to the onset of symptoms, rather than the infrequent neuroimaging currently practiced (12, 175). More research into the efficacy and utility of frequent neurologic assessments compared to the burden on patients is needed.

It is unclear whether carnitine supplementation provides any protection for cardiac tissue

(126). Baumgartner [2014, 130] recommend at least yearly ECGs in PA patients to determine QTc interval and regular 24-hr Holter monitoring to check for ventricular arrhythmias, with the specific interval between measurements based on individual needs/circumstances (11, 12).

There should be a low threshold to investigate PA patients for pancreatitis as this is a life- threatening complication that is more common in PA patients than in the general pediatric population (26, 88, 156). Measurement of amylase and lipase is recommended on a yearly basis. The standard of care for pancreatitis is appropriate in the PA patient population (12).

Though there is a suspicion of immunologic complications in the PA patient population

there is no increased risk to these patients from vaccination (96, 131).

A proposed addition to the standard of care is antioxidant supplementation, such as tiron,

trolox, resveratrol, and/or MitoQ. There is the possibility of increased oxidative stress in

this patient population (12, 58). Any true clinical efficacy from this treatment is unknown

as no large studies have been performed.

It is possible that PA patients suffer from metabolic derangement even prenatally. In

addition to the presence of PA metabolites in amniotic fluid, two fetuses with PA showed

large concentrations of odd-chain fatty acids, likely due to aberrant propionyl-CoA

metabolism, in utero, in various tissues (211). It must be noted that the transplacental

gradient of free amino acid transport is in favor of the fetus, so that a PA suffering fetus

sees very high concentrations of propionyl-CoA generating amino acids (181).

Alternatively, the high concentration of odd-chain fatty acids may be protective, acting as

non-toxic fetal storage of propionyl-CoA. High propionyl-CoA and/or odd-chain fatty acids may explain the common occurrence of neonatal metabolic crisis, as the rapid increase in catabolism in the neonate may cause increased production of propionyl-CoA through breakdown of odd-chain fatty acid stores, see Figure 1.2 (211). As fatty acid profiling is not common in PA metabolite panels, concentrations of these metabolites in plasma are unknown. It is possible that prenatal diagnosis of PA may be important in ameliorating symptoms. No studies have been conducted to assess the amount of prenatal injury in PA, nor on the effects of prenatal treatment for the PA fetus.

1.2.5 Patient Outcomes Post Newborn Screening

PA patients detected by newborn screening (NBS) showed minor improvement in non-

fatal outcomes, when treated upon identification as compared to PA patients diagnosed

based on clinical findings, and subsequent metabolic analysis. Mortality rate, however,

has decreased greatly in PA patients identified by NBS, with a large cohort of 55 total PA

patients studied by Grunert [2012, 41-49] showing no deaths in the neonatal period when

patients were identified by NBS. Overall, they showed a mortality rate of 8% in early

onset patients. Detection of PA by NBS, however, did not prevent neonatal metabolic decompensations. This is likely due to the fact that early onset PA patients are typical

Figure 1.2. β-oxidation of Odd-Chain Fatty Acids. n is an odd number.

symptomatic by the time a positive NBS result of PA is received. Interestingly, in 2/3rds

of the neonatally asymptomatic patients examined, NBS detection did not prevent a

subsequent metabolic crisis, suggesting that NBS detection does little to prevent the

initial metabolic crises PA patients suffer, or the resulting damage. Furthermore, NBS

detection did not change the number of metabolic decompensations suffered. Overall,

NBS detection had no significant improvement on the physiological complications seen

in PA compared to identification by clinical presentation. It must be noted that the

average age of participants in the study was approximately four years of age. NBS

detection of PA has only been implemented for approximately 15 years, as such, this

limits the ability to draw conclusive inferences on the long-term effectiveness of NBS

detection of PA (68).

Estimate of mortality from PA were as high as 40%, when the disease was first classified.

This number has steadily declined with advances in treatment and detection. Even with

the most aggressive treatment strategies, however, as of 2015 mortality from PA has been

reported to be as high as 17%. At least half of these deaths occur in the neonatal stage

(35).

1.3 Methylmalonic Acidemia

1.3.1 Overview

Methylmalonic Acidemia (MMA) was first described in 1967 in two severely ill infants

who presented with extremely high concentrations of methylmalonate in blood and urine

as well as metabolic ketoacidosis and developmental retardation (149, 192). It is

characterized by defective conversion of methylmalonyl-CoA to succinyl-CoA, and while

pernicious anemia is a known cause of increased methylmalonate, MMA patients are

distinct in the multiple times greater excretion of methylmalonate. Additionally, MMA

patients do not present with the hematologic and neurologic sequelae typical of

nutritional cobalamin deficiency (8, 38, 149). MMA, like PA, is an autosomal recessive

disease, with most patients presenting as compound heterozygotes (86, 110, 214). The

incidence of MMA has been estimated to be between 1:48,000 to 1:100,000, with reports

suggesting it is as common as PA (42, 120, 124).

Methylmalonyl-CoA is essentially only synthesized in the mammal from propionyl-CoA

(50). In 1955, methylmalonate, via methylmalonyl-CoA, was shown to be an intermediate in conversion of propionate to succinate (55, 93). The mechanism involves

formation of S-methylmalonyl-CoA by PCC, which is then racemized to R-

methylmalonyl-CoA by methylmalonyl-CoA epimerase (MCEE, EC 5.1.99.1). Lastly,

methylmalonyl-CoA mutase (MUT, EC 5.4.99.2) catalyzes the isomerization of R-

methylmalonyl-CoA to succinyl-CoA, see Figure 1.1 (129). MMA is closely related to

PA in clinical defect, biochemical perturbations, and resulting phenotype.

1.3.1.1 Methylmalonyl-CoA Epimerase and Methylmalonyl-CoA Mutase

There are a number of distinct impairments of methylmalonyl-CoA metabolism that can cause MMA. MMA patients can have a defect in MUT, which can either produce a complete deficiency, designated mut0, or a partial deficiency, designated mut-. More

rarely, the defect is in MCEE. Alternatively, the defect can be in the synthesis of the

active form of cobalamin, due to loss of function of either the mitochondrial reductase or

the adenosyltransferase. Finally, defects of abnormal cytosolic or lysosomal metabolism

of cobalamin can result in MMA. Despite the many possible defects that cause abnormal

methylmalonyl-CoA metabolism all are classified as MMA disorders. This work focuses

on MMA resulting from defects in MUT or MCEE (124).

1.3.1.1.1 Methylmalonyl-CoA Epimerase

Methylmalonyl-CoA epimerase, also known as methylmalonyl-CoA racemase, is encoded by the MCEE gene, and catalyzes the racemization of S-methylmalonyl-CoA to

R-methylmalonyl-CoA. Regions of this gene are high conserved, suggesting physiological importance (64).

Mutations in MCEE, may account for approximately 2% of all case of MMA, and patients seem to be predominantly homozygous for the mutation (64). One possibility for the low occurance of reported disease causing MCEE mutations is that a subset of these patients are misidentified (48). Another possibility for the low prevalence of pathologic mutations in this gene is the questionable physiological necessity of the enzyme.

Mazumder [1962, PC53-PC55] showed, in vitro, that racemization of methylmalonyl-

CoA occurs in the absence of MCEE, and Montgomery [1983, 1937-1947] further confirmed racemization of methylmalonyl-CoA in the absence of epimerase in the rat

(129, 136). The presence of disease causing mutations does suggest, however, at least partial requirement of MCEE.

Mutations in MCEE are a very rare cause of MMA, with a number of clinically non- pathologic mutations documented. Many of the patients affected with MMA with a documented mutation in MCEE were from consanguineous mating, which adds the possibility of confounding recessive diseases (48, 64).

1.3.1.1.2 Methylmalonyl-CoA Mutase

Methylmalonyl-CoA mutase catalyzes the isomerization of R-methylmalonyl-CoA to succinyl-CoA. It is encoded by the MUT gene and is a homodimer of identical subunits which requires 1 mol of cobalamin per mole of subunit (55). Mutations in MUT leading to complete loss of function account for approximately 2/3rds of MMA patients (106, 137,

213, 214). Mutations resulting in partial loss of activity result in an enzyme having activity as low as 2% compared to control. Clinically significant MMA has been documented with up to 75% preserved enzyme activity compared to controls.

Furthermore, mutations can cause a hundreds- to thousands-fold increase in Km for cobalamin, and increased instability of the enzymes and/or binding (137, 213, 214). Most pathologic mutations occur in the cobalamin binding domain explaining the altered affinity for cobalamin and the decreased activity of the enzyme (124).

Though activity of this enzyme is mitochondrial, the subunit is synthesized as a cytoplasmic precursor, as is PCC, prior to being transported into the mitochondria in an energy dependent manor (51). Failure of MUT uptake and/or cleavage is an extremely rare mutation resulting in MMA. Though possible, there are no documented cases of similar PCC uptake failure resulting in PA (52, 109).

1.3.2 Signs and Symptoms

Despite the specific defect, there is great similarity in clinical presentation across MMA disorders, there is also significant overlap with the presentation of PA as they are both disorders of propionyl-CoA metabolism. Briefly, at onset of clinical symptoms patients

25 typically present similarly to PA, with clinical symptomology associated with organic acidemias such as lethargy, failure to thrive, vomiting, and dehydration. Those with full deficiency of methylmalonyl-CoA mutase typically present earliest in life with 90% of these patients presenting clinically in the first month of life (128). As with PA, MMA infants are normal appearing at birth, and have periods of metabolic decompensation triggered by stress (221).

1.3.2.1 Renal Complications

One report suggests that greater than 65% of MMA patients suffer from reduced or greatly reduced glomerular filtration rate (210). The common progression is that of proximal and/or distal tubular dysfunction advancing to chronic tubulointerstitial nephritis and end stage renal disease (125). Post-OLT MMA patients remain at risk for renal complications, ultimately needing dialysis or transplant (105, 147, 210). A common complication of renal failure in this patient population is anemia, and should be suspected when renal failure presents (119). It is estimated that kidney transplant can restore up to

18% of MUT activity, though in one study 3 out of 4 patients showed signs of chronic kidney disease in the new organ within 55 months post-transplant (23).

Importantly, renal impairment is an early complication of MMA and has been seen within the first six months of life (149). Frequent monitoring of kidney function, via measurement of creatinine and glomerular filtration rate, as well as regular imaging of the kidneys, is recommended to allow for early treatment (124). Some reports suggest, however, that renal failure from MMA has poor correlation between creatinine and

glomerular filtration rate. This may be due to the reduced muscle mass and growth rate in

this patient population. This provides additional challenge for the early detection and

treatment of renal disease in MMA (125, 210). Measures of renal growth and serum

methylmalonate have been suggested as an alternative, as Kruszka [2013, 990-996]

showed that renal growth in MMA patients was as low as 1/3rd that of normal controls.

Broadly, renoprotective strategies in the pediatric population is a poorly investigated area

(105).

1.3.2.2 Neurological Complications

Neurological complications have been associated with MMA, including, intellectual

impairment or regression, metabolic stroke-like episodes manifesting as movement disorders or paralysis, and optic nerve atrophy. As with PA, the basal ganglia is most susceptible to damage (124). Reports suggest that the risk of metabolic stroke-like episodes in this patient population is between 17-30% (10, 80). Interestingly, rather than being a uniform process, distinct segments of the globus pallidus and substantia nigra of the basal ganglia are affected, suggesting that regional sensitivity is a factor in the as yet unknown mechanism of infarct. Furthermore, these sensitive regions were affected sequentially, providing additional evidence of varying vulnerability in the brain to damage by MMA (6). In a report from Harting [2008, 368-378] all four of their patients showed brainstem and cerebellar changes and/or atrophy (74). Altered myelination and subcortical changes in the white matter seem to be an increasingly common documented complication associated with MMA (74, 163). MMA patients suffer from altered brain maturation in the form of myelination delay, noted in approximately 48% of the patients

studied in one report (11 out of 23), as well as immature, abnormal, and/or delayed brain

patterning (24, 74).

Intellectual disability seems to be variable in MMA. In a retrospective study, only 50% of

MMA patients with the most severe form, complete loss of function of MUT, had an IQ below 80 (10). The mean IQ of MMA, however, is 85, which is low normal. It is likely that rather than a direct effect of enzyme deficiency, the recurrent metabolic attacks and hyperammonemia lead to intellectual impairment (148). The long term effects of MMA detection by NBS on IQ are unknown.

Ophthalmologic findings and optic nerve atrophy associated with MMA are increasingly being reported (203). Regular ophthalmology evaluations are recommended in this patient population (124).

As further proof that transplant, liver and/or kidney, does not cure disease, development of lesions in the brain independent of metabolic decompensations and after successful transplant occur. This is likely due to the trapping of metabolites in the CNS from inability to cross the blood-brain-barrier. Methylmalonate remained high in the CNS of

MMA patients post-OLT (30, 90, 149, 207).

1.3.2.3 Additional Complications

Immune abnormalities are also common in this disorder with greater than 50% of patient history including leukopenia and/or thrombocytopenia (50). Pancytopenia has also been reported (127). This would have severe effects on patient susceptibility to infection, a

trigger of metabolic decompensation. Specifically, the occurrence of bone marrow

abnormalities have been associated with metabolic crises in MMA, though it is unknown

whether the cytopenias occur prior to, or as a result of, metabolic crisis. Evidences seems

to suggest that a toxic metabolite of MMA is responsible as peripheral counts recover

after normalization of the metabolic state (119). Monitoring of bone marrow indices and

overall bone health is recommended in this patient population (124).

Pancreatitis is a documented complication in MMA but with an unknown incidence rate.

As with PA, because of the non-specific presentation of initial pancreatitis symptoms,

that of vomiting and abdominal pain, the clinician should have a low threshold for

investigation (124).

Infant and child growth and development are decreased in the MMA patient population, though likely as a result of chronic illness and restrictive protein diet than as a direct effect of the disease. It is likely that renal failure exacerbates protein deficiency to a greater extent than what is predicted by diet alone. MMA infants may be less than three standard deviations below normal for both length and weight (31, 124).

1.3.3 Biochemical Parameters

MMA is characterized by normal serum cobalamin concentrations and huge concentrations of methylmalonate in blood and urine (50). MMA is a disorder affecting metabolism of propionyl-CoA so the vast majority of precursors of PA also accumulate in MMA, such as propionylcarnitine, 3HP, and methylcitrate. Additionally C4- dicarboxylic carnitines, namely methylmalonylcarnitine, are present (124).

Measurements of methylmalonate concentration and/or the specific deficiency do not

correlate with disease severity. As with PA, however, secondary metabolites do show good correlation with disease severity (50, 221). Of note, a possible confounder of metabolite measurement and disease is variable renal function, which is known to have an influence on plasma methylmalonate (105, 124). Creatinine is abnormal in MMA and

is likely not the best measure of renal function in the MMA patient population (105).

As with PA, MMA patients also present with acidosis, hypoglycemia, hyperglycinemia,

and hyperammonemia (50). MMA patients also tend to develop hyperuricemia, especially

during metabolic crisis and possibly as a result of the effects of toxic metabolites on renal

function (221).

1.3.4 Diagnosis and Treatment

MMA is part of the recommended uniform newborn screening panel, and early detection

may allow for presymptomatic treatment. Initial diagnosis is dependent upon organic acid

analysis in plasma and/or urine by mass spectrometry. Identification of the specific defect

in MMA is dependent on the evaluation of cobalamin uptake and formation of activated

cobalamin, oxidation and utilization of labeled propionate, measures of enyme activity in

cells, and/or genetic identification of the defect (50, 124). Use of skin fibroblasts for

biochemical testing is considered the gold standard. A complete overview of the clinical

workup for a newborn suspected to have MMA is presented in the review by Manoli

[2016, 1993-2007] and separately in the review by Fowler [2008, 350-360] (57, 124).

It is possible that MMA patients suffer from metabolic derangement even prenatally, as

plasma and urine gestational concentrations of MMA are many times greater than

normal. Gestational exposure to toxic metabolites of MMA may explain the initial

metabolic episode that occurs soon after birth (31). Prenatal treatment of MMA is not

currently an area of investigation.

Treatment of the acute life-threatening symptoms of a metabolic decompensation episode in MMA does not differ greatly from PA. Treatment of MMA once the initial metabolic decompensation episode has been managed involves initiation of a protein restricted diet and supplementation with cobalamin, as a subset of MMA patients are cobalamin responsive (50). Though no standard treatment protocol has been developed for cobalamin therapy in MMA, recommended strategies include supplementation with approximately 1.0mg hydroxocobalamin either intramuscularly or intravenously every day for 1-2 weeks. A greater than 50% reduction in methylmalonate production/plasma concentration should be considered a positive response (57, 105).

In cobalamin-responsive patients, cobalamin administration can considerably reduce the disease burden (81). Carnitine supplementation has similar effects and mechanism of action as in PA. It results in increased acylcarnitine accumulation, especially propionylcarnitine, with concomitant reduction in urine methylmalonate and methylcitrate (167). Antibiotics have shown effect in improving symptoms, by reducing the amount of propionate precursor from the gut (5, 99). Long-term management is similar to PA, with the possible addition of periodic hydroxocobalamin intramuscular injections (124).

Antioxidant supplementation to treat optic nerve atrophy is currently being investigated.

Treatment of a single patient with Coenzyme Q10 and Vitamin E showed clear efficacy,

however subsequent studies suggest effectiveness is variable and patient dependent (124,

159, 203). A role for antioxidant supplementation in the management of renal failure is

also under investigation (125).

Even with aggressive treatment, the first presentation of MMA symptoms in the newborn is life-threatening, termed catastrophic decompensation. This is in part because initial

treatment is often non-specific for MMA, or the disease is misdiagnosed (39, 124).

OLT in MMA patients provides protection against metabolic decompensations but not the renal failure and other sequelae (113, 143). Additionally, kidney or combined liver and kidney transplant are emerging avenues of treatment, but more research into the efficacy are needed (124). The frequency of metabolic decompensations improves with liver transplant, but methylmalonate concentration, and a number of other biochemical parameters, remains high, likely from continued extrahepatic production (90, 147, 188).

Isolated kidney transplant is likely not efficacious in this patient population, as no valid reports have shown any great benefit from isolated kidney transplant. The effects of organ transplant on survival are unknown, but protein restriction should continue post- transplant (124, 188).

1.3.5 Patient Outcomes Post Newborn Screening

NBS of MMA, as with PA, uses propionylcarnitine as the primary analyte assayed. To differentiate MMA, and improve sensitivity, use of secondary analytes and

32 primary:secondary analyte ratios, such as C3/C2 and C3/C16, are recommended (116).

Also, similar to PA, a limitation in determining long-term outcomes of NBS is the lack of long-term follow-up in this patient population, for example in one study the oldest patient detected by NBS was 6.7 years old at the time of the study (47).

One clear effect of NBS on the progression of MMA is the decrease in basal ganglia injury that would lead to movement disorders or paralysis in the patient (47, 77, 104).

More broadly, global changes to the brain in MMA patients seem to be decreased with early detection by NBS. In the study by Radmanesh [2008, 1054-1061], four children with MMA were detected by newborn screening and three had normal imaging results while the forth had only “mild, ventricular dilation”; in total 17 out of 52 children diagnosed with MMA showed ventricular dilation. It is important to note that atrophy and dilation are hard to diagnose in patients under 2 as changing brain:head circumference can distort normal anatomy (163). Early management of hyperammonemia due to newborn screening seems to be an important aspect of increased IQ in MMA, as treatment after onset of symptoms does not result in improved neurocognition (148).

Chapter 2

Metabolism of Propionate

2.1 Normal Metabolism of Propionate

Propionyl-CoA, the CoA ester of propionate, is (i) an intermediate in the degradation of

various amino acids, namely isoleucine, valine, methionine, and threonine, (ii) the end

product of the β-oxidation of odd-chain fatty acids, and (iii) a catabolite of cholesterol side chains, of thymine, and of uracil (82, 173). Propionyl-CoA is converted to S- then R- methylmalonyl-CoA, through fixation of carbon dioxide. Methylmalonyl-CoA is then reversibly isomerized to succinyl-CoA, which enters anaplerotically into the CAC, see

Figure 2.1 (12, 79, 82). Propionyl-CoA can replace acetyl-CoA as a primer of fatty acid synthesis, generating odd-chain fatty acids. Propionate itself is a gluconeogenic compound as it can be converted to PEP via oxaloacetate. (50).

There are multiple sources of propionate in the body. Protein catabolism accounts for a maximum of approximately 70% of total propionate, while intestinal microflora can account for approximately 20% of propionate production, though the contribution from gut bacteria is heavily dependent on the individual’s microbial profile (200). Importantly,

the vast majority of propionate formed from intestinal fermentation is cleared during the

first pass of portal vein blood through the normal liver (162). In normal individuals, the

systemic plasma concentration of propionate is typically less than 10 µM (161).

Figure 2.1 Summary of Major Propionyl-CoA Metabolic Pathway in the Liver. The numbers refer to the following enzymes: 1, propionyl-CoA carboxylase; 2, methylmalonyl-CoA epimerase; 3, methylmalonyl-CoA mutase. Kasumov, T. et al. Arch. Biochem Biophys. 463(1): 110-117, 2007.

2.2 Metabolism of Propionate and Metabolic Toxicity in Disorders of Propionyl-

CoA Metabolism

In PA and MMA there is a buildup of both propionate and its amino acid precursors.

These propionyl-CoA precursors were shown to directly cause vomiting and ketoacidosis

(5). Increased propionyl-CoA from amino acids and fatty acid oxidation is a major reason to avoid fasting in disorders of propionyl-CoA metabolism (199, 200).

Propionate has varied effects on the CNS. Propionate interferes in intermediate filament phosphorylation, as shown in astrocyte populations. Propionate is also thought to be directly toxic to white matter through preferential metabolism to propionyl-CoA by glial cells, leading to buildup of toxic metabolites in this subset of CNS cells, namely astrocytes and oligodendrocytes. Propionate was shown to affect cerebral gene expression through interference with histone acetylation. While it is unlikely that neurons can directly activate propionate to form propionyl-CoA, formation of propionyl-CoA from amino acid precursors in this cell population is a possibility. Overall, though propionate enters the brain, global cerebral metabolism of propionate directly is low

(142).

In the muscle of PA patients, there is a disruption of energy metabolism as well as structural changes to the mitochondria. Furthermore, propionyl-CoA, but not propionate, inhibits the pyruvate dehydrogenase complex, the α-ketoglutarate dehydrogenase complex, and complex III in vitro (21, 25, 179). High concentrations of propionate resulted in a decrease in liver acetyl-CoA (191). It has been proposed that altered propionyl-CoA:free CoA ratio in PA may promote the inactive state of the pyruvate dehydrogenase complex, as opposed to direct competitive inhibition (21, 155). Though the free CoA concentration has known effects on pyruvate dehydrogenase complex,

Matsuishi [1991, 244-253] showed respiratory inhibition by propionate when 61% of the concentration of free CoA seen in control was present in isolated mitochondria (128). It is unclear the extent to which the inhibitory effects of propionyl-CoA, especially on pyruvate dehydrogenase complex, contribute to energy failure, seen as decreased ATP and phosphocreatine in patients, and also as increased lactate due to increased anaerobic

respiration (179). Propionyl-CoA may also decrease NADH cytochrome c reductase

activity in both cardiac and skeletal muscle, causing complex I deficiency (126). The

exact mechanism of energy failure is still an area of debate.

There was also inhibition of succinyl-CoA synthetase in the presence of propionate,

though the data seems to suggest that the inhibition is due to a subsequent metabolite

rather than propionate itself. This may also affect GTP dependent metabolism (194). One

metabolite from propionate that may be responsible for this inhibition, 3HP, is discussed

in subsequent chapters.

Methylcitrate, a metabolite formed as a result of propionyl-CoA condensation with

oxaloacetate by citrate synthase, likely has an effect on CAC metabolism (2, 3, 37). It is

important to note that the rate of utilization of propionyl-CoA by citrate synthase is likely

very low under normal conditions. In homologous enzymes from pig, propionyl-CoA

showed 0.1% of the rate of utilization, compared to acetyl-CoA, by citrate synthase; though both isomers of methylcitrate were produced even under physiologic conditions

(27, 190). Methylcitrate is suspected to use the mitochondrial citrate transporter to pass between cytoplasm and mitochondria to act as an inhibitor in both cytoplasmic and mitochondrial processes. It is known to inhibit citrate synthase, aconitase, and isocitrate dehydrogenase, which may cause transient increases in acetyl-CoA resulting in increased ketogenesis. Furthermore, alterations in citrate availability may lead to dysregulation of glycolysis. Methylcitrate is also a known inhibitor of phosphofructokinase, see Figure 2.2

(2, 3, 37).

Figure 2.2 Sites of Effect by Methylcitrate. The numbers refer to the following: 1, phosphofructokinase; 2, citrate synthase; 3, aconitase (note: aconitase showed non-competitive inhibition); 4, isocitrate dehydrogenase; 5, mitochondrial citrate transporter; 6, acetyl-CoA carboxylase (note: acetyl-CoA carboxylase was activated by methylcitrate) Cheema-Dhadli, L. et al. Pediat. Res. 9: 905-908, 1975.

Multiple mechanisms to explain the hyperglycinemia in PA and MMA have been

proposed. Isocitrate may accumulate in disorders of propionyl-CoA metabolism due to inhibition of the CAC and may subsequently be converted to glyoxylate and succinate, and succinate may then be converted to glycine (2, 3). Also, an enzyme converting

glyoxylate to glycine has been identified in mammalian liver (141, 198). However,

14 multiple studies have shown decreased release of CO2 from radiolabeled glycine in

liver, suggesting that the major mechanism of hyperglycinemia in PA and MMA is

decreased activity of the glycine cleavage system, rather than increased synthesis (182,

197). A small study on a single PA patient showed reduced protein concentrations,

resulting secondarily in decreased activity, of the various enzymes in the glycine

cleavage system. These findings bring into question whether hyperglycinemia is due to

decreased transcription and/or translation of the cleavage system enzymes as opposed to

an inhibitory action on one or more enzymes in the system directly. It is unknown

whether decreased enzyme activity of the glycine cleavage system was due to decreased

protein synthesis or increased protein degradation in the patient (138). Alternatively, it

has been shown in normal fibroblasts that incubation with tiglic acid, a metabolite that

accumulates in PA and MMA, inhibits glycine conversion to serine, without a similar

inhibition on serine to glycine conversion (78). Finally, the hyperglycinemia may be due to altered nitrogen balance in disorders of propionyl-CoA metabolism (50).

There is a likely buildup of odd-chain fatty acids in PA and MMA, but it is unknown whether this buildup occurs due to decreased catabolism of these fatty acids or increased synthesis due to the high propionyl-CoA concentration (79). It is known that propionate decreases palmitate oxidation, which is relieved by carnitine supplementation (21). It has also been shown that propionate inhibits ketogenesis from short and medium chain fatty acids. Carnitine supplementation relieves the inhibition of propionate on ketogenesis, but has no effect on ketogenesis independent of propionate. This suggests that carnitine affects ketogenesis indirectly, by overcoming the carnitine drain from propionate/propionyl-CoA metabolism (20). Additionally, carnitine increases pyruvate oxidation when in the presence of propionate (21). However, because neither

measurements of propionate nor of propionyl-CoA were conducted in these studies there is a small possibility of an unknown effect of carnitine supplementation acting to relieve

39 the inhibition, rather than carnitine acting via the classic mechanism of propionate/propionyl-CoA removal.

Carnitine conjugates with propionyl-CoA to increase transport out of the mitochondria, resulting in excretion in urine and increasing the free CoA pool. This results in carnitine deficiency in PA and MMA patients and is the clinical rationale for carnitine supplementation (67, 164). It is unclear as to the effectiveness of directly increasing CoA concentrations in PA and MMA, as compared to decreasing propionyl-CoA concentrations. Measured increases in hepatocyte CoA concentrations, via agents such as clofibrate which increase total CoA and carnitine acetyltransferase activity, were shown to relieve inhibition on pyruvate oxidation, a possible measure of toxicity, yet also resulted in increased propionyl-CoA (19, 187). The increase in propionyl-CoA is likely a direct result of increased CoA substrate from the treatment. Direct increases in CoA may actually worsen disease. Furthermore, propionylcarnitine synthesis only increased 2.5 fold compared to the 20 fold gross induction of hepatocyte carnitine acetyltransferase activity. This suggests that in the presence of high propionate, propionylcarnitine production is already at or near maximal efficiency (19). This is unsurprising as high doses of carnitine are given to PA and MMA patients in order to relieve carnitine deficiency, rather than to directly increase propionylcarnitine formation (20).

Livers of cobalamin deficient rats were shown to have an altered CoA metabolism with increased CoA biosynthesis, possible increases in intramitochondrial CoA, decreased cytosolic CoA, decreased acetyl-CoA, and buildup of propionyl-CoA and methylmalonyl-CoA. This is suggestive that complex metabolic changes in the total CoA pools occur in PA and MMA (22). It has been shown that even numbered long-chain

acylcarnitines increase CoA biosynthesis and it is possible that propionylcarnitine acts in

a similar manner (115). However, supplementation in an attempt to increase free CoA,

rather than decrease carnitine deficiency, again, may worsen outcome. Though it would

increase free CoA, this may result in increased propionyl-CoA and subsequent toxic metabolites, by providing additional substrate. This would lead to continued inhibition of acetyl-CoA production, CoA trapping, and no large increase in propionylcarnitine excretion (133). Further studies on the effectiveness of increasing CoA directly in disorders of propionyl-CoA metabolism are needed.

PA and MMA can be classified as CoA Sequestration, Toxicity, and Redistribution

(CASTOR) diseases. In order to function in biochemical reactions most organic acids must first be activated by forming a CoA thioester. This activation is hindered by the

CASTOR phenomenon. CASTOR occurs due to the buildup of acyl-CoA species such as propionyl-CoA, methylmalonyl-CoA, and 3HP-CoA, a metabolite discussed in more depth in subsequent chapters, and the secondary decrease in acetyl-CoA and free CoA.

This is of additional note to the clinician during treatment. Many drugs are formulated as organic acids for the ingested compound and/or gain activity via conversion to an organic acid within the body. The presence of CASTOR can greatly affect activity of these drugs.

Uninformed drug treatment of a patient with a CASTOR phenotype can result in Reye syndrome-like episodes (133).

Chapter 3

Metabolism of Methylmalonate

3.1 Normal Metabolism of Methylmalonate

Methylmalonate, the free acid of methylmalonyl-CoA, is involved in propionyl-CoA

metabolism. As stated previously, propionyl-CoA is converted to S- then R- methylmalonyl-CoA and R-methylmalonyl-CoA is then reversibly isomerized to succinyl-CoA. Methylmalonate is largely produced by the liver and kidneys, though there is modest contribution to body concentration of methylmalonate by skeletal muscle. The brain produces low amounts of methylmalonate normally, and the blood-brain-barrier is impenetrable to dicarboxylic acids such as methylmalonate (31, 101). There is evidence of a free methylmalonate shunt in mammals that bypasses methylmalonyl-CoA epimerase, see Figure 3.1. Though there is evidence of spontaneous racemization, the presence of disease causing mutations in MCEE confirm that this parallel metabolic process is not sufficient to handle the accumulation of methylmalonyl-CoA in a normal diet (136).

Physiologically, the concentration of methylmalonate is 0.27 µM in plasma and 0.59 µM in cerebrospinal fluid (130). Mitochondria have a very high affinity for methylmalonate and concentrations of methylmalonate are 3 to 9 times higher in the mitochondrial matrix than in the cytosol or other cellular compartments (202).

Methylmalonate

Figure 3.1 Presence of a Methylmalonate Shunt in the Propionyl-CoA Metabolic Pathway in the Liver. The numbers refer to the following enzymes: 1, propionyl-CoA carboxylase; 2, methylmalonyl-CoA epimerase. Adapted from Kasumov, T. et al. Arch. Biochem Biophys. 463(1): 110-117, 2007.

3.2 Metabolism of Methylmalonate and Metabolic Toxicity in Disorders of

Propionyl-CoA Metabolism

Methylmalonate seems to have multiple effects on energy homeostasis. In additional to

CoA trapping by methylmalonyl-CoA, it is known that methylmalonyl-CoA can inhibit pyruvate carboxylase, likely disrupting gluconeogenesis (205). Methylmalonate can directly inhibit the mitochondrial malate shuttle, similarly affecting gluconeogenesis.

Additionally, methylmalonate is transported by the carboxylate carrier systems for malate. Halperin [1971, 2276-2282] also showed that methylmalonate inhibits succinate

dehydrogenase (71). Other groups, however, suggest that rather than direct inhibition of succinate dehydrogenase, methylmalonate instead inhibits succinate transport (132).

Methylmalonate also acts as a competitive inhibitor for β-hydroxybutyrate

dehydrogenase and lactate dehydrogenase, further disrupting energy homeostasis.

Interestingly, methylmalonate inhibited lactate conversion to pyruvate but not the reverse, resulting in unimpeded lactate accumulation (49, 171).

In one study, methylmalonate inhibited bone marrow cell growth and development in a concentration dependent manner (84).

Methylmalonate, and methylmalonyl-CoA, metabolism is involved in the generation of free radicals. It has been shown that free radicals generated by methylmalonate, inhibit

creatine kinase, an energy buffering enzyme that helps to maintain ATP/ADP, especially

in high-energy requiring tissues such as the brain (178). One study showed that

methylmalonate decreases antioxidant stores in the brain, implying an increase in free

radical formation (56). As the optic nerve is very susceptible to oxidative damage, this

mechanism may be an explanation for the noticeable incidence of optic nerve atrophy in

MMA.

Chapter 4

Metabolism of 3-Hydroxypropionate

4.1 Normal Metabolism of 3-Hydroxypropionate

3HP is formed by the enzyme 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4) through an alternate mechanism of propionyl-CoA metabolism. Specifically, propionyl-CoA is reduced to acrylyl-CoA, subsequently acrylyl-CoA is hydrated to 3HP-CoA, and finally hydrolysis of 3HP-CoA to 3HP (2, 3, 185). 3HP can then reversibly generate malonate

semialdehyde via 3HP dehydrogenase (EC 1.1.1.59). This reversible step in 3HP

metabolism uses NAD+/NADH as a cofactor, see Figure 4.1. Although many organic

acids must be esterified to their CoA esters to have a metabolically active role, Den

[1959, 1666-1671] showed that 3HP dehydrogenase, purified from pig kidney, has no

activity on 3HP-CoA (45). 3HP is a normal metabolite present at low concentrations

physiologically, yet we could only find measurements of the urinary excretion of 3HP.

Physiologic concentrations of 3HP in urine are in the range of 3-10 mmol/mol creatinine,

whereas in PA and MMA urinary 3HP is multiple times higher in patient urine with

fluxuations likely representing changes in patient metabolic state (28). 3HP is also

produced by gut microflora, however the exact contribution of the gut to 3HP

concentration in either the normal or disease state is unknown (160). The roles of 3HP

and/or 3HP-CoA in PA and MMA are not well established, nor are the toxic effects of

these metabolites.

Propionyl-CoA

Acrylyl-CoA

Figure 4.1 Mechanism of 3HP Metabolism. Note: Formation of β-alanine from malonate semialdehyde has been documented for both 4-aminobutyrate aminotransferase and (S)-3-amino-2-methylpropionate transaminase, using glutamate as the amino acid nitrogen donor and alpha-ketoglutarate as the product ketone.

Malonate semialdehyde acts as a branch point for 3HP metabolism with metabolism of malonate semialdehyde generating either acetyl-CoA or β-alanine. Scholem [1983, 81-

85] first showed that catabolism of malonate semialdehyde to acetyl-CoA, with release of the C1 carboxyl carbon, occurs independently of malonate or malonyl-CoA in humans, refuting earlier hypotheses that 3HP is first metabolized to these intermediates prior to formation of acetyl-CoA (2, 3, 174). Original studies showed the presence of an enzyme termed malonate semialdehyde dehydrogenase (EC 1.2.1.18), which functioned to

convert malonate semialdehyde to acetyl-CoA. However, Goodwin [1989, 14965-14971] showed that malonate semialdehyde is metabolized to acetyl-CoA via methylmalonate- semialdehyde dehydrogenase (EC 1.2.1.27), and that in mammals the two are the same enzyme. They further showed that malonate semialdehyde is the preferred substrate of methylmalonate-semialdehyde dehydrogenase (63).

Studies have shown that in mammals, β-alanine is reversibly transaminated to malonate

semialdehyde, with either pyruvate or α-ketoglutarate as a nitrogen acceptor, and that

carbon from β-alanine is also incorporated into acetyl-CoA (75, 107, 158). β-alanine

metabolism is discussed in greater detail in subsequent chapters. Under normal

physiological conditions, the metabolism of propionate to 3HP, acetyl-CoA, and β-

alanine is a minor but constitutively active metabolic pathway for propionyl-CoA in

mammals (50, 174).

4.2 Metabolism of 3-Hydroxypropionate and Metabolic Toxicity in Disorders of

Propionyl-CoA Metabolism

There is little information on the metabolic effects and metabolic toxicity of 3HP, nor is there much information on the toxic effects of 3HP in the setting of PA or MMA. It has,

however, been shown that the production of 3HP is directly related to the severity of PA

(2, 3). Though no explanation was given, urinary 3HP concentration was also reported to

be positively associated with the severity of intellectual deficiency in PA patients (145).

Propionate has known toxicity to glial cells but it is unknown whether 3HP is similarly

toxic. As 3HP is a major metabolite in PA and MMA, 3HP production may represent a

significant portion of total propionyl-CoA metabolism in PA and MMA. This would be in stark contrast to the normal distribution of propionyl-CoA carbon, whereby propionyl-

CoA is largely acting as an anaplerotic substrate (2, 3). 3HP has been suggested as a possible biomarker for PA, however, more studies are needed investigating 3HP concentration in patients before conclusions can be drawn on the utility of 3HP to assess

PA patient status (2).

While 3HP directly contributes to the development of metabolic acidosis in PA and

MMA, there is evidence of an independent toxic effect of 3HP (222). 3-hydroxypropionic aciduria, caused by aberrant gut bacteria metabolism of unknown origin, was seen in four severely ill infants (160). Three of the infants were able to be evaluated clinically, and two had no observed confounding medical problems. Of these two infants, one presented with metabolic acidosis, hyperammonemia, and urea cycle abnormalities at post-natal day

7. The infant continued to deteriorate until death at post-natal day 9. The other infant presented with liver dysfunction, symptoms suggestive of hyperammonemia, and renal dysfunction at 77 days of age. Gross developmental retardation was noted in the second patient. 3HP was greatly elevated in the urine of all patients, while no other PA or MMA metabolites, nor metabolites of any other identified metabolic disease, were seen in excess (160). The clinical presentations of these infants is highly suggestive of a direct toxic effect by 3HP, though it must be noted that the initial cause of the aberrant intestinal microflora metabolism was not found. It is likely that 3HP is at least in part a direct contributor to the various clinical symptoms seen in PA and MMA.

The metabolites associated with 3HP catabolism likely also have toxicity in PA and

MMA. 3HP-CoA, in addition to contributing to CoA trapping in PA and MMA patients,

is suspected to have a toxic effect on FoxP2, a protein expressed in both the fetal and

adult brain, and other organs. It is necessary for speech and language development (152).

The inhibition of 3HP-CoA on FoxP2 was only shown in bacteria, and though highly

conserved across species, more research into the exact effect of 3HP-CoA on FoxP2, and

other proteins, is needed. Malonate semialdehyde, is likely very reactive towards many

macromolecules. Aldehydes are known to spontaneously crosslink proteins, inactivate

many enzymes, and act as mutagens (7). Malonate semialdehyde is known to be very

unstable, which also makes it difficult to study (166). Acrylyl-CoA has been shown to

dimerize with CoA in vitro, with unknown effects (154).

The metabolism of propionate to 3HP, and 3HP to non-toxic acetyl-CoA, is thought to be

a saturable process and is in all likelihood inadequate to dispose of all the propionyl-CoA

generated in a normal diet. As such, this pathway would be severely overloaded in PA.

Overload of this pathway would necessarily lead to accumulations of propionyl-CoA,

3HP-CoA, 3HP, and the other associated intermediates. Also, 3-

hydroxypropionylcarnitine has tentatively been identified in both the CSF and urine,

though the metabolism and effects of this compound have not been investigated, and no

studies have been done investigating the presence or effect of this metabolite in PA or

MMA (18, 123). Overload of the pathway and accumulation of these metabolites would

result in a worsening of any toxic effects (54). It is unknown whether this metabolic pathway is constantly overloaded in PA and MMA or if, because of various treatment

strategies including a low protein diet, it is only overloaded during episodes of metabolic

decompensation.

Chapter 5

Hyperammonemia in Disorders of Propionyl-CoA Metabolism

5.1 Ammonia Homeostasis

The regulation of ammonia concentration by the liver can be simplified to consisting of

(i) ammonium uptake, from either the blood or breakdown of amino acids such as

glutamine, and (ii) the conversion of ammonium to urea or glutamine. Figure 5.1 details

the sources of nitrogen in the urea cycle. Many studies have shown that part of the

normal regulatory response to acidotic conditions, like those seen in PA and MMA, is a

decrease in ureagenesis. Also present under acidotic conditions is an increase in

glutamine as an alternate storage of ammonia that does not affect blood pH (92, 117, 135,

150, 151, 157). An important aspect of the urea/glutamine relationship is the zonation of

the liver into zones 1, 2, and 3. Urea cycle enzymes are found in all hepatic zones, though

mostly concentrated in zone 1, and are in all hepatocytes, except those immediately

surrounding the outflowing pericentral venule, zone 3. Instead, glutamine synthetase (EC

6.3.1.2) is located in these cells and is limited to only those cells immediately

surrounding the pericentral venule. The distribution of these enzymes results in a

situation whereby urea is able to be produced, and blood glutamine hydrolyzed, at almost

every point in hepatic transit, whereas glutamine production occurs only at the end of

transit (4). Although glutamine synthetase is localized to only the innermost part of zone

3, Gebhardt [1983, 567-570] report a high synthesizing capacity and suggest that limiting

factors for glutamine synthesis are metabolite delivery and energy availability, rather than

enzyme concentration or distribution (60). In PA there is an absence of glutamine

increase in the presence of hyperammonemia, differentiating it from urea cycle disorders.

The mechanism preventing the glutamine response is unknown (41). In a small patient

study, Al-Hassnan [2003,89-91] showed no correlation between plasma glutamine and

plasma ammonia in PA, suggesting that there is a severe disruption in nitrogen

homeostasis in PA. Indeed, glutamine concentration may be below the normal range even

in metabolically compensated PA patients (1, 17, 43, 101, 144, 175, 204).

Figure 5.1 Overview of the Urea Cycle. The numbers refer to the following enzymes: 1, Carbamoyl phosphate synthetase I (CPSI); 2, Ornithine Transcarbamoylase; 3, Argininosuccinate synthetase; 4, Argininosuccinate lyase; 5, Arginase. Chhabra N. Biochemistry for Medics. 2015.

Lund [1971, 653-660] first showed that there is an apparent gradient in ammonia concentration between the liver and blood, with the liver having approximately 23x more ammonium than blood (118). It is possible that a damaged liver can directly contribute to blood ammonium levels by releasing its large pool of ammonium in the setting of PA.

5.2 ATP Utilization in Ureagenesis

Ureagenesis is an energy demanding process requiring four ATP equivalents per molecule of urea produced (4). Two of the four ATP equivalents are required to form carbamyl phosphate, the rate limiting step in ureagenesis (53, 144, 176, 204). It has been suggested that the energy deficit suspected in PA and MMA would decrease the ability of the urea cycle to respond to increased ammonia, in conjunction with the inhibition of carbamyl phosphate synthesis by propionyl-CoA, discussed subsequently (69). It was briefly suspected that acidosis altered energy homeostasis however, Kashiwagura [1984,

237-243] showed that energy balance is preserved under acidotic conditions despite an observed decrease in ureagenesis (92). The role of ATP in the development of hyperammonemia in PA and MMA is not well established.

5.3 Dysregulation of the Urea Cycle in Disorders of Propionyl-CoA Metabolism

Hyperammonemia is a life-threatening complication of PA and MMA (41). Interestingly, aspartate, which supplies nitrogen for urea formation, was found to be elevated in PA

(41, 175). Ornithine and citrulline concentrations, both urea cycle intermediates, were

widely variable in PA patients, while arginine was almost uniformly decreased in the

patient population examined (41, 175). This suggests dysregulation of the urea cycle,

altered nitrogen balance, and/or increased amino acid breakdown in the setting of PA and

MMA.

Propionate, as propionyl-CoA, has well documented effects on ureagenesis. In the setting

of PA and MMA, one suggested mechanism of propionate effect is inhibition of the urea

cycle via a reduction in N-acetylglutamate (NAG) concentration. NAG is required for

activation of mitochondrial carbamoyl phosphate synthetase I (CPSI, EC 6.3.4.16), the

rate limiting enzyme in the urea cycle (177). Propionyl-CoA can substitute for acetyl-

CoA as the substrate of N-acetylglutamate synthetase (NAGS, EC 2.3.1.1), forming N-

th propionylglutamate. However, propionyl-CoA slows the Vmax of this enzyme to 1/25 the

rate of activity it has for acetyl-CoA. Affinity of NAGS for propionyl-CoA is three times less than NAGS affinity for acetyl-CoA (69, 184). Propionyl-CoA acts as a competitive

inhibitor of NAGS; when propionyl-CoA was the substrate, NAGS showed

approximately 7% activity compared to acetyl-CoA (69, 219). The affinity of CPSI for

N-propionylglutamate is ten times less than for NAG, and N-propionylglutamate does not likely form physiologically. Due to the accumulation of propionyl-CoA in PA and MMA, however, binding of N-propionylglutamate to CPSI may occur (45, 69, 191). Stewart

[1980, 484-492] noted only a 28% decrease in CPSI activity due to the inhibition by N-

propionylglutamate. They suggested that this is likely too small to directly cause

hyperammonemia and instead would lower the overall capacity of the urea cycle to regulate ammonia. The change in NAG concentration by propionyl-CoA, however, was not examined (191).

CPSI may be directly inhibited in PA and MMA. While propionate likely only has

indirect inhibition of CPSI, as even at 10mM concentrations no inhibition on CPSI

activity by propionate was shown; propionyl-CoA has an inhibitory effect on CPSI by as

much as 45% at 1mM concentrations, with a linear increase in inhibition as propionyl-

CoA concentration increased (184). Proof of concept of the inhibition of CPSI in PA is

the effectiveness of N-carbamylglutamate, Carbaglu, on relieving hyperammonemia.

Carbaglu, a synthetic analogue of NAG, functions by substituting for NAG (172).

Ammonia levels increase alongside increases in PA and MMA metabolites, namely, propionate, propionylglycine, 3HP, and methylcitrate (172, 204). Hyperammonemia and coma can rarely present in the absence of severe ketoacidosis in PA (17). Additionally, while free carnitine deficiency has been reported as a causal factor in hyperammonemia, ammonia levels have been shown to increase prior to a clinically relevant decrease in free carnitine concentration (14, 204). Free carnitine deficiency is likely only a contributing factor to, rather than a direct cause of, hyperammonemia in PA and MMA (204). As there is a small pool of ammonia physiologically, small changes in nitrogen metabolism can easily result in steep changes in ammonia concentration. Decreased capacity of the urea cycle to handle nitrogen presents a possible explanation for the hyperammonemia seen in

PA and MMA (191). As the phenotype of nitrogen metabolism and hyperammonemia described in PA differs from classic urea cycle defects, it is likely that, rather than one obvious effect on the urea cycle, hyperammonemia in PA and MMA is due to a confluence of metabolic effects. Hyperammonemia in disorders of propionyl-CoA is a complex and broad topic. Our initial investigations into the cause of hyperammonemia in

PA and MMA quickly showed that this aspect of disease would require significant effort

54 and resources. As such we were not able to devote significant time to investigate changes in urea metabolism or to directly measure ammonia in our models.

Chapter 6

Metabolism of β-alanine

6.1 Normal Metabolism of β-Alanine

β-alanine is involved in CoA metabolism, it is a product of uracil metabolism, and importantly is a product of aspartate catabolism by gut microbes, see Figure 6.1. The contribution of the gut microbiome to β-alanine concentration and toxicity in PA and

MMA is unknown, though it is likely that intestinal flora act as an additional source of β- alanine independent of 3HP metabolism (121). Though an amino acid, β-alanine is not found in proteins. Most of the naturally occurring β-alanine is instead found as the dipeptide carnosine. Β-alanine metabolism is active in various tissues, including brain and liver. Furthermore, β-alanine is easily transported across these organs, though via different mechanisms (44, 75, 103, 174).

β-alanine can be reversibly transaminated to form malonate semialdehyde, which is further metabolized via decarboxylation to acetyl-CoA. β-alanine therefore has a clear connection to propionate and 3HP metabolism and likely plays a role in PA and MMA

(45, 107). β-alanine has not been previously investigated in connection to disorders of propionyl-CoA metabolism, though studies have shown that β-alanine accumulates in glial cells, the predominant neuronal cell type damaged in PA (103, 142).

β-alanine, though having unknown neuronal function(s), is considered a neurotransmitter as it adheres to all the requirements of a neurotransmitter: it is found naturally in the

CNS, is released by electrical stimulation, has binding sites, and has activity on neuronal excitability. Structurally, β-alanine is the intermediate between glycine and GABA, both

Carnosine Anserine

β-alanine-Histidine Dipeptidase

Histidine Methyl-Histidine

Pantothenate β-alanine Uracil Ketone

Transaminase activity

Amino acid Malonate Semialdehyde CoA + NAD+ Methylmalonate Semialdehyde Dehydrogenase

CO2 + NADH Acetyl-CoA

Figure 6.1 Overview of β-alanine Metabolism. The metabolism shown is limited to mammalian systems. Note: β-alanine forms pantothenate via mammalian gut microbes. Aspartate is catabolized to β-alanine by mammalian gut microbes.

of which are classified as inhibitory neurotransmitters. It is likely that β-alanine functions in a similar inhibitory role (103). It has been proposed that β-alanine acts as a post- synaptic inhibitor by hyperpolarizing the neuronal membrane. There also seems to be regional dependency of β-alanine content within the brain and the physiologic

57 concentration within the brain ranges from 0.03 to 0.08 mM (44, 201). The greatest concentration of β-alanine seems to be within the midbrain normally, which is the area associated with vision, hearing, motor control, sleep/wake, and arousal. The cortex has half the concentration of the midbrain, and the cerebellum has the lowest concentration

(44). How, and if, this distribution is perturbed in the setting of disorders of propionyl-

CoA metabolism is unknown.

The production and metabolism of β-alanine is likely both complex and poorly regulated.

Kikugawa [1988, 345-348] showed that propionate has multiple effects on β- ureidopropionase (EC 3.5.1.6), the final enzyme in uracil degradation to β-alanine.

Specifically, β-ureidopropionase catalyzes the cleavage of β-ureidopropionate to β- alanine. Propionate seems to act as both an allosteric activator of the enzyme and as a competitive inhibitor to β-ureidopropionate. β-ureidopropionase likely also catalyzes the production of β-ureidopropionate from β-alanine, which differs from propionate by the addition of a terminal carbamoylamino group. It is unknown if any metabolite is formed from metabolism of propionate by β-ureidopropionase (95). Overall, we could find no description of the effects of propionate on β-alanine metabolism in vivo.

6.2 Metabolism of β-alanine and Metabolic Toxicity in Disorders of Propionyl-CoA

Metabolism

β-alanine has been used as a supplement in exercising individuals in an attempt to maintain pH homeostasis due to the buffering capacity of carnosine and anserine, dipeptides of β-alanine and histidine or methylhistidine metabolism, respectively (98).

While the effectiveness of β-alanine supplementation on exercise tolerance are debatable,

β-alanine toxicity is known (46). Doses of β-alanine of 10mg/kg body weight or greater have been shown to induce paresthesia in humans in a dose dependent manner (73).

Flushing has also been reported with β-alanine ingestion (140). Additionally, elevated levels of carnosine and/or anserine in the plasma, urine, and tissues may be associated with mental retardation, possibly due to the concomitant increase in β-alanine concentration in the CSF (13, 180). It must be noted that the concentration of β-alanine in

PA and MMA has not been assessed in either the plasma, liver, CSF, brain or any other organs of patients.

The drug aminooxyacetate, a transaminase blocker, is being investigated as a cancer therapeutic but it has been shown to result in increased concentrations of β-alanine in urine, plasma, liver, heart, kidney, and a number of other organs in rats (16, 108, 189).

Pantothenic acid, a precursor of CoA, is produced by the microbial condensation of β- alanine and pantoate, and can be transported across the blood-brain barrier (62). This may worsen CoA trapping in the brain, by providing additional substrate for propionyl-CoA, methylmalonyl-CoA, and 3HP-CoA formation. Formation of β-alanyl-CoA in PA and

MMA is unknown. Changes to propionyl-CoA, 3HP-CoA, and other CoA ester concentrations from β-alanine supplementation are similarly unknown. Furthermore, as

β-alanine transamination is a mitochondrial process, mitochondrial changes due to high

β-alanine concentration are possible (94). The mechanism for β-alanine toxicity has yet to be elucidated, though it is known that β-alanine transamination is a saturable process

(146).

Chapter 7 Research Plan

7.1 Overview

The following research plan builds upon findings in the literature: (i) 3HP is generated from propionate and present in PA and MMA both pre- and post-OLT (2, 3, 185), (ii)

3HP forms malonate semialdehyde and acetyl-CoA, where independently it was shown that malonate semialdehyde formed from β-alanine generates acetyl-CoA (45, 174), (iii) the likelihood of altered nitrogen metabolism and the life-threatening hyperammonemia present in disorders of propionyl-CoA metabolism (41), and (iv) novel findings on the metabolism of propionate, 3HP, and β-alanine which occurred during analysis. PA and

MMA are characterized as diseases of toxic compound accumulation, and as such, changes in metabolism and toxicity due to PA and MMA associated metabolites likely cause many of the clinical symptoms (9). The effects of 3HP, and the effects of 3HP and propionate combined, on metabolism in the normal liver were unknown. Also, we could find no data on the relationship of β-alanine to symptoms of either PA or MMA, nor to metabolism or toxicity associated with these diseases. Indeed we could find no literature connecting β-alanine to disorders of propionyl-CoA metabolism at all. Acetyl-CoA represents an available mechanism of disposal of 3HP, and by extension propionyl-CoA.

The inference from acylcarnitine profiles that propionyl-CoA and the associated metabolites accumulate suggests that metabolism in disorders of propionyl-CoA metabolism is more complex than what has been described previously and therefore deserved further research. Additionally, it is important to understand the metabolism of

PA and MMA metabolites by the normal liver as OLT, though not curative, is an

increasingly supported treatment strategy for PA.

The investigation of the above required experiments to be conducted in ex vivo perfused

rat livers, and in vivo experiments involving intraperitoneal injections and gavages. The

ex vivo liver allowed for exact control and manipulation of substrate seen by the liver and

detailed accounting of substrate release from the liver, while the in vivo model allowed

for whole body studies of metabolism. The use of variously labeled substrates allowed us to follow the fates of the carbons, hydrogens, and nitrogens of these substrates under changing metabolic conditions, as well as determine flux and activity of the various metabolic pathways probed.

7.2 Metabolism of 3HP and Propionate in Perfused Rat Livers: Summary of

Stragegy and Methods

7.2.1 Characterizing the Metabolism of 3HP

Our interest in the metabolism and possible toxicity of 3HP arose from the unexpected labeling of CAC intermediates in ex vivo rat heart perfusions detailed in a previously published study from our lab (94). The increase in CAC intermediates was initially assumed to be the result of propionate generating 3HP. 3HP is excreted in high concentrations in the urine of PA and MMA patients, and is a known metabolite of propionate. It accumulates in disorders of propionyl-CoA metabolism and is produced by a variety of cell types (12, 218). Given that other PA and MMA associated metabolites have known effects on metabolism, and the structural similarity between propionate and

3HP, it is likely that 3HP is also an important metabolite contributing to the pathobiology of disorders of propionyl-CoA metabolism (2, 3, 37).

The liver is the major site of metabolism of propionate, though extrahepatic production is

a contributing factor to disease. Extrahepatic production of PA and MMA metabolites

from continued enzyme deficiency is the likely reason that OLT is not curative and

various groups have independently confirmed that all the enzymes necessary for

complete 3HP metabolism are present in the liver (2, 3, 45, 63, 75, 107, 158, 185).

Despite this however, experiments describing the metabolism and effects of 3HP in

mammals are lacking. The effects of 3HP on the normal liver are unknown. Therefore,

we sought to better characterize the metabolism of 3HP by the normal liver under various

conditions.

3HP is normally not found at high concentrations in the absence of high propionate.

Because of this, we also had to consider changes to metabolism that occur when both of

these compounds are present. Having data on the individual effects of 3HP and

propionate, however, would allow us to parse out individual contributions seen from

these combined studies, as well as changes due to any combinative or synergistic effects.

Furthermore, we had to consider the differences in metabolic environment seen in our

normal livers as compared to the livers of PA and MMA patients lacking enzyme

activity. It must be noted that this experimental model sought to characterize the post-

OLT PA or MMA patient, one with normal liver PCC activity in the presence of

continued extrahepatic production 3HP from high propionate concentrations. We

hypothesized that metabolism of 3HP and/or propionate is as follows:

1. 3HP is ultimately metabolized to non-toxic acetyl-

CoA, despite both ongoing CoA trapping and the

described accumulation of 3HP in PA.

2. 3HP has toxic metabolic effects, similar to those

ascribed to propionate.

3. Propionate metabolism to 3HP is likely, at least to

some extent, reversible.

4. 3HP and propionate have a greater effect on

metabolism than either compound alone and that

this setting most closely mimics the metabolism

seen in PA, including the presence of CoA trapping.

We also felt it necessary to investigate energy balance in our model, given the proposed energy deficit in PA. We further sought to confirm the involvement of reducing equivalents in the metabolism of 3HP. This was of particular note given the suspected perturbations of energy homeostasis in PA. As 3HP metabolism should be dependent on

[NAD]/[NADH] ratios, the oxidation of ethanol to acetaldehyde and ultimately acetate in the liver results in increased [NADH]. This should, and did inhibit, acetyl-CoA production from 3HP, yet similar inhibition was also seen with high β-alanine. This suggests a possible role for β-alanine in 3HP metabolism. The plan to investigate β- alanine metabolism is detailed in subsequent sections.

7.2.2 Investigating the Metabolism of 3HP and/or Propionate

A search of the literature showed that aspects of 3HP metabolism have been described

previously but no attempts to document the metabolism of 3HP directly have been made.

We hypothesized that various steps of 3HP metabolism were reversible. We also proposed that 3HP metabolism was saturated and inhibited in disorders of propionyl-CoA metabolism due to the increased burden.

13 13 13 13 To test these hypotheses we used [ C3]3HP, [1- C]3HP, [2- C]3HP, [3- C]3HP

substrates to follow the three carbons of 3HP. As 3HP should be ultimately converted to

acetyl-CoA, it was understood that in order to follow each of the carbons of 3HP to

determine loss and rearrangement, variously labeled substrates must be employed. It had

been shown previously that as 3HP is converted from the three carbon 3HP compound to

the two carbon acetyl moiety of acetyl-CoA, loss of the first carbon occurs as CO2 (45,

107). The rearrangement of the other carbons was described using the differently labeled

3HP compounds. The metabolism of unlabeled 3HP in deuterated water gave further

information on the metabolism of 3HP, though keto-enol tautomerization, or

interconversion, must be accounted for in this type of experimentation and analysis.

The use of labeled metabolites also allowed for the discovery and characterization of

novel metabolites involved in 3HP and propionate metabolism.

β-alanine is known to be metabolized to malonate semialdehyde, an intermediate in 3HP

metabolism. This led us to hypothesize that β-alanine has a role in 3HP metabolism and

may have a role in PA and MMA toxicity. Indeed β-alanine metabolism may have

independent toxic effects, and as such also warranted further investigation.

7.2.3 Health Relevance

The metabolism of 3HP and propionate in PA and MMA is poorly understood, as seen by the limited success of current treatment strategies such as OLT, biotin and carnitine supplementation, and protein restriction to fully ameliorate either the clinical findings or symptoms. Furthermore, the measurement of metabolites that accumulate as a direct result of enzyme deficiency, namely propionylcarnitine and 3HP, do not correlate well with disease presentation, severity, or progression (179, 222). Indeed, despite what is accepted as adequate treatment, outside of an OLT, PA and MMA patients show chronic progressive damage and decline. It has been shown that organ failure can occur even in patients deemed metabolically stable (183). PA and MMA, despite being two of the most common inborn errors of metabolism, are diseases with a poorly understood metabolism and pathophysiology.

7.2.4 Strategy

It was unknown whether the previously stated metabolic perturbations are due solely to the toxic effects of one substrate or, more likely, are the result of the combinative effects of multiple substrates. Our strategy, therefore, required a method to determine both the individual effects of our perfused compounds, as well as changes that occurred due to any additive and/or synergistic effects. We accomplished this in ex vivo rat livers by perfusing variously labeled substrates both individually and in combination. We analyzed our data using a combination of metabolomics and mass isotopomer techniques outlined below.

Overall, our data confirmed that (i) 3HP is metabolized to acetyl-CoA as well as being

reversibility esterified to 3HP-CoA and that (ii) out of all of our experimental conditions,

3HP in combination with propionate best mimics the metabolic changes seen in PA.

7.2.4.1 Perfused Liver Experiments

To characterize metabolism of 3HP and the metabolic changes of PA, livers from

overnight fasted male Sprague-Dawley rats were excised and perfused with recirculating bicarbonate buffer containing fatty acid free bovine serum albumin, 4mM glucose

13 without insulin and the following substrates in various combinations: (i) [ C3]propionate

13 13 13 (M3 propionate); (ii) [ C3]3HP (M3 3HP); (iii) [1- C1]3HP; (iv) [2- C1]3HP; (v) [3-

13 C1]3HP; and (vi) nothing (control). The perfusate was gassed with 95% O2 + 5%

unlabeled CO2 and was sampled at variable lengths of time, depending on the nature of the experiment, and livers were quick frozen at the end of the protocol.

7.2.4.2 Analytical Procedures

The concentrations and mass isotopomer distributions of the various metabolites in liver perfusate and tissue were assayed by GC-MS typically as trimethylsilyl, tert- butyldimethylsilyl, or pentafluorobenzyl derivatives, unless otherwise specified, using analogously labeled compounds as internal standards. The concentrations and mass isotopomer distributions of acyl-CoA esters were assayed using the methods described by

Zhang (220). Optimization of the various protocols and GC-MS conditions occurred as necessary to obtain adequate purification and separation of analytes.

7.2.4.2.1 Ultraviolet-Visible Absorption Spectroscopy

To determine the concentrations of adenine nucleotides, energy balance, and redox state

of our ex vivo quick frozen perfused rat livers, samples from the same livers were

deproteinized in perchloric acid, neutralized, and enzymatically assayed on a Spectra

Max M2 spectrophotometer. Analysis proceeded in a similar manner as outlined by

Williamson [1969, 434-513] (215).

7.2.4.2.2 Nuclear Magnetic Resonance Spectroscopy

To determine the positional labeling of acetyl-CoA the 13C labeling of C-4 and C-5 in

liver glutamate was assayed by 13C-NMR spectroscopy on the same overnight fasted

male Sprague-Dawley rats as above. Samples were also deionized to remove ionic

constituents allowing for analysis. The spectra were acquired using an AVANCE III HD

500 MHz spectrometer (Bruker Instruments, Inc. Billerica, MA).

Chemical shifts were referenced to spectra of standards with the pH adjusted to match

that of the liver extracts.

7.3 Metabolism of β-alanine: Summary of Strategy and Methods

7.3.1 Characterizing and Investigating the Metabolism of β-alanine

We could find no literature directly investigating the connection between β-alanine and disorders of propionyl-CoA metabolism. Malonate semialdehyde, like many aldehydes is toxic and very reactive, but is metabolized to β-alanine and/or acetyl-CoA (7, 45). Also,

there are known toxic effects from β-alanine supplementation, as was described in

previous sections. We suspect that β-alanine plays an as yet unknown role in both PA and

MMA and sought to better characterize the toxic effects of β-alanine supplementation.

Overall, β-alanine metabolism deserves further attention. Our initial research involving β- alanine, 3HP, and/or propionate led to numerous potential areas of investigation. We thus limited ourselves to only undertake initial inquiries into the three major findings, (i) effects of β-alanine on CoA synthesis, (ii) novel pathways of β-alanine metabolism, and

(iii) effects of β-alanine on neurotransmitter balance.

13 15 To test these hypotheses we used β-alanine and [ C3, N]β-alanine to probe mammalian metabolism under various conditions. Separately, we also injected rats with a non- specific transaminase inhibitor, aminooxyacetate, which has known effects on β-alanine metabolism. We then gavaged these rats with either: (i) saline or (ii) a load of β-alanine.

As the concentration of β-alanine in PA patients is unknown we based our studies on both

the physiological concentrations of β-alanine and previous studies of β-alanine

supplementation conducted by colleagues. Toxic effects have been noted by β-alanine

concentrations at or above 5mM (72).

7.3.2 Public Health Relevance

β-alanine is a metabolite that is produced, and likely accumulates, in PA and MMA, with

as yet unknown metabolic effects. It has known toxic effects, including paresthesia and

flushing when taken as a supplement. Additionally, it has ill-defined neurologic

function(s). Understanding the metabolism of β-alanine, and associated metabolites, and

68 the role of β-alanine metabolism in toxicity is vital to gaining a greater understanding of the disorders of propionyl-CoA metabolism as a whole, and finding more effective treatment strategies. β-alanine metabolism is an area of research that doesn’t seem to have been studied at all with regards to PA or MMA. In addition, since β-alanine is freely available as a supplement, the potential exists for β-alanine toxicity to become a serious public health problem.

7.3.3 Strategy

The aforementioned aspects of β-alanine metabolism required extensive testing and analysis of various tissues in order to achieve a satisfactory description of β-alanine effects and metabolism. In addition to production and metabolism by the liver, the effects of β-alanine on the muscle, both cardiac and skeletal, and the brain cannot be ignored.

We therefore tested our hypotheses in live rats under various conditions and with (i) oral gavage boluses of β-alanine, or (ii) perfused ex vivo rat livers. We obtained our data using a combination of metabolomics and mass isotopomer analysis using similar methodology as described previously.

7.3.3.1 Perfused Liver Experiments

To characterize the metabolism of β-alanine, livers from overnight fasted male Sprague-

Dawley rats were excised and perfused in a similar manner as noted previously, however,

13 15 β-alanine and [ C3, N]β-alanine (M4 β-alanine) were perfused.

7.3.3.2 In vivo Experiments

To determine the metabolism of β-alanine on various tissues, and notably β-alanine’s potential role in neurotransmitter chemistry, overnight fasted male Sprague-Dawley rats were divided into multiple groups and injected intraperitoneally with either saline or 0.09 mmol/kg aminooxyacetate. Rats were then temporarily re-housed, based on consultations

with colleagues, to determine the ideal protocol for maximal effectiveness of

aminooxyacetate. A group of rats were subsequently given an oral gavage of either: (i)

0.45 mmol/kg β-alanine or (ii) saline (control). Rats were given the gavage at 120 min

post intraperitoneal injection, and were killed at specific intervals until the conclusion of

the experiment, 240 min post intraperitoneal injection. For every group, at the time of kill

the following were collected: (i) whole blood, (ii) liver, (iii) heart, (iv) muscle, and (v)

whole brain.

7.3.3.3 Analytical Procedures

The concentrations and mass isotopomer distributions of various metabolites in blood, and the various tissues collected were assayed by a similar procedure as described previously.

Chapter 8 Publications

8.1 Overview The hypotheses, observations, and conclusions presented in Chapter 7 are reported in the following three papers, which are included in this thesis.

8.1.1 Wilson, K.A., Han, Y., Zhang, M., Hess, J., Chapman, K.A., Cline, G.W.,

Tochtrop, G.P., Brunengraber, H., Zhang, G-F. Interrelations between 3-

hydroxypropionate and propionate metabolism in rat liver: Relevance to disorders

of propionyl-CoA metabolism. Am. J. Physiol. Endocrinol. Metab. Article In

Press.

8.1.2 Wilson, K.A., Bedoyan, J., Zhang, G-F., Venditti, C.P., Brunengraber, H.

Maleate, a nephrotoxic plasma biomarker of propionic and methylmalonic

acidemias in humans and mice. (To be submitted to Mol Genet Metab. 2017).

8.1.3 Wilson, K.A., Hess, J., Zhang, M., Chapman, K.A., Derave W., Zhang, G-F,

Brunengraber, H., Tochtrop, G.P. Metabolism of β-alanine in the perfused rat

liver and live rats: Stimulation of CoA synthesis and carboxylation to 2-

(aminomethyl)-malonate. (To be submitted to Am. J. Physiol. Endocrinol. Metab.

2017).

8.1.1

Interrelations between 3-hydroxypropionate and propionate metabolism in rat liver: Relevance to disorders of propionyl-CoA metabolism

Kirkland A. Wilson1, Yong Han2, Miaoqi Zhang1, Jeremy Hess2, Kimberly A. Chapman3,

Gary W. Cline4, Gregory P. Tochtrop2, Henri Brunengraber1 and Guo-Fang Zhang5

Running title: 3-Hydroxypropionate metabolism in rat liver

Depts of Nutrition1, Chemistry2, Case Western Reserve University, Cleveland OH 44106, Children's National Medical Center and George Washington University3, Washington DC 20010, Dept of Internal Medicine4, Yale University, New Haven CT 06519 , and Division of Endocrinology, Metabolism and Nutrition5, Dept of Medicine, Duke Molecular Physiology Institute, Duke University, Durham NC 27701

Corresponding author: Henri Brunengraber, Department of Nutrition, Case Western

Reserve University, 10900 Euclid Ave. WG-48; Cleveland OH 44106-4954. Tel: (216)

368-6548. Fax: (216) 368-6846. Email: [email protected]

Abstract

Propionate, 3-hydroxypropionate (3HP), methylcitrate, related compounds and ammonium

accumulate in body fluids of patients with disorders of propionyl-CoA metabolism, such as propionic acidemia. Although liver transplantation alleviates hyperammonemia, high concentrations of propionate, 3HP and methylcitrate persist in body fluids. We hypothesized that conserved metabolic perturbations occurring in transplanted patients result from the simultaneous presence of propionate and 3HP in body fluids. We investigated the interrelations of propionate and 3HP metabolism in perfused livers from normal rats using metabolomic and stable isotopic technologies. In the presence of propionate, 3HP or both, we observed the following metabolic perturbations. First, the citric acid cycle (CAC) is overloaded, but does not provide sufficient reducing equivalents to the respiratory chain to maintain the homeostasis of adenine nucleotides. Second, there is major CoA trapping in the propionyl-CoA pathway, and a tripling of liver total CoA within 1 hr. Third, liver proteolysis is stimulated. Fourth, propionate inhibits the conversion of 3HP to acetyl-CoA and its oxidation in the CAC. Fifth, some propionate and some 3HP are converted to nephrotoxic maleate by different processes. Our data have implication for the clinical management of propionic acidemia. They also emphasize the perturbations of liver intermediary metabolism induced by supraphysiological i.e., mM concentrations of labeled propionate used to trace intermediary metabolism, in particular inhibition of CAC flux and major decreases in the [ATP]/[ADP] and [ATP]/[AMP] ratios.

Keywords: 3-Hydroxypropionate, propionate, propionic acidemia, liver, metabolomic and stable isotopic analysis 73

Introduction

In patients with inherited disorders of propionyl-CoA metabolism (propionic and methylmalonic acidemia), propionate (81), 3-hydroxypropionate (3HP) (5),

+ propionylcarnitine (86), heptadecanoylcarnitine (50), methylcitrate (6) and often NH4 (30,

79) accumulate in body fluids. There is much information on the normal metabolism of propionate. This gluconeogenic substrate, formed by intestinal fermentations, is mostly cleared in a single passage of portal vein blood through the normal liver (from 0.2 to 0.005 mM (66)). Propionate concentration in systemic plasma is very low (< 10 μM) (65). In the liver, propionate is converted sequentially to propionyl-CoA, S-methylmalonyl-CoA, R-

methylmalonyl-CoA, and anaplerotic succinyl-CoA. Because cataplerosis must balance anaplerosis, carbon from propionate leaves the citric acid cycle (CAC) as phosphoenolpyruvate which is converted to glucose, lactate and pyruvate. Labeled forms

14 13 of propionate, e.g., [ C]- and [ C3]propionate, have been used extensively to probe

pathways of intermediary metabolism in liver and heart (15, 41, 46, 75). Propionyl-CoA is

also formed in most organs by the catabolism of four amino acids: isoleucine, methionine,

threonine and valine, as well as the catabolism of odd-chain fatty acids (70). In the heart, a

non-gluconeogenic organ, anaplerosis from propionyl-CoA is balanced by the release of

CAC intermediates, mostly malate (41).

There is much less information on the mammalian metabolism of 3HP which is present at

very low concentrations in normal human plasma and urine (34). Small amounts of 3HP are formed in two secondary pathways of γ-hydroxybutyrate degradation in liver (92). In disorders of propionyl-CoA metabolism, the mechanism of formation of 3HP involves

reduction of propionyl-CoA to acrylyl-CoA, followed by hydration of acrylyl-CoA to 3HP-

CoA, and hydrolysis to 3HP (5). 3HP is converted to acetyl-CoA via malonic semialdehyde

(25, 44, 73). The latter is reversibly transaminated to β-alanine, a product of uracil degradation. Additional pathways of 3HP disposal via a methylcitrate shunt and via modified β-oxidation have been identified in microorganisms (61), plants (35) and C. elegans (84). These pathways may be present in humans (84).

Disorders of propionyl-CoA metabolism result mostly from a defect in propionyl-CoA

carboxylase or methylmalonyl-CoA mutase. The latter can be deficient or insensitive to

vitamin B12 activation (51). These patients show high plasma concentrations of propionate

(81), 3HP (5), methylcitrate (6), propionylcarnitine (86), glycine and ammonium. Multiple

organ damage has been related to inhibition of oxidative phosphorylation, mitochondrial

damage, accumulation of compounds that are toxic at high concentrations (especially CoA

esters, the concentrations of which are reflected by the concentrations of carnitine esters)

(4, 13, 60, 74). One major complication of disorders of propionyl-CoA metabolism is

hyperammonemia which is triggered by excess protein intake or stimulation of protein

degradation by catabolic stress (30, 79). Although patients excrete normal amounts of urea,

it appears that normal ureagenic flow occurs at a supraphysiological and often toxic

+ concentration of NH4 in body fluids. There is evidence that steady state hyperammonemia

results from inadequate activation of N-acetylglutamate synthetase (1, 23, 78). Patients are

treated with a low-protein diet and with N-carbamylglutamate, an activator of N-

acetylglutamate synthase (2, 78, 82). However the management of the disease is difficult

because of the frequency of life-threatening decompensation episodes (18, 72). Except for

the vitamin B12 responsive forms of methylmalonic acidemia, the prognosis for the

patients is poor (8).

In patients with propionic acidemia, liver transplantation usually prevents

hyperammonemia, but does not cure the disease in most cases: high concentrations of

propionate, 3HP, methylcitrate and propionylcarnitine persist in plasma (19, 20, 24, 71,

72). Also, kidney failure develops in about one-half of the transplanted patients (20). It

appears that 3HP, derived from amino acid catabolism in peripheral tissues, is released in

plasma and exerts a toxic effect on the transplanted liver. This results in an impairment of

the disposal of propionate formed by intestinal fermentations, and propionyl-CoA derived from amino acids. We hypothesized that some of the metabolic perturbations occurring in the liver of these patients are triggered by the simultaneous presence of propionate and

3HP in body fluids. The goal of the present study was to characterize the interrelations of propionate and 3HP metabolism in normal rat livers perfused with clinically-relevant mM concentrations of one or both substrates, unlabeled or 13C-labeled. Our strategy was based

on substrate balance and on following the spread of 13C through the metabolome,

concentrating on CoA esters, the CAC, carboxylic acids and glucose. We used

13 13 [ C3]propionate and [ C3]3HP, not as tracers of pathways of intermediary metabolism, but as substrate loads to identify their fates and the metabolic perturbations induced by them. The data presented below support the interrelations of propionate and 3HP metabolism outlined in Scheme 8.1.

Material and Methods

Materials

Sigma-Aldrich-Isotec supplied most chemicals, enzymes and the following isotopically

2 13 2 labeled compounds: H2O (99.8%), [ C3]propionate, [ H9]pentanoic acid, potassium

13 13 13 [ C]cyanide, 2-bromo-[ C2]ethanol, 2-bromo-[1- C]ethanol and 2-bromo-[2-

13C]ethanol. A standard of malonic semialdehyde was prepared by hydrolysis of the ethyl

ester diethylacetal of malonic semialdehyde (ethyl 3,3-diethylpropionate) (43). An internal

2 2 standard of [ H9]pentanoyl-CoA was prepared from [ H9]pentanoic as in (42). The

sensitive carbonyl derivation agent, N-[2-(Aminooxy)ethyl]-N,N-dimethyl-1- dodecylammonium iodide (52) was kindly provided by Dr. Teresa Fan.

13 13 3-Hydroxy-[1- C]propionic acid lactone was prepared by reacting 2-bromo-[ C2]ethanol

with K13CN, followed by alkaline hydrolysis of 3-hydroxy-[1-13C]propanenitrile,

13 1 acidification and extraction of the 3-hydroxy-[1- C3]propionic acid lactone. H-NMR

13 13 (D2O, 400MHz): 3.65 (dt,2H, -CH2OH), 2.31 (dt, 2H, COO-CH2-). C-NMR (D2O,

100MHz): 180.8 (13COO).

13 3-Hydroxy-[ C3]propionic acid lactone was prepared by reacting ethyl bromo -

13 13 13 [ C2]acetate with K CN to form ethyl-2-[ C3]cyanoacetate which was oxidized with

13 PtO2 to ethyl 3-amino-[ C3]propionate. The latter was treated with sodium nitrite.

13 1 Acidification and extraction yielded 3-hydroxy-[ C3]propionic acid lactone. H-NMR

13 13 13 (D2O, 400MHz): 3.65(ddt, 2H, - CH2OH), 2.32(ddt, 2H, - CH2). C-NMR (D2O,

13 13 13 100MHz): 180.3(d, COO), 59.8 (d, CH2OH), 40.2(dd, - CH2-).

3-Hydroxy-[2-13C]propionic acid lactone was prepared from ethyl bromo-[2-13C]acetate

1 and KCN as described above. H-NMR (D2O, 400MHz): 3.63(m, 2H, -CH2OH), 2.34(dm,

13 13 13 2H, - CH2). C-NMR (D2O, 100MHz): 40.5(- CH2-).

3-Hydroxy-[3-13C]propionic acid lactone was prepared from ethyl bromoacetate and

13 1 13 K CN as described above. H-NMR (D2O, 400MHz): 3.68 (d, 2H, CH2OH-), 2.32(m,

13 13 2H, -CH2-). C-NMR (D2O, 100MHz): 60.9 ( CH2OH).

The purity of the labeled lactones was verified by GC-MS and NMR. Before use, the

unlabeled and labeled 3HP lactones were hydrolyzed with 1.1 equivalents of NaOH at 80°C

for 1 h.

Perfused liver experiments

All animal protocols were approved by the IACUC Committee of Case Western Reserve

University. Male Sprague-Dawley rats were fed with Prolab Isopro RMH 3000 irradiated

chow containing 13 ppm of pantothenic acid as calcium acid. Livers from overnight-fasted

rats (160-180 g) were perfused (11) for 60 min with recirculating bicarbonate buffer containing 4% dialyzed, fatty acid-free, bovine serum albumin and 4 mM glucose. To the

13 basic perfusate, we added either nothing (controls), 2 mM 3-hydroxy-[ C3]propionate, 5

13 13 mM [ C3]propionate, 5 mM [ C3]propionate + 2 mM 3-hydroxypropionate, or 5 mM

13 propionate + 2 mM 3-hydroxy-[ C3]propionate (n = 5 or 6 in each group).

To trace the fate of individual carbons of 3-hydroxypropionate, single livers were perfused

with 2 mM 3-hydroxy-[1-13C]propionate, 2 mM 3-hydroxy-[2-13C]propionate, or 2 mM

3-hydroxy-[3-13C]propionate. To test for the reversibility of the transamination of β-

alanine to malonate semialdehyde, two livers were perfused with recirculating buffer made

2 in 100% H2O with or without 2 mM unlabeled 3HP. To test for the production of maleate from methylmalonate, one liver was perfused with 2 mM methylmalonate.

At the end of the experiments, all livers were quick-frozen and kept in liquid N2 until 78

analysis.

Analytical Procedures

The concentration and labeling pattern of propionate was assayed on 0.2 ml perfusate by

NH3 negative chemical ionization of the pentafluorobenzyl derivative (77). Carboxylic

acids (27), 3HP, CAC intermediates, methylcitrate, maleate, pantothenate and β-alanine

were assayed on 0.4 g of liver or 0.5 ml perfusate by GC-MS as TBDMS derivatives. An

Agilent Technologies Model 6890N chromatograph with an Agilent Technologies Model

5973 mass selective detector was equipped with a Varian 60 m × 0.25 mm capillary

column. The inlet temperature was set to 280°C. Helium was used as the carrier gas at a

flow rate of 1.3 ml/min for the column. Oven temperature was programmed at an initial

temperature of 80°C with an isothermal delay of 1 minute. The temperature was increased

at 5°C/min to 250°C, then at 10°C/min to 300°C. Retention times of unlabeled compounds

and m/z monitored (M-57) were: 3HP (21.6 min, m/z = 261), β-alanine (23.0 min, m/z =

260), maleate (24.6 min, m/z = 287), succinate (25.6 min, m/z = 289), fumarate (26.3 min,

m/z = 287), malate (32.4 min, m/z = 419), citrate (39.5 min, m/z = 591), pantothenate (40.3

min, m/z = 504). Methylcitrate (40.2 min) was quantitated via m/z = 473 (14). For the

corresponding labeled analytes the m/z of all possible mass isotopomers were monitored.

Acyl-CoAs were assayed by LC-MS/MS as previously described on 0.5 g of liver (49, 91).

Assays of CoA ester concentrations were run with calibration curves of unlabeled standards

2 spiked with [ H9]pentanoyl-CoA prepared from the acid (91). Because a standard of 3HP-

CoA is not commercially available, the concentrations of 3HP-CoA were calculated with

2 a propionyl-CoA/[ H9]pentanoyl-CoA calibration curve.

The concentrations of liver adenine nucleotides, lactate, pyruvate, β-hydroxybutyrate and acetoacetate were assayed enzymatically in neutralized perchloric acid extracts of 0.4 g of liver liver (89) on a Spectra Max M2 spectrophotometer (Molecular Devices, Sunnyvale,

CA). The concentrations of amino acids in the final liver perfusate were assayed on a

Hitachi L8800a amino acid analyzer with post-column ninhydrin derivatization.

The 13C labeling of C-4 and C-5 of liver glutamate was assayed by 13C-NMR spectroscopy

on neutralized perchloric acid extracts of 1 g liver, deionized on a Chelex column. Spectra

were acquired with an AVANCE III HD 500-MHz spectrometer (Bruker Instruments, Inc.

Billerica, MA) with TR=1.0s and Waltz-16 broadband proton decoupling. Chemical shifts

were referenced to spectra of standards with pH adjusted to match that of the liver extracts

(22). The (dry weight)/(wet weight) ratio was assayed on 0.2 g of liver.

Calculations and statistics

Measured mass isotopomer distributions, expressed as mol percent, were corrected for

natural enrichment (29, 54). Statistical differences were assayed by t test using the Prism

software.

Results

Uptake and release of substrates by perfused livers

Livers were perfused with initial concentrations of 5 mM propionate or/and 2 mM 3HP,

labeled or unlabeled. Thus, we covered the range of systemic plasma concentrations

measured in propionic acidemia patients, even after liver transplant (5, 81).

In control livers perfused without propionate or 3HP, we could not detect the release of

propionate, but we detected a low linear release of 3HP (0.86 nmol·min−1· (g. dry wt)−1) with an accumulation of 3 μM after 1 hr. The rate of propionate uptake decreased by half in the presence of 3HP (Table 8.1, rows 2, 4-6).

Table 8.1. Rates of uptake and release of substrates. Livers were perfused for 60 min 13 with 4 mM glucose and either nothing (Control), 2 mM [ C3]3HP (M3 3HP), 5 mM 13 13 [ C3]propionate (M3 propionate), 2 mM 3HP and 5 mM [ C3]propionate (M3 propionate 13 + M0 3HP), or 2 mM [ C3]3HP and 5 mM propionate (M0 propionate + M3 3HP). Data are presented in nmol min-1 (g dry weight)-1 as mean ± SD. The release of 3HP by livers perfused with unlabeled propionate + M3 3HP (row 5) was calculated from the decrease in labeled 3HP in the perfusate.⋅ ⋅ Italicized values for 3HP release (rows 4 and 5) are estimates (see text). *Significantly different from control (p<0.05). ^ Significantly different from M3 3HP (p<0.05). ‡Significantly different from M3 propionate (p<0.05). Substrate(s) Propionate 3HP uptake by 3-hydroxypropionate uptake by liver liver released from liver Row nmol min-1 g-1 nmol min-1 g-1 nmol min-1 g-1 Number 1 Control (n=5) ⋅ ⋅ ⋅ ⋅ 0.86⋅ ± 0.15⋅ 2 M3 propionate (n=5) 3840 ± 1300 15 ± 7.3* 3 M3 3HP (n=6) 806 ± 149 4 M3 Propionate + M0 1890 ± 950‡ 947 ± 476 8 ± 1* 3HP (n=5) 5 M0 Propionate + M3 1720 ± 650‡ 1517 ± 411^ 38 ± 12* 3HP (n=5) 6 Propionate + 3HP, 1810 ± 790‡ 1232 ± 516 Pooled data (n=10)

The basal rate of 3HP uptake was only 20% of that of propionate uptake (rows 3, 2). The

variability of the rate of 3HP uptake increased in the presence of propionate (CV= 42%,

row 6 (n = 10) vs 18%, row 3 (n = 5)). The low control rate of 3HP release increased 18

times in the presence of 5 mM M3 propionate (Table 8.1, rows 1, 2). When propionate and

3HP were both present (rows 4, 5), we could still estimate a range of 3HP release from (i)

its rate of labeling (in perfusions with M3 propionate + unlabeled 3HP, row 4), or (ii) de- labeling (in perfusions with unlabeled propionate + M3 3HP, row 5). Although these two estimates differed, overall the net rate of 3HP release by the liver was less than 3% of the rate of propionate uptake (Table 8.1, last column). The perfusate concentrations of CAC intermediates and 2-methylcitrate were too low to be measurable with precision.

Metabolite concentrations and crossover analyses

Table 8.2 shows liver metabolite concentrations expressed either as nmol/(g dry wt), or as

relative concentrations vs control. Addition of 3HP to the perfusate had no impact on the

concentrations of propionyl-CoA, methylmalonyl-CoA, succinyl-CoA, acetyl-CoA and

free CoA. In contrast, propionate induced major trapping of CoA in propionyl-CoA +

methylmalonyl-CoA + succinyl-CoA, the concentrations of which increased 250-, 120-

and 2-fold, respectively (rows 1-3). Acetyl-CoA and free CoA concentrations decreased by one-third and one-half, respectively. In the presence of both propionate and 3HP, CoA trapping was decreased, except for succinyl-CoA, a CAC intermediate, the concentration of which increased 4.5 times compared to control (row 3). 3HP-CoA was detected in control livers perfused with only 4 mM glucose. Its concentration increased 5 and 8-fold when 3HP or propionate + 3HP were added to the perfusate. The total concentration of

propionate or 5 mMpropionate 2 mMand [ 3HP different from control (p<0.05), ^ from (p<0.05), ^ fromdifferent control 3HP M3 (p<0.05), (Control), 2 mM nothing [ Table 8. 2. Metabolite 2. Metabolite concentrationsdry weight). (nmol/g 13 C 3 ]3HP (M3 3HP), 5 mM 3HP), 5 ]3HP (M3 [ 13 C 3 ]propionate (Propionate + 3HP). Data presented are as mean ± SD. *Significantly 13 C

‡from M3 Propionate Detected ‡from(p<0.05). ND Propionate M3 Not = 3 ]propionate (M3 Propionate), 2 mM Propionate), ]propionate (M3 [

Livers perf used for 60 min with 4 mM glucose and either for4 mM 60 mineither used with and glucose 13 C 3 ]3HP and 5 mM]3HP 5 and

CoA species assayed (including free CoA), was not affected by 3HP, but was tripled by propionate and by propionate + 3HP, respectively (row 8). Thus, the total pool of CoA was rapidly and markedly increased by the presence of propionate, or propionate + 3HP, but not by 3HP alone. Surprisingly, the increase in total CoA concentration induced by propionate ± 3HP was much greater than the pantothenate pool in control livers (row 9).

Thus, it appears that the liver contains some pool(s) of pantothenate or pantothenate precursor (s). Note that the chow fed to the rats was enriched with calcium pantothenate.

Also, plant and animal components of the chow contain pantothenate. This finding will require further investigation.

The concentrations of most CAC intermediates, except fumarate, were increased by 3HP, propionate, or propionate + 3HP (Table 8.2). The total concentration of CAC intermediates assayed, including succinyl-CoA, increased 2.5 fold with 3HP, 4.7 fold with propionate, and 2 fold with propionate + 3HP (rows 10-14). 2-Methylcitrate, formed by the condensation of propionyl-CoA and oxaloacetate (10, 85), was not detected in control livers or in livers perfused with 3HP, but was detected in livers perfused with propionate or propionate + 3HP (row 15).

The concentration of β-alanine was significantly increased by 3HP or 3HP + propionate

(row 18). This is compatible with β-alanine being a side-product of 3HP metabolism, via transamination of malonic semialdehyde (44, 73) (Scheme 8.1).

Figure 8.1 shows crossover plots comparing liver concentrations of intermediates of the propionyl-CoA pathway and of the CAC between groups of perfusions. In Figures 8.1, A,

B and C, metabolite concentrations are plotted relative to control livers (perfused with

SCHEME 8.1. Pathways of propionate and 3-hydroxypropionate metabolism in rat liver.

only 4 mM glucose). In the presence of propionate (Figure 8.1B), the concentrations of all intermediates are higher, or much higher than in control livers. This reflects a general overload of the propionyl-CoA pathway and the CAC. In contrast, in the presence of 3HP alone, a clear crossover occurs between succinyl-CoA and succinate (Figure 8.1A). Also, the concentrations of CAC intermediates were all greater than control. This reflects (i) an impairment of the disposal of endogenous intermediates of the propionyl-CoA pathway, and (ii) an overload of the CAC, albeit less than with propionate. In the presence of both propionate and 3HP, the plot appears similar to what was observed with propionate alone.

This reflects again a general overload of the propionyl-CoA pathway and the CAC.

However, when the metabolite concentrations measured in livers perfused with propionate

+ 3HP are plotted relative to the concentrations measured in the presence of propionate

alone (Figure 8.1D), the presence of 3HP decreased all relative concentrations assayed,

except for succinyl-CoA and malate. This reflects a decreased overload by propionate of the propionyl-CoA pathway and the CAC (which was not clearly visible in Figure 8.1B).

This was also reflected by the decrease in propionate uptake by 3HP (Table 8.1).

FIGURE 8.1. Crossover analyses of liver metabolite concentrations. Relative concentrations calculated vs controls in panels A (N = 5 in control; N = 6 in M3 3HP group), B (N = 5 in control; N = 5 in M3 propionate group), C (N = 5 in control; N = 10 in propionate + 3HP group), and vs data from livers perfused with M3 propionate in panel D (N = 5 in M3 propionate group; N = 10 in propionate + 3HP group). Note the different logarithmic vertical scales for panels B and C.

The profiles of adenine nucleotide concentrations in control livers (Table 8.2, rows 25-32) show the expected values of the [ATP]/[AMP] ratio (11, 88), energy charge (7) and mass

action ratio of the myokinase reaction (31). In the presence of 3HP, the pool of adenine

nucleotides was doubled and the [ATP]/[AMP] ratio was halved, compared to controls. In

the presence of propionate, the [ATP]/[ADP] and [ATP]/[AMP] ratios and the energy

charge decreased to very low levels, indicative of a major perturbation in energy

metabolism. In the presence of both propionate and 3HP, the adenine nucleotide pool was

somewhat less perturbed than with propionate alone.

The redox ratios [lactate]/[pyruvate], [BHB]/[AcAc] and [glycerol-3-

P]/[dihydroxyacetone-P] in control livers (Table 8.2, lines 39-41) are similar to values we

previously reported for livers from fasted rats perfused with 4 mM glucose (see Table IV

of (11)). The ratios [lactate]/[pyruvate] and [BHB]/[AcAc] reflect the [NADH]/[NAD+]

ratios in cytosol and mitochondria, respectively (87). In livers perfused with 3HP,

propionate or propionate + 3HP, the ratios remained in the physiological range, in spite of

the overload of the CAC (Figure 8.1). In the presence of 3HP alone, the total pool of

glycerol-P and dihydroxyacetone-P increased three fold compared to control, with a significant reduction of the [glycerol-3P]/[dihydroxyacetone-P] ratio (which was not reflected in the [lactate]/[pyruvate] and [BHB]/[AcAc] ratios. Because glycerol-P dehydrogenase is present in both cytosol and mitochondria, this may reflect some overload of the glycerol-3-P shuttle.

Labeling of metabolites

Figure 8.2 shows the time profiles of M3 labeling of 3HP released by livers perfused with

M3 propionate (Figure 8.2A), M3 propionate + unlabeled 3HP (Figure 8.2B), and unlabeled propionate + M3 3HP (Figure 8.2C). Figure 8.2A shows a clear precursor-to- product relationship between M3 propionate and M3 3HP. The M3 enrichment of 3HP stabilized at about 82%, i.e. below the 97% M3 enrichment of propionate. This reflects the generation of some unlabeled 3HP from endogenous sources, probably from ß-alanine derived from uracil catabolism (Scheme 8.1). This is confirmed by the detection of 3HP in control livers (Table 8.2). Figures 8.2B and 8.2C show that the release of 3HP from endogenous sources is not inhibited by exogenous 3HP. In livers perfused with M3 propionate + unlabeled 3HP (Figure 8.2B), the large pool of 3HP becomes slightly labeled from M3 propionate. Also, in livers perfused with unlabeled propionate + M3 3HP (Figure

8.2C), the labeling of the large pool of exogenous 3HP becomes steadily diluted by the production of unlabeled 3HP. The data of Figure 8.2C were used to calculate ranges of

3HP release (Table 8.1, rows 4 and 5).

FIGURE 8.2. Mass isotopomer distribution of 3HP released in perfusate. Livers were perfused with 5 mM M3 propionate (panel A, N = 5), 5 mM M3 propionate + 2 mM unlabeled 3HP (panel B, N = 5), or 5 mM unlabeled propionate + 2 mM M3 3HP (panel C, N = 5).

The labeling patterns of CoA esters are presented as mass isotopomer distributions (MIDs)

in Figure 8.3. Each panel shows the data for one CoA ester, identified on the Y scale. In each panel, each cluster of mass isotopomer enrichment (M1, M2, M3) compares data from livers perfused with M3 3HP (light blue bars), M3 propionate (orange bars), M3 propionate

+ unlabeled 3HP (red bars), and unlabeled propionate + M3 3HP (dark blue bars). In livers

perfused with M3 3HP, 3HP-CoA was strongly M3 labeled as expected (Figure 8.3D). The

intermediates of the propionyl-CoA pathway were poorly labeled, mostly in M1 and M2

(Figure 8.3, A, B, C). This reflects retrograde labeling via the CAC (68), and excludes a

direct precursor-to-product relationship between 3HP and propionyl-CoA. Acetyl-CoA

was 13% M2 labeled, while malonyl-CoA ws 10% M2 labeled without any M3 labeling

(Figure 8.3E and F). The absence of M3 malonyl-CoA demonstrates that 3HP is not

converted to malonyl-CoA, as occurs in some microorganisms (80). Malonyl-CoA was

10% M2 labeled from M2 acetyl-CoA.

To determine which carbons of 3HP are transferred to C-1 and C-2 of acetyl-CoA, we

perfused livers with 3HP labeled on different carbons. In one perfusion with [1-13C]3HP,

acetyl-CoA was unlabeled (not shown). This demonstrates that carbon 1 of 3HP is lost in

its conversion to acetyl-CoA via malonate semialdehyde dehydrogenase. Thus, C-1 of 3HP was lost in the conversion of malonic semialdehyde to acetyl-CoA. Then, in single

13 13 13 perfusions with [ C3]3HP, [3- C]3HP and [2- C]3HP, we assayed (i) the enrichment of

acetyl-CoA by LC-MS/MS (3), and (ii) the labeling of the C-4+5 moiety of glutamate (a

13 13 proxy of acetyl-CoA (39)) by C-NMR. In livers perfused with M3 [ C3]3HP, acetyl-CoA

was M2 labeled, as expected (Figure 8.3E). The NMR C4 glutamate resonance

FIGURE 8.3. Mass isotopomer distributions of CoA esters. Each panel shows the data for one CoA ester, identified on the Y scale. In each panel, each cluster of mass isotopomer enrichment (M1, M2,… Mn) compares data from livers perfused with 2 mM M3 3HP (light blue, N = 6), 5 mM M3 propionate (orange, N = 5), 5 mM M3 propionate + 2 mM unlabeled 3HP (red, N = 5), and 5 mM unlabeled propionate + 2 mM M3 3HP (dark blue, N = 5).

appeared as a doublet with a 13C- 13C J-coupling of 51 Hz, as would be expected from the coupling of 13C-labeling at C5 to C4. In one liver perfused with [3-13C]3HP, acetyl-CoA

was M1 labeled. The NMR C4 glutamate resonance again appeared as a doublet with a

13C- 13C J-coupling of 51 Hz, consistent with 13C-labeling at C5 (Figure 8.4). In one liver

perfused with [2-13C]3HP, acetyl-CoA was M1 labeled. The NMR C4 glutamate resonance

appeared as a singlet, indicative of 13C-labeling at C4, but not at C5. Thus, because C-5

and C-4 of glutamate correspond to C-1 and C-2 of acetyl-CoA, respectively, C-1 and C-2

of acetyl-CoA derive from C-3 and C-2 of 3HP, respectively (Figure 8.4 and Scheme 8.2).

FIGURE 8.4. NMR spectra of glutamate in livers perfused with 2 mM [2-13C]3HP, [3- 13 13 C]3HP or [ C3]3HP. The fate of the 13C-label of [2-13C]3HP and [3-13C]3HP was determined from the proton- decoupled 13C-NMR spectrum of glutamate carbon 4 from the perchloric acid extracts of the perfused livers. LC-MS/MS analysis of the glutamate C4-C5 fragment (3) indicated only M1 enrichment for livers perfused with either [2-13C]3HP and [3-13C]3HP. Glutamate 13 C4-C5 fragment in livers perfused with [ C3]3HP had mass isotopomers of M1 and M2. Upper spectrum: The singlet for glutamate C4 is consistent with 13C-labeling in C4, but not in C5, for the singly-labeled glutamate isotopomers in livers perfused with [2-13C]3HP. Middle spectrum: The doublet for glutamate C4 with 13C-13C spin coupling (1JCC) of 51 Hz, with negligible singlet resonance, is consistent with 13C-labeling predominately at C5 for the singly-labeled glutamate isotopomers in livers perfused with [3-13C]3HP. Lower spectrum: The doublet for glutamate C4 with 13C-13C spin coupling (1JCC) of 51 Hz is consistent with 13C-labeling at C4 and C5 for the doubly-labeled glutamate 13 isotopomers in livers perfused with [ C3]3HP.

SCHEME 8.2. Transfer of C-2 and C-3 of 3-hydroxypropionate to C-4 and C-5 of

glutamate via acetyl-CoA.

In livers perfused with 97% M3 propionate, propionyl-CoA and methylmalonyl-CoA were

about 80% M3 labeled (Figures 8.3A and B), reflecting the production of unlabeled

propionyl-CoA from endogenous sources (Scheme 8.1). The small M2 and M1

enrichments of propionyl-CoA and methylmalonyl-CoA reflect retrograde labeling from the CAC. The MID of succinyl-CoA labeled from M3 propionate (Figure 8.3C) reflects recycling and dilution of propionate label through multiple cycles of the CAC. This recycling results in a decrease in M3 succinyl-CoA enrichment (compared to methylmalonyl-CoA) and the formation of M2, M4 and M1 succinyl-CoA. The low

labeling of acetyl-CoA (11% M2, 3% M1) reflects the indirect labeling via (i) malate →

malic enzyme → pyruvate → pyruvate dehydrogenase → acetyl-CoA, (ii) oxaloacetate →

PEP → pyruvate → acetyl-CoA, and (iii) oxaloacetate → PEP →→ glucose →→ pyruvate

→ acetyl-CoA.

In perfusions with M3 propionate + unlabeled 3HP, the MID of succinyl-CoA was very different from that measured in perfusions with M3 propionate alone (Figure 8.3C, red vs orange bars). The M3 enrichment of succinyl-CoA was very high, with minuscule enrichment of the other mass isotopomers. The absence of M2 succinyl-CoA demonstrates that the presence of 3HP prevents the dilution and recycling of propionate label in the CAC past citrate, isocitrate or α-ketoglutarate (the latter two were not assayed). Flux through α- ketoglutarate dehydrogenase must be greatly decreased compared to perfusions with M3 propionate alone. Note that, in the perfusions with M3 propionate + unlabeled 3HP, the labeling of succinyl-CoA (Figure 8.3C, red bar) does not match those of succinate, malate and fumarate (Figures 8.5B-D, red bars). We have no explanation for this discrepancy.

Lastly, in livers perfused with unlabeled propionate + M3 3HP, very little labeling was detected in the assayed CoA esters, except for 3% M2 acetyl-CoA (Figure 8.3). Thus, propionate decreased the conversion of 3HP to acetyl-CoA (Figure 8.3E, light blue vs dark blue bar).

Figure 8.5 shows the MIDs of relevant non-CoA metabolites in liver tissue. In the presence of M3 3HP, the main labeling of CAC intermediates is the M2 labeling of citrate: about

10% (Figure 8.5A), similar to the M2 labeling of acetyl-CoA (Figure 8.3E). The low M2 and M1 labeling of succinate, fumarate and malate result from (i) recycling

FIGURE 8.5. Mass isotopomer distributions of non-CoA metabolites. Each panel shows data for one metabolite, identified on the Y scale. In each panel, each cluster of mass isotopomer enrichment (M1, M2,… Mn) compares data from livers perfused with 2 mM M3 3HP (light blue, N = 6), 5 mM M3 propionate (orange, N = 5), 5 mM M3 propionate + 2 mM unlabeled 3HP (red, N = 5), and unlabeled 5 mM propionate + 2 mM M3 3HP (dark blue, N = 5).

of label from the acetyl moiety of citrate with partial loss of label in the oxaloacetate to

PEP to pyruvate cycle, and (ii) the reversibility of the reactions linking oxaloacetate and

succinate. Thus the data clearly show that carbon from 3HP enters the CAC only in the

reaction from acetyl-CoA to citrate. 3HP is therefore not anaplerotic. In contrast, in livers

perfused with M3 propionate, the MID of succinyl-CoA (Figure 8.3C) and CAC intermediates (Figures 8.5, A-D) is consistent with the entry of propionate label into the

cycle mostly via anaplerosis and to a smaller extent as acetyl-CoA (Figure 8.3E). When

unlabeled 3HP was added to M3 propionate, there was very little changes in the MID of

citrate. In perfusions with M3 propionate without or with unlabeled 3HP, the MIDs of

malate were very similar in spite of the fact that the uptake of M3 propionate was decreased

by half by 3HP (Table 8.1 rows 2 and 4). Thus, the presence of unlabeled 3HP prevented

the dilution of malate labeling from M3 propionate by unlabeled substrate(s), presumably

glucose. This is further evidence of inhibition of CAC flux by the addition of 3HP. Lastly,

in livers perfused with M3 3HP + unlabeled propionate, there was very little labeling of

CAC intermediates, probably because of (i) the dilution by unlabeled carbon from

anaplerotic propionate, and/or (ii) the inhibition of 3HP catabolism by propionate (see

below).

Figure 8.6 shows the MID of perfusate glucose at the end of the 1 h experiments. With M3

3HP, with or without unlabeled propionate, (light and dark blue bars), there was very little

label in glucose, as expected from non-gluconeogenic 3HP. With M3 propionate alone

(orange bars), the MID of glucose included all M1 to M6 isotopomers, as expected. With

M3 propionate + unlabeled 3HP, the labeling of glucose was decreased, as shown by the shift to mostly M1 and M2 mass isotopomers (red bars). The average labeling of glucose carbon, calculated from the MIDs, decreased from 17% to 9%. This may result from the decreased uptake of M3 propionate (Table 8.1), some glycogen breakdown, and some gluconeogenesis from glycerol derived from intrahepatic lipolysis (90).

FIGURE 8.6. Mass isotopomer distributions of glucose. Data are from final perfusates of livers perfused with the indicated substrates, i.e., 2 mM M3 3HP (light blue, N = 6), 5 mM M3 propionate (orange, N = 5), 5 mM M3 propionate + 2 mM unlabeled 3HP (red, N = 5), and 5 mM unlabeled propionate + 2 mM M3 3HP (dark blue, N = 5).

FIGURE 8.7. Comparison of M2 enrichments of citrate, acetyl-CoA and C-1+2 of acetoacetate. Data are from livers perfused with 2 mM M3 3HP (N = 6) vs 5 mM unlabeled propionate + 2 mM M3 3HP (N = 5).

Figure 8.7 presents the key labeling data that illustrate the inhibition of 3HP metabolism

by propionate. First, propionate decreased 3.9-fold the conversion of M3 3HP to acetyl-

CoA, although propionate is not a sizeable precursor of acetyl-CoA. Second, propionate

decreased 41-fold the incorporation of the acetyl moiety of 3HP into citrate, that is more

than the conversion of 3HP to acetyl-CoA. The greater labeling of C-1+2 of acetoacetate compared to acetyl-CoA reflects the channeling of acetyl group preferentially toward acetoacetate rather than citrate. This is similar to the channeling of acetyl groups from fatty acids toward acetoacetate (26). The labeling of C-1+2 of acetoacetate decreased 3.9 times when M0 propionate was added to M3 3HP, confirming that propionate (i) inhibits the conversion of M3 3HP to M2 acetyl-CoA, and/or (ii) dilutes the M2 acetyl-CoA derived from M3 3HP. Thus, the data of Figure 8.7 clearly demonstrate the inhibition of 3HP metabolism by propionate.

Production and labeling of maleate

Maleate, the trans isomer of fumarate, was not detected in control livers, but was found in livers perfused with either propionate, 3HP, propionate + 3HP, or methylmalonate (Table

8.2, row 16; Fig 8.9). Maleate was clearly distinguished from fumarate by retention time and fragmentation pattern (not shown). Interconversion of fumarate and maleate did not occur under our conditions of GC-MS analysis. The accumulation of maleate in the recirculating perfusate (Figure 8.9) paralleled the tissue concentrations (Table 8.2, row 16).

In livers perfused with M3 propionate, the MIDs of fumarate and maleate were markedly different from each other in the liver tissue and in the perfusate. Fumarate was M1 to M4 labeled because of multiple cycling in the CAC (Figures 8.8A and 8.5C). In contrast,

maleate was only M3 labeled, with no unlabeled (Figures 8.8B and 8.5E). In one perfusion

with M3 propionate + 2 mM unlabeled acrylate, the M3 labeling of perfusate maleate was

progressively diluted, reflecting some production of unlabeled maleate from acrylate

(Figure 8.8C). The data demonstrate that a secondary pathway of propionate metabolism, not the CAC, generates maleate, the molecule of which contains the 3 carbons of propionate + 1 additional carbon. The data also support the view that, in mammalian tissue,

the conversion of propionyl-CoA to 3HP-CoA occurs via acrylyl-CoA (6).

Note that M3 3HP forms M3 3HP-CoA, but does not form M3 propionyl-CoA, just very

small amounts of M1 and M2 propionyl-CoA (Figure 8.3A). This is evidence that, in

Scheme 8.1, (i) the reaction converting acrylyl-CoA to 3HP-CoA is not reversible, while

(ii) the reaction between propionyl-CoA and acrylyl-CoA is reversible (based on Figure

8.8C, showing that the M3 labeling of maleate formed from M3 propionate is diluted by

unlabeled acrylate). We were not able to identify unstable acrylyl-CoA in livers, even when perfused with acrylate.

The following data demonstrate that maleate is also formed from 3HP, without involvement of propionyl-CoA. First, in livers perfused with M3 3HP, maleate was substantially M2 labeled in the tissue and in the perfusate (Figures 8.5E and 8.8D). Second, in single livers perfused with 3HP labeled with 13C on carbon 1, 2 or 3, maleate was unlabeled from [1-13C]3HP, 10.5% M1 labeled from [2-13C]3HP, and 9.9% M1 labeled

from [3-13C]3HP (not shown). This indicates that the molecules of maleate derived from

3HP contain carbons 2 and 3 of 3HP (not carbon 1). In contrast, the molecules of maleate

derived from propionate contain all 3 carbons of propionate (Figure 8.8B).

FIGURE 8.8. Mass isotopomer distributions (MID) of fumarate and maleate released by perfused livers. Panel A: MID of fumarate labeled from 5 mM M3 propionate. Panels B to F: MID of maleate labeled from: 5 mM M3 propionate (panel B), 5 mM M3 propionate in the presence of 2 mM unlabeled acrylate (panel C), 2 mM M3 3HP (panel D), 5 mM M3 propionate in the presence of 2 mM unlabeled 3HP (panel E), or 2 mM M3 3HP in the presence of 5 mM unlabeled propionate (panel F).

FIGURE 8.9. Relative concentration of maleate released by perfused livers. Relative concentrations of maleate released by livers perfused with 4 mM glucose + nothing (control, grey, N = 5), 2 mM M3 3HP (blue, N = 6), 5 mM M3 propionate (orange, N = 5), 5 mM propionate + 2 mM 3HP (green, N = 10), or 2 mM methylmalonate (purple, N = 1). The concentration profiles for controls and the M3 3HP groups are superposed.

In livers perfused with both propionate and 3HP, alternately labeled, the MID of maleate released in the perfusate is more complex than in the presence of a single labeled substrate:

M3 propionate or M3 3HP. The complexity results from the partial conversion of propionate to 3HP resulting in the formation of either (i) a secondary substrate/tracer, i.e.,

M3 3HP from M3 propionate, or (ii) unlabeled 3HP from unlabeled propionate. In the first

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case, when unlabeled 3HP is added to M3 propionate (Figure 8.8E), the M3 maleate

isotopomer derived from M3 propionate is diluted from 95 to 80%. The M2 isotopomer of

maleate derives from M3 3HP formed from M3 propionate. (compare Figures 8.8B and E).

In the second case, when unlabeled propionate is added to M3 3HP (Figure 8.8F), the

labeled maleate isotopomers derived from M3 3HP are diluted by unlabeled maleate

derived from unlabeled propionate (compare Figures 8.8F and D). One might wonder why, in the perfusions with M3 propionate alone, we did we not detect M2 maleate by the sequence M3 propionate  M3 3HP  M2 maleate? This is probably because, the very small concentration of M3 3HP (derived from M3 propionate alone) were insufficient to form detectable M2 maleate.

Labeling of β-alanine

β-Alanine was M3 labeled mostly from M3 3HP and M3 3HP + unlabeled propionate

(Figure 8.5F, light blue and dark blue bars). This is compatible with the partial equilibration of label from 3HP and β-alanine via reversible transamination of malonate semialdehyde (73) (Scheme 8.1). Very little label from M3 propionate was found in β-

alanine (Figure 8.5F, orange bars), probably via the conversion of propionate to 3HP (Table

8.1) and 3HP-CoA (Figure 8.3D). Overall, at least 90% of β-alanine derived from

endogenous unlabeled sources, presumably uracil (Figure 8.5F).

To verify the reversibility of the transamination of malonate semialdehyde to β-alanine, we

2 ran two liver perfusions in 100% H2O buffer containing 4 mM glucose. In one of the

perfusions, we added 2 mM unlabeled 3HP. In the latter liver, β-alanine was labeled 18.7%

M1, 62.5% M2, 1.6% M3 and 0.24% M4. In the absence of 3HP, liver β-alanine was

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labeled 24% M1, 42.2% M2, 3.7% M3 and 0.1% M4. The M1 and M2 enrichments of β- alanine are compatible with the exchanges of hydrogen atoms that occur during transamination. The M3 and M4 enrichments of β-alanine reflect the exchanges of hydrogen atoms that occur both in the transamination reaction and in the reversible reactions between uracil and β-alanine (53) (Scheme 8.1). Although we could assay standards of malonate semialdehyde with the sensitive carbonyl reagent kindly made available by Dr. Teresa Fan (52) we could not detect this very unstable compound (43, 69,

73) in liver.

Proteolysis and ureagenesis

Isolated livers perfused without nitrogenous substrates release amino acids and urea (32).

This reflects tissue proteolysis, amino acid catabolism and ureagenesis. We measured the final perfusate concentrations of urea and amino acids, and expressed all data in

(microequivalents of N)·(g dry wt)−1 ·h−1 (Table 8.3). The total release of amino acids was

significantly increased by 3HP and by propionate + 3HP. Ureagenesis was doubled by

propionate, but not by 3HP or 3HP + propionate. The fraction of total N release that was

found in urea was increased by propionate and decreased by propionate + 3HP. Overall,

3HP and propionate, singly or in combination, induced protein breakdown.

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values shown in the table comparisons are against control. weight) Table control (p<0.05). ^ Significantly different from different Significantly (p<0.05). ^ ‡Significantlyfromcontrol 3HPdifferent M3 (p<0.05). (p<0.05). Propionate M3 p mM alternately propionate labeled (Propionate + 3HP). Data presented are as mean and nothingeither mMglucose (Control), 2 [ 8. - 1 3. Release of amino acids and urea perfused from rat livers, expressed as (microequivalents of N)·(g dry ·hr - 1 . The calculations are based on final perfusate concentrations 13 C 3 ]3HP (M3 3HP), 5 mM 3HP), 5 ]3HP (M3 [

13 at 60 min. were at livers perfused Rat 4 mM with C 3 ]propionate (M3 Propionate),]propionate (M3 2 5 mM + 3HP ± SD. *Significantly different from

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Discussion

Compensation of CoA sequestration

Mitchell et al. have emphasized that CoA sequestration, toxicity or redistribution

(CASTOR) occurs “in many hereditary and acquired conditions in which the degradation

of CoA esters is impaired” (56). The implication of this concept is that large trapping of

CoA in either a metabolite that is not disposed of by a defective enzyme, or in a xenobiotic-

CoA ester, would interfere with some CoA-depending reactions and induce metabolic

perturbations. Our data illustrate some aspects of the CASTOR concept. In livers perfused

with 3HP, the profile of CoA ester concentrations assayed was similar to that of control

livers, except for a five-fold increase in the very small pool of 3HP-CoA (Table 8.2, row

7). Thus, in the presence of 3HP alone, there was no substantial CoA trapping in the livers.

In contrast, in livers perfused with propionate or propionate + 3HP, there were large increases in the concentrations of the CoA esters of the propionyl-CoA pathway. This resulted in a tripling of the total concentration of the CoA species assayed. Free CoA was halved only in the presence of propionate alone (Table 8.2, row 6). Although we did not assay long-chain acyl-CoAs, their total concentration in liver is similar to that of acetyl-

CoA, even in the presence of oleate (11, 12). Therefore, in livers perfused with propionate

or propionate + 3HP, there was a rapid reaction to CoA trapping, resulting in the tripling

of the total CoA pool assayed. Thus, the trapping of CoA does not necessarily result in a

decrease in free CoA, as implied in the CASTOR concept (56).

The increase in total CoA implies a very rapid activation of the pantothenate kinase

pathway, forming new CoA molecules from the liver store of dietary pantothenate or

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unidentified pantothenate precursors. Recent reports from the Jackowski group (47, 48, 67)

suggest that concentrations of acylcarnitines, acylethanolamides, as well as the expression

of pantothenate kinase and Nudix isoforms should be evaluated in livers perfused with

propionate ± 3HP.

Modulation of the CAC by 3HP, propionate or both

The labeling of CAC intermediates from M3 3HP (Figures 8.5, A-D, light blue bars) confirms the oxidation of acetyl-CoA derived from 3HP. From these data, it would seem that 3HP is simply a precursor of acetyl-CoA. However, the crossover analysis (Figure

8.1A) shows that the CAC is overloaded in the presence of 3HP, in spite of the small contribution of 3HP to acetyl-CoA (13%, Figure 8.3E). Also, intermediates of the endogenous propionyl-CoA pathway are prevented from entering the CAC, as shown by

(i) the crossover between succinyl-CoA and succinate (Figure 8.1A), and (ii) the increases in the [succinate]/[succinyl-CoA] and [malate]/[succinyl-CoA] ratios, compared to control

(Table 8.2). An unlikely explanation of the CAC overload would be the inhibition by methylcitrate of the rates of CO2 generating reactions (isocitrate dehydrogenase, α- ketoglutarate dehydrogenase (21)). This is because the Ki of the inhibitions by methylcitrate range from 1 to 8 mM (21), while the methylcitrate concentrations we measured in liver are very low (Table 8.2, row 15).

In the presence of M3 propionate, the labeling pattern of intermediates of the propionyl-

CoA pathway and of the CAC (Figures 8.3 and 8.5, orange bars) reproduce studies conducted previously (68). However, the crossover analysis (Figure 8.1B) and the individual concentrations (Table 8.2) show major overload of the CAC. This probably

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results in part of the high rate of entry of anaplerotic carbon into the CAC, relative to the

rate of acetyl-CoA entry. Still, the rate of anaplerosis from propionate (i.e., the uptake of

propionate) is about balanced by an equivalent rate of cataplerosis in the form of glucose

and lactate-bound carbon (not shown). The major perturbation of the adenine nucleotide

profile (Table 8.2) and the non-affected [lactate]/[pyruvate] and [BHB]/[AcAc] ratios

reflect a low flux of reducing equivalents from the engorged CAC to the respiratory chain.

However, the presence of M2 and M1 mass isotopomers of succinyl-CoA (Figure 8.3C)

demonstrates the operation of the α-ketoglutarate dehydrogenase reaction (in contrast to what occurs in the presence of propionate + 3HP, as discussed below). These isotopomers can only derive from the complete operation of the CAC, albeit at a low rate.

In the presence of propionate and 3HP, the CAC and the propionyl-CoA pathway remain overloaded, with a very high concentration of succinyl-CoA (Table 8.2), but to a lesser degree than in the presence of propionate alone (Figures 8.1C and D). Also, the MID of succinyl-CoA (with very little M2 and M1 components, Figure 8.3C, red bars) reflects a strong inhibition of the CAC between citrate and succinyl-CoA, which did not occur with

M3 propionate alone (Figure 8.3C, orange bars).

What are the fates of the carbon of 3HP?

In livers perfused with M3 3HP alone, the substrate contributes about 13% to acetyl-CoA flux (Figure 8.3E), and enters the CAC, as M2 citrate (Figures 8.5A and 8.7). Although the

CAC is overloaded (Figure 8.1A), 3HP contributes to the production of CO2 (as shown by the labeling pattern of succinyl-CoA (Figure 8.3C) and by the loss of C-1 of 3HP in the conversion of malonic semialdehyde to acetyl-CoA (Scheme 8.2). Another fate of acetyl

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groups derived from M3 3HP is the production of labeled ketone bodies (Figure 8.7).

However, the liver concentrations of ketone bodies were not increased by propionate, 3HP,

or both (Table 8.2, rows 35-36). When unlabeled propionate is added to M3 3HP, the

labeling of acetyl-CoA and citrate markedly decrease (Figures 8.3E and 8.5A, dark blue

bars). Also, very little of 13C from M3 3HP ends up in glucose (Figure 8.6, dark blue bars).

Only traces of β-alanine were found in the final perfusate. We have not identified any large

unknown peaks in the metabolomic profiles run on liver tissue and perfusate in these

experiments. The fates of the bulk of the 3HP carbon taken up are thus not clear and should

be further investigated.

Relevance to disorders of propionyl-CoA metabolism

Consider first the production of maleate from propionate and from 3HP (Figure 8.8, B to

F). Maleate is not a physiological metabolite in mammals. Injection of maleate to rats

impairs kidney function and induces glucosuria (76). Also, the maleate salts of some drugs

are nephrotoxic (28). Maleyl-CoA reacts with amino acids and proteins of the renal brush

border, and uncouples sodium-dependent solute transport (58). Also, maleyl-CoA reacts with free CoA to form a stable inert thioether, a form of CoA sequestration (62, 63). Our

data show that maleate is formed from propionate and from 3HP by different processes.

While all carbons from M3 propionate are transferred to maleate, only carbons 2 and 3 of

3HP (or 3HP-CoA) are transferred to maleate.

The formation of M3 maleate from M3 propionate suggests a carboxylation process,

probably of propionyl-CoA, followed by unknown reaction(s) leading to maleate. Wakil

had reported that acetyl-CoA carboxylase can carboxylate propionyl-CoA to form

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methylmalonyl-CoA, at a rate 60% that of the carboxylation of acetyl-CoA (83). This

secondary reaction of acetyl-CoA carboxylase is probably stimulated by the very high

concentration of propionyl-CoA in livers perfused with propionate ± 3HP (Table 8.2).

Because acetyl-CoA carboxylase is cytosolic, methylmalonyl-CoA formed in the cytosol

has different labeling pattern and fate(s) than methylmalonyl-CoA formed by

mitochondrial propionyl-CoA carboxylase. It is not clear how maleate could be formed

from cytosolic methylmalonyl-CoA. However, the release of maleate in one perfusion with

2 mM methylmalonate (Figure 8.9) is compatible with the conversion of some

methylmalonyl-CoA to maleate.

Alterations in kidney function (tubulo-interstitial nephritis) can occur in both propionic

acidemia (20, 45) (even after liver transplantation (20)) and methylmalonic acidemia (59).

In both conditions, maleate released by the liver (9) could concentrate in kidney tubules

and contribute to the degradation of kidney function. In methylmalonic acidemia, the liver

concentration of methylmalonyl-CoA is probably higher than in propionic acidemia. If part of this methylmalonyl-CoA were to be converted to maleate, this might explain the greater frequency of kidney problems in methylmalonic acidemia compared to propionic acidemia.

Our data seem to explain why propionic acidemia patients are not cured by liver

transplantation (19, 20, 24, 71, 72). The new liver should be able, initially, to dispose of

propionate formed by intestinal fermentations. However, peripheral tissues which cannot

dispose of propionyl-CoA derived from amino acid catabolism release 3HP and propionate.

These progressively accumulate and induce the metabolic perturbations we observed in

perfused rat livers. The presence of 3HP decreases the capacity of the liver to dispose of

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propionate (Table 8.1). This leads to CoA trapping and overload + inhibition of the CAC.

The stimulation of proteolysis in livers perfused with propionate, 3HP or both (i) increases

the release of essential amino acids that are precursors of propionyl-CoA (Table 8.3), and

(ii) evokes the propensity of propionic acidemia patients to go into proteolytic crises

resulting in hyperammonemia. The latter is explained by the inhibition of N-

acetylglutamate synthetase by propylated effectors (78, 79). Then, a normal rate of

ureagenesis is presumably achieved at supra-physiological and toxic concentrations of ammonium in body fluids. Promising clinical trials with N-carbamylglutamate, an activator of N-acetylglutamate synthetase, are currently conducted (2, 78, 82).

The total pool of liver adenine nucleotides was markedly increased in the presence of 3HP, propionate or both (Table 8.2, row 28). The ratios of adenine nucleotide concentrations, the energy charge and the mass action ratio of the myokinase reaction were also perturbed

(rows 29-32). These findings are not reflected by an overload of the respiratory chain because the redox indicator ratios [lactate]/[pyruvate] and [BHB]/[AcAc] remained in the oxidized range. The overloaded CAC did not release enough reducing equivalents to be oxidized in the respiratory chain and restore the balance of the adenine nucleotides pools.

It appears that, in our short-term experiments (1 h), the reduction in CAC flux was balanced by a reduction in the flux of oxidative phosphorylation without hyperreduction of the mitochondrial [NADH]/[NAD+] ratio. The inhibition of the CAC flux was probably not

caused by the formation of the very low concentrations of methylcitrate measured in livers

(Table 8.2, row 15). Note however that in mice with propionic acidemia, high

concentrations of methylcitrate accumulate in body fluids (33, 57). In mice and humans

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with methylmalonic acidemia, a large fraction of methylcitrate is released by muscle and

may affect the metabolism of other organs (16).

The excretion of methylcitrate and methylsuccinate in propionic acidemia patients is viewed by some clinicians as a cataplerotic drain which inhibits CAC operation. It was suggested in meeting discussions that decompensated patients could benefit from treatment with a suitable sodium-free anaplerotic substrate which is not a propionyl-CoA precursor.

We recently reported that the dicarboxylate dodecanedioate forms anaplerotic succinyl-

CoA in mitochondria of perfused rat livers (38). We suggested that the glycerol dodecanedioate ester, investigated by others as a nutrient (55), could be used as an anaplerotic agent for the acute treatment of propionic acidemia. However, the present study shows that in livers perfused with propionate + 3HP, there is an accumulation of succinyl-

CoA (Table 8.2) which traps CoA and inhibits the α-ketoglutarate dehydrogenase reaction

(Figure 8.3C). In the light of our present data, we retract our suggestion (38) to use dodecanedioate for the acute treatment of propionic acidemia.

13 Use of [ C3]propionate to trace pathways of intermediary metabolism

Labeled propionate has been used extensively to trace gluconeogenesis, CAC flux and

anaplerosis in rodents and humans. [14C]Propionate is a true tracer which does not perturb

13 the processes being traced (46). In contrast, [ C3]propionate is often administered in

amounts which greatly increase the very low concentration of endogenous propionate and

change the dynamics of the traced processes (36, 37, 40). Perry et al. (64) have recently

documented a number of perturbations of liver metabolism in rats after oral or intravenous

13 administration of large doses of [ C3]propionate: increase in hepatic glucose production

110 and in a number of metabolic rates, accumulation of CAC intermediates and of propionyl-

13 CoA. In the present study, we imposed mM concentrations of [ C3]propionate and/or

13 [ C3]3HP, not to trace normal liver metabolism, but to induce and trace metabolic perturbations relevant to disorders of propionyl-CoA metabolism. Thus tracer doses and loading doses of labeled compounds should be used for different applications.

In conclusion, the goal of the present study was to simulate the metabolic status of the liver of propionic acidemia patients who did not fully benefit from liver transplantation. Our data demonstrate that the simultaneous presence of propionate and 3HP in normal livers causes major metabolic perturbations which mimic those observed in the propionic acidemia patients. The next stages in this study will attempt to (i) explain the observed perturbations which are likely caused, at least in part, by the accumulation of CoA esters, especially 3HP-CoA (Table 8.2) and likely carnitine esters which are probably toxic at high concentrations (74), (ii) identify the mechanisms that trigger the rapid CoA synthesis in the presence of propionate ±3HP, (iii) shed light on the fates of the 3HP carbons, and (iv) identify the mechanism of maleate formation. Such studies will be conducted in normal livers, hepatocytes and mitochondria from propionic acidemia patients (17) and patient cultured cells.

Acknowledgments

We thank the Case Mouse Metabolic Phenotyping Center for help with the liver perfusion experiments. We thank Dr. Charles Venditti for constructive comments on our manuscript. 111

We thank Dr. Teresa Fan for supplying us with the sensitive carbonyl reagent synthesized

by her group.

Grants

This work was supported by the NIH (Roadmap grant R33DK070291, and training grant

5 T32HL105338 (fellowship to KAW)) and K08DK105233 (KAC).

Disclosures

The authors declare that they have no conflicts of interest with the context of this article.

Author contributions

K.A.W. helped with experimental design, conducted most of the experiments and analyses and computed the data. Y. H. and J. H. synthesized labeled 3-hydroxypropionate. M.Z. helped with the analyses. K.A.C. helped with data interpretation. G.W.C conducted NMR analyses and helped with experimental design. G.P.T. designed and oversaw the chemical syntheses. G.F.Z. made substantial contributions to experimental conception and design and manuscript preparation. H.B. conceived the idea for the project, helped with experimental design, and wrote the manuscript with contributions from all other authors.

References

1. Abacan M and Boneh A. Use of carglumic acid in the treatment of hyperammonaemia during metabolic decompensation of patients with propionic acidaemia. Molecular Genetics and Metabolism 109: 397-401, 2013. 2. Ah Mew N, McCarter R, Daikhin Y, Lichter-Konecki U, Nissim I, Yudkoff M, and Tuchman M. Augmenting ureagenesis in patients with partial carbamyl

112

phosphate synthetase 1 deficiency with N-carbamyl-L-glutamate. J Pediatr 165: 401-403 e403, 2014. 3. Alves TC, Pongratz RL, Zhao X, Yarborough O, Sereda S, Shirihai O, Cline GW, Mason G, and Kibbey RG. Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle. Cell Metab 22: 936-947, 2015. 4. Amaral AU, Cecatto C, Castilho RF, and Wajner M. 2-Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria. J Neurochem 137: 62-75, 2016. 5. Ando T, Rasmussen K, Nyhan WL, and Hull D. 3-hydroxypropionate: significance of -oxidation of propionate in patients with propionic acidemia and methylmalonic acidemia. Proc Natl Acad Sci U S A 69: 2807-2811, 1972. 6. Ando T, Rasmussen K, Wright JM, and Nyhan WL. Isolation and identification of methylcitrate, a major metabolic product of propionate in patients with propionic acidemia. J Biol Chem 247: 2200-2204, 1972. 7. Atkinson DE. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7: 4030-4034, 1968. 8. Baumgartner MR, Horster F, Dionisi-Vici C, Haliloglu G, Karall D, Chapman KA, Huemer M, Hochuli M, Assoun M, Ballhausen D, Burlina A, Fowler B, Grunert SC, Grunewald S, Honzik T, Merinero B, Perez-Cerda C, Scholl-Burgi S, Skovby F, Wijburg F, MacDonald A, Martinelli D, Sass JO, Valayannopoulos V, and Chakrapani A. Proposed guidelines for the diagnosis and management of methylmalonic and propionic acidemia. Orphanet J Rare Dis 9, 2014. 9. Bergstrom T, Greter J, Levin AH, Steen G, Tryding N, and Wass U. Propionyl-CoA carboxylase deficiency: case report, effect of low-protein diet and identification of 3-oxo-2-methylvaleric acid 3-hydroxy-2-methylvaleric acid, and maleic acid in urine. Scand J Clin Lab Invest 41: 117-126, 1981. 10. Brandange S, Josephson S, Mahlen A, Morch L, Sweetman L, and Vallen S. Stereochemistry of the methylcitric acids formed in the citrate synthase reaction with propionyl-CoA. Acta Chem Scand B 31: 628-630, 1977. 11. Brunengraber H, Boutry M, and Lowenstein JM. Fatty-Acid and 3-Beta- Hydroxysterol Synthesis in Perfused Rat-Liver - Including Measurements on Production of Lactate, Pyruvate, Beta-Hydroxybutyrate, and Acetoacetate by Fed Liver. Journal of Biological Chemistry 248: 2656-2669, 1973. 12. Brunengraber H, Boutry M, and Lowenstein JM. Fatty-Acid, 3-Beta- Hydroxysterol, and Ketone Synthesis in Perfused Rat-Liver - Effects of (-)- Hydroxycitrate and Oleate. Eur J Biochem 82: 373-384, 1978. 13. Brusque AM, Rosa RB, Schuck PF, Dalcin KB, Ribeiro CAJ, Silva CG, Wannmacher CMD, Dutra CS, Wyse ATS, Briones P, and Wajner M. Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid. Neurochem Int 40: 593-601, 2002. 14. Busch M, Stein G, Poppitz W, Hein G, and Muller A. Validated capillary gas chromatographic-mass spectrometric assay to determine 2-methylcitric acid I and II levels in human serum by using a pulsed splitless injection procedure. J Chromatogr B 775: 215-223, 2002.

113

15. Carvalho RA, Jones JG, McGuirk C, Sherry AD, and Malloy CR. Hepatic gluconeogenesis and Krebs cycle fluxes in a CCl4 model of acute liver failure. Nmr Biomed 15: 45-51, 2002. 16. Chandler RJ, Sloan J, Fu H, Tsai M, Stabler S, Allen R, Kaestner KH, Kazazian HH, and Venditti CP. Metabolic phenotype of methylmalonic acidemia in mice and humans: the role of skeletal muscle. BMC Med Genet 8: 64, 2007. 17. Chapman KA, Collado MS, Figler RA, Hoang SA, Armstrong AJ, Cui WX, Purdy M, Simmers MB, Yazigi NA, Summar ML, Wamhoff BR, and Dash A. Recapitulation of metabolic defects in a model of propionic acidemia using patient- derived primary hepatocytes. Molecular Genetics and Metabolism 117: 355-362, 2016. 18. Chapman KA, Gropman A, MacLeod E, Stagni K, Summar ML, Ueda K, Mew NA, Franks J, Island E, Matern D, Pena L, Smith B, Sutton VR, Urv T, Venditti C, and Chakrapani A. Acute management of propionic acidemia. Molecular Genetics and Metabolism 105: 16-25, 2012. 19. Chapman KA, Summar ML, and Enns GM. Propionic acidemia: To liver transplant or not to liver transplant? Pediatr Transplant 16: 209-210, 2012. 20. Charbit-Henrion F, Lacaille F, McKiernan P, Girard M, de Lonlay P, Valayannopoulos V, Ottolenghi C, Chakrapani A, Preece M, Sharif K, Chardot C, Hubert P, and Dupic L. Early and Late Complications After Liver Transplantation for Propionic Acidemia in Children: A Two Centers Study. Am J Transplant 15: 786-791, 2015. 21. Cheema-Dhadli S, Leznoff CC, and Halperin ML. Effect of 2-methylcitrate on citrate metabolism: implications for the management of patients with propionic acidemia and methylmalonic aciduria. Pediatr Res 9: 905-908, 1975. 22. Cline GW, LePine RL, Papas KK, Kibbey RG, and Shulman GI. C-13 NMR isotopomer analysis of anaplerotic pathways in INS-1 cells. Journal of Biological Chemistry 279: 44370-44375, 2004. 23. Coude FX, Sweetman L, and Nyhan WL. Inhibition by Propionyl-Coenzyme-a of N-Acetylglutamate Synthetase in Rat-Liver Mitochondria - Possible Explanation for Hyper-Ammonemia in Propionic and Methylmalonic Acidemia. J Clin Invest 64: 1544- 1551, 1979. 24. de Baulny HO, Benoist JF, Rigal O, Touati G, Rabier D, and Saudubray JM. Methylmalonic and propionic acidaemias: Management and outcome. J Inherit Metab Dis 28: 415-423, 2005. 25. Den H, Robinson WG, and Coon MJ. Enzymatic Conversion of Beta- Hydroxypropionate to Malonic Semialdehyde. Journal of Biological Chemistry 234: 1666-1671, 1959. 26. Des Rosiers C, David F, Garneau M, and Brunengraber H. Nonhomogeneous Labeling of Liver Mitochondrial Acetyl-Coa. Journal of Biological Chemistry 266: 1574- 1578, 1991. 27. Des Rosiers C, Didonato L, Comte B, Laplante A, Marcoux C, David F, Fernandez CA, and Brunengraber H. Isotopomer Analysis of Citric-Acid Cycle and Gluconeogenesis in Rat-Liver - Reversibility of Isocitrate Dehydrogenase and Involvement of Atp-Citrate Lyase in Gluconeogenesis. Journal of Biological Chemistry 270: 10027-10036, 1995. 114

28. Everett RM, Descotes G, Rollin M, Greener Y, Bradford JC, Benziger DP, and Ward SJ. Nephrotoxicity of pravadoline maleate (WIN 48098-6) in dogs: evidence of maleic acid-induced acute tubular necrosis. Fundam Appl Toxicol 21: 59-65, 1993. 29. Fernandez CA, DesRosiers C, Previs SF, David F, and Brunengraber H. Correction of C-13 mass isotopomer distributions for natural stable isotope abundance. J Mass Spectrom 31: 255-262, 1996. 30. Filipowicz HR, Ernst SL, Ashurst CL, Pasquali M, and Longo N. Metabolic changes associated with hyperammonemia in patients with propionic acidemia. Mol Genet Metab 88: 123-130, 2006. 31. Goldberg RN, Tewari YB, and Bhat TN. Thermodynamics of enzyme- catalyzed reactions - a database for quantitative biochemistry. Bioinformatics 20: 2874- 2877, 2004. 32. Green M and Miller LL. Protein Catabolism and Protein Synthesis in Perfused Livers of Normal and Alloxan-Diabetic Rats. Journal of Biological Chemistry 235: 3202- 3208, 1960. 33. Guenzel AJ, Hofherr SE, Hillestad M, Barry M, Weaver E, Venezia S, Kraus JP, Matern D, and Barry MA. Generation of a hypomorphic model of propionic acidemia amenable to gene therapy testing. Mol Ther 21: 1316-1323, 2013. 34. Guneral F and Bachmann C. Age-Related Reference Values for Urinary Organic-Acids in a Healthy Turkish Pediatric Population. Clinical Chemistry 40: 862- 868, 1994. 35. Halarnkar PP and Blomquist GJ. Comparative Aspects of Propionate Metabolism. Comp Biochem Phys B 92: 227-231, 1989. 36. Jensen PR, Peitersen T, Karlsson M, in't Zandt R, Gisselsson A, Hansson G, Meier S, and Lerche MH. Tissue-specific Short Chain Fatty Acid Metabolism and Slow Metabolic Recovery after Ischemia from Hyperpolarized NMR in Vivo. Journal of Biological Chemistry 284: 36077-36082, 2009. 37. Jin ES, Szuszkiewicz-Garcia M, Browning JD, Baxter JD, Abate N, and Malloy CR. Influence of Liver Triglycerides on Suppression of Glucose Production by Insulin in Men. J Clin Endocr Metab 100: 235-243, 2015. 38. Jin Z, Bian F, Tomcik K, Kelleher JK, Zhang GF, and Brunengraber H. Compartmentation of Metabolism of the C12-, C9-, and C5-n-dicarboxylates in Rat Liver, Investigated by Mass Isotopomer Analysis: ANAPLEROSIS FROM DODECANEDIOATE. J Biol Chem 290: 18671-18677, 2015. 39. Jones JG, Hansen J, Sherry AD, Malloy CR, and Victor RG. Determination of acetyl-CoA enrichment in rat heart and skeletal muscle by 1H nuclear magnetic resonance analysis of glutamate in tissue extracts. Anal Biochem 249: 201-206, 1997. 40. Jones JG, Solomon MA, Sherry AD, Jeffrey FMH, and Malloy CR. C-13 NMR measurements of human gluconeogenic fluxes after ingestion of [U-C- 13]propionate, phenylacetate, and acetaminophen. Am J Physiol-Endoc M 275: E843- E852, 1998. 41. Kasumov T, Cendrowski AV, David F, Jobbins KA, Anderson VE, and Brunengraber H. Mass isotopomer study of anaplerosis from propionate in the perfused rat heart. Arch Biochem Biophys 463: 110-117, 2007.

115

42. Kasumov T, Martini WZ, Reszko AE, Bian F, Pierce BA, David F, Roe CR, and Brunengraber H. Assay of the concentration and C-13 isotopic enrichment of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA by gas chromatography-mass spectrometry. Anal Biochem 305: 90-96, 2002. 43. Kedishvili NY, Goodwin GW, Popov KM, and Harris RA. Mammalian methylmalonate-semialdehyde dehydrogenase. Method Enzymol 324: 207-218, 2000. 44. Kupiecki FP and Coon MJ. The Enzymatic Synthesis of Beta- Aminoisobutyrate, a Product of Valine Metabolism, and of Beta-Alanine, a Product of Beta-Hydroxypropionate Metabolism. Journal of Biological Chemistry 229: 743-754, 1957. 45. Lam C, Desviat LR, Perez-Cerda C, Ugarte M, Barshop BA, and Cederbaum S. 45-Year-old female with propionic acidemia, renal failure, and premature ovarian failure; late complications of propionic acidemia? Mol Genet Metab 103: 338-340, 2011. 46. Landau BR, Schumann WC, Chandramouli V, Magnusson I, Kumaran K, and Wahren J. C-14-Labeled Propionate Metabolism in-Vivo and Estimates of Hepatic Gluconeogenesis Relative to Krebs Cycle Flux. American Journal of Physiology 265: E636-E647, 1993. 47. Leonardi R, Rehg JE, Rock CO, and Jackowski S. Pantothenate Kinase 1 Is Required to Support the Metabolic Transition from the Fed to the Fasted State. Plos One 5, 2010. 48. Leonardi R, Rock CO, Jackowski S, and Zhang YM. Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine. P Natl Acad Sci USA 104: 1494-1499, 2007. 49. Li QL, Zhang SH, Berthiaume JM, Simons B, and Zhang GF. Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl- dephospho-CoAs. J Lipid Res 55: 592-602, 2014. 50. Malvagia S, Haynes CA, Grisotto L, Ombrone D, Funghini S, Moretti E, McGreevy KS, Biggeri A, Guerrini R, Yahyaoui R, Garg U, Seeterlin M, Chace D, De Jesus VR, and la Marca G. Heptadecanoylcarnitine (C17) a novel candidate biomarker for newborn screening of propionic and methylmalonic acidemias. Clin Chim Acta 450: 342-348, 2015. 51. Manoli I, Sloan JL, and Venditti CP. Isolated Methylmalonic Acidemia. In: GeneReviews(R), edited by Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJH, Bird TD, Ledbetter N, Mefford HC, Smith RJH and Stephens K. Seattle (WA), 1993. 52. Mattingly SJ, Xu T, Nantz MH, Higashi RM, and Fan TWM. A carbonyl capture approach for profiling oxidized metabolites in cell extracts. Metabolomics 8: 989- 996, 2012. 53. Mentre F, Steimer JL, Sommadossi JP, Diasio RB, and Cano JP. A Mathematical-Model of the Kinetics of 5-Fluorouracil and Its Catabolites in Freshly Isolated Rat Hepatocytes. Biochem Pharmacol 33: 2727-2732, 1984. 54. Millard P, Letisse F, Sokol S, and Portais JC. IsoCor: correcting MS data in isotope labeling experiments. Bioinformatics 28: 1294-1296, 2012. 55. Mingrone G, Castagneto-Gissey L, and Mace K. Use of dicarboxylic acids in type 2 diabetes. Brit J Clin Pharmaco 75: 671-676, 2013. 116

56. Mitchell GA, Gauthier N, Lesimple A, Wang SP, Mamer O, and Qureshi I. Hereditary and acquired diseases of acyl-coenzyme A metabolism. Molecular Genetics and Metabolism 94: 4-15, 2008. 57. Miyazaki T, Ohura T, Kobayashi M, Shigematsu Y, Yamaguchi S, Suzuki Y, Hata I, Aoki Y, Yang X, Minjares C, Haruta I, Uto H, Ito Y, and Muller U. Fatal propionic acidemia in mice lacking propionyl-CoA carboxylase and its rescue by postnatal, liver-specific supplementation via a transgene. J Biol Chem 276: 35995-35999, 2001. 58. Mohuczy-Dominiak D, Pacanis A, Rogulski J, and Angielski S. Significance of CoA-transferase in the reaction of maleate with amino acids and proteins. Acta Biochim Pol 30: 39-49, 1983. 59. Molteni KH, Oberley TD, Wolff JA, and Friedman AL. Progressive renal insufficiency in methylmalonic acidemia. Pediatr Nephrol 5: 323-326, 1991. 60. Murphy GE, Lowekamp BC, Zerfas PM, Chandler RJ, Narasimha R, Venditti CP, and Subramaniam S. Ion-abrasion scanning electron microscopy reveals distorted liver mitochondrial morphology in murine methylmalonic acidemia. J Struct Biol 171: 125-132, 2010. 61. Otzen C, Bardl B, Jacobsen ID, Nett M, and Brock M. Candida albicans Utilizes a Modified beta-Oxidation Pathway for the Degradation of Toxic Propionyl- CoA. Journal of Biological Chemistry 289: 8151-8169, 2014. 62. Pacanis A and Rogulski J. Studies on chemical and enzymatic synthesis of maleyl-CoA. J Biol Chem 256: 13030-13034, 1981. 63. Pacanis A, Strzelecki T, and Rogulski J. Effects of maleate on the content of CoA and its derivatives in rat kidney mitochondria. J Biol Chem 256: 13035-13038, 1981. 64. Perry RJ, Borders CB, Cline GW, Zhang XM, Alves TC, Petersen KF, Rothman DL, Kibbey RG, and Shulman GI. Propionate Increases Hepatic Pyruvate Cycling and Anaplerosis and Alters Mitochondrial Metabolism. Journal of Biological Chemistry 291: 12161-12170, 2016. 65. Powers L, Osborn MK, Yang DW, Kien CL, Murray RD, Beylot M, and Brunengraber H. Assay of the Concentration and Stable-Isotope Enrichment of Short- Chain Fatty-Acids by Gas-Chromatography Mass-Spectrometry. J Mass Spectrom 30: 747-754, 1995. 66. Puchowicz MA, Bederman IR, Comte B, Yang D, David F, Stone E, Jabbour K, Wasserman DH, and Brunengraber H. Zonation of acetate labeling across the liver: implications for studies of lipogenesis by MIDA. Am J Physiol 277: E1022-1027, 1999. 67. Ramaswamy G, Karim MA, Murti KG, and Jackowski S. PPARalpha controls the intracellular coenzyme A concentration via regulation of PANK1alpha gene expression. J Lipid Res 45: 17-31, 2004. 68. Reszko AE, Kasumov T, Pierce BA, David F, Hoppel CL, Stanley WC, Des Rosiers C, and Brunengraber H. Assessing the reversibility of the anaplerotic reactions of the propionyl-CoA pathway in heart and liver. Journal of Biological Chemistry 278: 34959-34965, 2003.

117

69. Robinson WG and Coon MJ. Synthesis of Malonic Semialdehyde, Beta- Hydroxypropionate, and Beta-Hydroxyisobutyrate. Methods in Enzymology 6: 549-557, 1963. 70. Roe CR, Sweetman L, Roe DS, David F, and Brunengraber H. Treatment of cardiomyopathy and rhabdomyrolysis in long-chain fat oxidation disorders using an anaplerotic odd-chain triglyceride. J Clin Invest 110: 259-269, 2002. 71. Romano S, Valayannopoulos V, Touati G, Jais JP, Rabier D, de Keyzer Y, Bonnet D, and de Lonlay P. Cardiomyopathies in Propionic Aciduria are Reversible After Liver Transplantation. J Pediatr-Us 156: 128-134, 2010. 72. Saudubray JM, Touati G, Delonlay P, Jouvet P, Schlenzig J, Narcy C, Laurent J, Rabier D, Kamoun P, Jan D, and Revillon Y. Liver transplantation in propionic acidaemia. Eur J Pediatr 158: S65-S69, 1999. 73. Scholem RD and Brown GK. Metabolism of Malonic Semialdehyde in Man. Biochem J 216: 81-85, 1983. 74. Schwab MA, Sauer SW, Okun JG, Nijtmans LGJ, Rodenburg RJT, van den Heuvel LP, Drose S, Brandt U, Hoffmann GF, Ter Laak H, Kolker S, and Smeitink JAM. Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins. Biochem J 398: 107-112, 2006. 75. Sherry AD, Malloy CR, Roby RE, Rajagopal A, and Jeffrey FMH. Propionate Metabolism in the Rat-Heart by C-13 Nmr-Spectroscopy. Biochem J 254: 593-598, 1988. 76. Silverman M and Huang L. Mechanism of maleic acid-induced glucosuria in dog kidney. Am J Physiol 231: 1024-1032, 1976. 77. Tomcik K, Ibarra RA, Sadhukhan S, Han Y, Tochtrop GP, and Zhang GF. Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications. Anal Biochem 410: 110-117, 2011. 78. Tuchman M, Caldovic L, Daikhin Y, Horyn O, Nissim I, Nissim I, Korson M, Burton B, and Yudkoff M. N-carbamylglutamate markedly enhances ureagenesis in N- acetylglutamate deficiency and propionic acidemia as measured by isotopic incorporation and blood biomarkers. Pediatr Res 64: 213-217, 2008. 79. Tuchman M and Yudkoff M. Blood levels of ammonia and nitrogen scavenging amino acids in patients with inherited hyperammonemia. Mol Genet Metab 66: 10-15, 1999. 80. Vagelos PR. Propionic acid metabolism. IV. Synthesis of malonyl coenzyme A. J Biol Chem 235: 346-350, 1960. 81. Van den Berg H, Boelkens MT, and Hommes FA. A case of methylmalonic and propionic acidemia due to methylmalonyl-CoA carbonylmutase apoenzyme deficiency. Acta Paediatr Scand 65: 113-118, 1976. 82. Van Leynseele A, Jansen A, Goyens P, Martens G, Peeters S, Jonckheere A, and De Meirleir L. Early treatment of a child with NAGS deficiency using N-carbamyl glutamate results in a normal neurological outcome. Eur J Pediatr 173: 1635-1638, 2014. 83. Waite M and Wakil SJ. Studies on the mechanism of fatty acid synthesis. XII. Acetyl coenzyme A carboxylase. J Biol Chem 237: 2750-2757, 1962. 84. Watson E, Olin-Sandoval V, Hoy MJ, Li CH, Louisse T, Yao V, Mori A, Holdorf AD, Troyanskaya OG, Ralser M, and Walhout AJM. Metabolic network

118 rewiring of propionate flux compensates vitamin B12 deficiency in C-elegans. Elife 5, 2016. 85. Weidman SW and Drysdale GR. The biosynthesis of methylcitrate. Biochem J 177: 169-174, 1979. 86. Wikoff WR, Gangoiti JA, Barshop BA, and Siuzdak G. Metabolomics identifies perturbations in human disorders of propionate metabolism. Clin Chem 53: 2169-2176, 2007. 87. Williamson DH, Lund P, and Krebs HA. The redox state of free nicotinamide- adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J 103: 514- 527, 1967. 88. Williamson JR and Corkey BE. Assay of citric acid cycle intermediates and related compounds--update with tissue metabolite levels and intracellular distribution. Methods Enzymol 55: 200-222, 1979. 89. Williamson JR Corkey, B.E. Assay of citric acid cycle intermediates and related compounds by fluorometri enzyme methods. Methods Enzymol 13: 80, 1969. 90. Yang L, Kasumov T, Kombu RS, Zhu SH, Cendrowski AV, David F, Anderson VE, Kelleher JK, and Brunengraber H. Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern. J Biol Chem 283: 21988-21996, 2008. 91. Zhang GF, Kombu RS, Kasumov T, Han Y, Sadhukhan S, Zhang JY, Sayre LM, Ray D, Gibson KM, Anderson VA, Tochtrop GP, and Brunengraber H. Catabolism of 4-Hydroxyacids and 4-Hydroxynonenal via 4-Hydroxy-4-phosphoacyl- CoAs. Journal of Biological Chemistry 284: 33521-33534, 2009. 92. Zhang GF, Sadhukhan S, Ibarra RA, Lauden SM, Chuang CY, Sushailo S, Chatterjee P, Anderson VE, Tochtrop GP, and Brunengraber H. Metabolism of gamma-hydroxybutyrate in perfused rat livers. Biochem J 444: 333-341, 2012.

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8.1.2

Draft to be submitted to Molecular Genetics and Metabolism

Maleate, a nephrotoxic plasma biomarker of propionic and methymalonic acidemias

in humans and mice

Kirkland A. Wilsona, Jirair Bedoyanb, Guo-Fang Zhangc,

Charles P. Vendittid, and Henri Brunengrabera*

a Department of Nutrition, Case Western Reserve University, Cleveland OH 44106, USA b Department of Pediatrics, Case Western Reserve University, Cleveland OH 44106,

USA c Division of Endocrinology, Metabolism and Nutrition, Dept of Medicine, Duke

Molecular Physiology Institute, Duke University, Durham NC 27701, USA d National Human Genome Research Institute4, National Institutes of Health, Bethesda,

MD 20892-4472, USA

Correspondence to: H. Brunengraber, Department of Nutrition, Case Western Reserve

University, 10900 Euclid Ave. WG-48; Cleveland OH 44106-4954. Email: [email protected]

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Abbreviations: MMA, methylmalonic acidemia; PA, propionic acidemia.

Acknowledgements: This work was supported by the NIH (Roadmap grant

R33DK070291, and training grant 5 T32HL105338 (fellowship to KAW)).

Author contributions: K.A.W. helped with experimental design, conducted all analyses and computed the data. J.B. contributed to data intepretation and provided plasma samples from a MMA patient. C.P.V. contributed to the design of the project and provided plasma samples from humans and mice affected with PA and MMA. G.F.Z. made substantial contributions to experimental conception and design. H.B. conceived the idea for the project, helped with experimental design, and wrote the manuscript with contributions from all other authors.

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Abstract

Propionate, 3-hydroxypropionate and methylmalonate accumulate to pathological concentrations in body fluids of patients affected by propionic acidemia (PA) and methylmalonic acidemia (MMA). We recently identified maleate as a side product of the metabolism of propionate, 3-hydroxypropionate and methylmalonate in rat liver. Maleate the cis-isomer of fumarate is nephrotoxic. This raises the possibility that deterioration of renal function in PA and MMA patients is related to the formation of maleate. To test this hypothesis, we assayed concentrations of maleate, propionate, 3-hydroxypropionate and methylmalonate in the plasma of patients and of mice with PA and MMA. We found a clear correlation between the concentrations of maleate and methylmalonate in humans and in mice. Our data open the way to investigating the role of maleate in the alterations of renal function in PA and MMA.

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1. Introduction

Some patients with severe cases of propionic acidemia (PA) or methylmalonic acidemia

(MMA), especially those with recurrent hyperammonemia, are treated with liver

transplantation (3; 4; 9; 14). Although this treatment leads to decrease in hyperammonemic

crises, it does not cure the disease. Additionally, these patients may develop renal

dysfunction and failure. After liver transplantation, peripheral tissues continue to release

toxic amounts of propionate, 3-hydroxypropionate (3HP), methylcitrate, odd-chain acylcarnitine esters and related compounds which, presumably, affect the transplanted liver

(5). We recently hypothesized that metabolic perturbations of the transplanted liver could be investigated in normal rat livers perfused with mM concentrations of propionate and

3HP (12). Using a combination of metabolomics and stable isotopic techniques, we found that (i) the citric acid cycle is overloaded, with almost complete flux inhibition when propionate and 3HP are both present, (ii) adenine nucleotide pools are perturbed (low

[ATP]/[AMP]), (iii) CoA is trapped in the propionyl-CoA pathway, and there is a tripling of liver total CoA within 1 hr, (iv) propionate inhibits the conversion of 3HP to acetyl-CoA and its oxidation in the CAC.

Unexpectedly, we identified a non-physiological compound, maleate, which is absent in control livers, but is formed in livers perfused with either 3HP, propionate, 3HP + propionate, or methylmalonate (12). Maleate, the cis-isomer of the physiological fumarate, was well separated from fumarate on capillary columns used for GC-MS analyses. Maleate is a known nephrotoxic agent (11). Maleyl-CoA reacts with amino acids and proteins of the renal brush border, and uncouples sodium-dependent solute transport (8). Also, maleyl-

CoA reacts with free CoA to form a stable inert thioether, a form of CoA sequestration (6).

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Concern about the renal toxicity of maleate was raised in 2014 because of illegal

adulteration of starch with maleic anhydride in Taiwan (1).

The identification of maleate in our perfused rat liver experiments evoked the possibility

that maleate plays a role in the deterioration of renal function observed in some PA patients

(especially after liver transplantation) and in some methylmalonic acidemia (MMA)

patients. The goals of the present study were to (i) search for maleate in plasma of humans

and mice affected with PA or MMA, and (ii) to investigate possible correlations between

concentrations of maleate and concentrations of propionate, 3HP and methylmalonate.

2. Materials and methods

2.1. Human plasma samples

Most de-identified plasma samples from patients with PA, MMA or from healthy relatives

were obtained from the National Human Genome Research Institute, National Institutes of

Health. These samples fall under exemption 4 as characterized by the Human Subjects

Protection and Inclusion guidelines 45 CFR Part 46. Two de-identified plasma samples from one MMA patient were obtained from the Center for Inherited Disorders of Energy

Metabolism of Case Western Reserve University, under the same exemption.

2.2. Mouse plasma samples

Plasma samples from mice with propionic acidemia, methylmalonic acidemia or heterozygote control mice were obtained from the National Human Genome Research

Institute4, National Institutes of Health (2; 10).

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2.3. Analytical Procedures

2 Plasma samples (20 μl) were spiked with 250 nmol of 4-hydroxy-[ H6]butyrate internal

2 standard (prepared by alkaline hydrolysis of 4-hydroxy-[ H6]butyrolactone (Sigma) with

1.1 equivalent of NaOH at 80̊C for 1 hr). After deproteinization with 500 μl acetonitrile, centrifugation and evaporation of the extract under N2, the residue was reacted with 50 μl of N-methyl-N-(tert-butyldimethyl)trifluoroacetamide for 1 hr at 70̊C. GC-MS analyses were conducted under (i) electron ionization for the assays of maleate, methylmalonate and 3-hydroxypropionate, and (ii) ammonia positive chemical ionization for the assay of propionate. Because of the wide range of analyte concentrations, the samples were run once with the front inlet set to splitless mode, and a second time with the front inlet in split mode.

For the electron ionization assays, an Agilent Technologies Model 6890N chromatograph with an Agilent Technologies Model 5973 mass selective detector was equipped with a

Varian FactorFour 60 m × 0.25 mm capillary column. The inlet temperature was set to

280°C. Helium was used as the carrier gas at a flow rate of 1.3 ml/min for the column.

Oven temperature was programmed at an initial temperature of 80°C with an isothermal delay of 1 minute. The temperature was increased at 5°C/min to 250°C, then at 10°C/min to 300°C. Retention times of analytes and m/z monitored (M-57) were: 3HP (21.6 min, m/z

= 261), methylmalonate (23.3 min, m/z = 289), maleate (24.6 min, m/z = 287), fumarate

(26.3 min, m/z = 287). Under these chromatographic conditions, maleate was well separated from its isomer fumarate.

For the ammonia positive chemical ionization assay of propionate, a twin GC-MS instrument was equipped with an Agilent J&W 30 m x 0.25 mm capillary column. The inlet temperature was set at 280̊C. Helium was used as a carrier gas at a flow rate of 2.1

125

ml/min for the column. Oven temperature was programmed at an initial temperature of 80̊C

with an isothermal delay of 1 min. The temperature was increased at 10̊C/min to 300̊C,

then held at 300̊C for 10 min. The retention time of propionate was 4.9 min, the m/z

+ + monitored were 189 (M + H) for quantification, and 206 (M + NH4) for verification.

Figure 8.10. Calibration Curve of Maleate Concentration.

Assays of analytes were run with calibration curves of unlabeled standards spiked with of

2 4-hydroxy-[ H6]butyrate. In order to accurately measure analyte concentrations across the expected large range of patient concentrations two separate calibration curves were prepared for each analyte: (i) a calibration curve of 0-50 nmol run in splitless mode, and

(ii) a calibration curve of 0-1000 nmol run in split mode. Fig. 8.10 shows a calibration

curve of maleate concentration in the 0 to 50 nmol range.

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Table 8.4. Metabolite concentrations in plasma samples from humans (µM). Plasma samples were taken from parents of MMA or PA patients as parental controls (Control), patients with confirmed MMA who had low plasma methylmalonate (MMA), MMA patients who had high plasma methylmalonate (MMA A), or patients with confirmed PA who had an unknown patient status at the time of sampling (PA). Group data are also presented as mean ± SD. *Significantly different from control (p<0.05), ^ from MMA (p<0.05). Row # Patient ID Maleate Methylmalonate 3-Hydroxypropionate Propionate 1 Control 1 1.19 15.6 8.35 4.12 2 Control 2 2.91 9.84 5.36 2.13 3 Control 3 1.52 12.4 4.34 3.34 4 Control 4 1.31 19.6 7.47 3.7 5 Control 5 1.88 15.5 3.71 3.24 6 Mean ± SD 1.76 ± 0.69 14.6 ± 3.68 5.85 ± 2.00 3.31 ± 0.74 7 MMA 1 4.65 533 136.6 108 8 MMA 2 0.056 6.73 3.85 51 9 MMA 3 0.7 14.6 7.15 112 10 MMA 4 0.036 10.8 12.3 81.3 11 MMA 5 0.69 15.7 13.9 114 12 Mean ± SD 1.23 ± 1.94 116 ± 233 34.8 ± 57.1 93.3 ± 27.1* 13 MMA A 1 61.7 14099 256 101 14 MMA A 2 12.1 3055 70.9 62.3 15 MMA A 3 12.1 1425 313.5 188 16 MMA A 4 3.53 2078 59.9 120 17 MMA A 5 14.4 2805 154.5 183 18 Mean ± SD 20.8 ± 23.3 4692 ± 5297 171 ± 112*^ 131 ± 54*^ 19 PA 1 16.6 4.7 77.7 191 20 PA 2 0.24 5.89 19.2 130 21 PA 3 8.15 14.25 373.8 103 22 PA 4 4.09 5.51 165.4 126 23 Mean ± SD 7.27 ± 7.01 7.59 ± 4.47* 159 ± 155 138 ± 38*

Table 8.5. Plasma samples from single patient taken as patient metabolic status changed. Plasma was sampled from a single MMA patient both when they were clinically stable (stable) and when the patient suffered a metabolic decompensation episode (decompensated).

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Patient ID Row # Metabolites MMA (Stable) MMA (Decompensated) 1 Maleate 1.21 17.1 2 Methylmalonate 99.3 2013 3 3-Hydroxypropionate 8.73 21.9 4 Propionate 74.8 105 5 Methylmalonylcarnitine 1.69 2.19 6 Propionylcarnitine 31.46 57.08 7 Total acylcarnitine 44.96 145.19

3. Results

Table 8.4 presents the concentrations of maleate, methylmalonate, 3HP and propionate in plasmas of MMA and PA patients, as well as in control subjects. The MMA patients are presented in two groups with very high, or low, methylmalonate concentrations. One

MMA patient was studied twice, when she was stable, and when she started to

decompensate (Table 8.5).

Control subjects had the expected very low plasma concentrations of propionate (< 10 μM,

(7), Table 8.4). Their concentrations of 3HP (< 10 μM) and methylmalonate (< 20 μM)

were low, but we do not know of reference concentrations of 3HP and methylmalonate in

control subjects. Their concentrations of maleate were less than 3 μM.

In PA patients, the plasma concentrations of propionate and 3HP were much higher than

in controls, but lower than concentrations reported in patients with decompensated PA.

Also, the concentration of maleate ranged from 0.2 to 17 μM.

In MMA patients with low methylmalonate concentrations, the maleate concentrations

were similar to those of controls. In MMA patients with high methylmalonate

concentrations, the maleate concentrations were higher, but not significally higher than in

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controls. In all groups, the concentrations of maleate were very variable between subjects.

One MMA patient was tested twice, once when stable and once when she started to

decompensate. Decompensation caused a 15-fold increase in maleate concentration, and a

20-fold increase in methylmalonate concentration (Table 8.5). The tripling of total acylcarnitine concentration probably reflects CoA trapping in her cells.

Fig. 8.11 shows attempts to correlate the concentrations of maleate with those of

methylmalonate, 3HP and propionate in the human subjects. We found a good correlation

between methylmalonate and maleate concentrations (R2 = 0.90, Fig. 8.11A), but no

correlation between maleate and either 3HP or propionate (Figs 8.11B and C).

Table 8.6 presents the concentrations of maleate, methylmalonate, 3HP and propionate in

plasmas of MMA-/- and PA -/- mice as well as in their respective heterozygote controls.

The MMA -/- mice had concentrations of maleate, methylmalonate and 3HP significantly

higher than their controls. The PA mice had concentrations of maleate, methylmalonate

and 3HP higher, but not significantly higher than their controls. There was a good

correlation between the concentrations of maleate and methylmalonate (R2 = 0.86, Fig

8.12A), and a moderate correlation between the concentrations of maleate and 3HP (R2 =

0.68. Fig. 8.12B). There was no correlation between maleate and propionate concentrations

(Fig. 8.12C).

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(B); or propionate (C). correlations confirmed the(PA), time whounknownat methylmalonate3 - an PA had status with (A); of sampling plasma who high (MMA had methylmalonate patients (MMA), with methylmalonate MMA patients A), or who low plasma had Maleate concentrations fromsamples plasma taken from parents of MMA or PA patients (Control), patients with confirmed MMA Figure

8.11 .

Correlation of Maleate to Metabolites Related to Disorders of Propionyl

- CoA Humans. Metabolism in

hydroxypropionate

130 with methylmalonate (A); 3 for MMA mutation (MMA for - MMA Maleate concentra tions fromsamples plasma taken frommice, either heterozygous for MMA mutation (MMA +/ Figure 8.12 .

Correlation of Maleate to Metabolites Related to Disorders of Propionyl - / hydroxy - ), heterozygousmutation ), for(PA PA+/ propionate (B); or propionate (B); or (C). propionate - ), or homozygous for PA mutation (PA), homozygousmutation for or PA-

- CoA Metabolism in Mice. / - ), correlations - ), homozygous ),

131

Table 8.6. Metabolite concentrations in plasma samples from mice (µM). Plasma samples taken from mice, either heterozygous for MMA mutation (MMA +/-), homozygous for MMA mutation (MMA -/-), heterozygous for PA mutation (PA +/-), or homozygous for PA mutation (PA -/-). Group data are also presented as mean ± SD. *Significantly different from heterozygous control (p<0.05). Row # Sample ID Maleate Methylmalonate 3-Hydroxypropionate Propionate 1 MMA +/- 1 0.231 14.9 16.7 102 2 MMA +/- 2 0.324 16.8 30.6 122 3 MMA +/- 3 0.383 31.8 55.2 167 4 Mean ± SD 0.31 ± 0.08 21.2 ± 9.25 34.2 ± 19.5 131 ± 33 5 MMA -/- 1 11.8 256 74.2 72.9 6 MMA -/- 2 9.02 250 73.5 109 7 MMA -/- 3 16.3 281 110 72.4 8 Mean ± SD 12.4 ± 3.7* 262 ± 17* 85.9 ± 20.9* 84.8 ± 21.0 9 PA +/- 1 0.266 35.5 24.9 133 10 PA +/- 2 0.501 26.1 17.3 80.2 11 PA +/- 3 8.54 143 112 69.7 12 PA +/- 4 0.397 24.9 19.2 104 13 PA +/- 5 0.477 10.4 19.3 86.5 14 PA +/- 6 10.8 195 67.7 86.8 15 PA +/- 7 10.9 258 73.4 89.0 16 PA +/- 8 0.393 9.73 25.1 102 17 PA +/- 9 0.389 28.7 40.6 43.4 18 PA +/- 10 0.674 16.5 25.6 121 19 Mean ± SD 3.33 ± 4.70 74.8 ± 90.1 42.6 ± 31.8 91.5 ± 25.4 20 PA -/- 1 4.69 126 75.6 130 21 PA -/- 2 11.4 211 77.9 110 22 PA -/- 3 5.25 119 67.4 109 23 PA -/- 4 14.3 162 54.3 110 24 PA -/- 5 7.82 128 49.3 146 25 PA -/- 6 4.05 115 58.3 95.6 26 PA -/- 7 11.5 140 56.4 114 27 PA -/- 8 19.1 286 122 99.2 28 PA -/- 9 9.17 178 69.8 116 29 PA -/- 10 7.28 103 53.4 129 30 Mean ± SD 9.46 ± 4.74 157 ± 56 68.5 ± 21.2 116 ± 15*

132

4. Discussion

The goal of this short report is to draw attention on a new marker of PA and MMA, maleate,

which may play a role in the deterioration of renal function in some of the patients. When

interpreting data on plasma maleate concentrations, one should keep in mind that maleate

is a reactive molecule which has probably a short half-life in plasma because of a high

fractional first-pass uptake by organs. This is inferred and demonstrated by the following.

First, maleate is more reactive than its isomer fumarate because of steric and electronic

strains imparted from the cis-geometry of the double bond. Second, maleate is a known

nephrotoxic compound (11). Third, maleyl-CoA reacts with amino acids and proteins of

the renal brush border and uncouples sodium-dependent solute transport (8). Fourth, maleyl-CoA reacts with free CoA to form a stable inert thioether (6). Fiftth, we did not find reports of identification of maleate in urinary organic acid profiles of PA and MMA patients, or of patients with other inborn metabolic disorders. This, although the concentration of carboxylic acids in urine is often higher than in plasma. This must reflect the trapping of maleate molecules in the kidney tubules. Sixth, it is likely that part of the maleate formed in a given organ, such as the liver, is trapped in the same organ and is not released in the plasma. Lastly, a direct evidence of a rapid first-pass uptake of maleate is an apparent volume of distribution of 120% of body weight in rats given an intravenous injection of maleate at 6 or 60 mg/kg (0.05 or 0.5 mmol/kg) (13). These doses induced peak plasma maleate concentrations of 0.9 and 9 μM, respectively. Baseline plasma maleate concentrations in these rats were about 0.02 μM. For the above reasons, plasma concentrations of maleate in the μM range should not be viewed as innocuous: they reflect the tip of the iceberg of maleate traffic in the body.

133

Further research will involve epidemiological investigations of maleate concentrations in plasma of MMA and PA patients, in relation to renal function. Also, mechanistic studies on the formation and disposal of maleate will be conducted in mouse models to identify strategies to prevent maleate nephrotoxicity.

134

Reference List

1. Baoren, J. Contamination of maleic anhydride modified starch in food. Taiwan

Food and Drug Administration. 2017. mddb.apec.org/Documents/2014/SCSC/WKSP/14_scsc_wksp_011.pdf

Ref Type: Internet Communication

2. Chandler RJ, Zerfas PM, Shanske S, Sloan J, Hoffmann V, DiMauro S and

Venditti CP. Mitochondrial dysfunction in mut methylmalonic acidemia. Faseb J 23:

1252-1261, 2009.

3. Chapman KA, Summar ML and Enns GM. Propionic acidemia: to liver transplant or not to liver transplant? Pediatr Transplant 16: 209-210, 2012.

4. Charbit-Henrion F, Lacaille F, McKiernan P, Girard M, de LP,

Valayannopoulos V, Ottolenghi C, Chakrapani A, Preece M, Sharif K, Chardot C,

Hubert P and Dupic L. Early and late complications after liver transplantation for propionic acidemia in children: a two centers study. Am J Transplant 15: 786-791, 2015.

5. de Baulny HO, Benoist JF, Rigal O, Touati G, Rabier D and Saudubray JM.

Methylmalonic and propionic acidaemias: management and outcome. J Inherit Metab Dis

28: 415-423, 2005.

6. Mohuczy-Dominiak D, Pacanis A, Rogulski J and Angielski S. Significance of

CoA-transferase in the reaction of maleate with amino acids and proteins. Acta Biochim

Pol 30: 39-49, 1983.

7. Powers L, Osborn MK, Kien CL, Murray RD and Brunengraber H. Assay of the concentration and stable isotope enrichment of short-chain fatty acids by gas chromatography-mass spectrometry. J Mass Spectrom 30: 747-754, 1995.

135

8. Rosenberg LE and Segal S. Maleic acid-induced inhibition of amino acid transport

in rat kidney. Biochem J 92: 345-352, 1964.

9. Saudubray JM, Touati G, Delonlay P, Jouvet P, Schlenzig J, Narcy C, Laurent

J, Rabier D, Kamoun P, Jan D and Revillon Y. Liver transplantation in propionic acidaemia. Eur J Pediatr 158 Suppl 2: S65-S69, 1999.

10. Senac JS, Chandler RJ, Sysol JR, Li L and Venditti CP. Gene therapy in a murine model of methylmalonic acidemia using rAAV9-mediated gene delivery. Gene

Ther 19: 385-391, 2012.

11. Silverman M and Huang L. Mechanism of maleic acid-induced glucosuria in dog kidney. Am J Physiol 231: 1024-1032, 1976.

12. Wilson KA, Han Y, Zhang M, Hess J, Chapman KA, Cline GW, Tochtrop GP,

Brunengraber H and Zhang G-F. Interrelations between 3-hydroxypropionate and propionate metabolism in rat liver: Relevance to disorders of propionyl-CoA metabolism.

Am J Physiol (submitted): 2017.

13. Wu C, Chen HC, Luo YS, Chiang SY and Wu KY. Pharmacokinetics and bioavailability of oral single-dose maleic acid in biofluids of Sprague-Dawley rats. Drug

Metab Pharmacokinet 31: 451-457, 2016.

14. Yorifuji T, Kawai M, Mamada M, Kurokawa K, Egawa H, Shigematsu Y,

Kohno Y, Tanaka K and Nakahata T. Living-donor liver transplantation for propionic acidaemia. J Inherit Metab Dis 27: 205-210, 2004.

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8.1.3 Draft to be submitted to American Journal of Physiology-Endocrinology and Metabolism

Metabolism of ß-alanine in the perfused rat liver and live rats:

Stimulation of CoA synthesis, and carboxylation to 2-(aminomethyl)-malonate

Kirkland A. Wilson1, Jeremy P. Hess2, Miaoqi Zhang1, Kimberly A. Chapman3,

Wim Derave4, Guo-Fang Zhang5, Henri Brunengraber1* and Gregory P. Tochtrop2

Departments of Nutrition1 and Chemistry2, Case Western Reserve University, Cleveland OH 44106, Children's National Medical Center and George Washington University3, Washington DC 20010, Division of Endocrinology, Metabolism and Nutrition5, Dept of Medicine, Duke Molecular Physiology Institute, Duke University, Durham NC 27701, and Department of Movement and Sports Sciences , Ghent University, B-9000 Belgium.

Corresponding author: Henri Brunengraber, Department of Nutrition, Case Western

Reserve University, 10900 Euclid Ave. WG-48; Cleveland OH 44106-4954. Tel: (216)

368-6548. Fax: (216) 368-6846. Email: [email protected]

137

Abbreviations: AMM, 2-(aminomethyl)-malonate; AOA, aminooxyacetate;

Keywords: metabolomics, neurotransmission, paresthesia, exercise physiology, pyruvate carboxylase, aminooxyacetate

Acknowledgements: This work was supported by the NIH (Roadmap grant

R33DK070291 and grant RO1CA196643 to HB, grant 5 RO1 CA157735 to GPT, and training grant 5 T32HL105338 (fellowship to KAW)).

Author contributions: K.A.W. helped with experimental design, conducted most analyses and computed the data. J.H. synthesized AMM. MZ helped with the analyses. KC ran the amino acid analyses and contributed to data intepretation. WD contributed to the design of the project. G.F.Z. made substantial contributions to experimental conception and design. H.B. conceived the idea for the project, helped with experimental design, and wrote the manuscript with contributions from all other authors. GT designed the syntheses of unlabeled and labeled AMM.

138

Abstract

ß-Alanine is a common metabolite found in the degradation of 3-hydroxypropionate and

uracil. It is also a component of the dipeptides carnosine and anserine which act as pH

buffers in muscle. Some athletes ingest ß-alanine to increase the pH buffering capacity of their muscle and enhance athletic performance. Ingestion of ß-alanine often causes paresthesias, possibly linked to the neurotransmitter-like structure of ß-alanine. As a follow-up to a report on 3-hydroxypropionate metabolism in rat liver (Wilson, K.A. Am.

15 13 J. Physiol. in press), we perfused livers with ß-[ N, C3]alanine. Metabolomic and

isotopic assays revealed a marked accumulation of CoA by ß-alanine, but without

incorporation of label. We identified a new compound, 2-(aminomethyl)-malonate

(AMM), formed by carboxylation of ß-alanine by pyruvate carboxylase. We treated live rats with ß-alanine ± aminooxyacetate to raise the ß-alanine concentration in body fluids.

This resulted in marked increases in the concentrations of (i) ß-alanine and AMM in plasma, liver, muscle, heart and brain, and (ii) N-acetylaspartate, GABA and α- aminobutyrate in brain. Thus, ß-alanine ingestion leads to major perturbations in neurotransmitter metabolism in rats. In humans ingesting ß-alanine, the potential impact of

this compound on neurotransmission should be considered.

139

Introduction

ß-Alanine (3-aminopropanoate) is an amino acid not found in proteins. It is a common

catabolite of 3-hydroxypropionate (3HP) and of uracil (Scheme 8.3). It is degraded by

transamination to malonic semialdehyde which undergoes decarboxylation to acetyl-CoA.

The latter is oxidized in the citric acid cycle (CAC). Inhibition of transaminases by

aminooxyacetate (AOA) leads to increases in plasma and urine ß-alanine concentrations in

mice (10).

ß-Alanine is also reversibly incorporated into the dipeptides carnosine and anserine which

are present at high concentrations in muscle. The dipeptides act as pH buffers during bouts

of muscular exercise. For this reason, some athletes ingest ß-alanine before strenuous

exercise, with the expectation that the dietary supplement will increase the intensity and

duration of their performance (2). A frequently reported side effect of ß-alanine ingestion is the unexplained development of unpleasant paresthesias (1; 5; 7). The similarity between the chemical structure of ß-alanine and those of some neurotransmitters suggests that ß-alanine can interfere with some neurotransmission processes (15).

Lastly, ß-alanine is part of the CoA molecule and of its precursor pantothenate (vitamin

B5). The latter is not synthesized in mammalian cells, but is absorbed from the diet.

Pantothenate can be ingested as a calcium salt, or as pantothenol which is oxidized to pantothenate via liver alcohol and aldehyde dehydrogenases.

We recently reported a study on the metabolism of mM concentrations of 3HP in perfused rat livers (17). 3HP and propionate accumulate in body fluids of patients with

140

Scheme 8.3 .

Relationship of β - alanine metabolism to pathwaysalanine and to metabolism 3 propionate of - hydroxypropionate metabolism metabolism hydroxypropionate

in ratin liver.

141

propionic acidemia or methylmalonic acidemia. These inborn disorders of metabolism

result from deficiencies in propionyl-CoA carboxylase and methylmalonyl-CoA mutase,

13 respectively (11; 14). In livers perfused with 3-hydroxy-[ C3]propionate (M3 3HP), the

concentration of ß-alanine was 3-fold increased compared to control livers. In addition, ß-

alanine was 9% M3 labeled, reflecting partial isotopic equilibration of ß-alanine and

malonic semialdehyde (Scheme 8.3). Lastly, acetyl-CoA was 13% M2 labeled confirming the degradation of 3HP.

The initial goal of the present study was to examine the reversibility of the reactions between 3HP and ß-alanine. This was tested in livers perfused with M4 ß-[15N,

13 C3]alanine. Besides confirming the expected reaction reversibilities, the study revealed

two unknown processes: the stimulation of CoA synthesis by ß-alanine, and the

carboxylation of ß-alanine to a novel neurotransmitter-like compound: 2-(aminomethyl)-

malonate (AMM) (Scheme 8.3). In vivo experiments revealed major perturbations of

neurotransmitter metabolism in rats gavaged with ß-alanine.

142

Materials and Methods

Materials

Sigma-Aldrich-Isotec supplied most chemicals, enzymes and the following isotopically

13 13 13 15 labeled compounds: NaH CO3, K CN, ethyl bromo-[ C2]acetate and ß-[ N,

13 15 C3]alanine. Cambridge Isotopes Laboratories supplied another batch of ß-[ N,

13 15 13 C3]alanine. Both batches of ß-[ N, C3]alanine, when assayed by GC-MS as TBDMS derivatives, had the same unexpected mass isotopomer distribution: 90?% M4 and 4-5%

M2 (after correction of the raw data for natural enrichment ). The manufacturer explained

15 13 13 15 13 that ß-[ N, C3]alanine was synthesized from K C N and ethyl-2-bromo-[ C2]acetate.

15 13 The M2 component of ß-[ N, C3]alanine resulted from the presence of some unlabeled

13 15 15 13 KCN in the K C N. We decided to use these batches of ß-[ N, C3]alanine, but to take

13 into consideration the M2 form when interpreting the data. 3-Hydroxy-[ C3]propionate

13 13 was synthesized from K CN and ethyl bromo-[ C2]acetate as described previously (17).

2-(Aminomethyl)-malonic acid was synthesized by the following steps: Diethyl 2- cyanomalonate was prepared by reacting ethyl chloroformate and ethyl cyanoacetate in

33% potassium carbonate in acetone. The reaction was quenched with water and acetone was removed under reduced pressure. The resulting aqueous solution was extracted with dichloromethane before the aqueous solution was acidified and extracted with diethyl ether. The resulting red oil was distilled (.5mmHg, 108̊C) to obtain compound as a clear

1 oil. H NMR (500MHz, CDCl3): major tautomer: 4.48 (s, 1H), 4.36 (q, J=5, 4H), 1.37 (t,

J=5, 6H) minor tautomer: 4.48 (s, 1H), 4.43(q, J=5, 4H), 1.42 (t, J=, 6H). . 13C NMR

(500MHz, CDCl3): : 160.72, 111.68, 62.60, 44.62, 13.58.

𝛿𝛿

143

Diethyl 2–aminomethylmalonate hydrochloride was prepared by reductive hydrogenation

of diethyl 2-cyanomalonate in an H2 charged pressure vessel utilizing 10% by weight

platinum oxide in an ethanolic HCl solution. Crude mixture was filtered through cotton to

remove catalyst. Solvent was removed under reduced pressure to yield the amine product

1 as a fine white powder. H NMR (500MHz, D2O): 4.21 (t, J=5Hz, 4H), 3.45 (s, 2H), 1.20

13 (t, J=5Hz, 6H). C NMR (500MHz, D2O): 168.11𝛿𝛿 , 63.65, 48.98, 36.96, 13.06.

𝛿𝛿 Disodium 2-aminomethylmalonate hydrochloride. The ethyl ester was added to 3.5M aq.

NAOH and stirred at room temperature. Following incubation period, solution was

1 13 lyophilized to a white crystalline solid. H NMR (500MHz, D2O): 3.36 (s, 2H). C NMR

(500MHz, H2O/D2O): 175.14, 53.16, 47.83. 𝛿𝛿

13 𝛿𝛿 13 Ethyl [1,2,3- C3) cyanoacetate was prepared via substitution utilizing [1,2- C2]

bromoacetic acid and (13C) potassium cyanide under basic conditions. This was acidified and dried under reduced pressure to form a solid product. Solid was suspended in ethanol and filtered. The carboxylic acid intermediate in ethanol was esterfied with sulfuric acid

and extracted with water and ethyl acetate to afford the product as yellow oil.

13 Disodium [1,2,3- C3] 2-aminomethylmalonate hydrochloride was prepared as above,

13 substituting [1,2,3- C3] ethyl cyanoacetate.

Perfused liver experiments.

All animal experiments were approved by the IACUC Committee of Case Western Reserve

University. Male Sprague-Dawley rats were fed with Prolab Isopro RMH 3000 irradiated

chow containing 13 ppm of pantothenic acid as calcium acid. Livers from overnight-fasted

rats (160-180 g) were perfused (4) for 60 min with recirculating bicarbonate buffer

144

containing 4% dialyzed, fatty acid-free, bovine serum albumin and 4 mM glucose. In some

15 13 experiments, the buffer also contained 2 mM ß-[ N, C3]alanine.

One liver was perfused with non-recirculating, albumin-free bicarbonate buffer containing

4 mM glucose and 2 mM unlabeled ß-alanine; after 15 min, the unlabeled bicarbonate in the buffer was replaced by 40% 13C-labeled bicarbonate. Samples of perfusate were taken

at regular intervals and quick-frozen. At the end of all liver perfusion experiments, the

organ was quick-frozen and kept in liquid N2 until analyses.

In vivo experiments

Four groups of overnight-fasted rats were injected intraperitoneally at zero time with either

0.6 ml saline (groups 1 and 2) or 10 mg AOA/kg (groups 3 and 4) (Scheme 8.4). At 120

min, they were gavaged with 1 ml saline/kg (group 1 and 3), or 450 μmol ß-alanine/kg in

the same volume of saline (groups 2 and 4).

The rats were sacrificed one at a time at different times from 0 to 240 min, indicated by the small vertical arrows in Scheme 8.4.

Analytical Procedures

The concentrations and labeling patterns of CoA esters, CAC intermediates, 3HP, ß- alanine, adenine nucleotides, lactate, pyruvate, β-hydroxybutyrate, acetoacetate and urea were assayed by GC-MS and LC-MS/MS as described previously (17). The concentration of AMM was assayed by GC-MS as a TMS or TBDMS derivative formed at room temperature. The concentrations of amino acids in the final liver perfusates were assayed on a Hitachi L8800a amino acid analyzer with post-column ninhydrin derivatization.

The carboxylation of ß-alanine by pyruvate carboxylase (Sigma) was assayed at pH 8.0

145

Scheme 8.4 . Protocol for .Protocol in vivo rat experiments .

146

and 30°C by a modification of the method of Warren (16). The reaction mixture (3 ml)

contained 134 mM triethanolamine, 5mM MgSO4, 0.05mM acetyl-CoA (or propionyl-

CoA), 1mM ATP, 15mM KHCO3, 2µM magnesium acetate, 1µM EDTA, and varying concentrations of β-alanine. The reaction was started with pyruvate carboxylase. Aliquots of the reaction mixture (75 μL), taken every min for 10 min, were treated with acetonitrile.

After centrifugation and evaporation of the extract, the residue was reacted with TBDMS before GC-MS analysis (17). The retention time of AMM was 34.2 min and the m/z monitored was 460 (M - 15) for identification and 418 (M-57) for quantitation.

Calculations and statistics

Measured mass isotopomer distributions, expressed as mol percent, were corrected for natural enrichment . Statistical differences were assayed by t test using the Prism software.

147

Results

Uptake and release of substrates

15 13 In livers perfused with 2 mM of ß-[ N, C3]alanine, the substrate was taken up at a rate

of 40 ± 11 (SD) (μEquivalents of N) (g dry wt)−1 (hr)-1 (Figure 8.13A). In the same

livers, the release of urea N was 3.2 times⋅ that of ⋅control livers perfused without ß-

alanine (Fig 8.13A and Table 8.7, row 19). The increase in urea N induced by ß-alanine

was 5.5 times the uptake of ß-alanine N (Table 8.7, row 26). This was evidence that

Figure 8.13. Concentration and mass isotopomer distribution of select nitrogenous compounds released by the perfused liver. Livers were perfused with 2mM β- 13 15 [ C3, N]alanine. Panel A: Uptake of β-alanine (β-alanine) and release of AMM (AMM) and urea into perfusate by the liver. Release of urea into perfusate is by livers perfused with either: (i) 4mM glucose + 2mM β-alanine, Urea (β-alanine). or (ii) 4mM glucose + nothing, Urea (control). Panel B: Mass isotopomer distribution of AMM labeled from β- 13 15 [ C3, N]alanine. Concentration of AMM was calculated with a β-alanine/γ- 2 [ H6]hydroxybutyrate. Panel C: Mass isotopomer distribution of urea labeled from β- 13 15 15 13 15 [ C3, N]alanine. Panel D: Calculated distribution of N from β-[ C3, N]alanine in mitochondrial (represented at NH4) and cytoplasmic (represented as Aspartate nitrogen) nitrogen pools. N=5.

148

Table 8.7. Release of amino acids and urea from livers, in nitrogen equivalents (nanoequivalent of N·g dry weight-1·hr-1). The compounds were assayed in the perfusate after 60 min of recirculating perfusions with 4mM glucose and either nothing 15 13 (Control), or 2mM [ N1, C3]β-alanine (M4 β-alanine). *Significantly different from control (p<0.05). Row Control M4 β-alanine # (n=5) (n=5) 1 Aspartate 63.8 ± 42.6 60.7 ± 81.8 2 Threonine 941 ± 351 1620 ± 420* 3 Serine 379 ± 245 643 ± 248 4 Glutamate 2820 ± 1150 6790 ± 3260* 5 Glycine 6150 ± 1070 7650 ± 1510 6 Alanine 766 ± 151 929 ± 238 7 AABA 1220 ± 400 1350 ± 730 8 Valine 3140 ± 1060 3790 ± 950 9 Methionine 509 ± 139 427 ± 409 10 Cystine 346 ± 127 390 ± 345 11 Isoleucine 2020 ± 1670 2330 ± 990 12 Leucine 4000 ± 1440 5290 ± 1570 13 Tyrosine 518 ± 120 980 ± 210* 14 Phenylalanine 1650 ± 400 1850 ± 370 15 Ornithine 1850 ± 310 1090 ± 380* 16 Lysine 5580 ± 1070 7490 ± 1040* 17 Histidine 4080 ± 860 4590 ± 1060 18 Glutamine 5400 ± 3890 17790 ± 6650* 19 Urea 19980 ± 7990 47300 ± 37790 20 Amino Acids (total) 41420 ± 11250 65076 ± 9884* 21 Amino Acids precursors of Propionyl- 6600 ± 2690 8170 ± 2610 CoA 22 Essential Amino Acids (total) 21910 ± 6020 27400 ± 5780 23 Total Nitrogen (total) 61400 ± 17370 104080 ± 38274

24 (Amino Acids Precursors of Propionyl- 0.15 ± 0.03 0.12 ± 0.02* CoA)/(total Amino Acids) 25 (Urea release)/(Total N release) 0.32 ± 0.03 0.38 ± 0.17

ß-alanine activated proteolysis and ureagenesis in liver. This was confirmed by the assay

of the balance of amino acids and urea concentrations in the final perfusate, both

expressed as (nanoequivalents of N)(g dry wt)-1 hr-1 (Table 8.7, rows 20-25). The effect

149

of 15N-labeled ß-alanine on proteolysis is similar to that of α-[15N]alanine reported by

Brosnan et al (3). Also, the distribution of M1 and M2 urea isotopomers formed from ß-

15 13 [ N, C3]alanine (Figure 8.13C) is similar to what was reported by Brosnan et al for α-

[15N]alanine. Using their equations, the distribution of urea mass isotopomers allowed to

15 + calculate the enrichment profiles of mitochondrial NH4 and cytosolic aspartate (Figure

8.13D). These were similar to those reported for α-[15N]alanine. As pointed out by

Brosnan et al, the proteolytic effect of α-[15N]alanine supplies aspartate that carries the

second N used to form urea . In conclusion, the fates of the N of ß-alanine and α-alanine are similar in rat liver.

Reversibility of reactions linking 3HP and ß-alanine

15 13 Figure 8.14 compares the labeling pattern of ß-[ N, C3]alanine with those of

metabolites that could become labeled, directly or indirectly, from the M4 substrate via

the reactions of Scheme 8.3. The substantial M3 labeling of 3-hydroxypropionate (3HP),

but absence of labeling of 3HP-CoA shows that the conversion of 3HP-CoA to 3HP is

15 13 not reversible. Although 3HP was labeled from 2 mM ß-[ N, C3]alanine, its

concentration was not increased compared to controls (Table 8.8, row 2). Also the

concentration of 3HP-CoA was decreased compared to controls (row 13).Thus, the

reversibility of the reactions linking 3HP and ß-alanine is limited to partial isotopic

equilibration, without net backward flux toward propionyl-CoA. This is consistent with

the mostly M1 labeling of methylmalonyl-CoA and propionyl-CoA which result from

retrograde labeling of the propionyl-CoA pathway via succinyl-CoA (13).

150 labeling pat labeling group ofaddition carboxyl a onto [ 8.14 Figure 13 C 3 , 15 N] alanine generates (i) M3 3 terns of AMM and aspartate. . Mass isotopomer distributions of metabolites β - - alanine. The confirmalanine. conversiondata the of β hydroxypropionate acetyl M2 hydroxypropionate (3HP),

in livers perfused with 2 mM - CoA, citrate of M2 and by loss - alanine to acetyl β - [ 15 N, - CoA. Note the very different CoA. very the Note 13 C 3 ]alanine. 15 N or (ii) M4 AMM by

β -

151

Table8.8. Metabolite Concentrations (nmol/g dry weight). Livers were perfused for 60 13 15 min with 4mM glucose and either nothing (Control) or 2mM [ C3, N1] β-alanine (M4 β-alanine). Data are presented as mean ± SD. * Significantly different from control 2 (p<0.05). ^ [3HP-CoA] was calculated with the [propionyl-CoA]/[ H9]pentanoyl-CoA] concentration curve. Row # Control M4 β-alanine (n=5) (n=5) 1 β-alanine 507 ± 224 2850 ± 1430* 2 3HP 16.2 ± 7.3 19.5 ± 5.7 3 AMM (relative) 0.44 ± 0.30 26.7 ± 4.9* 4 Aspartate (relative) 22.0 ± 6.5 28.6 ± 18.0 5 Uracil 136 ± 73 157 ± 153 6 Succinate 714 ± 333 1090 ± 550 7 Fumarate 152 ± 37 140 ± 79 8 Malate 334 ± 128 344 ± 141 9 Citrate 252 ± 106 336 ± 223 10 CAC Intermediates Assayed 1450 ± 390 1960 ± 970 (total) 11 Pantothenate 46.2 ± 29.4 73.7 ± 44.7 12 Free CoA 144 ± 17 1330 ± 460* 13 Acetyl-CoA 92.1 ± 42.7 672 ± 344* 14 3HP-CoA^ 1.4 ± 0.08 0.41 ± 0.36* 15 Propionyl-CoA 1.25 ± 0.34 7.76 ± 4.15* 16 Methylmalonyl-CoA 2.18 ± 1.67 11.8 ± 3.7* 17 Succinyl-CoA 48.3 ± 27.2 214 ± 71* 18 Malonyl-CoA 6.5 ± 3.5 43.5 ± 20.4 19 CoA Species Assayed (total) 266 ± 54 2250 ± 800* 20 ATP 2860 ± 1402 3150 ± 1610 21 ADP 1720 ± 602 2410 ± 170* 22 AMP 190 ± 151 91 ± 59* 23 ATP + ADP + AMP 4770 ± 295 5650 ± 1550 24 [ATP]/[ADP] 1.82 ± 0.68 1.32 ± 0.70 25 [ATP]/[AMP] 15.5 ± 2.9 47.6 ± 32.8 (p=0.06) 26 Energy Charge 0.77 ± 0.06 0.76 ± 0.07 27 Myokinase Mass Action Ratio 1.09 ± 0.53 2.21 ± 1.16 (p=0.08) 28 Lactate 1230 ± 1120 640 ± 460 29 Pyruvate 3520 ± 840 830 ± 150* 30 3-Hydroxybutyrate (BHB) 1930 ± 1840 280 ± 144 (p=0.08) 31 Acetoacetate (AcAc) 2690 ± 1160 1740 ± 190 32 α-Glycerol-phosphate (αGP) 1780 ± 950 1490 ± 1120 33 Dihydroxyacetone-P (DHAP) 776 ± 646 840 ± 336 34 [Lactate]/[Pyruvate] 0.53 ± 0.47 0.88 ± 0.87 35 [BHB]/[AcAc] 0.79 ± 0.68 0.17 ± 0.09 (p=0.07) 36 [αGP]/[DHAP] 3.45 ± 3.15 1.93 ± 1.18

152

Metabolic effects of ß-alanine

The M2 labeling of acetyl-CoA and citrate (Figure 8.14) reflect the conversion of ß- alanine to acetyl-CoA via malonic semialdehyde dehydrogenase and the processing of acetyl-CoA in the CAC (Scheme 8.3). The metabolism of ß-alanine labels, but does not overload, the pool of CAC intermediates (Table 8.8, row 10). This contrasts with the massive overloading of CAC intermediates by 3HP, propionate or both as we showed previously (17)).

In the presence of 2 mM M4 ß-alanine, the concentrations of all CoA esters assayed were markedly increased (except for 3HP-CoA) (Table 8.8, rows 12 to 19). The total concentration of the CoA esters assayed increased 8.5 fold (row 19). Remarkably, there was no detectable labeling of the CoA esters. Also, the concentration of liver pantothenate was not affected by the load of M4 ß-alanine (row 11). Liver pantothenate remained unlabeled. Thus, the presence of 2 mM ß-alanine rapidly increased total CoA accumulation from unknown CoA source(s). In our previous study (17), 5 mM propionate

± 2 mM 3HP tripled the pool of CoA esters assayed, without changing the liver pool of pantothenate.

ß-Alanine did not affect the total pool of adenine nucleotides, but almost significantly increased the [ATP]/[AMP] ratio (Table 8.8, rows 20 to 25). Also, ß-alanine decreased the concentration of pyruvate, without affecting the [lactate]/[pyruvate] ratio (Table 8.8, rows 28, 29, 34). The [BHB] / [AcAc] and [α-glycerophosphate]/[ DHAP] ratios were not affected. Thus, β-alanine does not affect parameters of energy and redox metabolism.

Evidence of carboxylation of ß-alanine

GC-MS analysis of the extract of a liver perfused with 2 mM unlabeled ß-alanine

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revealed an unknown peak with m/z 44 amu above that of ß-alanine. This compound X

was not detected in control livers, and was not listed in the NIST database of mass

15 13 spectra. In another liver perfused with M4 ß-[ N, C3]alanine, compound X had a m/z

44 amu above that of M4 ß-alanine. The M4 enrichment of compound X, released in the

perfusate, remained over 90% during the experiment (Figure 8.13B). The data suggested

that the molecule of compound X (i) contains the 3 carbon atoms and the N atom of ß-

alanine, and (ii) is formed by addition of CO2 to ß-alanine. Compound X seemed to be an

isomer of aspartate. However, on the GC-MS trace of the liver extract, aspartate and compound X have different retention times and fragmentation patterns (Figure 8.15).

13 If derived from carboxylation of ß-alanine, compound X should be labeled from CO2.

We perfused one liver with non-recirculating, albumin-free bicarbonate buffer containing

4 mM glucose and 2 mM unlabeled ß-alanine (infused through a syringe pump). After 15

min, the perfusion buffer was switched to a bicarbonate buffer containing 40%

13 NaH CO3. Compound X was detected in the effluent perfusate. When unlabeled

13 bicarbonate in the buffer was switched to 40% NaH CO3, compound X became 25% M1

labeled (Figure 8.16). Urea in the effluent perfusate was also 25% M1 labeled (not

13 shown). Since the labeling of urea from NaH CO3 is a proxy of the labeling of

mitochondrial CO2, the data demonstrate that compound X is formed by carboxylation of

ß-alanine. The lower enrichments of urea and compound X compared to the enrichment

of inflowing bicarbonate result from (i) the gassing of the perfusate with 95% O2 + 5% unlabeled CO2, and (ii) the production of unlabeled mitochondria CO2 from unlabeled

glucose and endogenous substrates.

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Figure 8.15. Electron ionization mass spectra of TBDMS derivatives of: AMM in a liver perfused with unlabeled ß-alanine (A), AMM formed in a liver perfused with M+4 15 13 13 ß-[ N, C3]alanine (B), AMM formed in a liver perfused with 40% NaH CO3 (C), AMM formed in vitro by reaction of ß-alanine with pyruvate carboxylase (D), synthetic AMM (E), and aspartate (F). The peaks of AMM and aspartate were resolved (not shown). The main ion of each compound is M-57. The mass of the molecular ions was confirmed by positive NH3 chemical ionization (not shown).

155

Figure 8.16. Evidence that AMM is formed by carboxylation of β-alanine. A rat liver was perfused with non-recirculating bicarbonate buffer containing 4 mM glucose + 2 mM of unlabeled β-alanine. At 15 min, the unlabeled NaHCO3 in the buffer was replaced by 13 40% NaH CO3. The data show the mass isotopomer distribution of AMM in the effluent perfusate. M0 = unlabeled; M1 = singly 13C-labeled.

The above data show that the molecule of compound X has a global formula of C4H7NO4

with two carboxyls (one derived from CO2) and an amino group. There is only one possible molecule with such composition, in addition to aspartate, i.e., 2-(aminomethyl)-

malonate (AMM, Scheme 8.5). The identity of compound X with AMM was confirmed

by chemical synthesis and NMR (see Materials). The GC-MS retention time and

electron ionization mass spectrum of the TBDMS derivative of synthetic AMM are

identical to those of the compound detected in liver perfused with unlabeled ß-alanine

(Figure 8.15).

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SCHEME 8.5. 2-aminomethylmalonate (AMM), an isomer of aspartate formed by addition of CO2 onto β-alanine. Reversible carboxylation of β-alanine to AMM results in identical carbons on carbon-2, either of which can become carbon-1 of β-alanine.

We hypothesized that AMM is formed by a side-reaction of pyruvate carboxylase, the only mammalian enzyme that carboxylates a compound which is not a CoA ester or a peptide. We incubated purified pyruvate carboxylase (Sigma) with ß-alanine in a buffer

containing ß-alanine + ATP + bicarbonate + acetyl-CoA (a pyruvate carboxylase activator). GC-MS analysis of the reaction medium revealed the formation of AMM

(Figure 8.15D). The reaction also occurs when acetyl-CoA is replaced by propionyl-CoA

(not shown). Detailed kinetics of ß-alanine carboxylation by pyruvate carboxylase will be

presented in a future report.

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In vivo experiments with ß-alanine

ß-Alanine is a neurotransmitter-like compound (15). So is AMM. The production of

AMM by rat livers perfused with ß-alanine evoked the paresthesias reported by some

athletes after ß-alanine ingestion (1; 5; 7). We hypothesized that AMM might be formed

in some tissues after ingestion of ß-alanine. We conducted in vivo experiments on 4 groups of rats treated as outlined in Scheme 8.3. We used two strategies to load body fluids with ß-alanine: stomach gavage with ß-alanine (with controls gavaged with saline), and pre-treatment with an intraperitoneal injection of AOA, a transaminase inhibitor.

AOA inhibits the conversion of ß-alanine to malonic semialdehyde (Scheme 8.3) leading

to increase in plasma ß-alanine concentration (10). ß-Alanine was given at a dose

corresponding to the dose ingested by some human athletes before exercise. This protocol

afforded 8 conditions during which we assayed the concentrations of ß-alanine, AMM,

3HP and pantothenate in the plasma, liver, skeletal muscle, heart and brain of the rats.

We also assayed compounds of related interest: (ii) CoA and CoA esters in the tissues,

and (ii) N-acetylaspartate, α-aminobutyrate, and γ-aminobutyrate in brain. Note that in

Figures 8.17 to 8.22, each point represents one rat killed at that time.

In control rats injected and gavaged with saline, the concentrations of ß-alanine and

AMM remained at baseline in plasma, liver, skeletal muscle and brain (blue traces in

Figures 8.17 to 8.22), with some variations in heart ß-alanine concentration (Figure

8.20D). In rats pre-injected with AOA and gavaged with saline, the concentration of ß-

alanine increased in plasma and liver (Figures 8.17A and 8.18A), but not in muscle, heart

and brain (Figures 8.20A and D, and 8.21A). In the same rats, the concentration of AMM

remained at baseline in plasma, muscle, heart and brain (Figures 8.17C, 8.20C and F, and

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8.21C), but increased in liver (Figure 8.18C).

In rats pre-injected with saline and gavaged with ß-alanine at 120 min, the concentrations of ß-alanine increased in plasma, liver muscle and brain (Figures 8.17A, 8.18A, 8.20A and 8.21A), but not in heart (Figure 8.20D). Pre-treatment with AOA potentiated the increase in ß-alanine concentration after ß-alanine gavage (Figures 8.17A, 8.18A, 8.20A and 8.21A).

In the brain, after gavage with ß-alanine, the concentrations of AMM, N-acetylaspartate,

α-aminobutyrate and GABA were markedly increased (Figures 8.21C-F). The increases in AMM, α-aminobutyrate and GABA were potentiated by pre-treatment with AOA

(Figures 8.21C-F). The concentrations of pantothenate and CoA esters in the brain were increased by ß-alanine gavage, and potentiated by pre-treatment with AOA (Figure

8.22A-E). Also, gavage with ß-alanine increased the concentration of 3HP in plasma, liver, muscle, heart and brain (Figures 8.17B, 8.18B, 8.20B and E, 8.21B).

Lastly, the concentrations of pantothenate, free CoA, acetyl-CoA, propionyl-CoA, 3HP-

CoA and total CoA assayed were increased by ß-alanine (Figure 8.19A-F). Addition of

AOA to ß-alanine increased the concentration of pantothenate, free CoA, acetyl-CoA, and total CoA (Figures 8.19A-C, and F), but not propionyl-CoA and 3HP-CoA (Figure

8.19D and E).

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Figure 8.17. Metabolite concentrations in PLASMA of rats injected with AOA or saline and subsequently gavaged with β-alanine or saline. Saline or AOA was injected IP. Control (blue) indicates rats injected and gavaged with saline. β-alanine (yellow) indicates rats injected with saline and gavaged with β-alanine. AOA (orange) indicates rats injected with AOA and gavaged with saline. AOA + β-alanine (grey) indicates rats injected with AOA and gavaged with β-alanine. Rats were sacrificed and plasma collected at indicated times (N=1). Panel A: Concentration of β-alanine in mM. Panel B: Concentration of 3HP in mM. Panel C: Concentration of AMM relative to a metabolomic 2 internal standard ([ H4]L-alanine). Panel D: Concentration of pantothenate in mM. Note the different y-axis scales.

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1 ([ with gavaged saline. saline. Saline or alanine or AOA was injected IP. Saline or AOA was injected IP. Control (blue) indicates rats injected and 8.18 Figure Rats were sacrificed and livers were quick frozen indicated at times (N=1). Panel A: Concentration of β with AOAgavaged saline. β + AOA and with injected . Panel B: . Panel wtConcen tration nmol*g ofdry 3HP in 2 H 4 ]L - alanine). Note the different y . Metabolite concentrations in LIVER of rats injected with AOA or saline and subsequently gavaged with β - alanine (yellow)indicates rats injected withsaline and gavaged with - axis scales.

- 1 . Panel C: Concentration of AMM relative to a metabolomic internal standard - alanine (grey) indicates rats injected with AOA and gavaged with β β - alanine. AOA (orange) indicates rats - alanine in µ mol*g wt dry - alanine. β - -

161 species in Propionyl Concentration ofCoA µ Free in AOAgavaged saline. with AOA and β + saline. with saline. Saline or alanine or AOA was injected IP. Saline or AOA was injected IP. Control (blue) indicates rats injected and gavaged 8.19 Figure sacrificed and - µ CoA n in mol*g dry wtmol*g dry . Metabolite concentrations in LIVER of rats injected with AOA or saline and subsequently gavaged with β - livers werefrozen indicated livers quick A: at Panel µ times Concentrationin (N=1). of Pantothenate alan ine (yellow) indicates rats injected with saline and gavaged with β mol*g wt dry - 1 . Note the the y - . Note different mol*g wt dry - 1 . Panel E: Concentration E: of. Panel 3HP - - 1 alanine (grey) indicates rats injected with AOA gavaged and with β . Panel C: Concentration C: Concentration of. Panel Acetyl axis scales.

- CoA in nmol*g dry wt nmol*g dry CoA in - CoA in µ in CoA - alanine. AOA (orange) indicates rats injected - 1 mol*g wt dry . Panel F: Concentration of . PanelTotal Concentration CoA F: - 1 . Panel D: Concentration D: Concentration of. Panel mol*g dry wtmol*g dry - alanine.were Rats - 1 . Panel B: . Panel β -

162

of AMM relative D,E,F concentrationsHEART in are sacrifice d and livers were quick frozen indicated at times (N=1). Panels A,B,C concentrations are in skeletal y different the AOA saline. with β + AOA gavaged and saline. saline. 8 Figure Saline or AOA was injected IP. Saline or AOAwas injected IP. Control (blue) indicates rats injected and gavaged with β - .20 alanine (yellow) indicates rats injected with saline and gavaged with β .Metabolite concentratio - axis scales. to a metabolomic internal standard ([

muscle. Panelofmuscle. A,D: Concentration ß - ns in rats injected with AOA or saline and subsequently gavaged with with gavaged subsequently and saline or with AOA injected rats in ns - alanine (grey) AOAalanine indicates rats with (grey) β injected with gavaged and 2 H 4 ]L - alanine) . Panel C,F: Concentration . Panel C,F:wt Concentration of dry 3HP nmol*g in alanine in µ - alanine. AOA (orange) indicates rats injected with mol*g wt dry - 1 . Panel B,E: Panel . . Panels MUSCLE - alanine. Rats were β - alanine or or alanine Concentration Concentration - 1 . Note . Note

163

µmol*g wt dry D alanine). Panel sacrificed and AOA saline. with β + AOA gavaged and saline. or saline. 8.21 Figure Concentration of 3HP in nmol*g dry wtConcentration nmol*g ofdry 3HP in β - alan Saline or AOA was injected IP. Saline or AOA was injected IP. Control (blue) indicates rats injected and gavaged with . Metabolite concentrations in BRAIN of rats injected with AOA or saline and subsequently gavaged with ine (yellow) indicates rats injected with saline and gavaged with β livers werefrozen indicated livers quick A: at Panel times Concentration (N=1). of β - 1 . Panel F . Panel : Concentration of: N : Concentration of: 4 - - acetylaspartate in µmol*g dry wt - 1 - . Panel C: Concentration of AMM relative to a metabolomic internal standard ([ alanine (grey) AOAalanine indicates rats with (grey) β injected with gavaged and aminobutyrate (GABA) µmol*gwtaminobutyrate in dry - 1 . Panel E . Panel - alanine. AOA (orange) indicates rats injected with : Concentration: of 2 - - 1 . Note the the y - . Note different - alanine in µmol*gwt in dry alanine aminobutyrate (AABA) in - alanine. Rats were axis scales. - 1 . Panel B: 2 H

4 ]L β - - alanine alanine

164

Concentration of Pr wt nmol*g B: Concentration in of CoA Panel Free dry different y saline. with saline. Saline or alanine or AOA was injected IP. Saline or AOA was injected IP. Control (blue) indicates rats injected and gavaged 8.22 Figure with AOA and gavaged AOAgavaged saline. with AOA and β + were sacrifice liversquickd and were indicatedPanel times A: Concentration at (N=1). of frozen µ Pantothenate in - axis scales. 3HP . Metabol β - alanine (yellow) indicates rats injected with saline and gavaged with β opionyl ite ite concentrations in BRAIN of rats injected with AOA or saline and subsequently gavaged with - - CoA wt nmol*g dry in CoA was not detected. not CoA was - alanine (grey) indicates rats injected with AOA gavaged and with β - 1 . Panel E: Concentration of Total CoA species in nmol*g of in . Panel Totalwt Concentration CoAE: dry species - 1 . Panel C: Concentration of Acetyl - alanine. AOA (orange) indicates rats injected - CoA wt nmol*g dry in - - 1 mol*g dry wtmol*gdry alanine. Rats . Panel D: Panel . - 1 . Note the the . Note β - - 1 .

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Discussion

The initial goal of this study was clearly defined: to explore the reversibility of the

reactions linking 3HP to ß-alanine. This was meant to be a small final component of our

previous study on the interrelations of propionate and 3HP metabolism in rat liver (17).

However, the data of our liver perfusions with M4 ß-alanine generated new lines of

research unrelated to the original research plan, i.e., (i) stimulation of CoA synthesis by

ß-alanine, (ii) carboxylation of ß-alanine to a neurotransmitter-like compound, and (iii) metabolic effects of in vivo treatment with ß-alanine. This led us to separate the data of our investigations with M4 ß-alanine from the already large report on propionate and

3HP metabolism (17). As a result, some of the data of our control liver perfusions are presented in both reports (left column of Tables 8.7 and 8.8).

15 13 The major increase in total CoA concentration induced by ß-[ N, C3]alanine (Table

8.8) is puzzling because (i) the CoA molecules are not labeled by M4 β-alanine, (ii) the

pool of liver pantothenate does not decrease and remains unlabeled, and (iii) we did not

detect pantothenol in the liver. Thus, ß-alanine likely triggers the conversion of hidden

pool(s) of pantothenate precursor to pantothenate or to a closely related CoA precursor.

We had also observed increases in total CoA species assayed in livers perfused with

propionate ± 3HP (17), and in livers perfused with levulinate (4-ketopentanoate) (8). An

explanation of the accumulation of CoA by β-alanine will require extensive additional

investigations.

The identification of AMM as a new carboxylated metabolite of β-alanine illustrates the

potential of the association of metabolomics and stable isotope technology for the

discovery of new compounds and of the pathways in which they are involved (18). Since

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β-alanine is a physiological compound, and pyruvate carboxylase activity is found in

many cell types, especially liver and kidney, it is likely that AMM plays a role in the

metabolism of neurotransmitters. This is supported by the detection of AMM in plasma

and tissues from control rats which were not treated with β-alanine or AOA (Figures

8.17C, 8.18C, 8.20C and F, and 8.21C). Also, the loading of body fluids with β-alanine,

AOA or both leads to major increases in AMM concentration in brain (Figure 8.21C), and in perturbations of brain concentrations of N-acetylaspartate, α-aminobutyrate and

GABA (Figures 8.21D-F). The neurotransmitter storm induced by high concentrations of

β-alanine in plasma evokes the paresthesias reported by athletes trying to improve their performance by ingesting large doses of β-alanine. After intragastric administration of β- alanine, the plasma β-alanine concentration increases rapidly in the systemic circulation

(Figure 8.17A). The wave of β-alanine entering the systemic circulation reaches not only muscle tissue (the intended target), but also the central nervous system where it wreaks havoc with neurotransmitter concentrations (Figures 8.21D-F) and presumably functions.

During muscular exercise, the concentration of muscle pyruvate is lowered to the μM range by the increases in the [NADH]/[NAD+] and the [lactate]/[pyruvate] ratios. Then,

anaplerosis of the CAC by pyruvate carboxylation is low because the Km of pyruvate

carboxylase for pyruvate is about 0.4 mM. When a high concentration of muscle β-

alanine is set by pre-exercise ingestion, this high concentration of β-alanine probably

competes with the low pyruvate concentration for carboxylation. Thus, β-alanine is likely

interfering with some reactions of intermediary metabolism in muscle. The use of β-

alanine to improve athletic performance should be critically revisited.

In recent years, AOA has been tested as an anti-cancer agent because inhibition of

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transaminases decreases the anaplerotic flux of glutamine carbon to the CAC of the

cancer cells (6; 9; 12). Likely side-effects of this therapy would be an increase in plasma

β-alanine (10), an increase in AMM formation, perturbations of neurotransmitter balance, and possibly the induction of paresthesias. These side-effects should be weighed in relation to the benefits expected from the interference of AOA (or other transaminase inhibitor) on tumor metabolism.

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Reference List

1. Bellinger PM and Minahan CL. Performance effects of acute beta-alanine induced paresthesia in competitive cyclists. Eur J Sport Sci 16: 88-95, 2016.

2. Blancquaert L, Everaert I and Derave W. Beta-alanine supplementation, muscle carnosine and exercise performance. Curr Opin Clin Nutr Metab Care 18: 63-70, 2015.

3. Brosnan JT, Brosnan ME, Yudkoff M, Nissim I, Daikhin Y, Lazarow A, Horyn

O and Nissim I. Alanine metabolism in the perfused rat liver. Studies with (15)N. J Biol

Chem 276: 31876-31882, 2001.

4. Brunengraber H, Boutry M and Lowenstein JM. Fatty acid, 3-beta- hydroxysterol, and ketone synthesis in the perfused rat liver. Effects of (--)- hydroxycitrate and oleate. Eur J Biochem 82: 373-384, 1978.

5. Decombaz J, Beaumont M, Vuichoud J, Bouisset F and Stellingwerff T. Effect of slow-release beta-alanine tablets on absorption kinetics and paresthesia. Amino Acids

43: 67-76, 2012.

6. Hao Y, Samuels Y, Li Q, Krokowski D, Guan BJ, Wang C, Jin Z, Dong B, Cao

B, Feng X, Xiang M, Xu C, Fink S, Meropol NJ, Xu Y, Conlon RA, Markowitz S,

Kinzler KW, Velculescu VE, Brunengraber H, Willis JE, LaFramboise T,

Hatzoglou M, Zhang GF, Vogelstein B and Wang Z. Oncogenic PIK3CA mutations reprogram glutamine metabolism in colorectal cancer. Nat Commun 7: 11971, 2016.

7. Harris RC and Stellingwerff T. Effect of beta-alanine supplementation on high- intensity exercise performance. Nestle Nutr Inst Workshop Ser 76: 61-71, 2013.

8. Harris SR, Zhang GF, Sadhukhan S, Murphy AM, Tomcik KA, Vazquez EJ,

169

Anderson VE, Tochtrop GP and Brunengraber H. Metabolism of levulinate in

perfused rat livers and live rats: conversion to the drug of abuse 4-hydroxypentanoate. J

Biol Chem 286: 5895-5904, 2011.

9. Korangath P, Teo WW, Sadik H, Han L, Mori N, Huijts CM, Wildes F, Bharti

S, Zhang Z, Santa-Maria CA, Tsai H, Dang CV, Stearns V, Bhujwalla ZM and

Sukumar S. Targeting Glutamine Metabolism in Breast Cancer with Aminooxyacetate.

Clin Cancer Res 21: 3263-3273, 2015.

10. Kurozumi Y, Abe T, Yao WB and Ubuka T. Experimental beta-alaninuria

induced by (aminooxy)acetate. Acta Med Okayama 53: 13-18, 1999.

11. Manoli I, Sloan JL and Venditti CP. Isolated Methylmalonic Acidemia. 1993.

12. Qing G, Li B, Vu A, Skuli N, Walton ZE, Liu X, Mayes PA, Wise DR,

Thompson CB, Maris JM, Hogarty MD and Simon MC. ATF4 regulates MYC-

mediated neuroblastoma cell death upon glutamine deprivation. Cancer Cell 22: 631-644,

2012.

13. Reszko AE, Kasumov T, Pierce BA, David F, Hoppel CL, Stanley WC, Des

Rosiers C and Brunengraber H. Assessing the reversibility of the anaplerotic reactions

of the propionyl-CoA pathway in heart and liver. J Biol Chem 278: 34959-34965, 2003.

14. Shchelochkov OA, Carrillo N and Venditti C. Propionic Acidemia. 1993.

15. Tiedje KE, Stevens K, Barnes S and Weaver DF. Beta-alanine as a small molecule neurotransmitter. Neurochem Int 57: 177-188, 2010.

16. Warren GB and Tipton KF. Pig liver pyruvate carboxylase. Purification, properties and cation specificity. Biochem J 139: 297-310, 1974.

17. Wilson KA, Han Y, Zhang M, Hess J, Chapman KA, Cline GW, Tochtrop GP,

170

Brunengraber H and Zhang G-F. Interrelations between 3-hydroxypropionate and propionate metabolism in rat liver: Relevance to disorders of propionyl-CoA metabolism.

Am J Physiol (in press): 2017.

18. Zhang G-F, Sadukhan S, Tochtrop G and Brunengraber H. Metabolomics, pathway regulation and pathway discovery. J Biol Chem 286: 32631-23635, 2011.

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Chapter 9 Discussion, Implications, and Future Directions

9.1 CoA Trapping in Disorders of Propionyl-CoA Metabolism

9.1.1 Discussion and Conclusions

In livers perfused with propionate and 3HP we observed that propionate induces major

CoA trapping in the propionyl-CoA anaplerotic pathway, specifically in the CoA esters:

propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA. Our findings illustrate some aspects of the CASTOR phenotype described in PA and MMA, specifically the accumulation of various CoA esters. This would result in impairment of CoA-dependent reactions (133). This would induce various metabolic perturbations and is likely a contribution to the metabolic derangements noted in the studies. Interestingly, liver pantothenate concentration was not changed. Also, though we showed that 3HP can be reversibly metabolized to 3HP-CoA, the vast majority of the small 3HP-CoA pool that accumulated in our combined propionate + 3HP perfusions was generated from propionate not 3HP. These data indicate that propionate alone is the major contributor to

CoA trapping in disorders of propionyl-CoA metabolism. Though we detected an increase in the concentration of 3HP-CoA, the total pool of 3HP-CoA was small in relation to the other CoA esters assayed. Because we did not have a standard of 3HP-CoA available, we could not determine the toxicity or metabolic effects of this metabolite.

We saw large increases in the total CoA pool, measured as the summation of all CoA esters assayed, possibly due to rapid stimulation of the CoA generating pantothenate pathway by propionate ± 3HP. This is of interest given the literature on regulation of

172

CoA production. Halvorsen [1982, 211-215] showed that propionyl-CoA is a strong inhibitor of pantothenate kinase, whereas Leonardi [2010, e11107] showed that long chain acylcarnitines activate pantothenate kinase (72, 114). Propionylcarnitine, as a short

chain acylcarnitine, was not specifically shown to have activity (114). Our data show that

in our model, rather than decreased CoA from inhibition of CoA synthesis by propionyl-

CoA, CoA accumulates. This is of particular note because it suggests that the pantothenate pathway can be stimulated in the acute setting, under conditions which account for whole organ metabolism, as compared to previous isolated enzyme kinetic studies (59, 72, 165). Fasting conditions are known to increase CoA but were not likely to have a significant effect between our fasted control rat livers and the fasted experimental rat livers (114). Additionally, NAD/NADH ratios were not significantly different

suggesting that altered redox state effects on CoA, and related metabolism, were not at

play in our experimental conditions (114).

9.1.2 Future Directions

The mechanism by which propionate ± 3HP stimulates the rapid activation of CoA

synthesis, in the acute setting, needs to be investigated further. It is unknown whether

propionylcarnitine stimulates pantothenate kinase, as has been shown with

palmitoylcarnitine. It must also be noted that we used supraphysiologic concentrations of propionate, 5mM as have been seen in PA and MMA, as compared to the 0.02mM concentrations of propionyl-CoA used by Halvorsen [1982, 211-215] (72). The pantothenate pathway is a multiezymatic system, thus it is possible that, in the ex vivo

liver, inhibition of pantothenate kinase by propionyl-CoA is of minor importance

173

compared to the activation by propionate ± 3HP (72, 114). There also must be a hidden

pool of pantothenate precursor that is rapidly mobilized to generate new CoA during CoA trapping in the acute setting. Effects of propionate and 3HP on CoA metabolism and discovery of novel precursors of pantothenate merit further investigation.

It would be interesting to obtain liver acyl-CoA concentrations, measured in PA and

MMA patients both pre- and post-OLT. Also, expanded studies on CoA biosynthesis could determine the role of propionate ± 3HP and the effects of the CoA esters and carnitine compounds of these metabolites in the setting of PA and MMA.

The metabolic effects of 3HP-CoA are unknown but likely also play a role in the metabolic perturbations seen in disorders of propionyl-CoA metabolism. We did not have access to a standard of 3HP-CoA with which to conduct experiments, nor could we find concentrations for this metabolite in PA or MMA patients in the literature. The toxicity of

3HP-CoA was therefore not investigated in these studies and thus merits further examination. Similarly, the suspected metabolite 3-hydroxypropionylcarnitine was not investigated to any great extent.

9.2 Modulation of the CAC in Disorders of Propionyl-CoA Metabolism

9.2.1 Discussion and Conclusions

The CAC was overloaded by propionate ± 3HP, as was the propionyl-CoA anaplerotic pathway. Our data show that 3HP, or a metabolite, impaired both the utilization and disposal of these intermediates. Specifically 3HP, or a metabolite of 3HP, inhibited the

uptake of propionate, the dilution of propionate by CAC precursors such as glucose,

174 prevented propionyl-CoA, as succinyl-CoA, from entering the CAC, and prevented the recycling of propionate into the CAC past citrate, isocitrate, or α-ketoglutarate. The activity of citrate synthase was preserved. This occurs despite the small amount of carbon contributed by 3HP directly to the CAC, non-anaplerotically, via acetyl-CoA. The overload of the CAC by 3HP is possibly due, at least in part, to a decrease in the CO2 generating reactions by methylcitrate. However in our model the modest accumulation of methylcitrate likely does not completely explain the CAC inhibition, and thus merits further investigation (37). Regardless, in the context of PA and MMA, this suggests that the CAC in the patient, rather than being under a cataplerotic drain by methylcitrate and methylsuccinate, is instead overloaded and inhibited as a result of PA and MMA metabolites. Inhibition of the CAC would lead to similar effects as if the CAC was deficient in intermediates, but would require a different strategy for treatment (186).

Liver energy metabolism was severely perturbed under our conditions. The total pool of adenine nucleotides is increased, likely in response to the changing energy status of the liver. The observed changes in energy status are not explained by an overload of the

NAD pools with reducing equivalents nor by an impairment of the respiratory chain itself. If this occurred, the NAD/NADH ratio should increase, but this did not occur.

Rather, the redox status suggests a low flux of reducing equivalents from the overloaded

CAC to the respiratory chain. As a result, the major cause of acute energy deficit in disorders of propionyl-CoA metabolism may be due to CAC disruption, rather than direct inhibition of the respiratory chain by propionyl-CoA. Direct inhibition and mitochondrial disfunction likely still play a large role in the chronic presentation of PA and MMA (126,

175

128). Under our conditions we seem to replicate the energy deficit in PA and MMA (43,

175).

As noted previously, ureagenesis was not explored to any great extend and requires further investigation.

After OLT, peripheral tissues continue to produce and release PA and MMA metabolites which progressively accumulate. We show metabolic derangement of the CAC by these

PA metabolites in the normal liver, a model of the post-OLT PA and MMA patient.

Additionally, 3HP was shown to decrease the ability of a liver with normal PCC, MUT, and MCEE activity to dispose of propionate, thus at least in part replicating PA and

MMA in the normal liver. The long term viability of a normal liver in the metabolic environment of PA and MMA should be an area of future investigation as treatment of disorders of propionyl-CoA metabolism by OLT is new. Early cases of PA patients receiving liver transplants had short follow-up periods, or patients were lost to follow-up

(34, 91, 206). For example, the average length of follow-up in one retrospective study was only 7.3 years. Patients at the time of last follow-up showed normal biochemical indices of liver viability (206). The success of OLT in treatment of PA is well- documented, however treatment with OLT should not result in a relaxation of patient care by the clinician. Furthermore, our data, which shows multiple metabolic perturbations from 3HP and propionate in the normal liver, suggest that the health of the new liver should be closely monitored.

9.2.2 Future Directions

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We showed preserved activity of citrate synthase, an enzyme reportedly inhibited by methylcitrate (37). Additionally the concentration of methylcitrate we reported was low.

It is possible that the degree of methylcitrate inhibition, in the ex vivo liver model we used, was markedly different than those originally reported in the in vitro setting. It is also possible that there are additional mechanisms of inhibition caused by high concentrations of propionate and 3HP. The acute inhibition of the CAC that occurs in disorders of propionyl-CoA metabolism deserves further investigation in multiple models of disease state. Similarly, the inhibition of the CAC by 3HP alone, or a related metabolite such as 3HP-CoA, deserves further investigation. It is of importance to determine the exact site of inhibition in the CAC past citrate that occurs.

In the PA and MMA patient it is likely that both methylcitrate and methylsuccinate do act as cataplerotic substances, but we suggest additional explanations for the modulation of the CAC. We propose that complex changes occur in PA and MMA to disrupt the CAC, more than what can be explaned by methylcitrate alone. We further suggest that careful attention be paid to the liver of post-OLT PA and MMA patients for signs of metabolic derangement, and that these patients continue to be followed in a long term prospective manner. Plasma concentrations of PA and MMA metabolites in the post-OLT patient would likely provide insight into the metabolic status of the liver, based on our data.

Long term retrospective analysis of post-OLT PA and MMA patients would also yield valuable data. Additionally, as little patient concentration data has been obtained for 3HP, this metabolite may be a potential biomarker for PA and MMA related to disease status and patient outcome.

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Of additional note, there are a number of other metabolic diseases that affect the amino

acids that are ultimately metabolized to propionyl-CoA, such as, famously, Maple syrup

urine disease which results in buildup of the relevant amino acids isoleucine and valine as well as a number of branched chain organic acidurias which result from enzyme deficiencies in metabolic steps prior to propionyl-CoA. These upstream deficiencies result in varied metabolic profiles depending on the specific enzyme but the overall clinical picture is largely similar to PA and MMA suggesting overlap in mechanism of action in organic acidemias and diseases of toxic compound accumulation. The continued disruption of ketone body metabolism in these disorders is a possible mechanism to explain the similar overlap in clinical presentation, as is the continued sequestration of carnitine by various compounds. The structural similiarities of a number of compounds discussed previously to compounds that accumulate in these various organic acidemias, such as 2-methyl-3-hydroxybutyrate and 2-methylacetoacetate, also cannot be ignored.

Though these various organic acidemias do not result in accumulation of propionyl-CoA the similarities should not be ignored. In assessing PA and MMA moveing forward it will be important to relate discovery to these early amino acids and the disorders that result from the various upstream processes that are none the less related to propionyl-CoA metabolism (97, 208).

9.3 Possible Fates of the Carbon of 3HP

9.3.1 Discussion and Conclusions

We showed that the first carbon in the conversion of 3HP to acetyl-CoA is lost when the intermediate malonate semialdehyde is metabolized by methylmalonate semialdehyde

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dehydrogenase; the C-3 and C-2 of 3HP correspond to C-1 and C-2 of the acetyl-CoA moiety, respectively. Acetyl-CoA formed then either enters the CAC or is channeled into ketone bodies, in a mechanism similar to what was previously described with acetyl-CoA generated from fatty acids (170). Propionate, however, decreases the conversion of 3HP to acetyl-CoA, citrate, and acetoacetate. Though we showed decrease carbon contribution from 3HP into acetyl-CoA and subsequent metabolites we could not discern where the carbon of 3HP accumulates. We could not identify an unknown compound that accumulated 3HP carbon using our GC-MS or LC-MS/MS techniques. It is possible that a volatile compound is formed along a novel metabolic pathway. We did not measure carnitine profiles. We also showed that propionate decreased the entrance of acetyl-CoA from 3HP into the CAC to a greater extent than it does for the formation of ketone bodies. The decrease in 3HP carbon incorporated into ketone bodies can be explained solely by the decrease in 3HP carbon incorporated into acetyl-CoA when propionate was present.

Our data shows that propionate inhibits the disposal of 3HP to non-toxic acetyl-CoA.

Though the energy status of the liver was altered and the metabolism of 3HP to acetyl-

CoA involves two dehydrogenases, the normal redox state of the liver precludes the lack of oxidizing equivalents, namely NAD+, as the mechanism of inhibition on the 3HP

metabolic pathway by propionate.

9.3.2 Future Directions

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We could not determine the fate(s) of the bulk of the 3HP carbon when propionate was

additionally perfused. The metabolism of 3HP deserves further investigation to fully

elucidate the role 3HP plays in PA. We propose expanded and targeted metabolic

analysis of 3HP metabolism in the normal liver using 13C to track the labeled carbon

through the metabolome.

The mechanism by which propionate alters 3HP metabolism requires additional research.

It is possible, given the similar chemical structures of the two compounds, 3HP and

propionate, that propionate has direct inhibition on 3HP dehydrogenase. Overall, detailed

kinetic studies involving 3HP, propionate, and related acyl-CoAs and acylcarnitines on a number of enzymes are required.

9.4 Production of Maleate from Propionate and from 3HP

9.4.1 Discussion and Conclusions

Maleate, the trans enantiomer of fumarate, is not a physiologic metabolite in mammals,

though it does have documented renal toxicity and can induce glucosuria (134). Maleyl-

CoA, the activated CoA ester of maleate, is known to react with amino acids and proteins

of the renal brush border (153). Maleyl-CoA also reacts with free CoA to form additional

thioesters (15, 153). We showed that maleate is formed from propionate, likely in the

cytoplasm, and independently by 3HP via novel pathways of metabolism. This formation

is independent of the CAC. The production of maleate from propionate would likely

occur via a mechanism involving carboxylation of propionyl-CoA to methylmalonyl-

CoA, then subsequent as yet unknown metabolic reactions to form maleate. Furthermore

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we detected maleate in both mouse models of PA and MMA and in PA and MMA patient

samples. Maleate was well correlated with methylmalonate in these samples lending

further support to the hypothesis: propionyl-CoAmethylmalonyl-CoAmaleate.

Also, if maleate forms the various CoA esters described in previous sections, it would, in addition to playing a role in deteriorating renal function, also be a contributing metabolite to CoA trapping seen in PA and MMA (15, 153).

9.4.2 Future Directions

Only one study reported maleate in a PA patient previously, and they could not separate maleate from fumarate (209). Thus, it was necessary to confirm the presence of this compound in human samples. Maleate is easily distinguishable from fumarate by GC-MS under our conditions, but even under conditions which result in the co-elution of maleate with fumarate, careful analysis of the fragmentation pattern should allow for the detection and analysis of maleate. Waite [1962, 2750-2757] reported that acetyl-CoA carboxylase can carboxylate propionyl-CoA to form cytosolic methylmalonyl-CoA (168). It however is not obvious to us how maleate could be formed from cytosolic methylmalonyl-CoA.

We have not yet determined the exact mechanism of maleate production from propionate or 3HP, though we did show that perfusion with methylmalonate also resulted in the production of maleate. The novel metabolism of propionate and 3HP to maleate requires further investigation. Specifically, we need to identify the various enzymes responsible for metabolism of propionyl-CoA to maleate as well as confirm the susepected role of acetyl-CoA carboxylase in maleate formation; the enzymes responsible for 3HP

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metabolism to maleate are similarly unknown It is interesting to note that there is

increased incidence of kidney disease in post-OLT PA patients, lending further support to

these claims (206). Evaluation of samples from PA and MMA patients, and murine models of PA and MMA, at different metabolic states, renal dysfunction, and both pre- and post-OLT, would also be valuable to establish a causal relationship.

9.5 Novel Metabolism of β-Alanine

9.5.1 Discussion and Conclusions

Though we saw an increase in total concentration of β-alanine when 3HP was perfused,

in livers ex vivo, only a minority of that β-alanine came from 3HP. Thus, β-alanine is not

a sink for 3HP, nor propionate, carbon. Additionally, the reversible metabolism of β-

alanine to 3HP is a minor metabolic pathway for β-alanine carbon. It seems that 3HP ±

propionate instead stimulates production/accumulation of β-alanine, by some unknown

mechanism.

β-alanine was taken up and metabolized by all organs investigated, and metabolism of β-

alanine, AMM, and 3HP, may suggest compartmentalization. This would result in

“hidden” pools of metabolites that do not mix with the easily measurable soluble pools of

metabolites seen in blood and urine. Furthermore, high β-alanine results in (i) increased

cytosolic and mitochondrial nitrogen pools, (ii) increased proteolysis to provide aspartate

nitrogen to the urea cycle, and (iii) increased production of ammonia. This results in

increased demand on the urea cycle. β-alanine should be avoided by patients with

disorders of propionyl-CoA metabolism.

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β-alanine is carboxylated from aspartate by intestinal microflora but we could find no

evidence of β-alanine carboxylation to the proposed compound, 2-(aminomethyl)-

malonate (AMM), alternatively called 2-(aminomethyl)-propanedioate, an isomer of

aspartate, by any organisms (180). We discovered this novel metabolite in rat liver and

confirmed that it is a direct product of the carboxylation of β-alanine by pyruvate

carboxylase. We also confirmed that it is distinct from aspartate in both retention time

and fragmentation pattern. While carboxylation of β-alanine generates AMM we detected

no production of the isomer aspartate from β-alanine.

9.5.2 Future Directions

The metabolic effects of β-alanine in the setting of PA deserve further investigation. In

order to do so, the concentration of β-alanine in both pre- and post-OLT PA patients

should be assayed. Additionally the combined effects of 3HP, propionate, and β-alanine need to be investigated. Compartmentalization of metabolism of propionate, β-alanine,

3HP, and related metabolites would result in hard to measure pools of PA and MMA metabolites. This may explain the poor association between PA and MMA patient statuses and metabolites directly associated with disorders of propionyl-CoA metabolism.

To investigate this would require more invasive sampling from PA and MMA patients than only blood and urine collection. Comparative analysis of metabolite profiles from stored excised livers of patients to blood and/or urine samples from those same patients would provide valuable data (17).

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The kinetics of pyruvate carboxylase for the reversible reaction involving β-alanine and

AMM under various conditions, acetyl-CoA vs propionyl-CoA, the presence of pyruvate,

etc. are unknown. The Km, Vmax, competition, and various other parameters all need to be

investigated for this novel enzymatic reaction. There has been little direct connection, or

investigation of the relationship between, β-alanine and propionic acidemia. β-Alanine

may be a potential biomarker of disease. We propose assaying the concentration of both

β-alanine and AMM in patient samples. Investigating the metabolism of AMM, and using

variously labeled AMM substrates, would greatly increase our understanding of the novel

compound and possible toxicity. The similar structure of AMM to aspartate suggests

substitution for aspartate by AMM in a number of neurotransmitter processes.

9.6 Altered CoA Metabolism from High β-Alanine

9.6.1 Discussion and Conclusions

Any effects on acetyl-CoA concentration by PA and MMA should not hinder carboxylation of β-alanine by pyruvate carboxylase in PA and MMA as propionyl-CoA, and possibly 3HP-CoA, can replace acetyl-CoA as a cofactor (205). Furthermore, β- alanine results in an increase in total CoA concentration, even in the absence of intestinal microflora metabolism. It is likely that β-alanine has a regulatory role in CoA metabolism, acting as an acute modulator of CoA synthesis, in addition to being a precursor of pantothenate in gut microbe metabolism. The increase in total CoA included an increase in both 3HP-CoA and propionyl-CoA even in the absence of a greater than physiological load of propionate. Interestingly, administration of aminooxyacetate prior to a load of β-alanine resulted in a decrease, or inhibition of the effect seen from β-

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alanine alone, to a number of PA and MMA metabolites in multiple organs. This occurs

even in the presence of continued high free CoA. Acetyl-CoA was also increased

suggesting that the effect is not a global effect on acyl-CoA synthesis.

9.6.2 Future Directions

The increase in propionyl-CoA paralleled the increase in free CoA, suggesting that the

increased free CoA was acting as substrate for propionate  propionyl-CoA. Overall the

increase in both 3HP-CoA and propionyl-CoA was likely due to both the increase in

propionyl-CoA amino acid precursors from increased proteolysis by β-alanine and from increased free CoA substrate. While the mechanism of action for β-alanine effects on proteolysis are clear, the need for aspartate to dispose of the additional nitrogen, the mechanism for β-alanine effects on CoA synthesis beyond intestinal microflora metabolism require further investigation. The effects of aminooxyacetate, a transaminase inhibitor, on metabolites related to PA and MMA was an unexpected finding for which we have no explaination. The effects of aminooxyacetate in the setting of disorders of propionyl-CoA metabolism, and the effects of increasing CoA, require much further

study.

9.7 Effects of High β-Alanine on Neurotransmitter Balance

9.7.1 Discussion and Conclusions

β-alanine administration caused increases to all neurotransmitters assayed, and the

possible direct neurotransmitter role(s) of β-alanine and/or AMM cannot be ignored.

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AMM may have effects on neurotransmission. The increase in these, and probably other

neurotransmitters, is likely due to non-specific transport by CNS transport proteins,

including GABA transport proteins, for β-alanine. Similarly, β-alanine has multiple associated receptor sites, including GABA-A and GABA-C receptors. Both of these

actions by β-alanine likely affect neurotransmitter balance (201). These transporters and receptors are found throughout the CNS, especially in glial cells, the primary cell type damaged in PA and MMA, and the primary cell type of β-alanine accumulation (103,

142, 201).

AABA is a compound that may have a role in glutathione compensation against oxidative stress. In mouse hearts it was shown that increased AABA concentration paralleled increased reactive oxygen species concentration, which suggests increased oxidative stress. However, a rise in AABA was also shown to increase glutathione, thus conferring a protective effect. The underlying mechanism for this increased in glutathione may involve AABA stimulation of serine biosynthesis secondarily resulting in an increase in the downstream product, glutathione. It is possible that a similar mechanism is found in brain (85).

9.7.2 Future Directions

The effects of β-alanine on neurotransmitter balance are an area requiring further investigation because parathesia from high β-alanine ingestition suggest likely toxic effects on the CNS, particularly as it is not regulated but is widely used by athletes to increase performance. An assessment of the effect of β-alanine on a broad range of neurotransmitters is needed. The identification of any direct neutrotransmitter activity by

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β-alanine are an area of ongoing investigation, though because no specific transmitter or

receptor has been identified for β-alanine, discovery and resolution of effects by β-

alanine alone are slow (201). AMM effects on the CNS are unknown but as AMM is an

isomer of aspartate, we suspect it has effects on aspartate mediated neurochemistry.

AMM effects on mammals require further study. A mechanism to explain the apparent

high vunerability of glial cells to damage in disorders of propionyl-CoA metabolism has

not yet been established. It is likely due to the combined effects of β-alanine, AMM, and

metabolites of PA and MMA, rather than β-alanine alone. Even large ingestion of β-

alanine alone does not seemingly result in glial cell damage. β-alanine and AMM

metabolism, and the relationship to propiony-CoA metabolism requires more research.

The brain is very sensitive to oxidative stress and the findings of changes to AABA

concentration demand further attention. It is possible that the rise in AABA in brain

signals an increase in oxidative stress due to β-alanine administration but whether the rise in AABA also resulted in a rise in glutathione requires additional investigation.

9.8 Summary and Implications

Our studies show why OLT is not curative for patients with disorders of propionyl-CoA

metabolism. Peripheral tissues cannot dispose of propionyl-CoA and instead release PA

and MMA metabolites, including 3HP, which accumulate and induce metabolic

perturbations even in the normal liver. Furthermore, the presence of 3HP hinders the normal liver from adequately disposing of propionate. The observed increased proteolysis in the presence of propionate suggests an additional role of propionate in causing

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metabolic decompensation in PA and MMA. Our studies were all conducted in the acute

setting, and we recognize the need to both verify our findings in human samples, and to

examine the role of propionate and 3HP in the chronic setting.

As has been described above, there are many unknowns and unresolved questions with

regards to disorders of propionyl-CoA metabolism. The mechanistic study of PA and

MMA and the effects of PA and MMA metabolites on the patient will lead to strategies to alleviate the metabolic perturbations associated with the disease. We suggest that such studies as proposed above be conducted in a variety of models, normal livers, hepatocytes from PA and MMA patients and various other patient cell lines, and animal models of disease (32, 70).

PA and MMA patients are now living longer due to increasing knowledge of the disease and better, more aggressive treatment strategies (156). Though, not curative, this equates to an adult population with largely unknown medical needs and symptoms due to the disease, the treatment, or a combination of both. One such example is the recent discovery of the link between carnitine supplementation, an aspect of PA and MMA management, and the development of atherosclerosis without the normal change in lipoproteins (66). As the proposed mechanism involves intestinal microflora, this discovery may necessitate a change in microbiotic management of the PA patient (66,

67). This is one such example of the previously unexplored area of research involving the adult PA patient population. Overall, our findings contribute to the understanding of disorders of propionyl-CoA metabolism, but more indepth and robust investigation is needed.

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Literature Cited 1. Al-Hassnan ZN, Boyadjiev SA, Praphanphoj V, Hamosh A, Braverman NE, Thomas GH, Geraghty MT. The relationship of plasma glutamine to ammonium and of glycine to acid-base balance in propionic acidaemia. [Online]. J Inherit Metab Dis 26: 89–91, 2003. http://www.ncbi.nlm.nih.gov/pubmed/12872849 [8 Jun. 2017]. 2. Ando T, Rasmussen K, Nyhan WL, Hull D. 3-hydroxypropionate: significance of -oxidation of propionate in patients with propionic acidemia and methylmalonic acidemia. [Online]. Proc Natl Acad Sci U S A 69: 2807–11, 1972. http://www.ncbi.nlm.nih.gov/pubmed/4507604 [8 Jun. 2017]. 3. Ando T, Rasmussen K, Wright JM, Nyhan WL. Isolation and identification of methylcitrate, a major metabolic product of propionate in patients with propionic acidemia. [Online]. J Biol Chem 247: 2200–4, 1972. http://www.ncbi.nlm.nih.gov/pubmed/5016650 [8 Jun. 2017]. 4. Atkinson DE, Bourke E. Metabolic aspects of the regulation of systemic pH. [Online]. Am J Physiol 252: F947-56, 1987. http://www.ncbi.nlm.nih.gov/pubmed/3296786 [8 Jun. 2017]. 5. Bain MD, Jones M, Borriello SP, Reed PJ, Tracey BM, Chalmers RA, Stacey TE. Contribution of gut bacterial metabolism to human metabolic disease. [Online]. Lancet (London, England) 1: 1078–9, 1988. http://www.ncbi.nlm.nih.gov/pubmed/2896913 [8 Jun. 2017]. 6. Baker EH, Sloan JL, Hauser NS, Gropman AL, Adams DR, Toro C, Manoli I, Venditti CP. MRI characteristics of globus pallidus infarcts in isolated methylmalonic acidemia. AJNR Am J Neuroradiol 36: 194–201, 2015. 7. Bar-Even A, Flamholz A, Noor E, Milo R. Rethinking glycolysis: on the biochemical logic of metabolic pathways. Nat Chem Biol 8: 509–17, 2012. 8. BARNESS LA, YOUNG D, MELLMAN WJ, KAHN SB, WILLIAMS WJ. Methylmalonate excretion in a patient with pernicious anemia. N Engl J Med 268: 144–6, 1963. 9. De Baulny HO, Benoist JF, Rigal O, Touati G, Rabier D, Saudubray JM. Methylmalonic and propionic acidaemias: Management and outcome. J Inherit Metab Dis 28: 415–423, 2005. 10. Baumgarter ER, Viardot C. Long-term follow-up of 77 patients with isolated methylmalonic acidaemia. [Online]. J Inherit Metab Dis 18: 138–42, 1995. http://www.ncbi.nlm.nih.gov/pubmed/7564229 [8 Jun. 2017]. 11. Baumgartner D, Scholl-Bürgi S, Sass JO, Sperl W, Schweigmann U, Stein J-I, Karall D. Prolonged QTc intervals and decreased left ventricular contractility in patients with propionic acidemia. J Pediatr 150: 192–7, 197.e1, 2007. 12. Baumgartner MR, Hörster F, Dionisi-Vici C, Haliloglu G, Karall D, Chapman KA, Huemer M, Hochuli M, Assoun M, Ballhausen D, Burlina A, Fowler B, Grünert SC, Grünewald S, Honzik T, Merinero B, Pérez-Cerdá C,

189

Scholl-Bürgi S, Skovby F, Wijburg F, MacDonald A, Martinelli D, Sass JO, Valayannopoulos V, Chakrapani A, Zeiher A, Bax J, Baumgartner H, Ceconi C, Dean V. Proposed guidelines for the diagnosis and management of methylmalonic and propionic acidemia. Orphanet J Rare Dis 9: 130, 2014. 13. BAXTER CF, ROBERTS E. Elevation of gamma-aminobutyric acid in brain: selective inhibition of gamma-aminobutyric-alpha-ketoglutaric acid transaminase. [Online]. J Biol Chem 236: 3287–94, 1961. http://www.ncbi.nlm.nih.gov/pubmed/13866006 [8 Jun. 2017]. 14. Bergman AJ, Van der Knaap MS, Smeitink JA, Duran M, Dorland L, Valk J, Poll-The BT. Magnetic resonance imaging and spectroscopy of the brain in propionic acidemia: clinical and biochemical considerations. Pediatr Res 40: 404– 9, 1996. 15. Bergstrøm T, Greter J, Levin AH, Steen G, Tryding N, Wass U. Propionyl- CoA carboxylase deficiency: case report, effect of low-protein diet and identification of 3-oxo-2-methylvaleric acid 3-hydroxy-2-methylvaleric acid, and maleic acid in urine. [Online]. Scand J Clin Lab Invest 41: 117–26, 1981. http://www.ncbi.nlm.nih.gov/pubmed/7313494 [8 Jun. 2017]. 16. Blancquaert L, Baba SP, Kwiatkowski S, Stautemas J, Stegen S, Barbaresi S, Chung W, Boakye AA, Hoetker JD, Bhatnagar A, Delanghe J, Vanheel B, Veiga-da-Cunha M, Derave W, Everaert I. Carnosine and anserine homeostasis in skeletal muscle and heart is controlled by β-alanine transamination. J Physiol 594: 4849–4863, 2016. 17. Boon L, Blommaart PJ, Meijer AJ, Lamers WH, Schoolwerth AC. Response of hepatic amino acid consumption to chronic metabolic acidosis. [Online]. Am J Physiol 271: F198-202, 1996. http://www.ncbi.nlm.nih.gov/pubmed/8760261 [8 Jun. 2017]. 18. Bouatra S, Aziat F, Mandal R, Guo AC, Wilson MR, Knox C, Bjorndahl TC, Krishnamurthy R, Saleem F, Liu P, Dame ZT, Poelzer J, Huynh J, Yallou FS, Psychogios N, Dong E, Bogumil R, Roehring C, Wishart DS. The Human Urine Metabolome. PLoS One 8: e73076, 2013. 19. Brass EP. Interaction of carnitine and propionate with pyruvate oxidation by hepatocytes from clofibrate-treated rats: importance of coenzyme A availability. [Online]. J Nutr 122: 234–40, 1992. http://www.ncbi.nlm.nih.gov/pubmed/1732464 [8 Jun. 2017]. 20. Brass EP, Beyerinck RA. Effects of propionate and carnitine on the hepatic oxidation of short- and medium-chain-length fatty acids. [Online]. Biochem J 250: 819–25, 1988. http://www.ncbi.nlm.nih.gov/pubmed/3134008 [8 Jun. 2017]. 21. Brass EP, Fennessey P V, Miller L V. Inhibition of oxidative metabolism by propionic acid and its reversal by carnitine in isolated rat hepatocytes. [Online]. Biochem J 236: 131–6, 1986. http://www.ncbi.nlm.nih.gov/pubmed/3790065 [8 Jun. 2017]. 22. Brass EP, Tahiliani AG, Allen RH, Stabler SP. Coenzyme A metabolism in

190

vitamin B-12-deficient rats. [Online]. J Nutr 120: 290–7, 1990. http://www.ncbi.nlm.nih.gov/pubmed/2319347 [8 Jun. 2017]. 23. Brassier A, Boyer O, Valayannopoulos V, Ottolenghi C, Krug P, Cosson MAA, Touati G, Arnoux JBB, Barbier V, Bahi-Buisson N, Desguerre I, Charbit M, Benoist JFF, Dupic L, Aigrain Y, Blanc T, Salomon R, Rabier D, Guest G, de Lonlay P, Niaudet P. Renal transplantation in 4 patients with methylmalonic aciduria: a cell therapy for metabolic disease. Mol Genet Metab 110: 106–10, 2013. 24. Brismar J, Ozand PT. CT and MR of the brain in disorders of the propionate and methylmalonate metabolism. [Online]. AJNR Am J Neuroradiol 15: 1459–73, 1994. http://www.ncbi.nlm.nih.gov/pubmed/7985563 [8 Jun. 2017]. 25. Brock M, Buckel W. On the mechanism of action of the antifungal agent propionate. Eur J Biochem 271: 3227–3241, 2004. 26. Burlina AB, Dionisi-Vici C, Piovan S, Saponara I, Bartuli A, Sabetta G, Zacchello F. Acute pancreatitis in propionic acidaemia. [Online]. J Inherit Metab Dis 18: 169–72, 1995. http://www.ncbi.nlm.nih.gov/pubmed/7564238 [8 Jun. 2017]. 27. C Davies SE, Frenkiel T, Chalmers RA, Iles RA. Propionyl-CoA inhibition of citrate synthase and methyl citrate production studied by 1H nuclear-magnetic- resonance spectroscopy [Online]. http://www.biochemsoctrans.org/content/ppbiost/15/1/150.full.pdf [8 Jun. 2017]. 28. Carrillo-Carrasco N, Chandler RJ, Venditti CP. Combined methylmalonic acidemia and homocystinuria, cblC type. I. Clinical presentations, diagnosis and management. J Inherit Metab Dis 35: 91–102, 2012. 29. Chace DH, DiPerna JC, Kalas TA, Johnson RW, Naylor EW. Rapid diagnosis of methylmalonic and propionic acidemias: quantitative tandem mass spectrometric analysis of propionylcarnitine in filter-paper blood specimens obtained from newborns. [Online]. Clin Chem 47: 2040–4, 2001. http://www.ncbi.nlm.nih.gov/pubmed/11673377 [8 Jun. 2017]. 30. Chakrapani A, Sivakumar P, McKiernan PJ, Leonard J V. Metabolic stroke in methylmalonic acidemia five years after liver transplantation. J Pediatr 140: 261– 3, 2002. 31. Chandler RJ, Sloan J, Fu H, Tsai M, Stabler S, Allen R, Kaestner KH, Kazazian HH, Venditti CP. Metabolic phenotype of methylmalonic acidemia in mice and humans: the role of skeletal muscle. BMC Med Genet 8: 64, 2007. 32. Chapman KA, Collado MS, Figler RA, Hoang SA, Armstrong AJ, Cui W, Purdy M, Simmers MB, Yazigi NA, Summar ML, Wamhoff BR, Dash A. Recapitulation of metabolic defects in a model of propionic acidemia using patient-derived primary hepatocytes. Mol Genet Metab 117: 355–62, 2016. 33. Chapman KA, Gropman A, MacLeod E, Stagni K, Summar ML, Ueda K, Mew NA, Franks J, Island E, Matern D, Pena L, Smith B, Sutton VR, Urv T, Venditti C, Chakrapani A. Acute management of propionic acidemia. Mol Genet

191

Metab 105: 16–25, 2012. 34. Chapman KA, Summar ML, Enns GM. Propionic acidemia: To liver transplant or not to liver transplant? Pediatr Transplant 16: 209–210, 2012. 35. Charbit-Henrion F, Lacaille F, McKiernan P, Girard M, de Lonlay P, Valayannopoulos V, Ottolenghi C, Chakrapani A, Preece M, Sharif K, Chardot C, Hubert P, Dupic L. Early and late complications after liver transplantation for propionic acidemia in children: a two centers study. Am J Transplant 15: 786–91, 2015. 36. Cheema-Dhadli S, Jungas RL, Halperin ML. Regulation of urea synthesis by acid-base balance in vivo: role of NH3 concentration. [Online]. Am J Physiol 252: F221-5, 1987. http://www.ncbi.nlm.nih.gov/pubmed/3812736 [8 Jun. 2017]. 37. Cheema-Dhadli S, Leznoff CC, Halperin ML. Effect of 2-methylcitrate on citrate metabolism: implications for the management of patients with propionic acidemia and methylmalonic aciduria. Pediatr Res 9: 905–8, 1975. 38. Childs B, Nyhan WL, Borden M, Bard L, Cooke RE. Idiopathic hyperglycinemia and hyperglycinuria: a new disorder of amino acid metabolism. I [Online]. Pediatrics 27: 522–538, 1961. http://pediatrics.aappublications.org/content/pediatrics/27/4/522.full.pdf [8 Jun. 2017]. 39. Ciani F, Donati MA, Tulli G, Poggi GM, Pasquini E, Rosenblatt DS, Zammarchi E. Lethal late onset cblB methylmalonic aciduria. [Online]. Crit Care Med 28: 2119–21, 2000. http://www.ncbi.nlm.nih.gov/pubmed/10890676 [8 Jun. 2017]. 40. Cohen RD, Woods HF. Disturbances of acid–base homeostasis. Oxford University Press. 41. Coude FX, Sweetman L, Nyhan WL. Inhibition by propionyl-coenzyme A of N- acetylglutamate synthetase in rat liver mitochondria. A possible explanation for hyperammonemia in propionic and methylmalonic acidemia. J Clin Invest 64: 1544–51, 1979. 42. Coulombe JT, Shih VE, Levy HL. Massachusetts Metabolic Disorders Screening Program. II. Methylmalonic aciduria. [Online]. Pediatrics 67: 26–31, 1981. http://www.ncbi.nlm.nih.gov/pubmed/7243433 [8 Jun. 2017]. 43. Davison JE, Davies NP, Wilson M, Sun Y, Chakrapani A, McKiernan PJ, Walter JH, Gissen P, Peet AC. MR spectroscopy-based brain metabolite profiling in propionic acidaemia: metabolic changes in the basal ganglia during acute decompensation and effect of liver transplantation. Orphanet J Rare Dis 6: 19, 2011. 44. DeFeudis F V, Martin del Rio R. Is beta-alanine an inhibitory neurotransmitter? [Online]. Gen Pharmacol 8: 177–80, 1977. http://www.ncbi.nlm.nih.gov/pubmed/23341 [8 Jun. 2017]. 45. DEN H, ROBINSON WG, COON MJ. Enzymatic conversion of beta-

192

hydroxypropionate to malonic semialdehyde. [Online]. J Biol Chem 234: 1666–71, 1959. http://www.ncbi.nlm.nih.gov/pubmed/13672942 [8 Jun. 2017]. 46. Derave W, Ozdemir MS, Harris RC, Pottier A, Reyngoudt H, Koppo K, Wise JA, Achten E. beta-Alanine supplementation augments muscle carnosine content and attenuates fatigue during repeated isokinetic contraction bouts in trained sprinters. J Appl Physiol 103: 1736–43, 2007. 47. Dionisi-Vici C, Deodato F, Röschinger W, Rhead W, Wilcken B. “Classical” organic acidurias, propionic aciduria, methylmalonic aciduria and isovaleric aciduria: Long-term outcome and effects of expanded newborn screening using tandem mass spectrometry. J Inherit Metab Dis 29: 383–389, 2006. 48. Dobson CM, Gradinger A, Longo N, Wu X, Leclerc D, Lerner-Ellis J, Lemieux M, Belair C, Watkins D, Rosenblatt DS, Gravel RA. Homozygous nonsense mutation in the MCEE gene and siRNA suppression of methylmalonyl- CoA epimerase expression: a novel cause of mild methylmalonic aciduria. Mol Genet Metab 88: 327–33, 2006. 49. Dutra JC, Dutra-Filho CS, Cardozo SE, Wannmacher CM, Sarkis JJ, Wajner M. Inhibition of succinate dehydrogenase and beta-hydroxybutyrate dehydrogenase activities by methylmalonate in brain and liver of developing rats. [Online]. J Inherit Metab Dis 16: 147–53, 1993. http://www.ncbi.nlm.nih.gov/pubmed/8487494 [8 Jun. 2017]. 50. Fenton WA, Gravel RA, Rosenblatt DS. Disorders of Propionate and Methylmalonate Metabolism [Online]. http://www.medgen.mcgill.ca/Files/DavidRosenblattOnline/ch94.pdf [8 Jun. 2017]. 51. Fenton WA, Hack AM, Helfgott D, Rosenberg LE. Biogenesis of the mitochondrial enzyme methylmalonyl-CoA mutase. Synthesis and processing of a precursor in a cell-free system and in cultured cells. [Online]. J Biol Chem 259: 6616–21, 1984. http://www.ncbi.nlm.nih.gov/pubmed/6144679 [8 Jun. 2017]. 52. Fenton WA, Hack AM, Kraus JP, Rosenberg LE. Immunochemical studies of fibroblasts from patients with methylmalonyl-CoA mutase apoenzyme deficiency: detection of a mutation interfering with mitochondrial import. [Online]. Proc Natl Acad Sci U S A 84: 1421–4, 1987. http://www.ncbi.nlm.nih.gov/pubmed/2881300 [8 Jun. 2017]. 53. Filipowicz HR, Ernst SL, Ashurst CL, Pasquali M, Longo N. Metabolic changes associated with hyperammonemia in patients with propionic acidemia. Mol Genet Metab 88: 123–30, 2006. 54. Finch L, Frederick CB. Rate and route of oxidation of acrylic acid to carbon dioxide in rat liver. [Online]. Fundam Appl Toxicol 19: 498–504, 1992. http://www.ncbi.nlm.nih.gov/pubmed/1426707 [8 Jun. 2017]. 55. FLAVIN M, ORTIZ PJ, OCHOA S. Metabolism of propionic acid in animal tissues. [Online]. Nature 176: 823–6, 1955. http://www.ncbi.nlm.nih.gov/pubmed/13272701 [8 Jun. 2017].

193

56. Fontella FU, Pulrolnik V, Gassen E, Wannmacher CM, Klein AB, Wajner M, Dutra-Filho CS. Propionic and L-methylmalonic acids induce oxidative stress in brain of young rats. [Online]. Neuroreport 11: 541–4, 2000. http://www.ncbi.nlm.nih.gov/pubmed/10718311 [8 Jun. 2017]. 57. Fowler B, Leonard J V., Baumgartner MR. Causes of and diagnostic approach to methylmalonic acidurias. J Inherit Metab Dis 31: 350–360, 2008. 58. Gallego-Villar L, Pérez B, Ugarte M, Desviat LR, Richard E. Antioxidants successfully reduce ROS production in propionic acidemia fibroblasts. Biochem Biophys Res Commun 452: 457–61, 2014. 59. Garcia M, Leonardi R, Zhang Y-M, Rehg JE, Jackowski S. Germline deletion of pantothenate kinases 1 and 2 reveals the key roles for CoA in postnatal metabolism. PLoS One 7: e40871, 2012. 60. Gebhardt R, Mecke D. Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture. [Online]. EMBO J 2: 567–70, 1983. http://www.ncbi.nlm.nih.gov/pubmed/6138251 [8 Jun. 2017]. 61. Gibala MJ, Young ME, Taegtmeyer H. Anaplerosis of the citric acid cycle: role in energy metabolism of heart and skeletal muscle. Acta Physiol Scand 168: 657– 65, 2000. 62. Good P. Why Do Children With Propionic Acidemia Or Urea Cycle Disorders Rarely Show Autistic Behavior? Autism Open Access 3, 2014. 63. Goodwin GW, Rougraff PM, Davis EJ, Harris RA. Purification and characterization of methylmalonate-semialdehyde dehydrogenase from rat liver. Identity to malonate-semialdehyde dehydrogenase. [Online]. J Biol Chem 264: 14965–71, 1989. http://www.ncbi.nlm.nih.gov/pubmed/2768248 [8 Jun. 2017]. 64. Gradinger AB, Bélair C, Worgan LC, Li CD, Lavallée J, Roquis D, Watkins D, Rosenblatt DS. Atypical methylmalonic aciduria: frequency of mutations in the methylmalonyl CoA epimerase gene (MCEE). Hum Mutat 28: 1045, 2007. 65. Gravel RA, Lam KF, Scully KJ, Hsia Y. Genetic complementation of propionyl- CoA carboxylase deficiency in cultured human fibroblasts. [Online]. Am J Hum Genet 29: 378–88, 1977. http://www.ncbi.nlm.nih.gov/pubmed/195466 [8 Jun. 2017]. 66. Gregory JC, Buffa JA, Org E, Wang Z, Levison BS, Zhu W, Wagner MA, Bennett BJ, Li L, DiDonato JA, Lusis AJ, Hazen SL. Transmission of Atherosclerosis Susceptibility with Gut Microbial Transplantation. J Biol Chem 290: 5647–5660, 2015. 67. Grünert SC, Müllerleile S, De Silva L, Barth M, Walter M, Walter K, Meissner T, Lindner M, Ensenauer R, Santer R, Bodamer OA, Baumgartner MR, Brunner-Krainz M, Karall D, Haase C, Knerr I, Marquardt T, Hennermann JB, Steinfeld R, Beblo S, Koch H-G, Konstantopoulou V, Scholl- Bürgi S, van Teeffelen-Heithoff A, Suormala T, Sperl W, Kraus JP, Superti- Furga A, Schwab KO, Sass JO. Propionic acidemia: clinical course and outcome in 55 pediatric and adolescent patients. Orphanet J Rare Dis 8: 6, 2013.

194

68. Grünert SC, Müllerleile S, de Silva L, Barth M, Walter M, Walter K, Meissner T, Lindner M, Ensenauer R, Santer R, Bodamer OA, Baumgartner MR, Brunner-Krainz M, Karall D, Haase C, Knerr I, Marquardt T, Hennermann JB, Steinfeld R, Beblo S, Koch HG, Konstantopoulou V, Scholl- Bürgi S, van Teeffelen-Heithoff A, Suormala T, Sperl W, Kraus JP, Superti- Furga A, Schwab KO, Sass JO. Propionic acidemia: neonatal versus selective metabolic screening. J Inherit Metab Dis 35: 41–49, 2012. 69. Gruskay JA, Rosemberg LE. Inhibition of hepatic mitochondrial carbamoyl phosphate synthetase (CPSI) by acyl CoA esters: possible mechanisms of hyperammonemia in the organic acidemias. Pediatr Res 13: Abstract, 1979. 70. Guenzel AJ, Hofherr SE, Hillestad M, Barry MAM, Weaver E, Venezia S, Kraus JP, Matern D, Barry MAM. Generation of a hypomorphic model of propionic acidemia amenable to gene therapy testing. Mol Ther 21: 1316–23, 2013. 71. Halperin ML, Schiller CM, Fritz IB. The inhibition by methylmalonic acid of malate transport by the dicarboxylate carrier in rat liver mitochondria. A possible explantation for hypoglycemia in methylmalonic aciduria. J Clin Invest 50: 2276– 82, 1971. 72. Halvorsen O, Skrede S. Regulation of the biosynthesis of CoA at the level of pantothenate kinase. [Online]. Eur J Biochem 124: 211–5, 1982. http://www.ncbi.nlm.nih.gov/pubmed/7084227 [8 Jun. 2017]. 73. Harris RC, Tallon MJ, Dunnett M, Boobis L, Coakley J, Kim HJ, Fallowfield JL, Hill CA, Sale C, Wise JA. The absorption of orally supplied beta-alanine and its effect on muscle carnosine synthesis in human vastus lateralis. Amino Acids 30: 279–89, 2006. 74. Harting I, Seitz A, Geb S, Zwickler T, Porto L, Lindner M, Kölker S, Hörster F. Looking beyond the basal ganglia: the spectrum of MRI changes in methylmalonic acidaemia. J Inherit Metab Dis 31: 368–78, 2008. 75. HAYAISHI O, NISHIZUKA Y, TATIBANA M, TAKESHITA M, KUNO S. Enzymatic studies on the metabolism of beta-alanine. [Online]. J Biol Chem 236: 781–90, 1961. http://www.ncbi.nlm.nih.gov/pubmed/13712439 [8 Jun. 2017]. 76. Hayasaka K, Tada K. Effects of the metabolites of the branched-chain amino acids and cysteamine on the glycine cleavage system. [Online]. Biochem Int 6: 225–30, 1983. http://www.ncbi.nlm.nih.gov/pubmed/6679320 [8 Jun. 2017]. 77. Heidenreich R, Natowicz M, Hainline BE, Berman P, Kelley RI, Hillman RE, Berry GT. Acute extrapyramidal syndrome in methylmalonic acidemia: "metabolic stroke" involving the globus pallidus. [Online]. J Pediatr 113: 1022–7, 1988. http://www.ncbi.nlm.nih.gov/pubmed/3193307 [8 Jun. 2017]. 78. Hillman RE, Otto EF. Inhibition of glycine-serine interconversion in cultured human fibroblasts by products of isoleucine catabolism. Pediatr Res 8: 941–5, 1974. 79. Hommes FA, Kuipers JRG, Elema JD, Jansen JF, Jonxis JHP.

195

Propionicacidemia, a New Inborn Error of Metabolism. Pediatr Res 2: 519–524, 1968. 80. Hörster F, Baumgartner MR, Viardot C, Suormala T, Burgard P, Fowler B, Hoffmann GF, Garbade SF, Kölker S, Baumgartner ER. Long-term outcome in methylmalonic acidurias is influenced by the underlying defect (mut0, mut-, cblA, cblB). Pediatr Res 62: 225–30, 2007. 81. Hsia YE, Lilljeqvist AC, Rosenberg LE. Vitamin B12-dependent methylmalonicaciduria: amino acid toxicity, long chain ketonuria, and protective effect of vitamin B12. [Online]. Pediatrics 46: 497–507, 1970. http://www.ncbi.nlm.nih.gov/pubmed/5503685 [8 Jun. 2017]. 82. Hsia YE, Scully KJ, Rosenberg LE. Inherited propionyl-CoA carboxylase deficiency in “ketotic hyperglycinemia.” J Clin Invest 50: 127–130, 1971. 83. Huang CS, Sadre-Bazzaz K, Shen Y, Deng B, Zhou ZH, Tong L. Crystal structure of the alpha(6)beta(6) holoenzyme of propionyl-coenzyme A carboxylase. Nature 466: 1001–5, 2010. 84. Inoue S, Krieger I, Sarnaik A, Ravindranath Y, Fracassa M, Ottenbreit MJ. Inhibition of bone marrow stem cell growth in vitro by methylmalonic acid: a mechanism for pancytopenia in a patient with methylmalonic acidemia. Pediatr Res 15: 95–8, 1981. 85. Irino Y, Toh R, Nagao M, Mori T, Honjo T, Shinohara M, Tsuda S, Nakajima H, Satomi-Kobayashi S, Shinke T, Tanaka H, Ishida T, Miyata O, Hirata K-I. 2-Aminobutyric acid modulates glutathione homeostasis in the myocardium. Sci Rep 6: 36749, 2016. 86. Jansen R, Ledley FD. Heterozygous mutations at the mut locus in fibroblasts with mut0 methylmalonic acidemia identified by polymerase-chain-reaction cDNA cloning. [Online]. Am J Hum Genet 47: 808–14, 1990. http://www.ncbi.nlm.nih.gov/pubmed/1977311 [8 Jun. 2017]. 87. Jung T, Kolodecik T, Gorelick F. Extracellular acidification sensitizes the rat pancreatic acinar cell to cerulein-induced protease activation. In: Gastroenterology AGA CME. 2006, p. 383. 88. Kahler SG, Sherwood WG, Woolf D, Lawless ST, Zaritsky A, Bonham J, Taylor CJ, Clarke JT, Durie P, Leonard J V. Pancreatitis in patients with organic acidemias. [Online]. J Pediatr 124: 239–43, 1994. http://www.ncbi.nlm.nih.gov/pubmed/8301430 [8 Jun. 2017]. 89. Kakavand B, Schroeder VA, Di Sessa TG. Coincidence of long QT syndrome and propionic acidemia. Pediatr Cardiol 27: 160–161, 2006. 90. Kaplan P, Ficicioglu C, Mazur AT, Palmieri MJ, Berry GT. Liver transplantation is not curative for methylmalonic acidopathy caused by methylmalonyl-CoA mutase deficiency. Mol Genet Metab 88: 322–326, 2006. 91. Kasahara M, Sakamoto S, Kanazawa H, Karaki C, Kakiuchi T, Shigeta T, Fukuda A, Kosaki R, Nakazawa A, Ishige M, Nagao M, Shigematsu Y,

196

Yorifuji T, Naiki Y, Horikawa R. Living-donor liver transplantation for propionic acidemia. Pediatr Transplant 16: 230–234, 2012. 92. Kashiwagura T, Deutsch CJ, Taylor J, Erecińska M, Wilson DF. Dependence of gluconeogenesis, urea synthesis, and energy metabolism of hepatocytes on intracellular pH. [Online]. J Biol Chem 259: 237–43, 1984. http://www.ncbi.nlm.nih.gov/pubmed/6323402 [8 Jun. 2017]. 93. Katz J, Chaikoff IL. THE METABOLISM OF PROPIONATE BY RAT LIVER SLICES AND THE FORMATION OF ISOSUCCINIC ACID 1. J Am Chem Soc 77: 2659–2660, 1955. 94. de Keyzer Y, Valayannopoulos V, Benoist J-F, Batteux F, Lacaille F, Hubert L, Chrétien D, Chadefeaux-Vekemans B, Niaudet P, Touati G, Munnich A, de Lonlay P. Multiple OXPHOS deficiency in the liver, kidney, heart, and skeletal muscle of patients with methylmalonic aciduria and propionic aciduria. Pediatr Res 66: 91–5, 2009. 95. Kikugawa M, Fujimoto S, Mizota C, Tamaki N. Bisfunction of propionic acid on purified rat liver beta-ureidopropionase. [Online]. FEBS Lett 229: 345–8, 1988. http://www.ncbi.nlm.nih.gov/pubmed/3345846 [8 Jun. 2017]. 96. Klein NP, Aukes L, Lee J, Fireman B, Shapira SK, Slade B, Baxter R, Summar M. Evaluation of Immunization Rates and Safety Among Children With Inborn Errors of Metabolism. Pediatrics 127: e1139–e1146, 2011. 97. Knerr I, Weinhold N, Vockley J, Gibson KM. Advances and challenges in the treatment of branched-chain amino/keto acid metabolic defects. J Inherit Metab Dis 35: 29–40, 2012. 98. Ko R, Low Dog T, Gorecki DKJ, Cantilena LR, Costello RB, Evans WJ, Hardy ML, Jordan SA, Maughan RJ, Rankin JW, Smith-Ryan AE, Valerio LG, Jones D, Deuster P, Giancaspro GI, Sarma ND. Evidence-based evaluation of potential benefits and safety of beta-alanine supplementation for military personnel. Nutr Rev 72: 217–25, 2014. 99. Koletzko B, Bachmann C, Wendel U. Antibiotic therapy for improvement of metabolic control in methylmalonic aciduria. [Online]. J Pediatr 117: 99–101, 1990. http://www.ncbi.nlm.nih.gov/pubmed/2370620 [8 Jun. 2017]. 100. Kölker S, Cazorla AG, Valayannopoulos V, Lund AM, Burlina AB, Sykut- Cegielska J, Wijburg FA, Teles EL, Zeman J, Dionisi-Vici C, Barić I, Karall D, Augoustides-Savvopoulou P, Aksglaede L, Arnoux J-B, Avram P, Baumgartner MR, Blasco-Alonso J, Chabrol B, Chakrapani A, Chapman K, i Saladelafont EC, Couce ML, de Meirleir L, Dobbelaere D, Dvorakova V, Furlan F, Gleich F, Gradowska W, Grünewald S, Jalan A, Häberle J, Haege G, Lachmann R, Laemmle A, Langereis E, de Lonlay P, Martinelli D, Matsumoto S, Mühlhausen C, de Baulny HO, Ortez C, Peña-Quintana L, Ramadža DP, Rodrigues E, Scholl-Bürgi S, Sokal E, Staufner C, Summar ML, Thompson N, Vara R, Pinera IV, Walter JH, Williams M, Burgard P, Burgard P. The phenotypic spectrum of organic acidurias and urea cycle disorders. Part 1: the initial presentation. J Inherit Metab Dis 38: 1041–1057,

197

2015. 101. Kölker S, Sauer SW, Surtees RAH, Leonard J V. The aetiology of neurological complications of organic acidaemias--a role for the blood-brain barrier. J Inherit Metab Dis 29: 701-4-6, 2006. 102. Kølvraa S. Inhibition of the Glycine Cleavage System by Branched-Chain Amino Acid Metabolites. Pediatr Res 13: 889–893, 1979. 103. Komura J, Tamai I, Senmaru M, Terasaki T, Sai Y, Tsuji A. Brain-to-blood active transport of beta-alanine across the blood-brain barrier. [Online]. FEBS Lett 400: 131–5, 1997. http://www.ncbi.nlm.nih.gov/pubmed/9000528 [8 Jun. 2017]. 104. Korf B, Wallman JK, Levy HL. Bilateral lucency of the globus pallidus complicating methylmalonic acidemia. Ann Neurol 20: 364–6, 1986. 105. Kruszka PS, Manoli I, Sloan JL, Kopp JB, Venditti CP. Renal growth in isolated methylmalonic acidemia. (2013). doi: 10.1038/gim.2013.42. 106. Ktena YP, Paul SM, Hauser NS, Sloan JL, Gropman A, Manoli I, Venditti CP. Delineating the spectrum of impairments, disabilities, and rehabilitation needs in methylmalonic acidemia (MMA). Am J Med Genet Part A 167: 2075–2084, 2015. 107. KUPIECKI FP, COON MJ. The enzymatic synthesis of beta-aminoisobutyrate, a product of valine metabolism, and of beta-alanine, a product of beta- hydroxypropionate metabolism. [Online]. J Biol Chem 229: 743–54, 1957. http://www.ncbi.nlm.nih.gov/pubmed/13502336 [8 Jun. 2017]. 108. Kurozumi Y, Abe T, Yao WB, Ubuka T. Experimental beta-alaninuria induced by (aminooxy)acetate. [Online]. Acta Med Okayama 53: 13–8, 1999. http://www.ncbi.nlm.nih.gov/pubmed/10096733 [8 Jun. 2017]. 109. Ledley FD, Crane AM, Lumetta M. Heterogeneous alleles and expression of methylmalonyl CoA mutase in mut methylmalonic acidemia. [Online]. Am J Hum Genet 46: 539–47, 1990. http://www.ncbi.nlm.nih.gov/pubmed/1968706 [8 Jun. 2017]. 110. Ledley FD, Jansen R, Nham SU, Fenton WA, Rosenberg LE. Mutation eliminating mitochondrial leader sequence of methylmalonyl-CoA mutase causes muto methylmalonic acidemia. [Online]. Proc Natl Acad Sci U S A 87: 3147–50, 1990. http://www.ncbi.nlm.nih.gov/pubmed/1970180 [8 Jun. 2017]. 111. Lehnert W. Long-term results of selective screening for inborn errors of metabolism. [Online]. Eur J Pediatr 153: S9-13, 1994. http://www.ncbi.nlm.nih.gov/pubmed/7957394 [8 Jun. 2017]. 112. Lehnert W, Sperl W, Suormala T, Baumgartner ER. Propionic acidaemia: clinical, biochemical and therapeutic aspects. Experience in 30 patients. [Online]. Eur J Pediatr 153: S68-80, 1994. http://www.ncbi.nlm.nih.gov/pubmed/7957390 [8 Jun. 2017]. 113. Leonard J V. The management and outcome of propionic and methylmalonic acidaemia. [Online]. J Inherit Metab Dis 18: 430–4, 1995.

198

http://www.ncbi.nlm.nih.gov/pubmed/7494401 [8 Jun. 2017]. 114. Leonardi R, Rehg JE, Rock CO, Jackowski S. Pantothenate kinase 1 is required to support the metabolic transition from the fed to the fasted state. PLoS One 5: e11107, 2010. 115. Leonardi R, Rock CO, Jackowski S, Zhang Y-M. Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine. Proc Natl Acad Sci 104: 1494–1499, 2007. 116. Lindner M, Ho S, Kölker S, Abdoh G, Hoffmann GF, Burgard P. Newborn screening for methylmalonic acidurias--optimization by statistical parameter combination. J Inherit Metab Dis 31: 379–85, 2008. 117. Lueck JD, Miller LL. The effect of perfusate pH on glutamine metabolism in the isolated perfused rat liver. [Online]. J Biol Chem 245: 5491–7, 1970. http://www.ncbi.nlm.nih.gov/pubmed/5469177 [8 Jun. 2017]. 118. Lund P. Control of glutamine synthesis in rat liver. [Online]. Biochem J 124: 653– 60, 1971. http://www.ncbi.nlm.nih.gov/pubmed/4400073 [8 Jun. 2017]. 119. Macfarland S, Hartung H. Pancytopenia in a patient with methylmalonic acidemia [Online]. http://www.bloodjournal.org/content/bloodjournal/125/11/1840.full.pdf [8 Jun. 2017]. 120. MacMahon M. Requesting vitamin B12 and folate assays. [Online]. Lancet (London, England) 346: 973, 1995. http://www.ncbi.nlm.nih.gov/pubmed/7564764 [8 Jun. 2017]. 121. Macphee S, Weaver IN, Weaver DF. An Evaluation of Interindividual Responses to the Orally Administered Neurotransmitter β -Alanine. J Amino Acids 2013: 429847, 2013. 122. Maekawa K, Hirayama A, Iwata Y, Tajima Y, Nishimaki-Mogami T, Sugawara S, Ueno N, Abe H, Ishikawa M, Murayama M, Matsuzawa Y, Nakanishi H, Ikeda K, Arita M, Taguchi R, Minamino N, Wakabayashi S, Soga T, Saito Y. Global metabolomic analysis of heart tissue in a hamster model for dilated cardiomyopathy. J Mol Cell Cardiol 59: 76–85, 2013. 123. Mandal R, Guo AC, Chaudhary KK, Liu P, Yallou FS, Dong E, Aziat F, Wishart DS. Multi-platform characterization of the human cerebrospinal fluid metabolome: a comprehensive and quantitative update. Genome Med 4: 38, 2012. 124. Manoli I, Sloan JL, Venditti CP. Isolated Methylmalonic Acidemia [Online]. http://www.ncbi.nlm.nih.gov/pubmed/20301409 [8 Jun. 2017]. 125. Manoli I, Sysol JR, Li L, Houillier P, Garone C, Wang C, Zerfas PM, Cusmano-Ozog K, Young S, Trivedi NS, Cheng J, Sloan JL, Chandler RJ, Abu-Asab M, Tsokos M, Elkahloun AG, Rosen S, Enns GM, Berry GT, Hoffmann V, DiMauro S, Schnermann J, Venditti CP. Targeting proximal tubule mitochondrial dysfunction attenuates the renal disease of methylmalonic acidemia. Proc Natl Acad Sci U S A 110: 13552–7, 2013.

199

126. Mardach R, Verity MA, Cederbaum SD. Clinical, pathological, and biochemical studies in a patient with propionic acidemia and fatal cardiomyopathy. Mol Genet Metab 85: 286–90, 2005. 127. Matsui SM, Mahoney MJ, Rosenberg LE. The natural history of the inherited methylmalonic acidemias. N Engl J Med 308: 857–61, 1983. 128. Matsuishi T, Stumpf DA, Seliem M, Eguren LA, Chrislip K. Propionate mitochondrial toxicity in liver and skeletal muscle: acyl CoA levels. [Online]. Biochem Med Metab Biol 45: 244–53, 1991. http://www.ncbi.nlm.nih.gov/pubmed/1883630 [8 Jun. 2017]. 129. MAZUMDER R, SASAKAWA T, KAZIRO Y, OCHOA S. A new enzyme in the conversion of propionyl coenzyme A to succinyl coenzyme A. [Online]. J Biol Chem 236: PC53-5, 1961. http://www.ncbi.nlm.nih.gov/pubmed/13768681 [8 Jun. 2017]. 130. Melo DR, Kowaltowski AJ, Wajner M, Castilho RF. Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia. J Bioenerg Biomembr 43: 39–46, 2011. 131. Menni F, Chiarelli G, Sabatini C, Principi N, Esposito S. Vaccination in children with inborn errors of metabolism. Vaccine 30: 7161–7164, 2012. 132. Mirandola SR, Melo DR, Schuck PF, Ferreira GC, Wajner M, Castilho RF. Methylmalonate inhibits succinate-supported oxygen consumption by interfering with mitochondrial succinate uptake. J Inherit Metab Dis 31: 44–54, 2008. 133. Mitchell GA, Gauthier N, Lesimple A, Wang SP, Mamer O, Qureshi I. Hereditary and acquired diseases of acyl-coenzyme A metabolism. Mol Genet Metab 94: 4–15, 2008. 134. Mohuczy-Dominiak D, Pacanis A, Rogulski J, Angielski S. Significance of CoA-transferase in the reaction of maleate with amino acids and proteins. [Online]. Acta Biochim Pol 30: 39–49, 1983. http://www.ncbi.nlm.nih.gov/pubmed/6575532 [8 Jun. 2017]. 135. Monson JP, Henderson RM, Smith JA, Iles RA, Faus-Dader M, Carter ND, Heath R, Metcalfe H, Cohen RD. The mechanism of inhibition of ureogenesis by acidosis. [Online]. Biosci Rep 4: 819–25, 1984. http://www.ncbi.nlm.nih.gov/pubmed/6518274 [8 Jun. 2017]. 136. Montgomery JA, Mamer OA, Scriver CR. Metabolism of methylmalonic acid in rats. Is methylmalonyl-coenzyme a racemase deficiency symptomatic in man? J Clin Invest 72: 1937–47, 1983. 137. Morrow G, Revsin B, Clark R, Lebowitz J, Whelan DT. A new variant of methylmalonic acidemia-defective coenzyme-apoenzyme binding in cultured fibroblasts. [Online]. Clin Chim Acta 85: 67–72, 1978. http://www.ncbi.nlm.nih.gov/pubmed/25730 [8 Jun. 2017]. 138. Motokawa Y, Kikuchi G, Narisawa K, Arakawa T. Reduced level of glycine cleavage system in the liver of hyperglycinemia patients. [Online]. Clin Chim Acta

200

79: 173–81, 1977. http://www.ncbi.nlm.nih.gov/pubmed/890950 [8 Jun. 2017]. 139. Müller S, Falkenberg N, Mönch E, Jakobs C. Propionacidaemia and immunodeficiency. [Online]. Lancet (London, England) 1: 551–2, 1980. http://www.ncbi.nlm.nih.gov/pubmed/6102279 [8 Jun. 2017]. 140. Murphey WH, Lindmark DG, Patchen LI, Housler ME, Harrod EK, Mosovich L. Serum carnosinase deficiency concomitant with mental retardation. Pediatr Res 7: 601–6, 1973. 141. Nagata M, Ichiyama A, Takayama T, Oda T, Mugiya S, Ozono S. Assay of alanine:glyoxylate aminotransferase in human liver by its serine: glyoxylate aminotransferase activity. [Online]. Biomed Res 30: 295–301, 2009. http://www.ncbi.nlm.nih.gov/pubmed/19887726 [8 Jun. 2017]. 142. Nguyen NHT, Morland C, Gonzalez SV, Rise F, Storm-Mathisen J, Gundersen V, Hassel B. Propionate increases neuronal histone acetylation, but is metabolized oxidatively by glia. Relevance for propionic acidemia. J Neurochem 101: 806–14, 2007. 143. Nicolaides P, Leonard J, Surtees R. Neurological outcome of methylmalonic acidaemia. [Online]. Arch Dis Child 78: 508–12, 1998. http://www.ncbi.nlm.nih.gov/pubmed/9713004 [8 Jun. 2017]. 144. Nissim I. Newer aspects of glutamine/glutamate metabolism: the role of acute pH changes. [Online]. Am J Physiol 277: F493-7, 1999. http://www.ncbi.nlm.nih.gov/pubmed/10516271 [8 Jun. 2017]. 145. Nizon M, Ottolenghi C, Valayannopoulos V, Arnoux J-B, Barbier V, Habarou F, Desguerre I, Boddaert N, Bonnefont J-P, Acquaviva C, Benoist J-F, Rabier D, Touati G, de Lonlay P. Long-term neurological outcome of a cohort of 80 patients with classical organic acidurias. Orphanet J Rare Dis 8: 148, 2013. 146. Nutzenadel W, Scriver CR. Uptake and metabolism of beta-alanine and L- carnosine by rat tissues in vitro: role in nutrition. [Online]. Am J Physiol 230: 643– 51, 1976. http://www.ncbi.nlm.nih.gov/pubmed/1266966 [8 Jun. 2017]. 147. Nyhan WL, Gargus JJ, Boyle K, Selby R, Koch R. Progressive neurologic disability in methylmalonic acidemia despite transplantation of the liver. Eur J Pediatr 161: 377–9, 2002. 148. O’Shea CJ, Sloan JL, Wiggs EA, Pao M, Gropman A, Baker EH, Manoli I, Venditti CP, Snow J. Neurocognitive Phenotype of Isolated Methylmalonic Acidemia. Pediatrics 129: e1541–e1551, 2012. 149. Oberholzer VG, Levin B, Burgess EA, Young WF. Methylmalonic aciduria. An inborn error of metabolism leading to chronic metabolic acidosis. [Online]. Arch Dis Child 42: 492–504, 1967. http://www.ncbi.nlm.nih.gov/pubmed/6061291 [8 Jun. 2017]. 150. Oliver J, Bourke E. Adaptations in urea ammonium excretion in metabolic acidosis in the rat: a reinterpretation. [Online]. Clin Sci Mol Med Suppl 48: 515– 10, 1975. http://www.ncbi.nlm.nih.gov/pubmed/1056282 [8 Jun. 2017].

201

151. Oliver J, Koelz AM, Costello J, Bourke E. Acid-base induced alterations in glutamine metabolism and ureogenesis in perfused muscle and liver of the rat. [Online]. Eur J Clin Invest 7: 445–9, 1977. http://www.ncbi.nlm.nih.gov/pubmed/411675 [8 Jun. 2017]. 152. Otzen C, Bardl B, Jacobsen ID, Nett M, Brock M. Candida albicans Utilizes a Modified -Oxidation Pathway for the Degradation of Toxic Propionyl-CoA. J Biol Chem 289: 8151–8169, 2014. 153. Pacanis A, Strzelecki T, Rogulski J. Effects of maleate on the content of CoA and its derivatives in rat kidney mitochondria. [Online]. J Biol Chem 256: 13035– 8, 1981. http://www.ncbi.nlm.nih.gov/pubmed/7309749 [8 Jun. 2017]. 154. Patel SS, Walt DR. Substrate specificity of acetyl coenzyme A synthetase. [Online]. J Biol Chem 262: 7132–4, 1987. http://www.ncbi.nlm.nih.gov/pubmed/2884217 [8 Jun. 2017]. 155. Patel TB, DeBuysere MS, Olson MS. The effect of propionate on the regulation of the pyruvate dehydrogenase complex in the rat liver. [Online]. Arch Biochem Biophys 220: 405–14, 1983. http://www.ncbi.nlm.nih.gov/pubmed/6824332 [8 Jun. 2017]. 156. Pena L, Franks J, Chapman KA, Gropman A, Ah Mew N, Chakrapani A, Island E, MacLeod E, Matern D, Smith B, Stagni K, Sutton VR, Ueda K, Urv T, Venditti C, Enns GM, Summar ML. Natural history of propionic acidemia. Mol Genet Metab 105: 5–9, 2012. 157. Phromphetcharat V, Jackson A, Dass PD, Welbourne TC. Ammonia partitioning between glutamine and urea: interorgan participation in metabolic acidosis. [Online]. Kidney Int 20: 598–605, 1981. http://www.ncbi.nlm.nih.gov/pubmed/7343710 [8 Jun. 2017]. 158. PIHL A, FRITZSON P. The catabolism of C14-labeled beta-alanine in the intact rat. [Online]. J Biol Chem 215: 345–51, 1955. http://www.ncbi.nlm.nih.gov/pubmed/14392168 [8 Jun. 2017]. 159. Pinar-Sueiro S, Martínez-Fernández R, Lage-Medina S, Aldamiz-Echevarria L, Vecino E. Optic neuropathy in methylmalonic acidemia: the role of neuroprotection. J Inherit Metab Dis 33 Suppl 3: S199-203, 2010. 160. Pollitt RJ, Fowler B, Sardharwalla IB, Edwards MA, Gray RG. Increased excretion of propan-1,3-diol and 3-hydroxypropionic acid apparently caused by abnormal bacterial metabolism in the gut. [Online]. Clin Chim Acta 169: 151–7, 1987. http://www.ncbi.nlm.nih.gov/pubmed/3427776 [8 Jun. 2017]. 161. Powers L, Osborn MK, Yang D, Kien CL, Murray RD, Beylot M, Brunengraber H. Assay of the concentration and stable isotope enrichment of short-chain fatty acids by gas chromatography/mass spectrometry. J Mass Spectrom 30: 747–754, 1995. 162. Puchowicz MA, Bederman IR, Comte B, Yang D, David F, Stone E, Jabbour K, Wasserman DH, Brunengraber H. Zonation of acetate labeling across the liver: implications for studies of lipogenesis by MIDA. [Online]. Am J Physiol

202

277: E1022-7, 1999. http://www.ncbi.nlm.nih.gov/pubmed/10600790 [8 Jun. 2017]. 163. Radmanesh A, Zaman T, Ghanaati H, Molaei S, Robertson RL, Zamani AA. Methylmalonic acidemia: brain imaging findings in 52 children and a review of the literature. Pediatr Radiol 38: 1054–61, 2008. 164. Rafique M. Emerging trends in management of propionic acidemia. [Online]. Arq Bras Endocrinol Metabol 58: 237–42, 2014. http://www.ncbi.nlm.nih.gov/pubmed/24863085 [8 Jun. 2017]. 165. Ramaswamy G, Karim MA, Murti KG, Jackowski S. PPARalpha controls the intracellular coenzyme A concentration via regulation of PANK1alpha gene expression. J Lipid Res 45: 17–31, 2004. 166. Robinson WG, Coon MJ. [80] Synthesis of malonic semialdehyde, β- hydroxypropionate, and β-hydroxyisobutyrate. , p. 549–553. 167. Roe CR, Hoppel CL, Stacey TE, Chalmers RA, Tracey BM, Millington DS. Metabolic response to carnitine in methylmalonic aciduria. An effective strategy for elimination of propionyl groups. [Online]. Arch Dis Child 58: 916–20, 1983. http://www.ncbi.nlm.nih.gov/pubmed/6651329 [8 Jun. 2017]. 168. Rolfe DF, Brown GC. Cellular energy utilization and molecular origin of standard metabolic rate in mammals. [Online]. Physiol Rev 77: 731–58, 1997. http://www.ncbi.nlm.nih.gov/pubmed/9234964 [8 Jun. 2017]. 169. Romano S, Valayannopoulos V, Touati G, Jais J-P, Rabier D, de Keyzer Y, Bonnet D, de Lonlay P. Cardiomyopathies in Propionic Aciduria are Reversible After Liver Transplantation. J Pediatr 156: 128–34, 2010. 170. Des Rosiers C, David F, Garneau M, Brunengraber H. Nonhomogeneous labeling of liver mitochondrial acetyl-CoA. [Online]. J Biol Chem 266: 1574–8, 1991. http://www.ncbi.nlm.nih.gov/pubmed/1988437 [8 Jun. 2017]. 171. Saad LO, Mirandola SR, Maciel EN, Castilho RF. Lactate dehydrogenase activity is inhibited by methylmalonate in vitro. Neurochem Res 31: 541–8, 2006. 172. Saheki T, Tomomura M, Horiuchi M, Imamura Y, Tomomura A, Abdullah Abue Musa D, Kobayashi K. Abnormal gene expression causing hyperammonemia in carnitine-deficient juvenile visceral steatosis (JVS) mice. [Online]. Adv Exp Med Biol 420: 159–72, 1997. http://www.ncbi.nlm.nih.gov/pubmed/9286432 [8 Jun. 2017]. 173. Sass JO, Hofmann M, Skladal D, Mayatepek E, Schwahn B, Sperl W. Propionic acidemia revisited: a workshop report. Clin Pediatr (Phila) 43: 837–43, 2004. 174. Scholem RD, Brown GK. Metabolism of malonic semialdehyde in man. [Online]. Biochem J 216: 81–5, 1983. http://www.ncbi.nlm.nih.gov/pubmed/6418146 [8 Jun. 2017]. 175. Scholl-Bürgi S, Haberlandt E, Gotwald T, Albrecht U, Baumgartner Sigl S, Rauchenzauner M, Rostásy K, Karall D. Stroke-like episodes in propionic

203

acidemia caused by central focal metabolic decompensation. Neuropediatrics 40: 76–81, 2009. 176. Scholl-Bürgi S, Sass JO, Zschocke J, Karall D. Amino acid metabolism in patients with propionic acidaemia. J Inherit Metab Dis 35: 65–70, 2012. 177. Schreiber J, Chapman KA, Summar ML, Ah Mew N, Sutton VR, MacLeod E, Stagni K, Ueda K, Franks J, Island E, Matern D, Peña L, Smith B, Urv T, Venditti C, Chakarapani A, Gropman AL. Neurologic considerations in propionic acidemia. Mol Genet Metab 105: 10–5, 2012. 178. Schuck PF, Rosa RB, Pettenuzzo LF, Sitta A, Wannmacher CMD, Wyse ATS, Wajner M. Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid. Neurochem Int 45: 661–7, 2004. 179. Schwab MA, Sauer SW, Okun JG, Nijtmans LGJ, Rodenburg RJT, van den Heuvel LP, Dröse S, Brandt U, Hoffmann GF, Ter Laak H, Kölker S, Smeitink JAM. Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins. Biochem J 398: 107–12, 2006. 180. Scriver CR, Pueschel S, Davies E. Hyper-beta-alaninemia associated with beta- aminoaciduria and gamma-aminobutyricaciduaia, somnolence and seizures. N Engl J Med 274: 635–43, 1966. 181. Scriver CR, Rosenberg LE. Amino acid metabolism and its disorders. [Online]. Major Probl Clin Pediatr 10: 1–478, 1973. http://www.ncbi.nlm.nih.gov/pubmed/4768607 [8 Jun. 2017]. 182. Shafai T, Sweetman L, Weyler W, Goodman SI, Fennessey P V, Nyhan WL. Propionic acidemia with severe hyperammonemia and defective glycine metabolism. [Online]. J Pediatr 92: 84–6, 1978. http://www.ncbi.nlm.nih.gov/pubmed/619088 [8 Jun. 2017]. 183. Shetewy A, Shimada-Takaura K, Warner D, Jong CJ, Mehdi A-B Al, Alexeyev M, Takahashi K, Schaffer SW. Mitochondrial defects associated with β-alanine toxicity: relevance to hyper-beta-alaninemia. Mol Cell Biochem 416: 11– 22, 2016. 184. Shigesada K, Tatibana M. Enzymatic synthesis of acetylglutamate by mammalian liver preparations and its stimulation by arginine. [Online]. Biochem Biophys Res Commun 44: 1117–24, 1971. http://www.ncbi.nlm.nih.gov/pubmed/5160402 [8 Jun. 2017]. 185. Shimomura Y, Murakami T, Fujitsuka N, Nakai N, Sato Y, Sugiyama S, Shimomura N, Irwin J, Hawes JW, Harris RA. Purification and partial characterization of 3-hydroxyisobutyryl-coenzyme A hydrolase of rat liver. [Online]. J Biol Chem 269: 14248–53, 1994. http://www.ncbi.nlm.nih.gov/pubmed/8188708 [8 Jun. 2017]. 186. Silverman M, Huang L. Mechanism of maleic acid-induced glucosuria in dog kidney. [Online]. Am J Physiol 231: 1024–32, 1976. http://www.ncbi.nlm.nih.gov/pubmed/136203 [8 Jun. 2017].

204

187. Skrede S, Halvorsen O. Increased biosynthesis of CoA in the liver of rats treated with clofibrate. [Online]. Eur J Biochem 98: 223–9, 1979. http://www.ncbi.nlm.nih.gov/pubmed/467442 [8 Jun. 2017]. 188. Sloan JL, Manoli I, Venditti CP. Liver or Combined Liver-Kidney Transplantation for Patients with Isolated Methylmalonic Acidemia: Who and When? J Pediatr 166: 1346–1350, 2015. 189. Spector R, Johanson CE. Vitamin transport and homeostasis in mammalian brain: focus on Vitamins B and E. J Neurochem 103: 425–38, 2007. 190. Stern J. Oxalacetate transacetase (condensing enzyme, nitrogenase). In: The Enzymes, edited by Boyer PD, Lardy H, Myrick K. London: 1961, p. 367–380. 191. Stewart PM, Walser M. Failure of the normal ureagenic response to amino acids in organic acid-loaded rats. Proposed mechanism for the hyperammonemia of propionic and methylmalonic acidemia. J Clin Invest 66: 484–92, 1980. 192. Stokke O, Eldjarn L, Norum KR, Steen-johnsen J, Halvorsen S. Methylmalonic Acidemia a new inborn error of metabolism which may cause fatal acidosis in the neonatal period. Scand J Clin Lab Invest 20: 313–328, 1967. 193. Stork LC, Ambruso DR, Wallner SF, Sambrano JE, Moscinski LC, Wilson HL, McCabe ER. Pancytopenia in propionic acidemia: hematologic evaluation and studies of hematopoiesis in vitro. Pediatr Res 20: 783–8, 1986. 194. Stumpf DA, McAfee J, Parks JK, Eguren L. Propionate inhibition of succinate:CoA ligase (GDP) and the citric acid cycle in mitochondria. [Online]. Pediatr Res 14: 1127–31, 1980. http://www.ncbi.nlm.nih.gov/pubmed/6780967 [8 Jun. 2017]. 195. Surtees RA, Matthews EE, Leonard J V. Neurologic outcome of propionic acidemia. [Online]. Pediatr Neurol 8: 333–7, [date unknown]. http://www.ncbi.nlm.nih.gov/pubmed/1418175 [8 Jun. 2017]. 196. Sutton VR, Chapman KA, Gropman AL, MacLeod E, Stagni K, Summar ML, Ueda K, Ah Mew N, Franks J, Island E, Matern D, Peña L, Smith B, Urv T, Venditti C, Chakarapani A. Chronic management and health supervision of individuals with propionic acidemia. Mol Genet Metab 105: 26–33, 2012. 197. Tada K, Corbeel LM, Eeckels R, Eggermont E. A block in glycine cleavage reaction as a common mechanism in ketotic and nonketotic hyperglycinemia. Pediatr Res 8: 721–3, 1974. 198. Takada Y, Noguchi T. Characteristics of alanine: glyoxylate aminotransferase from Saccharomyces cerevisiae, a regulatory enzyme in the glyoxylate pathway of glycine and serine biosynthesis from tricarboxylic acid-cycle intermediates. [Online]. Biochem J 231: 157–63, 1985. http://www.ncbi.nlm.nih.gov/pubmed/3933486 [8 Jun. 2017]. 199. Thompson GN, Chalmers RA. Increased urinary metabolite excretion during fasting in disorders of propionate metabolism. Pediatr Res 27: 413–6, 1990. 200. Thompson GN, Walter JH, Bresson JL, Ford GC, Lyonnet SL, Chalmers RA,

205

Saudubray JM, Leonard J V, Halliday D. Sources of propionate in inborn errors of propionate metabolism. [Online]. Metabolism 39: 1133–7, 1990. http://www.ncbi.nlm.nih.gov/pubmed/2233273 [8 Jun. 2017]. 201. Tiedje KE, Stevens K, Barnes S, Weaver DF. β-Alanine as a small molecule neurotransmitter. Neurochem Int 57: 177–188, 2010. 202. Toyoshima S, Watanabe F, Saido H, Miyatake K, Nakano Y. Methylmalonic acid inhibits respiration in rat liver mitochondria. [Online]. J Nutr 125: 2846–50, 1995. http://www.ncbi.nlm.nih.gov/pubmed/7472665 [8 Jun. 2017]. 203. Traber G, Baumgartner MR, Schwarz U, Pangalu A, Donath MY, Landau K. Subacute bilateral visual loss in methylmalonic acidemia. J Neuroophthalmol 31: 344–6, 2011. 204. Tuchman M, Yudkoff M. Blood levels of ammonia and nitrogen scavenging amino acids in patients with inherited hyperammonemia. Mol Genet Metab 66: 10– 5, 1999. 205. Utter MF, Keech DB. Pyruvate Carboxylase I. NATURE OF THE REACTION* [Online]. J Biol Chem 238, 1963. http://www.jbc.org/content/238/8/2603.full.pdf [8 Jun. 2017]. 206. Vara R, Turner C, Mundy H, Heaton ND, Rela M, Mieli-Vergani G, Champion M, Hadzic N. Liver transplantation for propionic acidemia in children. Liver Transplant 17: 661–667, 2011. 207. Vernon HJ, Sperati CJ, King JD, Poretti A, Miller NR, Sloan JL, Cameron AM, Myers D, Venditti CP, Valle D. A detailed analysis of methylmalonic acid kinetics during hemodialysis and after combined liver/kidney transplantation in a patient with mut (0) methylmalonic acidemia. J Inherit Metab Dis 37: 899–907, 2014. 208. Vockley J, Zschocke J, Knerr I, Vockley CW, Gibson KM. Branched Chain Organic Acidurias. Online Metab. Mol. Bases Inherit. Dis. . 209. WAITE M, WAKIL SJ. Studies on the mechanism of fatty acid synthesis. XII. Acetyl coenzyme A carboxylase. [Online]. J Biol Chem 237: 2750–7, 1962. http://www.ncbi.nlm.nih.gov/pubmed/14004432 [8 Jun. 2017]. 210. Walter JH, Michalski A, Wilson WM, Leonard J V, Barratt TM, Dillon MJ. Chronic renal failure in methylmalonic acidaemia. [Online]. Eur J Pediatr 148: 344–8, 1989. http://www.ncbi.nlm.nih.gov/pubmed/2707280 [8 Jun. 2017]. 211. Wendel U, Baumgartner R, van der Meer SB, Spaapen LJ. Accumulation of odd-numbered long-chain fatty acids in fetuses and neonates with inherited disorders of propionate metabolism. Pediatr Res 29: 403–5, 1991. 212. West JA, Beqqali A, Ament Z, Elliott P, Pinto YM, Arbustini E, Griffin JL. A targeted metabolomics assay for cardiac metabolism and demonstration using a mouse model of dilated cardiomyopathy. Metabolomics 12: 59, 2016. 213. Willard HF, Rosenberg LE. Inherited deficiencies of human methylmalonyl CaA mutase activity: reduced affinity of mutant apoenzyme for adenosylcobalamin.

206

[Online]. Biochem Biophys Res Commun 78: 927–34, 1977. http://www.ncbi.nlm.nih.gov/pubmed/20894 [8 Jun. 2017]. 214. Willard HF, Rosenberg LE. Inherited Methylmalonyl CoA Mutase Apoenzyme Deficiency in Human Fibroblasts. J Clin Invest 65: 690–698, 1980. 215. Williamson JR, Corkey BE. [65] Assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods. , p. 434–513. 216. Wolf B. Evaluation of biotin responsiveness in cultured fibroblasts from patients with propionic acidemia: absence of response by structurally altered carboxylases. [Online]. Biochem Genet 17: 709–13, 1979. http://www.ncbi.nlm.nih.gov/pubmed/540014 [8 Jun. 2017]. 217. Wolf B. Reassessment of biotin-responsiveness in "unresponsive" propionyl CoA carboxylase deficiency. [Online]. J Pediatr 97: 964–6, 1980. http://www.ncbi.nlm.nih.gov/pubmed/7441430 [8 Jun. 2017]. 218. Wolf B, Hsia YE, Sweetman L, Gravel R, Harris DJ, Nyhan WL. Propionic acidemia: a clinical update. [Online]. J Pediatr 99: 835–46, 1981. http://www.ncbi.nlm.nih.gov/pubmed/7031206 [8 Jun. 2017]. 219. Wolf B, Hsia YE, Tanaka K, Rosenberg LE. Correlation between serum propionate and blood ammonia concentrations in propionic acidemia. [Online]. J Pediatr 93: 471–3, 1978. http://www.ncbi.nlm.nih.gov/pubmed/690769 [8 Jun. 2017]. 220. Zhang G-F, Kombu RS, Kasumov T, Han Y, Sadhukhan S, Zhang J, Sayre LM, Ray D, Gibson KM, Anderson VA, Tochtrop GP, Brunengraber H. Catabolism of 4-Hydroxyacids and 4-Hydroxynonenal via 4-Hydroxy-4- phosphoacyl-CoAs. J Biol Chem 284: 33521–33534, 2009. 221. Zwickler T, Haege G, Riderer A, Hörster F, Hoffmann GF, Burgard P, Kölker S. Metabolic decompensation in methylmalonic aciduria: which biochemical parameters are discriminative? 35: 797–806, 2012. 222. Zwickler T, Riderer A, Haege G, Hoffmann GF, Kölker S, Burgard P. Usefulness of biochemical parameters in decision-making on the start of emergency treatment in patients with propionic acidemia. J Inherit Metab Dis 37: 31–37, 2014.

207