METABOLISM OF LEVULINATE
AND
CONVERSION TO THE DRUG OF ABUSE
4-HYDROXYPENTANOATE
by
STEPHANIE R. HARRIS
Submitted in partial fulfillment of the requirements For the Degree of Doctor of Philosophy
Thesis Advisor: Henri Brunengraber, M.D., Ph.D.
Department of Nutrition
CASE WESTERN RESERVE UNIVERSITY
August 2011
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
STEPHANIE R. HARRIS
candidate for the ______Doctor of Philosophy degree *.
(signed) ______Edith Lerner, PhD (Chair of the committee)
______Henri Brunengraber, MD, PhD
______Colleen Croniger, PhD
______Paul Ernsberger, PhD
______Janos Kerner, PhD
______Michelle Puchowicz, PhD
(date) ______June 16, 2011
*We also certify that written approval has been obtained for any proprietary material contained therein.
ii
DEDICATION
I dedicate this work to my parents and to my husband, Paul. My parents have provided continuous love, encouragement, and guidance throughout my life. They have taught me
to set my goals high. My husband has been a source of strength and inspiration, and his dedication and enthusiastic support have helped me achieve this work.
iii
TABLE OF CONTENTS
Table of Contents………………………………………………………………………... iv
List of Tables…………………………………………………………………………....viii
List of Figures…………………………………………………………………………… ix
Acknowledgements…………………………………………………………………...... xi
List of Abbreviations…………………………………………………………………... xiii
Abstract………………………………………………………………………………..... xv
CHAPTER 1: FATTY ACID OXIDATION
1.1. Overview……………………………………………………………………………...1
1.2. Transport of Fatty Acids into Cells…………………………………………………..2
1.3. Activation of Fatty Acids…………………………………………………………….6
1.3.1. The Carnitine Palmitoyltransferase System…………………………………...7
1.4. Reactions of Mitochondrial β-oxidation……………………………………….……10
1.4.1. The Trifunctional Protein Complex…………………….…………………….13
1.4.2. Reversibility of 3-Ketoacyl-CoA Thiolase…………………………………...13
1.4.3. Peroxisomal β-oxidation……………………………………………………...15
1.4.4. Fates of Acetyl-CoA Derived from Fatty Acid Oxidation…………………...17
1.5. Fatty Acid Alpha- and Omega-Oxidations...………………………………………..22
1.6. Regulation of Mitochondrial β-oxidation…………………………………………...23
1.6.1. Regulation of CPT-I by Malonyl-CoA…………………….…………………23
1.6.2. Redox Regulation of Mitochondrial β-oxidation……………………………..28
1.6.3. Feedback Regulation of Mitochondrial β-oxidation………………………….28
1.6.4. The Role of Carnitine and Free CoA in Fatty Acid β-oxidation……………..29
iv
CHAPTER 2: 4-HYDROXYACID METABOLISM
2.1. Overview of What is Known About 4-Hydroxyacids ………...…………………....32
2.2. 4-Hydroxyacids: Products of Lipid Peroxidation (C9, C6).………………………...33
2.2.1. 4-Hydroxyacids: Drugs of Abuse (C4, C5).………………………………….35
2.3. Recent Development in 4-Hydroxyacid Metabolism ...…………………………….41
2.3.1. Nomenclature of Mass Isotopomers……………………………………. ……41
2.3.2. Pathways of 4-Hydroxyacid Catabolism.……………………………………..41
2.3.3. Formation of 4-Phosphoacyl-CoAs from 4-Hydroxyacids…………………...46
2.3.4. Trapping of CoA into Intermediates of 4-Hydroxyacid Catabolism………....52
CHAPTER 3: LEVULINIC ACID
3.1. Overview……………………………………………………………………….……56
3.2. Production and Industrial Applications of Levulinic Acid………………………….56
3.3. Levulinic Acid as a Calcium Salt ……………………………………………..…...58
3.4. Past Observations of Levulinate Metabolism……………………………………….61
CHAPTER 4: HEXANE TOXICITY
4.1. Overview…………………………………………………………………………….64
4.2. Metabolism of n-Hexane……………………… ……………………………………64
4.3. Hexane Toxicity via the Formation of Pyrrole Adducts…………………………….65
CHAPTER 5: RESEARCH PLAN
5.1. Overview…………………………………………………………………………….69
5.2. Part A: Metabolism of Levulinate ± Ethanol in Perfused Rat Livers and in Live Rats…………...…………………………………….………………………….70
v
5.2.1. Mechanisms for the Conversion of Levulinate to 4-Hydroxypentanoate…….70
5.2.2. Pathways of Levulinate Catabolism………………………………………….73
5.2.3. Public Health Relevance……………………………………………………...77
5.2.4. Strategy……………………………………………………………………….77
5.2.4.1. In Vivo Experiments…………………………………………………...78
5.2.4.2. Liver Perfusion Experiments…………………………………………...79
5.2.4.3. Pilot Enzyme Assays…………………………………………………...79
5.2.4.4. Analytical Procedures…………………………………………………..80
5.3. Part B: Metabolism of Calcium Levulinate………………………………………...83
5.3.1. Metabolism of Calcium Levulinate Given Enterally ± Ethanol………………83
5.3.2. Strategy…………………………………………………………………. ……85
5.3.2.1. Analytical Procedures…………………………………………………..85
CHAPTER 6: PUBLICATIONS
6.1. Overview…………………………………………………………………………….87
6.1.1. Harris, S.R., Zhang, G.F., Sadhukhan, S., Murphy, A.M., Tomcik, K.A., Vazquez, E.J., Anderson, V.E., Tochtrop, G.P., Brunengraber, H. Metabolism of levulinate in perfused rat livers and in live rats: Conversion to the drug of abuse 4-hydroxypentanoate. J. Biol. Chem. 286: 5895-5904, 2011. …………………………………………………………………...88 6.1.2. Harris, S.R., Zhang, G.F., Sadhukhan, S., Puchowicz, M.A., Anderson, V.E., Tochtrop, G.P., Brunengraber, H. Cyclical CoA esters derived from Levulinate. (To be submitted to J. Biol. Chem. July 2011) ………………………………………………………………….126
vi
CHAPTER 7: DISCUSSION, IMPLICATIONS, AND FUTURE DIRECTIONS
7.1. Reduction of Levulinate to 4-Hydroxypentanoate In Vivo and in Isolated Livers………………………………….…………………………………………..156
7.1.1. Discussion and Conclusions…...……………………………………………156
7.1.2. Future Directions………………………………………….…………………157
7.2. Mechanisms for the Conversion of Levulinate to 4-Hydroxypentanoate……... ….158
7.2.1. Discussion and Conclusions…………………………………………………158
7.2.2. Future Directions…………………………………………………………….160
7.3. Pathways of Levulinate Catabolism……………………………………………….162
7.3.1. Discussion and Conclusions…………………………………………………162
7.3.2. Future Directions…………………………………………………………….167
7.4. Metabolism of Oral Calcium Levulinate Ethanol………..…...………………..…171
7.4.1. Discussion and Conclusions…………………………………………………171
7.4.2. Future Directions………………………………………………………….…175
7.5. New Cyclical CoA Esters Derived from Levulinate…………………………….…177
7.5.1. Discussion and Conclusions…...……………………………………………177
7.5.2. Future Directions………………….…………………………………………180
7.6. Overall Summary and Implications……...... ……….……………………………..182
LITERATURE CITED……………………………………………………………….183
vii
LIST OF TABLES
CHAPTER 6
Table 6.1. Concentrations of Acyl-CoAs in Perfused Rat Livers…..…………………124
13 Table 6.2. Mass Isotopomer Distribution of Products of [ C5]levulinate Metabolism in Perfused Rat Livers.……………………………………………………..125
Table 6.3. Distribution of Labeled Mass Isotopomers of C7 CoA Esters and Acetyl-CoA in Rat Livers Perfused with Levulinate and Substrates Labeled with 13C, 15N or Both ………..…………………………………………………………155
viii
LIST OF FIGURES
CHAPTER 1
Fig 1.1. Mechanisms of Fatty Acid Transport into Cells…………………………...……5
Fig 1.2. The Carnitine Palmitoyltransferase System……………………………………..9
Fig 1.3. Mitochondrial β-oxidation Spiral and Sites of Regulation……………………..12
Fig 1.4. Lipogenesis from Ketone Bodies in the Liver………………………………….21
Fig 1.5. Regulation of CPT-I Activity…………………………………………………..27
CHAPTER 2
Fig 2.1. Structures of 4-Hydroxybutyrate and 4-Hydroxypentanoate…………………..40
Fig 2.2. Proposed Scheme for the Catabolism of 4-Hydroxyacids……………………..45
Fig 2.3. Fragmentation Patterns of 4-Phosphopentanoyl-CoA………………………....49
Fig 2.4. Accumulation of 4-Phosphoacyl-CoAs derived from 4-Hydroxyacids………..50
Fig 2.5. Concentrations of 4-Phosphobutyryl-CoA in the Brain and Liver of Mice Deficient in Succinic Semialdehyde Dehydrogenase….………………………51
Fig 2.6. Evidence of CoA Trapping in CoA Esters Derived from 4-Hydroxy-n-acids …………………………………………………………………………………55
CHAPTER 3
Fig 3.1. Biochemical Parameters in the Blood of Premature Babies Following Treatment with Calcium Levulinate……..………………………………………………..63
CHAPTER 4
Fig 4.1. Formation of a Dimethylpyrrole Adduct from 2,5-Diketohexane and an Amine…………………………………………………………………………..68
ix
CHAPTER 5
Fig 5.1. Proposed Scheme for the Catabolism of Levulinate…………………………...82
CHAPTER 6
Fig 6.1. Proposed Scheme for the Metabolism of Levulinate and 4-Hydroxypentanoate ………………………………………………………………………………..120
Fig 6.2. Profiles of Plasma Concentrations of Levulinate and 4-Hydroxypentanoate in Rats Infused Intravenously with Sodium Levulinate ± Ethanol …………….121
Fig 6.3. Effect of Ethanol on Levulinate Uptake and Metabolism in Perfused Rat Livers ………………………………………………………………………..122
13 13 Fig 6.4. Release of [ C]formate by Rat Livers Perfused with [ Cn]levulinate………123
Fig 6.5. Proposed Scheme for the Formation of Cyclical CoA Esters from Levulinate…………………………………………………………………….150
Fig 6.6. Concentrations of Levulinate and 4-Hydroxypentanoate in Plasma of Rats Gavaged with Calcium Levulinate ± Ethanol……………………………...... 151
Fig 6.7. Liver Concentrations of the Main C5 Acyl-CoAs Derived from Levulinate in Rats Gavaged with Calcium Levulinate ± Ethanol…………………………..152
Fig 6.8. Liver Concentrations of the C7 Cyclical CoA Esters Derived from Levulinate in Rats Gavaged with Calcium Levulinate ± Ethanol………….………………..153
Fig 6.9. Brain Concentrations of the Main C5 Acyl-CoAs Derived from Levulinate in Rats Gavaged with Calcium Levulinate ± Ethanol…………………………..154
CHAPTER 7
Fig 7.1. Trapping of Heart CoA by the Metabolites of Levulinate……………………169
Fig 7.2. Tentative Strategy for Measuring the Distribution of Levulinate Catabolism…………………………………………………………………...170
Fig 7.3. Scheme of the Formation of New Cyclical CoA Esters Derived from Levulinate...... …………………………………………………………179
x
ACKNOWLEDGEMENTS
The completion of this degree would not have been possible without the efforts,
direction, and support of my teachers, family, friends, and colleagues. First and
foremost, I would like to thank my professional mentor, Dr. Henri Brunengraber, for
giving me the educational opportunity of a lifetime. Dr. Brunengraber’s enthusiasm for
scientific research is truly infectious. His endless patience, ability to communicate and
teach, steady leadership, and his kindness are admirable qualities that make him the
quintessential advisor. In addition, he has provided an ideal environment for nurturing
my academic growth, self-confidence, and individual independence. I feel very fortunate
to have received my scientific training under the guidance of Dr. Brunengraber and will
forever be grateful for the countless hours he has spent mentoring me.
Secondly, I would like to thank Dr. Edith Lerner for her exceptional guidance as a
professional and as a teacher. Dr. Lerner and I have had numerous enlightening
discussions about coursework, careers, and potential academic opportunities, and I am
appreciative for all of her time and words of wisdom. I especially want to thank her for
seeing my potential and encouraging me to take additional steps in attaining a higher
education. I would also like to thank my dissertation committee members: Dr. Michelle
Puchowicz, Dr. Colleen Croniger, Dr. Paul Ernsberger, Dr. Janos Kerner, and Dr. Edith
Lerner. They have challenged my intellect, taught me how to critically evaluate my research, taken an active interest in my progress and growth as a scientist, and have provided encouragement and invaluable advice throughout my PhD work.
xi
Last, but certainly not least, I would like to thank all of my colleagues in the
Brunengraber Lab, in the Mouse Metabolic Phenotyping Center, and in the Department
of Chemistry. I have been extremely fortunate to work side-by-side with Dr. Guo-Fang
Zhang. His profound scientific expertise, patience, and pleasant demeanor have made my transition from clinical work to bench science enjoyable and as “painless” as possible. I am thankful for all of the time he has spent teaching me a variety of scientific techniques
and helping me “survive” through the difficulties/headaches of working with GC-MS and
LC-MS. John Koshy, Sophie Roussel-Kochheiser, Fred Allen, Edwin Vazquez, Sharon
Zhang, Shuang Deng, Dr. Rafael Ibarra, and Asha Lochan have also made significant
contributions to this research. Their assistance, technical support, enthusiasm, and
benevolence have allowed me to accomplish my goals for the research presented in this
thesis. They have become great friends and are a part of my support network, and have
also provided many laughs and memories, while exemplifying unwavering teamwork. In
addition, Dr. Gregory Tochtrop and Sushabhan Sadhukhan from the Department of
Chemistry have synthesized all of the labeled compounds used in my experiments. Dr.
Vernon Anderson from the Department of Biochemistry has spent a considerable amount
of time providing feedback on specific aspects of my project. Without their help and
guidance this project would not have been possible.
It has been a true pleasure working with all of the aforementioned individuals. I will
forever be indebted to all of their help and guidance and will benefit from all that I have
learned from them as I pursue future endeavors.
xii
LIST OF ABBREVIATIONS
AcAc acetoacetate
AcAc-CoA acetoacetyl-CoA
ACC acetyl-CoA carboxylase
ATP adenosine-5'-triphosphate
BHB β-hydroxybutyrate
BHP β-hydroxypentanoate
BHP-CoA β-hydroxypentanoyl-CoA
BKP β-ketopentanoate
CAC citric acid cycle
CoA coenzyme A
CPT carnitine palmitoyltransferase system
ER endoplasmic reticulum
ETOH ethanol
FA fatty acid
FABP fatty acid binding proteins
FAD flavin adenine dinucleotide
FFA free fatty acids
GC-MS gas chromatography-mass spectrometry
GHB 4-hydroxybutyrate
GHP 4-hydroxypentanoate
HHE 4-hydroxyhexenal
xiii
HNE 4-hydroxynonenal
LC-MS liquid chromatography-mass spectrometry
LEV levulinate
MID mass isotopomer distribution
MPE mol percent enrichment
NAD+ nicotinamide adenine dinucleotide (oxidized)
NADH nicotinamide adenine dinucleotide (reduced)
PC pyruvate carboxylase
PEP phosphoenolpyruvate
PK pyruvate kinase
xiv
Metabolism of Levulinate
And
Conversion to the Drug of Abuse 4-Hydroxypentanoate
Abstract
by
STEPHANIE R. HARRIS
The calcium salt of levulinate is used as an oral or intravenous source of calcium. We
hypothesized that (i) levulinate is converted to 4-hydroxypentanoate, a new drug of abuse, analog of 4-hydroxybutyrate, (ii) the formation of 4-hydroxypentanoate from levulinate is enhanced by ethanol oxidation, and (iii) the metabolism of levulinate results in CoA trapping in levulinate-derived CoA esters. We confirmed these hypotheses in perfused rat livers, live rats, and in liver subcellular preparations, using a combination of metabolomics and mass isotopomer analysis. Levulinate is reduced to (R)-4-hydroxypentanoate by cytosolic and mitochondrial dehydrogenases, which are NADPH- and NADH-dependent, respectively. A mitochondrial dehydrogenase or racemase system also forms (S)-4- hydroxypentanoate. We showed that levulinate is catabolized by three pathways to acetyl-
CoA, propionyl-CoA, and lactate. Also, levulinate metabolism leads to substantial accumulation of levulinyl-CoA, 4-hydroxypentanoyl-CoA and 4-phosphopentanoyl-CoA (a novel type of CoA ester). Ethanol stimulates (6 fold) the accumulation of 4- phosphopentanoyl-CoA, a potential neuromodulator, which might contribute to the brain toxicity of levulinate.
xv
In live rats given an oral gavage of calcium levulinate ± ethanol, we observed substantial
accumulation of levulinate-derived CoA esters (including 4-phosphopentanoyl-CoA) in the brain and liver. These esters remained longer in the brain than in the liver.
Lastly, we identified new fates of levulinate metabolism in the liver and brain. First, levulinyl-CoA is elongated with acetyl-CoA to 3,6-diketoheptanoyl-CoA, which is converted to two types of cyclical CoA esters: one with a cyclopentane ring and one with a pyrrole ring. The latter evokes pyrrole compounds formed in the brain by the binding of toxic gamma-diketones (such as 2,5-diketohexane) to the ε-nitrogen of lysine residues of proteins. These cyclical CoA esters were identified in rat livers perfused with levulinate, and in livers and brains from rats gavaged with calcium levulinate ± ethanol. Such compounds may contribute to the toxicity of levulinate. Overall, oral ingestion of calcium levulinate + ethanol is a public health concern since calcium-levulinate is freely available.
xvi
LITERATURE REVIEW:
CHAPTER 1
FATTY ACID OXIDATION
1.1. Overview
Fat, stored as triglycerides, is the main source of energy for the human body. Plasma
circulating fatty acids are primarily derived from (i) endogenous triglycerides stored in
adipose tissue, and (ii) exogenous triglycerides from the diet. Fatty acids, ingested as triglycerides, enter the stomach and are hydrolyzed to free fatty acids (FFA) and glycerol by lingual, gastric, and pancreatic lipases (1-4). Fatty acids derived from triglyceride hydrolysis are absorbed through intestinal mucosal cells, incorporated into new molecules of triglyceride, and packaged with proteins and phospholipids to form chylomicrons. The chylomicrons from the intestine enter the lymphatic system and are released into the blood of the left subclavian vein (1; 4). Most dietary fat (i.e. long-chain fatty acids) is absorbed into the lymph as just described. Short-chain and medium-chain fatty acids, which make up only a small portion of fat from the diet, are absorbed into the portal blood (5; 6).
Following entry of chylomicrons into the circulatory system, lipoprotein lipase hydrolyzes their triglyceride component and releases FFA and glycerol. Lipoprotein lipase is an enzyme mainly found on the endothelial surface of capillaries in heart and skeletal muscle, adipose tissue and the liver. Most of the FFA are taken up by the tissue
1
where the hydrolysis occurs and are stored or used as fuel in the tissue (7; 8). In adipose
tissue, FFA are re-esterified and stored as triglycerides. Lipolysis of triglycerides stored
in adipose tissues is a process catalyzed by hormone sensitive lipase. It provides FFA for
oxidation in tissues in need of energy (i.e. muscle), or re-esterification into triglycerides
(packaged mainly in very low density lipoproteins) in the liver (9). In the cardiac and
skeletal muscle and in the liver, FFA are oxidized via β-oxidation to yield acetyl-CoA, which enters the citric acid cycle (CAC) to generate energy for these tissues. In addition,
FFA are used by the liver to generate ketone bodies, which are oxidized in extrahepatic tissues (i.e. brain, muscle, or kidney) (4; 8). Overall, depending on metabolic demand and the specific type of tissue, fatty acids are either converted to triglycerides or membrane phospholipids, or oxidized for energy production.
1.2. Transport of Fatty Acids into Cells
Free fatty acids (derived from the diet, lipolysis of triglycerides, or de novo synthesis) are transported in the blood bound to albumin and are taken up by target tissues for storage or oxidation. In addition, fatty acids are transported from the liver and intestine in the form of triglycerides (in lipoproteins) and are released in the capillary endothelium at sites of utilization (10). Free fatty acids are hydrophobic molecules that, theoretically, should easily pass through a hydrophobic plasma membrane. It is generally assumed that fatty acid uptake occurs via passive diffusion (flip-flop) through the plasma membrane (10;
11). However, it is also believed that FFA exist solely as anions at a physiological pH
(12), and that the flip-flop of ionized fatty acids is very slow, making passive diffusion
slow (13; 14). Since rates of flip-flop are usually slower than rates of fatty acid oxidation
2
in organs with high fatty acid demand, passive diffusion alone may not account for the
total transport of FFA into cells (15). Therefore, more complex mechanisms have been
considered for fatty acid transport through plasma membranes.
The identification of a family of plasma membrane proteins that bind FFA in several
tissues suggests that fatty acid transport is also mediated by proteins (16-18). Fatty acid
translocase (FAT/CD36) is an integral transmembrane protein found on the surface of
many cell types including myocytes, adipocytes and platelets. CD36 null mice have
impaired fatty acid uptake and oxidation in heart, skeletal muscle, and adipose tissue
(19). In contrast, mice with CD36 overexpression in muscle have increased fatty acid
oxidation in response to muscle contraction (20). Fatty acid binding protein (FABPpm),
a membrane-associated protein expressed in a variety of tissues including heart, liver,
muscle, intestine, and adipose tissue, increases fatty acid uptake in cells (21).
Overexpression of FABPpm in rat muscle increases palmitate transport and oxidation
(22). Heart FABPpm null mice have impaired myocardial fatty acid uptake (23). In
addition, fatty acid transport proteins (FATP), a family of six closely related integral
membrane proteins, are expressed in all tissues with high fatty acid uptake and active
lipid metabolism (24). Insulin-stimulated fatty acid uptake is inhibited in FATP1-null
adipocytes, and greatly reduced in skeletal muscle of FATP1-null mice (25).
The above experimental evidence supports two mechanisms of transport of fatty acids
into cells: non-protein-mediated and protein-mediated transport. As illustrated in Figure
1.1, the first step in cellular uptake of fatty acids is the dissociation from the albumin-
3
fatty acid complex. This dissociation is facilitated by the interaction with albumin
receptor (ALB-R). Then, the cellular transport of fatty acids into the cellular membrane
involves either (i) passive diffusion (flip-flop) through the phospholipid bilayer (10; 11),
and/or (ii) protein mediated transport via integral transmembrane and membrane-
associated proteins (i.e. FAT/CD36, FABPpm, and FATP) (24; 26-28). Once inside the
cell, fatty acids, (free or bound to cytoplasmic fatty acid binding proteins (FABPc)),
diffuse to sites of utilization (i.e. mitochondria, peroxisomes, endoplasmic reticulum).
Free fatty acids are rapidly taken up from the blood by a variety of tissues. The rate of
uptake increases with the plasma concentration of FFA, which is mainly determined by
the rate of lipolysis. Adipose tissue lipolysis is regulated by hormone sensitive lipase
(HSL), the activity of which is primarily regulated by circulating concentrations of
insulin (inhibitory effect) and epinephrine (stimulatory effect) (29; 30). For example,
short-term fasting increases the rate of lipolysis as a result of a decrease in blood glucose
concentration (and therefore insulin), and an increase in epinephrine concentration. In
the fed state, the rate of lipolysis decreases as a result of increased insulin secretion by
pancreatic beta-cells in response to hyperglycemia. Overall, these hormones regulate
rates of lipolysis and circulating FFA concentrations, which are modulated by feeding,
exercise and states of stress (i.e. starvation, trauma, disease) (1; 4; 5; 31).
4
Figure 1.1. Mechanisms of Fatty Acid Transport into Cells.
(A) Dissociation of fatty acids from fatty acid-albumin complex (FA-ALB), facilitated by albumin receptor (ALB-R); (B) Passive diffusion (flip-flop) of fatty acids in their un- ionized form through phospholipid bilayer of the plasma membrane; (C) Transport of fatty acids in their ionized form into the plasma membrane via fatty acid translocase (CD36/FAT), fatty acid binding protein (FABPpm), and fatty acid transport protein (FATP). Modified from Schaffer, J.E., Am. J. Physiol. 282: E239-E246, 2002. (11)
5
1.3. Activation of Fatty Acids
The intracellular utilization of FFA begins with the enzymatic conversion of FFA to acyl-
CoAs (21). Specific acyl-CoA synthetases generate CoA esters from (i) straight-chain
saturated and unsaturated fatty acids, (ii) dicarboxylic fatty acids, and (iii) branched-
chain fatty acids. The activation consumes ATP and produces AMP and pyrophosphate
(32). The global reaction is:
R-COO- + CoASH + ATP → R-CO-SCoA + AMP + PPi-
The activation reaction is catalyzed by different acyl-CoA synthetases, with specificity for long, very long, medium, and short-chain FFA (33).
Short-chain fatty acids (C2-C5) and medium-chain fatty acids (C6-C12) can enter the
mitochondria directly and are activated to their corresponding CoA esters mostly in the mitochondrial matrix by acyl-CoA synthetases (i.e. ACSM1-3), which are located in the
inner mitochondrial membrane (34; 34-36). Acetate and propionate are also activated in
liver cytosol by short-chain acyl-CoA synthetase (37). Long-chain fatty acids (C12-C21) are activated to their corresponding CoA esters in the cytosol by long-chain acyl-CoA synthetases (i.e. five isoforms designated as ACS-1 and ACS3-6), which are located on the outer mitochondrial membrane but with the CoA binding domain exposed to the cytosol (33; 35). In addition, very long-chain fatty acids (>C22) are activated to acyl-
CoAs in the cytosol by a family of six very long-chain acyl-CoA synthetases (i.e. VLCS)
(38). Unlike short-chain and medium-chain fatty acids which do not require an active
6
transport mechanism to pass into the mitochondrial matrix, long-chain fatty acids require
the carnitine palmitoyltransferase system for transport as long-chain acyl-CoAs into the
mitochondrial matrix.
1.3.1. The Carnitine Palmitoyltransferease System
Before long-chain acyl-CoAs are oxidized in the mitochondria, they must first be
converted to acylcarnitines by the carnitine palmitoyltransferase (CPT) system for
transport into the mitochondrial matrix. The CPT system consists of three proteins:
carnitine palmitoyltransferase I (CPT-I), carnitine:acylcarnitine translocase (CACT), and
carnitine palmitoyltransferase II (CTP-II) (39). As illustrated in Figure 1.2, CPT-I,
located on the outer mitochondrial membrane, catalyzes the reaction of acyl-CoA with
carnitine, generating free CoA and acylcarnitine in the cytosol. Second, acylcarnitine is
translocated into the mitochondrial matrix by CACT, an integral inner mitochondrial
membrane protein. Third, CPT-II, located in the inner mitochondrial membrane,
catalyzes the reaction of acylcarnitine with the mitochondrial matrix pool of free CoA, to
regenerate acyl-CoA. The latter enters the β-oxidation pathway, while the released
carnitine returns to the extramitochondrial compartment for subsequent reutilization via
CPT-I (39; 40).
CPT-I, a key regulatory enzyme controlling the flux of long-chain fatty acids through β-
oxidation, exists in three human isoforms (41). These tissue specific isoforms are (i) a
liver isoform (L-CPT I) mainly found in the liver, (ii) a brain isoform (C-CPT I), and (iii)
a muscle isoform (M-CPT I) mostly found in skeletal and cardiac muscle (42-44). All
7
three CPT-I isoforms are associated with the outer mitochondrial membrane and bind
malonyl-CoA. Unlike CPT-I, there is only one human isoform of CPT-II which is bound
to the inner mitochondrial membrane (45; 46).
8
Figure 1.2. The Carnitine Palmitoyltransferase System.
Abbreviations: CPT-I, carnitine palmitoyltransferase-I; CACT, carnitine:acylcarnitine translocase; CPT-II, carnitine palmitoyltransferase-II; CAT, carnitine acetyltransferase. Modified from Oregon State University, Linus Pauling Institute Micronutrient Information Center. (http://lpi.oregonstate.edu/infocenter/othernuts/carnitine/transport.html)
9
1.4. Reactions of Mitochondrial β-oxidation
Beta-oxidation of activated short, medium, and long-chain fatty acids occurs within the
mitochondrial matrix. It degrades fatty acyl-CoAs by sequentially removing 2-carbon
units at a time from the β-carbon position of the fatty acyl-CoA molecule. The reactions
of β-oxidation are catalyzed by the sequential action of four enzyme families, each with
different substrate specificities for short, medium, and long-chain acyl-CoAs. The four
enzyme families are acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-
CoA dehydrogenase, and 3-ketoacyl-CoA thiolase (47).
As illustrated in Figure 1.3, the first step in β-oxidation involves the formation of a 2- enoyl-CoA ester from the corresponding saturated acyl-CoA. Dehydrogenation is catalyzed by acyl-CoA dehydrogenase, a mitochondrial enzyme directly linked to the
electron-transport chain by flavin adenine dinucleotide (FAD). The dehydrogenase exists
in four forms with specificity for chain length of acyl-CoA (47). In the second step (i.e.
hydration), 2-enoyl-CoA is converted to L-3-hydroxyacyl-CoA by enoyl-CoA hydratase,
which exists in three forms specific for acyl-CoA chain length (48). In the third step, L-
3-hydroxyacyl-CoA undergoes dehydrogenation to form 3-ketoacyl-CoA (and NADH).
This reaction is catalyzed by NAD+-dependent L-3-hydroxyacyl-CoA dehydrogenase,
which exists in two forms with overlapping chain length specificity (49). The final step
in β-oxidation is the thiolytic cleavage of 3-ketoacyl-CoA between the α and β carbons, catalyzed by 3-ketoacyl-CoA thiolase. The cleavage forms acetyl-CoA and a new acyl-
CoA with a chain length that is two carbons shorter. This process is repeated consecutively until the entire fatty acyl-CoA molecule is split into acetyl-CoA.
10
The overall reaction for one cycle of β-oxidation is:
+ Cn Acyl-CoA + FAD + H2O + NAD + CoASH → Cn-2 Acyl-CoA +
+ FADH2 + NADH + H + Acetyl-CoA
Each cycle of β-oxidation yields one mole of acetyl-CoA, one mole of NADH, and one
mole of FADH2. The acetyl group of acetyl-CoA is oxidized in the CAC, forming 2 CO2
+ 3 NADH + 1 FADH2 + 1 ATP. The reducing equivalents generated enter the
respiratory chain for production of ATP (32).
11
Figure 1.3. Mitochondrial β-oxidation Spiral and Sites of Regulation.
Abbreviations: UQred, reduced ubiquinone; UQox, oxidized ubiquinone; ETFox, oxidized ETF; ETFsq, ETF semiquinone; ETFhq, reduced ETF; Complex 1, NADH:ubiquinone oxidoreductase. Eaton, S., Biochem. J. 320: 345-357, 1996. (47)
12
1.4.1. The Trifunctional Protein Complex
The following enzymes involved in mitochondrial β-oxidation are monofunctional: (i)
short, medium, and long-chain acyl-CoA dehydrogenase, (ii) short-chain enoyl-CoA
hydratase, (iii) short-chain 3-hydroxyacyl-CoA dehydrogenase, and (iv) 3-ketoacyl-CoA
thiolase (50). Other enzymes are grouped in complexes (51; 52). The mitochondrial trifunctional protein (MTP) complex consists of three closely bound enzymes located in the inner mitochondrial membrane and is unique to long-chain fatty acid β-oxidation.
The MTP complex includes (i) long-chain 2-enoyl-CoA hydratase, (ii) long-chain 3- hydroxylacyl-CoA dehydrogenase (which are both located on the α-subunit of the MTP complex), and (iii) long-chain 3-ketoacyl-CoA thiolase (which is located on the β-subunit of the MTP complex) (53). The physical association of enzymes that catalyze sequential reactions allows for rapid metabolism of substrates (54).
1.4.2. Reversibility of 3-Ketoacyl-CoA Thiolase
3-Ketoacyl-CoA thiolase splits 3-ketoacyl-CoAs between the α and β carbons to form
acetyl-CoA and a new CoA ester that is 2 carbons shorter in chain length. The 3-
ketoacyl-CoA thiolase reaction is reversible, but with peculiar kinetics. For example,
consider the thiolytic cleavage of acetoacetyl-CoA (AcAc-CoA):
Acetoacetyl-CoA + CoA ↔ 2 Acetyl-CoA
The exchange of carbons 1 and 2 of AcAc-CoA with free acetyl-CoA is much more rapid
than the exchange of carbons 3 and 4 of AcAc-CoA with free acetyl-CoA (55). This is
13
because of the binding of the C-4 methyl of AcAc-CoA to the active site of the thiolase.
Similarly, in the thiolytic cleavage of odd-chain pentanoyl-CoA, the C-3+4+5 of
pentanoyl-CoA exchange more slowly with free propionyl-CoA than that of C-1+2 of pentanoyl-CoA with free acetyl-CoA (56).
Pentanoyl-CoA + CoA ↔ Propionyl-CoA + Acetyl-CoA
The different kinetics of exchange of the two acetyls of AcAc-CoA with free acetyl-CoA have implications for the interpretation of isotopic data in studies of fatty acid oxidation and ketogenesis (55). Although thiolase is a reversible reaction in vitro, physiologically this reversibility is unfavorable and only works if there is a pull on this reaction. For example, the kinetic properties of thiolase prevent C4 ketone body production in the liver
from the acetyl-CoA derived from glucose metabolism (i.e. AcAc-CoA from acetyl-CoA)
(55; 57). In addition, Deng et al. demonstrated that the production of C5 ketone bodies
from propionate is extremely low, showing that liver thiolase does not allow the
formation of 3-ketopentanoyl-CoA from propionyl-CoA and acetyl-CoA (56).
Thiolases not only function in the degradative reactions of fatty acid β-oxidation, but they
also function in pathways that lead to the synthesis or elongation of fatty acids (58; 59).
A cytosolic thiolase is involved in the synthesis of cholesterol from acetyl-CoA (60):
Acetyl-CoA → AcAc-CoA → HMG-CoA →→→→→ Cholesterol
14
It is also involved in the synthesis of both fatty acids and cholesterol from acetoacetate
(AcAc) in the liver (61; 62), developing brain (63), and mammary gland (64).
1.4.3. Peroxisomal β-oxidation
Another fatty acid β-oxidation system is present in the peroxisomes mainly for long-
chain and very long-chain fatty acids, branched-chain fatty acids, and long-chain
dicarboxylates. Mammalian peroxisomal β-oxidation has been extensively studied in the
liver (65), and to a lesser extent in the heart (66) and in brown adipose tissue (67).
Activation of very long-chain fatty acids and dicarboxylic acids to CoA esters is
catalyzed by two acyl-CoA synthetases: (i) a very long-chain acyl-CoA synthetase,
which is present in the peroxisomal membrane and in the endoplasmic reticulum (ER),
but not in the mitochondria, and (ii) a long-chain acyl-CoA synthetase (68). The
activation of dicarboxylic acids occurs only in the ER. In addition, the peroxisomal β-
oxidizing enzyme system consists of acyl-CoA oxidase, enoyl-CoA hydratase, 3-
hydroxyacyl-CoA dehydrogenase, and peroxisomal 3-ketoacyl-CoA thiolase (53).
There are several differences between mitochondrial and peroxisomal β-oxidation. First,
peroxisomal β-oxidation is independent of carnitine, so transport of long-chain acyl-CoA
into the peroxisome does not involve the CPT system as required for transport into the
mitochondria. Second, the initial step in the peroxisomal system is catalyzed by an acyl-
CoA oxidase, an enzyme that consumes oxygen and yields H2O2 (and heat) (69). In the
mitochondria, FAD-linked acyl-CoA dehydrogenases catalyze the initial step in β- oxidation and yield FADH2 (and ATP). Third, unlike the mitochondria, peroxisomes do
15
not have the CAC or electron transport chain. Therefore, acyl-CoAs are not completely
oxidized by the peroxisome and β-oxidation in the peroxisome does not lead to ATP production, but rather heat (70). Overall, it is believed that the main physiological function of peroxisomal β-oxidation is to shorten long-chain fatty acids (rather than to completely oxidize them) to produce better substrates for the mitochondrial β-oxidation system (71). However, Kasumov et al. recently demonstrated that octanoate undergoes two cycles of peroxisomal β-oxidation in rat liver (72). Thus, peroxisomal β-oxidation is not limited to long-chain fatty acids.
The products of peroxisomal β-oxidation, (i.e. acetyl-CoA and chain-shortened acyl-
CoAs) are transferred from the peroxisomes to the cytosol and/or mitochondria as carnitine esters by carnitine acetyltransferase. In the liver, but not in the heart (73),
peroxisomal acetyl-CoA is hydrolyzed to acetate which is released (74). Acylcarnitine is converted back to the corresponding acyl-CoA in the mitochondria for further oxidation
(71). In the heart, which lacks cytosolic carnitine acetyltransferase (75), it appears that
peroxisomal acetyl-CoA diffuses as such into the cytosol (73). When measured as a rate
of acetyl-CoA production, peroxisomal β-oxidation contributes to ~10% of the total cellular β-oxidation of palmitate in rat liver (76; 77). However, the β-oxidative capacity of the mitochondria (to generate acetyl groups) is much greater than that of the peroxisomes (i.e. mitochondrial oxidation of a C22:1 fatty acid yields 11 acetyl groups whereas the peroxisomal process yields 1-3 acetyl groups) (78-80).
16
1.4.4. Fates of Acetyl-CoA Derived from Fatty Acid Oxidation
Beta-oxidation is the main process by which fatty acids are oxidized. The acetyl-CoA
generated from β-oxidation provides a major source of energy in extrahepatic tissues,
such as heart and skeletal muscle. In the liver, some of the acetyl-CoA generated from mitochondrial β-oxidation is (i) oxidized to CO2 via citrate in the CAC, (ii) transferred to the cytosol via citrate and ATP-citrate lyase to generate cytosolic malonyl-CoA for fatty acid synthesis (61), and (iii) converted to acetate in the mitochondria (74). However, most of the acetyl-CoA generated from liver mitochondrial β-oxidation is used for ketogenesis via the hydroxymethylglutaryl-CoA (HMG-CoA) cycle in the liver (81).
C4 ketogenesis (derived from even-chain fatty acid degradation) begins with the
condensation of one molecule of AcAc-CoA with one molecule of acetyl-CoA to form
HMG-CoA. This condensation is catalyzed by HMG-CoA synthase. Second, HMG-
CoA lyase cleaves HMG-CoA to yield one molecule of AcAc, which equilibrates with β-
hydroxybutyrate (BHB) via BHB dehydrogenase (81; 82). AcAc and BHB (i.e. the C4 ketone bodies), diffuse through liver cell membranes, enter the bloodstream, and are taken up by extrahepatic tissues. Then, BHB is oxidized to AcAc by BHB dehydrogenase, and AcAc is converted back to AcAc-CoA by 3-ketoacid-CoA transferase. Last, AcAc-CoA thiolase cleaves AcAc-CoA into two molecules of acetyl-
CoA, which enter the CAC and are oxidized for energy (32; 81; 82). C5 ketone bodies
(i.e. β-hydroxypentanoate (BHP) and β-ketopentanoate (BKP)), are derived from odd-
chain fatty acid degradation and utilize the same enzymes as previously described for C4 ketogenesis in the HMG-CoA cycle in the liver (56).
17
Ketogenesis is regulated by: (i) the supply of fatty acids to the liver by adipose tissue
lipolysis (83; 84), (ii) the activity of the CPT system (i.e. β-oxidation of fatty acids) (85;
86), and (iii) the nutritional modulation of the latter two points (i.e. the energy status of
the liver) (87). For example, in the fasted state the following occurs: (i) insulin levels
decrease and glucagon/epinephrine levels increase, (ii) adipose tissue lipolysis increases
resulting in increased concentrations of circulating FFA, (iii) a greater fraction of FFA
entering the liver undergoes β-oxidation and a smaller fraction of FFA are used to form
TG, and (iv) the inhibition on CPT-I is removed (because insulin activates acetyl-CoA
carboxylase increasing malonyl-CoA which inhibits CPT-I activity) (81; 88; 89). As a
result, more FFA is oxidized to AcAc and BHB in the fasted state than in the fed state.
Overall, fasting decreases the synthesis of malonyl-CoA and redirects acetyl-CoA
towards ketogenesis. In the fed state, intracellular levels of malonyl-CoA increase and
inhibit CPT-I activity, driving metabolism in the direction of fatty acid and triglyceride
synthesis (31).
In addition, ketogenesis disposes of carbon derived from fatty acid β-oxidation that
cannot be oxidized to CO2 in the CAC because the need for ATP by the liver is satisfied.
Citric acid cycle activity is directly linked to ATP turnover. Since the flux through β-
oxidation often exceeds CAC flux, excess carbon from β-oxidation is exported from the
liver as ketone bodies. This explains why ketogenesis is decreased in hypermetabolic
states where the ATP requirement of the liver is increased (i.e. burn injuries), despite the stimulation of lipolysis by stress hormones (90; 91).
18
Both C4 and C5 ketone bodies are exported from the liver to extrahepatic tissues, and are
converted to acetyl-CoA (from C4 and C5) and to anaplerotic propionyl-CoA. During
starvation or when blood glucose is low, ketone bodies are transported from the liver for
utilization in extrahepatic tissues (i.e. muscle, heart, kidney and brain) as a source of
energy via oxidation to CO2 in the CAC (92-94). In addition, ketone bodies also
contribute to lipogenesis in the liver (61), developing brain (63), and lactating mammary
gland (64; 95). As shown in Figure 1.4, the mechanism which converts AcAc to
lipogenic acetyl-CoA is: (i) cytosolic activation of AcAc to AcAc-CoA by AcAc-CoA
synthetase, followed by entry into the HMG-CoA pathway for synthesis of sterols, and/or
(ii) thiolytic cleavage of cytosolic AcAc-CoA to acetyl-CoA by AcAc-CoA thiolase,
followed by conversion to malonyl-CoA for fatty acid synthesis (61). Endemann et al.
demonstrated that ketone bodies contribute between 19-80% of the carbon incorporated
into sterols, and up to 22% of the carbon incorporated into fatty acids, depending on the
metabolic state of the liver (61). The metabolism of ketone bodies has been extensively
reviewed by McGarry and Foster (81), Robinson and Williamson (82), and Balasse and
Fery (87).
The acetyl-CoA generated from peroxisomal β-oxidation can be hydrolyzed to acetate by
acetyl-CoA hydrolase, and exported to the cytosol as free acetate (74). In the cytosol
acetate can be activated and used for lipogenesis (in liver) and/or transported into the
mitochondria. In addition, the acetyl-CoA generated from peroxisomal β-oxidation can
be transported to the cytosol for transfer into the mitochondria for oxidation, or used in
the synthesis of cytosolic malonyl-CoA (73; 96). The latter is an intermediate of fatty
19
acid synthesis in lipogenic organs. In the heart, where there is no fatty acid synthesis,
malonyl-CoA is a dead-end metabolite which regulates CPT-I activity. The
concentration of heart malonyl-CoA is regulated by the activity of acetyl-CoA
carboxylase and malonyl-CoA dehydrogenase (73; 97; 98).
20
Figure 1.4. Lipogenesis from Ketone Bodies in the Liver.
The numbers refer to the following enzymes: 1, citrate synthase; 2, ATP-citrate lyase; 3, acetoacetyl-CoA thiolase; 4, BHB dehydrogenase; 5, acetoacetyl-CoA synthetase; 6, HMG-CoA synthase. Endemann, S., J. Biol. Chem. 257: 3434-3440, 1982. (61)
21
1.5. Fatty Acid Alpha- and Omega-oxidations
Minor pathways of fatty acid degradation include α-oxidation and ω-oxidation, which
occur in the peroxisomes and ER, respectively. The process of α-oxidation involves the
removal of one single carbon atom from the carboxyl end of a fatty acid and the
conversion of C-2 to a carboxyl. In mammals, α-oxidation is used to break down dietary
phytanic acid, which is a branched-chain fatty acid found in dairy products, ruminant
meat products, fish, and vegetable oils (99). The presence of the 3-methyl group on
phytanic acid prevents its degradation via normal β-oxidation (i.e. it cannot act as a
substrate for the third enzyme of mitochondrial β-oxidation, 3-hydroxylacyl-CoA
dehydrogenase), so degradation requires prior α-oxidation (99). The enzymes of α-
oxidation are localized in the peroxisome. The reactions of α-oxidation include: (i)
activation of phytanic acid to phytanoyl-CoA by long-chain acyl-CoA synthetase, (ii)
hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA by phytanoyl-CoA
hydroxylase, (iii) decarboxylation of 2-hydroxyphytanoyl-CoA to pristanal and formyl-
CoA by 2-hydroxyphytanoyl-CoA lyase, and (iv) dehydrogenation of pristanal to
pristanic acid by aldehyde dehydrogenase (100-102). Pristanic acid is activated to
pristanoyl-CoA and undergoes three cycles of peroxisomal β-oxidation to yield 4,8-
dimethylnonanoyl-CoA, propionyl-CoA and acetyl-CoA (103). Formyl-CoA, formed in
the α-oxidation step, is hydrolyzed to CoA and formate, which is oxidized to CO2 (104).
The process of ω-oxidation involves the conversion of the omega carbon of a fatty acid to
a primary alcohol, and then to a carboxyl group. The reactions of ω-oxidation include: (i)
hydroxylation of the omega carbon of a fatty acid to form an ω-hydroxyacid via
22
cytochrome P450 enzymes (which occurs in the smooth ER of liver and kidney cells
(105)), (ii) oxidation of the hydroxyl group to an aldehyde to form via alcohol
dehydrogenase, and (iii) oxidation of the aldehyde group to a carboxyl to form a
dicarboxylic acid via aldehyde dehydrogenase (106). The dicarboxylic acid is activated
to a CoA ester by acyl-CoA synthetase (which is present in the cytosol but facing the ER)
and then undergoes β-oxidation. Long-chain dicarboxylic acids formed via ω-oxidation,
undergo β-oxidation primarily in the peroxisome (107; 108). Omega-oxidation is also a minor catabolic pathway for medium-chain fatty acids, which generates medium-chain dicarboxylic acids (108).
1.6. Regulation of Mitochondrial β-oxidation
Once fatty acids are activated to fatty acyl-CoA, their subsequent oxidation is regulated at
three main sites: (i) transport into the cell, (ii) regulation of the CPT system, and (iii)
enzymatic regulation of β-oxidation within the mitochondria. Short-term and long-term
regulation of fatty acid oxidation can occur via substrate availability, allosteric effects,
and/or enzyme modification.
1.6.1. Regulation of CPT-I by Malonyl-CoA
The inhibitory effect of malonyl-CoA on CPT-I, plays an important role in regulating the
flux of fatty acids through mitochondrial β-oxidation. The regulation of CPT-I activity is
demonstrated in Figure 1.5. In the fed state, dietary fatty acids supply acetyl-CoA in the
mitochondria for oxidation in the CAC. However, some of these acetyl units are
transported to the cytosol via citrate/ATP citrate lyase or acetylcarnitine, and are used to
23
synthesize malonyl-CoA via acetyl-CoA carboxylase (ACC). ACC is the rate limiting
enzyme in fatty acid synthesis in the liver (109; 110). The cytosolic concentration of
malonyl-CoA is primarily regulated by the activities of ACC (synthesis) and malonyl-
CoA decarboxylase (MCD) (degradation). ACC gene expression is enhanced by insulin
(via dephosphorylation) and by a high carbohydrate/low fat diet (via decreased cAMP
levels) (111). Citrate (an allosteric activator) also activates ACC and contributes to
cytosolic acetyl-CoA, the substrate of ACC (112; 113). The activation of ACC results in
increased concentrations of malonyl-CoA. Malonyl-CoA binds to CPT-I and decreases
its activity, thereby preventing entry of long-chain fatty acids into the mitochondria for β-
oxidation (114). On the other hand, ACC is inhibited by an increase in long-chain acyl-
CoA concentrations (via allosteric inhibition) and by AMP-activated protein kinase
(AMPK) (via phosphorylation) (113; 115). AMPK can be activated under conditions
such as exercise (113; 115). The inactivation of ACC results in decreased concentrations
of malonyl-CoA, which removes the inhibition on CPT-I. Thus, a decrease in malonyl-
CoA facilitates the transport of long-chain fatty acids into the mitochondria (i.e.
movement of acyl-moieties from long-chain acyl-CoAs into the mitochondria via CPT-I)
for subsequent β-oxidation.
CPT-I has an inhibitor binding site (N-terminal domain) and catalytic binding site (C-
terminal domain) on the same side of the outer mitochondrial membrane exposed to the
cytosol (116). Thus the presence of binding sites for malonyl-CoA on CPT-I allows for
control of CPT-I activity. CPT-I exists in two isoforms, both with different kinetic and
regulatory properties (40). The muscle isoform (M-CPT I) is highly expressed in the
24
heart and skeletal muscle. The liver isoform (L-CPT I) is highly expressed in the liver
and kidney (42). L-CPT I has a much lower affinity (i.e. sensitivity) for malonyl-CoA than M-CPT 1 (i.e. IC50 = 2.7 – 50 μM versus IC50 = 0.01 – 0.1 μM, respectively) (117).
The hepatic regulation of mitochondrial β-oxidation at the level of CPT-I was first demonstrated by McGarry et al. (114). The inhibition of CPT-I by malonyl-CoA occurs in the fed state (81; 114). Aside from the cytosolic concentration of malonyl-CoA, several other factors affect the sensitivity of L-CPT I to malonyl-CoA inhibition in the liver. These factors include: (i) changes in membrane fluidity, which can increase L-
CPT I sensitivity to the inhibition of malonyl-CoA (118), (ii) phosphorylation of L-CPT
I, which can decrease its sensitivity to malonyl-CoA (119), and/or (iii) changes in L-CPT
I activity induced by changes in the expression of L-CPT I, as long-term insulin exposure can inhibit L-CPT I mRNA transcription (120). In addition, L-CPT I becomes more active, but less sensitive to inhibition by malonyl-CoA, in conditions that increase fatty acid oxidation, such as starvation, diabetes, and high fat feeding (121; 122).
The regulation of β-oxidation by modulation of CPT-I activity via malonyl-CoA in extrahepatic tissues is still debated. M-CPT I in heart and skeletal muscle is highly sensitive to malonyl-CoA (i.e. IC50 = 0.01 – 0.1 μM) (117). However, the concentration
of malonyl-CoA in these tissues (1 – 10 μM) is way higher than the IC50 (half maximal
inhibitory concentration) of malonyl-CoA on M-CPT I (110; 117). Thus, one could
assume that M-CPT I activity would be inhibited by physiological concentrations of
malonyl-CoA in these tissues (47). Despite the latter, β-oxidation occurs in the heart and
25
skeletal muscle, as both tissues obtain most of their energy requirements from fatty acids
(123). The fact that β-oxidation still proceeds in these tissues with M-CPT I might be
explained by the following. First, much of the physiological malonyl-CoA measured in
the heart is cytosolic; only a small fraction of the physiological malonyl-CoA is possibly
mitochondrial due to the very low rate of acetyl-CoA carboxylation by mitochondrial
propionyl-CoA carboxylase (124). However, there has not been direct evidence
supporting this hypothesis. Second, some of the cytosolic malonyl-CoA in both of these
tissues is bound to cytosolic binding proteins or to mitochondrial low affinity sites,
preventing physiological concentrations of malonyl-CoA from inhibiting M-CPT I (125).
Third, a malonyl-CoA insensitive form of M-CPT I exists and is located on the outer
mitochondrial membrane (126). Fourth, M-CPT I is not rate limiting for β-oxidation in
heart or skeletal muscle. Regardless, strong correlations between malonyl-CoA
concentration and rates of fatty acid oxidation have been demonstrated in the perfused
heart (97). In addition, β-oxidation is partly suppressed in the fed state (i.e. by
carbohydrates) in the perfused heart (123). The latter suggests that the slow transfer of
citrate across the mitochondrial membrane could provide enough malonyl-CoA for
inhibition of M-CPT I during the oxidation of pyruvate in the heart and muscle (123).
Last, unlike CPT-I, neither CACT nor CPT-II play a major role in the regulation of
mitochondrial β-oxidation (117; 121).
26
Figure 1.5. Regulation of CPT-I Activity.
The pathway shown in red shows sites for the inhibition of CPT-I activity in the liver. Abbreviations: CPT, carnitine palmitoyltransferase; CAT, carnitine acetyltransferase. Eaton, S., Biochem. J. 320: 345-357, 1996. (47)
27
1.6.2. Redox Regulation of Mitochondrial β-oxidation
As demonstrated in Figure 1.3, β-oxidation is linked to the respiratory chain at two steps:
(i) acyl-CoA dehydrogenase which is linked to ubiquinone via electron transfer
flavoprotein (ETF), and (ii) 3-hydroxyacyl-CoA dehydrogenase which is linked to
complex 1 via the [NADH]/[NAD+] ratio (47). The inhibition of either step can lead to
inhibition of β-oxidation. For example, ETF semiquinone (the partially reduced form of
ETF), can accumulate when the ubiquinone pool is low (i.e. in some chronic disease
conditions such as cancer or heart disease, or with statin use). ETF semiquinone is a
potent inhibitor of acyl-CoA dehydrogenase; this can result in inhibition of fatty acid
oxidation (127).
The oxidation of ethanol to acetaldehyde and acetate increases the mitochondrial
[NADH]/[NAD+] ratio in the liver. This increase inhibits the 3-hydroxyacyl-CoA
dehydrogenase reaction, thereby decreasing fatty acid oxidation (128; 129). An increase
in the [NADH]/[NAD+] ratio resulting from ethanol oxidation also inhibits reactions of
the CAC, such as the reactions catalyzed by isocitrate dehydrogenase and α-ketoglutarate
dehydrogenase (130). In addition, Guzman et al., demonstrated that ethanol inhibits
CPT-I activity and fatty acid oxidation in rat hepatocytes incubated with ethanol (131).
Thus redox control is an important regulator of mitochondrial β-oxidation.
1.6.3. Feedback Regulation of Mitochondrial β-oxidation
There is also feedback control (i.e. inhibition) on the β-oxidation system when the
products of a particular enzyme (i.e. reaction) accumulate. For example, 3-hydroxyacyl-
28
CoA dehydrogenases are subject to product inhibition by 3-keto-acyl-CoA esters (132).
Likewise, enoyl-CoA hydratase is similarly inhibited by its 3-hydroxyacyl-CoA products
(132; 133). Thus, accumulation of 3-ketoacyl-CoAs in the mitochondria inhibits both of these reactions of β-oxidation via a feedback mechanism.
The 3-ketoacyl-CoA thiolases are not inhibited by their acyl-CoA products. However, the inhibition of 3-ketoacyl-CoA thiolase (via acetyl-CoA) provides feedback inhibition on β-oxidation. Olowe et al. demonstrated that 3-ketoacyl-CoA thiolase is inhibited by high concentration ratios of [acetyl-CoA]/[free CoA] in heart muscle (134). Since the
[acetyl-CoA]/[free CoA] ratio depends on the metabolic demand of the tissue (i.e. the rate of the CAC), this suggested that fatty acid oxidation in heart muscle is partly controlled via the regulation of 3-ketoacyl-CoA thiolase by the [acetyl-CoA]/[free CoA] ratio (134).
If the inhibition of 3-ketoacyl-CoA thiolase by acetyl-CoA prevents disposal of acetyl-
CoA to the CAC, to ketogenesis, or to acetylcarnitine, then feedback inhibition on β-
oxidation would be a consequence (47). Figure 1.3 summarizes the key regulatory points
in mitochondrial β-oxidation.
1.6.4. The Role of Carnitine and Free CoA in Fatty Acid β-oxidation
Carnitine and CoA availability also regulate fatty acid oxidation. Three reactions in fatty
acid oxidation require CoA: (i) fatty acid activation, (ii) conversion of acylcarnitines to
acyl-CoAs via CPT-II, and (iii) thiolytic cleavage of Cn 3-ketoacyl-CoAs to acetyl-CoA
and a CoA ester that is two carbons shorter in chain length via 3-ketoacyl-CoA thiolase
(47). Since the mitochondrial pool of free CoA is small, depletion of free CoA can
29
inhibit both CPT-II and 3-ketoacyl-CoA thiolase, thus inhibiting β-oxidation. Garland et
al. demonstrated that 90 – 95% of the intramitochondrial CoA pool is acylated during
maximal β-oxidation flux, so only a small amount of free CoA sustains β-oxidation (135).
As previously discussed, the lack of mitochondrial CoA inhibits the 3-ketoacyl-CoA
thiolase reaction (134). Inhibition of the 3-ketoacyl-CoA thiolase reaction results in the
accumulation of 3-ketoacyl-CoAs, which provide feedback inhibition on the other
reactions of β-oxidation. The latter results in accumulation of more acyl-CoAs, further
trapping intramitochondrial free CoA. In addition, trapping of CoA can inhibit other
intramitochondrial reactions that are CoA-dependent (i.e. pyruvate dehydrogenase,
branched-chain ketoacid dehydrogenase, and α-ketoglutarate dehydrogenase) and effect
many reactions of mitochondrial oxidative metabolism (136).
Since carnitine is required for the transport of long-chain fatty acids into the
mitochondria, the supply of carnitine can also modulate long-chain fatty acid oxidation.
Carnitine is obtained through the diet and is synthesized from lysine and methionine in
various tissues (137). Although the whole-body turnover of carnitine is slow, the
turnover time of carnitine varies in different tissues (i.e. 1-2 hours in the liver versus 1-2
days in skeletal muscle) (138). Overall, when the carnitine supply decreases, long-chain
fatty acid oxidation is decreased, resulting in the accumulation of long-chain acyl-CoAs.
The supply of carnitine to β-oxidation is decreased in inborn defects of the plasma
membrane carnitine transporter, causing primary carnitine deficiency (139; 140). Also,
in some inborn defects of long-chain fatty acid oxidation (e.g. long-chain acyl-CoA
dehydrogenase deficiency), the urinary excretion of long-chain acyl-carnitine decreases
30
the availability of free carnitine (31; 141; 142). Last, during hemodialysis in renal
patients, much carnitine is lost in the dialysate. This can lead to defective β-oxidation of
long-chain fatty acids (143). These decreases in carnitine must be compensated by oral
administration of carnitine (144; 145).
31
CHAPTER 2
4-HYDROXYACID METABOLISM
2.1. Overview of What is Known About 4-Hydroxyacids
Gamma-hydroxyacids (4-hydroxyacids) are products of lipid peroxidation (C9, C6) or drugs of abuse (C4, C5). They are involved in different areas of mammalian metabolism.
For example, unsaturated 4-hydroxyacids are derived from 4-hydroxynonenal (C9) and 4- hydroxyhexenal (C6), which are products of lipid peroxidation of polyunsaturated fatty
acids (146). 4-Hydroxybutyrate (C4), a physiological neurotransmitter derived from 4- aminobutyrate (GABA), is used for the treatment of narcolepsy (147), but is also a drug of abuse (148). Last, 4-hydroxypentanaote (C5) is not formed in normal human
metabolism, but is also used as a drug of abuse (149). Although the metabolism of 4-
hydroxynonenal has been extensively studied in regards to binding to proteins and
glutathione, and conversion to 4-hydroxynonanoate (146; 150), the catabolism of its
carbon skeleton was only recently unraveled (151). In addition, the catabolism of 4-
hydroxypentanoate in mammalian cells was unknown prior to the work completed in this
thesis (152). Thus the metabolism of 4-hydroxyacids is a relatively new field.
Some microorganisms use 4-hydroxyacids (possibly derived from levulinate) to make
biopolymers with or without 3-hydroxyacids (153; 154). This field is not reviewed here.
32
2.1.1. 4-Hydroxyacids: Products of Lipid Peroxidation (C9, C6)
Lipid peroxidation refers to the oxidative degradation of lipids, which occurs under
conditions of oxidative stress. It is a process in which free radicals (i.e. reactive oxygen
species such as ˙OH and HO2) initiate oxidation by stealing electrons from lipids (155).
Lipid peroxidation most often affects polyunsaturated fatty acids (in cholesterol esters,
phospholipids, and triglycerides). The methylene (CH2) groups between the double
bonds have reactive hydrogens (156; 157). Lipid peroxidation initially yields lipid
hydroperoxides. These hydroperoxides then break down mostly into short-chain
aldehydes (such as 2-alkenals, 4-hydroxy-2-alkenals and ketoaldehydes) that have a wide
variety of damaging actions (157; 158). 4-Hydroxy-2-alkenals represent the most
prominent lipid peroxidation-specific aldehydes (150). For example, 4-hydroxynonenal
(HNE) and 4-hydroxyhexenal (HHE), are products of lipid peroxidation of ω6 and ω3 polyunsaturated fatty acids: HNE is derived from linoleic and arachidonic acid; HHE is derived from linolenic acid (146). Although other 4-hydroxyalkenals varying in chain length from 8 to 11 carbons are formed from the peroxidation of various unsaturated lipids (150; 159), the 9-carbon HNE is the most prominent. Because HNE has three functional groups, (i) aldehyde (CHO), (ii) alkene (C-2 = C-3 double bond), and (iii) a secondary alcohol (OH at chiral C-4) it is a highly reactive molecule (156).
HNE diffuses from the intracellular site of formation to the extracellular fluid, from where it can enter other cells and react with a variety of compounds causing oxidative injury (160). The aldehyde group of HNE is (i) oxidized to a carboxyl by aldehyde dehydrogenase forming 4-hydroxynonenoate (161), which can undergo β-oxidation
33
(151), (ii) reduced to an alcohol by alcohol dehydrogenase forming 1,4-dihydroxynonene
(an unreactive metabolite) (162), or (ii) forms Schiff bases with amino groups of peptides
and proteins, which cause alterations in cell signaling, as well as protein and DNA damage, enzyme modification and cytotoxicity (162-164). In addition, glutathione-S- transferase catalyzes the conjugation of HNE to glutathione, via the Michael addition (at
C-3) with the thiol group of glutathione, thereby preventing further nucleophilic addition
to this toxic compound (165; 166). Thus, conjugation with glutathione is a pathway for
the detoxification of HNE. However, adduct formation between HNE and proteins is
associated with numerous cytotoxic effects including inhibition of enzyme activity,
mitochondrial dysfunction, and impaired energy metabolism (summarized in Table 2 of
(167)). For example, covalent modification of proteins via the formation of HNE adducts with histidine, lysine, and cysteine (163; 168), are linked to inactivation of a variety of
susceptible enzymes such as glucose-6-phosphate dehydrogenase (169), glyceraldehyde-
3-phosphate dehydrogenase (170), glutathione reductase (171), and others (157). HNE
also interacts with (i.e. binds) and impairs the function of several cellular target proteins
such as neuronal glucose transporter GLUT 3 (172), the glutamate transporter GLT-1
(173), and others (157).
Low levels of HNE are normally found in mammalian tissues (0 - 0.7 μM) (167), but
HNE levels are increased (10 μM to 5 mM) in pathological conditions that increase
oxidative stress (and thus increase lipid peroxidation) (157). These conditions include
various cancers, vitamin E deficiency, inflammation, atherosclerosis and
neurodegenerative diseases (157). HNE is also a mediator of oxidative stress via
34
activation of apoptosis, cell death, and down regulation of the inflammatory response
(157). Although the exact role of HNE as a mediator of oxidative stress and its link to
specific human diseases are still somewhat debated, HNE-modified proteins have been
found in: (i) atherosclerosis (174), (ii) neurodegenerative diseases such as Alzheimer’s
and Parkinson’s disease (175; 176), and (iii) in tissues from patients with cancer (177),
cirrhosis (178), and diabetic nephropathy (179). The metabolism of HNE and the
mechanisms of HNE formation have been extensively reviewed by Esterbauer et al.
(150), Alary et al. (162), Shaur (180), Siems and Grune (160), and Schneider et al. (146).
2.1.2. 4-Hydroxyacids: Drugs of Abuse (C4, C5)
4-Hydroxypentanoate (GHP) and 4-hydroxybutyrate (GHB) are C5 and C4 4-
hydroxyacids respectively; they are also drugs of abuse. GHB (C4) is a physiological
neurotransmitter derived from 4-aminobutyrate (GABA) (148). Its known metabolism
proceeds via oxidation to succinic semialdehyde (SSA) and then to succinate, which is an
intermediate of the CAC (148; 181-183). Although the metabolism of GHB is also
thought to proceed via β-oxidation to yield keto acids and hydroxyacids, this has not been
experimentally confirmed (184; 185). Humans with an inborn error of SSA
dehydrogenase have high concentrations of GHB in plasma and body fluids and suffer
from mental retardation and seizures (186). SSA dehydrogenase deficient mice fail to
thrive and suffer from severe epileptic seizures, unless maintained on a ketogenic diet
(187; 188).
35
In the United States, the only legal use of GHB is for the treatment of narcolepsy in FDA-
approved clinical studies (147; 189; 190). However, GHB is also used as a recreational
drug of abuse for its alleged properties of being a strength enhancer, euphoriant, sedative
and aphrodisiac (147; 148). GHB impairs the capacity to exercise judgment for unknown
reasons, which is why it is often referred to and used as a “date rape” drug (148; 191;
192). Clinically, the effects of GHB are dose dependent. Low doses of GHB at 10
mg/kg induce euphoria and passivity in humans, whereas doses over 50 mg/kg can result
in coma, cardio-respiratory depression, and death (193; 194). Doses of 10-50 mg/kg of
GHB in rats cause memory deficits and behavioral changes, while doses over 300 mg/kg
can result in coma (195; 196). The median lethal dose of GHB in the rat is 1.7 g/kg
(148). The toxicity of GHB is increased by compounds that decrease its disposal, such as
alcoholic beverages, barbiturates and salicylates (i.e. aspirin). The latter decrease the
metabolism of GHB by inhibiting its conversion to SSA, mainly via inhibition of GHB
dehydrogenase (181).
Brain receptors for GHB have been extensively studied, but are beyond the scope of this
thesis. Briefly, the precise mechanisms by which GHB produces its therapeutic and abuse-related effects remain unclear (197). However, it is likely that multiple mechanisms contribute to the effects of GHB because it binds to multiple receptors in different regions in the brain (i.e. cerebral cortex and hippocampus) and is metabolized to and from GABA (197). Overall, it has been postulated that (i) at low doses, GHB binds to GHB receptors (which are excitatory), resulting in stimulatory properties, and (ii) at high doses (i.e. drug abuse), GHB activates GABAB receptors in the brain (which are
36
inhibitory), resulting in sedative properties (195; 198-202). GABAB receptor-mediated effects of GHB are secondary to (i) conversion of GHB into GABA, or (ii) GHB induced stimulation of GABA release (203; 204). Both actions would result in an increase in
GABA, which in turn binds to GABAB receptors causing hypnotic/sedative properties
(195). Although GHB binds to GABAB receptors, it is unclear whether the GABAB-like activity of GHB results from it binding directly to GABAB receptors or indirectly through
conversion to GABA (204; 205). In addition, although GHB does not appear to directly
interact with GABAA receptors, behavioral studies suggest that GHB still might exert
some effect through the GABAA receptor complex (likely via conversion to GABA)
(198; 206; 207).
Recently, the GHB receptor was cloned from both human and rat brains and
characterized as a G-protein coupled receptor that can modulate the electrical potential of
neurons through cyclic AMP and calcium signaling (208; 209). Only nanomolar and
micromolar concentrations of GHB are required to stimulate cAMP and calcium
signaling via the GHB receptor, respectively (210). However, higher concentrations of
GHB (in the millimolar therapeutic or drug abuse range) desensitize GHB receptor-
mediated calcium signaling (208; 210). In addition, in GABAB deficient mice, the
pharmacological effects of GHB disappear, while the binding of GHB to GHB receptors
is unaffected (211). Although the specific role of GHB receptors in the behavioral effects
of GHB remains elusive, the latter two points suggest that the GHB receptor may not be
involved in the response to high doses of GHB.
37
Since GHB is now a controlled substance in the United States, addicts are turning to GHP
as a drug of abuse for the same alleged properties as GHB (149). GHP is a C5 (4-methyl
substituted) analog of the four carbon GHB, which can be prepared by the alkaline
hydrolysis of the solvent 4-valerolactone (GHV) (149). See Figure 2.1 for their
structures. Unlike other analogs of GHB (i.e. gamma-butyrolactone and 1,4-butanediol)
that are metabolized to GHB via peripheral lactonases (212) and liver alcohol
dehydrogenase (213), GHP is not metabolized to GHB (197). However, GHP binds to
GHB receptors in the brain (but with a 2-fold lower affinity than GHB) and possibly to
GABAB receptors (197; 214). In addition, there is evidence that GHP shares similar
behavioral effects with GHB (i.e. sedation, muscle relations, and euphoria). Specifically,
(i) GHP is used as a recreational substitute for GHB (215), (ii) there are many anecdotal
comparisons found on the internet from addicts who use GHB versus GHP (197; 216-
218), and (iii) GHP is listed as the legal active ingredient in GHB-related dietary
supplements such as Tranquili-G (produced by Smartbodyz Nutrition and Avant Labs,
but recently discontinued) (216).
In C57/B16 mice, GHP dose-dependently decreased locomotor activity and produced
ataxia (i.e. lack of coordination of muscle movement), but was 10-fold less potent than
GHB (197). However, lethality was observed 24 to 48 hours after administration in 50%
of mice tested with a large dose of GHP (5600 mg/kg), whereas lethality was never observed following similar doses of GHB (197). Therefore, although the drug effects of
GHP appear to be less potent than those of GHB, there is some evidence that GHP is
38
more toxic than GHB. Thus, people who take GHP as a substitute of GHB, with the
intention of producing GHB-like drug effects, may require near toxic doses of GHP.
There is only one report related to GHP metabolism in mammalian cells. It was
identified in the urine of children treated for hypocalcemia with intravenous calcium
levulinate (4-ketopentanoate) (219). This showed that levulinate can be reduced to GHP in vivo. This topic is expanded on in Chapters 4 and 5. Although the metabolism of
GHP is unknown, Zhang et al. recently demonstrated that the catabolism of 4-hydroxy-n-
acids with five or more carbons involves degradation via two parallel pathways to yield
acetyl-CoA, propionyl-CoA and formate (151) (see next section).
39
Figure 2.1. Structures of 4-Hydroxybutyrate and 4-Hydroxypentaoate.
Abbreviations: GHB, 4-hydroxybutyrate; GHP, 4-hydroxypentanoate.
40
2.2. Recent Development in 4-Hydroxyacid Metabolism
2.2.1. Nomenclature of Mass Isotopomers
The following sections use the concept of mass isotopomers, which are chemically
identical compounds with the same number of labeled atoms regardless of their position in the molecule. The unlabeled molecules are designated at M0 or M. The labeled mass isotopomers are designated as M1, M2, M3…Mn, where n is the number of heavy atoms in the molecule. The designation of m1, m2, M3…mn refers to the isotopic enrichment of the corresponding isotopomer, expressed as mol percent.
2.2.2. Pathways of 4-Hydroxyacid Catabolism
Prior to the work completed by Zhang et al. in 2009 (151), there was virtually no information in the literature on the catabolism of 4-hydroxyacids with five or more carbons. This includes the saturated and unsaturated 4-hydroxyacids derived from HNE
and HHE. Since classical β-oxidation involves 3-ketoacyl-CoAs and 3-hydroxyacyl-
CoAs, it was previously unclear how the carbon skeleton of a 4-hydroxyacid could be
degraded. However, through a combination of mass isotopomer analysis (220) and
metabolomics (221; 222), Zhang et al. demonstrated that the catabolism of 4-
hydroxyacids with five or more carbons occurs via two mechanisms (151). The
mechanisms were confirmed by using 4-hydroxyacids labeled with 13C or 2H. As shown in Figure 2.2, 4-hydroxy-n-acids with five or more carbons are catabolized via two parallel pathways. The first pathway (i.e. pathway A), involves the isomerization of 4- hydroxyacyl-CoAs to 3-ketoacyl-CoAs, which are physiological β-oxidation intermediates, via the newly identified 4-phosphoacyl-CoAs (4-P-acyl-CoAs) (151). This
41
is followed by regular β-oxidation cycles producing (i) acetyl-CoA and propionyl-CoA
(from odd-chain 4-hydroxyacids) or (ii) acetyl-CoA (from even-chain 4-hydroxyacids).
The second pathway (i.e. pathway B starting at 4-hydroxyacyl-CoA) proceeds via a sequence of one cycle of β-oxidation, one α-oxidation step (223; 224), and then β- oxidation cycles. Pathway B leads to formate via the α-oxidation of 2-hydroxyacyl-CoA and formyl-CoA hydrolysis (104; 224), and either (i) acetyl-CoA and propionyl-CoA
(from even-chain 4-hydroxyacids) or (ii) acetyl-CoA (from odd-chain 4-hydroxyacids).
Overall, the degradation of 4-hydroxyacids with five or more carbons via these two
pathways yields acetyl-CoA, propionyl-CoA and formate (151). This pioneering work
demonstrated the complete oxidation of the carbon skeleton of HNE to acetyl-CoA,
propionyl-CoA and formate.
13 By perfusing livers with different labeling patterns of 4-hydroxy-[ Cn]nonanoate, Zhang
et al. calculated the contribution of pathway A versus pathway B to the catabolism of 4-
hydroxynonanoate, and thus determined the predominant pathway in 4-hydroxyacid
catabolism (151). Since both pathways of catabolism generate acetyl-CoA (i.e. pathways
A and B yield 3 and 4 acetyl-CoA respectively, with one acetyl-CoA being labeled in
13 each pathway), the use of 4-hydroxy-[3,4- C2]nonanoate produces one M1 or one M2
labeled acetyl-CoA. By measuring the labeling of acetyl-CoA and the labeling of the
three proxies of acetyl-CoA (i.e. (i) acetyl moiety of citrate (73; 225), (ii) C-1+2 acetyl of
BHB (72), and (iii) free acetate (72; 226)), the contributions of pathway A and B to the production of acetyl-CoA from the labeled 4-hydroxynonanoate can be calculated. For example, when Zhang et al. perfused livers with 4-hydroxy-[3-13C]nonanoate, which
42
forms M1 acetyl-CoA via pathway A, the M1 labeling of acetyl-CoA and its three
proxies were similar (151). However, when perfusing with 4-hydroxy-[3,4-
13 C2]nonanoate, which forms M2 acetyl-CoA via pathway A and M1 acetyl-CoA via
pathway B, the M2 labeling of acetyl-CoA and its three proxies were 5-6 times higher
than the M1 labeling (151). This demonstrated that pathway A is the predominant
pathway for the catabolism of 4-hydroxynonanoate. In addition, considering that the
peroxisome is the site of fatty acid α-oxidation (223), some of the reactions of 4-
hydroxyacid metabolism probably occur in the peroxisome. For example, [13C]formate is
formed from 4-hydroxynonanoate labeled on carbon 3 when catabolized via pathway B.
Given the high labeling of acetyl-CoA and its proxies in livers perfused with 4-hydroxy-
[3-13C]nonanoate (again which forms M1 acetyl-CoA via pathway A), Zhang et al.
concluded that pathway B is likely peroxisomal and that pathway A is mitochondrial
(151).
Zhang et al. also provided evidence for the interconversion of 4-hydroxyacids with their
4-ketoacid counterparts (i.e. 4-hydroxynonanoate and 4-ketononanoate), as well as for the interconversion of their CoA esters (i.e. 4-hydroxynonanoyl-CoA and 4-ketononanoyl-
CoA) in livers perfused with various 4-hydroxyacids (151). In addition, in an orientation liver perfusion with GHP, the 4-keto analog levulinate (4-ketopentanoate) was identified
(151). Note that GHP was found in the urine of children treated with calcium levulinate as previously mentioned (219). All of the latter findings suggest that there must be mechanisms (i.e. enzymes) in the liver that interconvert 4-hydroxyacids and 4- hydroxyacyl-CoAs with their 4-keto counterparts. Known cases of conversion of
43
hydroxyacid to ketoacid occur via dehydrogenases specific to 2- and 3-hydroxyacids.
For example, lactate dehydrogenase catalyzes the conversion of lactate to pyruvate. BHB dehydrogenase catalyzes the conversion of BHB to AcAc (227). In addition, S-3- hydroxyacyl-CoA dehydrogenase catalyzes the conversion of 3-hydroxyacyl-CoAs to 3- ketoacyl-CoAs in the β-oxidation pathway (49). However, there is no literature evidence of a dehydrogenase or enzyme system that catalyzes the conversion of (i) a 4- hydroxyacid to its 4-ketoacid counterpart, or (ii) a 4-hydroxyacyl-CoA to its 4-ketoacyl-
CoA counterpart.
Last, recent data by Zhang et al. revealed that GHB (i.e. the C4 4-hydroxyacid) is
metabolized by multiple processes, in addition to its previously reported metabolism in
the CAC via oxidation to succinate by SSA dehydrogenase (demonstrated by Kaufman et
al. (181-183)). Zhang et al. have demonstrated that GHB undergoes (i) β-oxdiation to
glycolyl-CoA + acetyl-CoA, (ii) β-oxidation via 4-phosphobutyryl-CoA, and (iii) two
parallel α-oxdiation processes starting from both ends of the molecule and forming 3-
hydroxypropionate and formate (manuscript submitted). However, the degradation of 4-
hydroxyacids via pathways A and B (as shown in Figure 2.2) is specific to 4-
hydroxyacids with five or more carbons (151).
44
Figure 2.2. Proposed Scheme for the Catabolism of 4-Hydroxyacids.
The scheme uses 4-hydroxynonanoate (C9) as an example. Carbons 3 and 4 of the substrate are colored in red and green, respectively, to facilitate the tracing of their fates through pathways A and B. Note that the doubly labeled substrate forms acetyl-CoA, part of which is doubly labeled (M2) via pathway A and singly labeled (M1) via pathway B. Formate, derived from carbon 3 of the substrate, is formed via pathway B. The compounds shown are 4-hydroxynonanoate (compound 1), 4-ketononanoate (compound 2), 4-hydroxynonanoyl-CoA (compound 3), 4-ketononanoyl-CoA (compound 4), 4- phosphononanoyl-CoA (compound 5), 4-phospho-2-ene-nonanoyl-CoA (compound 6), 3- hydroxy-4-phosphononanoyl-CoA (compound 7), enol form of 3-ketononanoyl-CoA (compound 8), 3-ketononanoyl-CoA (compound 9), 3-hydroxynonanoyl-CoA (compound 10), 2-hydroxyheptanoyl-CoA (compound 11), hexanoyl-CoA (compound 12), formate (compound 13), unlabeled acetyl-CoA (compound 14), singly labeled (M1) acetyl-CoA (compound 15), doubly labeled (M2) acetyl-CoA (compound 16), and propionyl-CoA (compound 17). Note that for even-chain 4-hydroxyacids with at least six carbons, pathway A leads to acetyl-CoA, whereas pathway B leads to acetyl-CoA, propionyl-CoA, and formate. Zhang, G.F., J. Biol. Chem. 284: 33521-33534, 2009. (151)
45
2.2.3. Formation of 4-Phosphoacyl-CoAs from 4-Hydroxyacids
In rat livers perfused with C4 to C11 4-hydroxyacids, LC-MS/MS analysis identified the
expected CoA esters of the substrates via the typical transition of the mass of parent
molecular ion to the product ions at m/z = 428 and 261 (151). The latter correspond to
the nucleoside and pantetheine fragments of CoA, and are the signature ions of CoA
(228). In addition to all expected CoA esters derived from C4 to C11 4-hydroxyacids,
Zhang et al. also identified unexpected CoA esters that: (i) migrate faster than the
expected esters on the C18 column, suggesting the presence of an additional polar group
when compared with usual CoA esters, (ii) contained all of the carbon and hydrogen atoms of the corresponding 4-hydroxyacids, when using 13C and 2H labeled substrates,
and (iii) were 80 Da greater than the corresponding 4-hydroxyacyl-CoAs (151). In addition, when comparing the fragmentation pattern of 4-hydroxyacyl-CoAs to the new unexpected CoA esters, Zhang et al. found that the two singly charged ion transitions were accompanied by the loss of m/z = 98, which is equivalent to phosphoric acid (151).
See Figure 2.3 for the fragmentation patterns of 4-phosphopentanoyl-CoA (as an example). All of the latter led to the hypothesis that the unknown CoA esters were 4- phosphoacyl-CoAs (4-P-acyl-CoAs). This was confirmed by (i) isolating 4-P-acyl-CoAs from rat livers perfused with 4-hydroxyacids using semi-preparative HPLC, and then (ii) identifying 4-P-acyl-CoAs by exact mass spectrometry and 31P-NMR (151).
As shown in Figure 2.4, all of the saturated C4 to C11 4-hydroxyacids formed 4-P-acyl-
CoAs, which accumulated in different concentrations in the liver (151). Note that the 4-
phosphopentanoyl-CoA (derived from the C5 4-hydroxyacid, GHP) accumulated to the
46
highest level. Unlike the C9 and C6 4-hydroxyacids (which derive from lipid peroxidation
products HNE and HHE) or C4 (which derives from GABA), the C5 4-hydroxyacid (i.e.
GHP) and thus 4-phosphopentanoyl-CoA are not physiological compounds. It is not
clear why, at equal C4 to C11 4-hydroxyacid concentration, 4-phosphopentanoyl-CoA is
the 4-P-acyl-CoA that accumulates the most in perfused rat livers. However, this may
result from (i) high activity of the kinase for 4-hydroxypentanoyl-CoA or (ii) some
constraint on the metabolism of 4-phosphopentanoyl-CoA via the reactions in pathway A
(Figure 2.2). Zhang et al., also found that 4-P-acyl-CoAs are only generated from 4-
hydroxyacids (and not 2-, 3-, or 5-hydroxy-n-acids), suggesting that 4-P-acyl-CoAs play
a specific role in the metabolism of 4-hydroxyacids with five or more carbons (i.e.
catabolism via pathway A in Figure 2.2) (151). As shown in the following reaction, 4-P-
acyl-CoAs are formed by the action of an unknown kinase on 4-hydroxyacyl-CoAs
(Figure 2.2, pathway A).
4-Hydroxy-n-acid + ATP + CoASH → 4-Phosphoacyl-CoA + AMP + PPi-
The phosphorylation of 4-hydroxyacyl-CoA is followed by dehydrogenation and hydration, forming 3-hydroxy-4-P-acyl-CoA, which is dephosphorylated to the enol form of 3-ketoacyl-CoA. Thus, the 4-P-acyl-CoAs are part of a series of reactions that isomerize 4-hydroxyacyl-CoAs to 3-ketoacyl-CoAs and 3-hydroxyacyl-CoAs (pathway
A in Figure 2.2), which are intermediates of fatty acid β-oxidation (151).
47
The fact that 4-phosphopentanoyl-CoA (4-P-GHP-CoA) derived from GHP accumulates
at much higher levels in the liver than 4-phosphobutyryl-CoA (4-P-GHB-CoA) derived
from GHB, is of particular interest. As mentioned previously, SSA dehydrogenase
deficient mice have high concentrations of plasma GHB and suffer from severe epileptic
seizures (187; 188). As shown in Figure 2.5, Zhang et al. found accumulation of 4-
phosphobutyryl-CoA in the liver and brain of mice unable to convert GHB to succinate
(SSA dehydrogenase deficient mice) (151). In fact, the concentration of 4-
phosphobutyryl-CoA in liver and brain of mutant (M) mice was about 10 and 50 times
higher, respectively, than in wildtype (W) mice. This suggests that 4-phosphobutyryl-
CoA accumulation may contribute to (i) the perturbations in brain metabolism in these
mice who experience severe epileptic seizures (229), (ii) the severe mental retardation in
patients with GHB aciduria (186), and/or (iii) the acute mental dysfunction in subjects
who ingest GHB as a drug of abuse (191). Since 4-phosphopentanoyl-CoA accumulates
in much higher concentrations in the liver when compared to 4-phosphobutyryl-CoA (as
shown in Figure 2.3), there is some potential that very high concentrations of 4-
phosphopentanoyl-CoA may also accumulate in the brain. Therefore, similar mental
dysfunction and/or perturbations in brain metabolism may occur in persons who ingest
GHP as an alternate drug of abuse to GHB (149). Although the exact toxicity of GHP in
humans is unknown, the data published by Zhang et al. suggest that GHP is potentially
more toxic than GHB. Thus it is possible that 4-phosphopentanoyl-CoA derived from
GHP might perturb some brain functions by acting as a neuromodulator.
48
Figure 2.3. Fragmentation Patterns of 4-Phosphopentanoyl-CoA.
Note the loss of phosphoric acid (98 amu) during the two MS/MS/MS fragmentations (A → C and D → F). Fragments A and D are specific to 4-phosphopentanoyl-CoA. Fragments B, C, E, F and G are common to 4-hydroxypentanoyl-CoA and 4- phosphopentanoyl-CoA. Fragments B and E are common to all acyl-CoAs. Zhang, G.F., J. Biol. Chem. 284: 33521-33534, 2009. (151)
49
Figure 2.4. Accumulation of 4-Phosphoacyl-CoAs derived from 4-Hydroxyacids.
Retention times and relative abundances of 4-phosphoacyl-CoAs assayed under identical LC-MS conditions in extracts of rat livers perfused with 2mM of C4 to C11 4- hydroxyacids (composite chromatogram). Note: C4 is the 4-phosphobutyryl-CoA derived from 4-hydroxybutyrate (GHB) and C5 is the 4-phosphopentanoyl-CoA derived from 4- hydroxypentanoate (GHP). Zhang, G.F., J. Biol. Chem. 284: 33521-33534, 2009. (151)
50
Figure 2.5. Concentrations of 4-Phosphobutyryl-CoA in the Brain and Liver of Mice Deficient in Succinic Semialdehyde Dehydrogenase.
Concentrations of 4-phosphobuytyrl-CoA in the brains (A) and liver (B) of wildtype (W), heterozygote (T), and homozygote or mutant (M) mice deficient in succinic semialdehyde dehydrogenase. Zhang, G.F., J. Biol. Chem. 284: 33521-33534, 2009. (151)
51
2.2.4. Trapping of CoA into Intermediates of 4-Hydroxyacid Catabolism
Zhang et al. demonstrated that the catabolism of 4-hydroxyacids with five or more
carbons results in the accumulation of CoA esters derived from 4-hydroxyacids (see
supplemental Figure 3S of (151)). As shown in Figure 2.6, the trapping of CoA in the
latter intermediates of 4-hydroxyacid catabolism resulted in a decrease in the liver
concentration of free CoA, acetyl-CoA, and malonyl-CoA (151). The trapping of CoA in
the metabolites of some drugs has been linked to the deleterious side effects of the drugs.
For example, valproic acid, a drug used in the treatment of epilepsy, is linked to steatosis
and acute hepatotoxicity secondary to CoA sequestration in valproyl-CoA and other
metabolites of this drug (230). CoA sequestration inhibits fatty acid oxidation (230-232).
Indeed, many drugs are organic acids or are metabolized to organic acids (i.e. such as
salicylic, pivalic, phenylbutyric and benzoic acids), and require CoA for their metabolism
(233). Some toxins are also metabolized to CoA esters, such as hypoglycin, a
mitochondrial poison found in unripe ackee nuts, which causes Jamaican vomiting
sickness upon ingestion (234; 235). In addition, the accumulation of short- and medium-
chain acyl-CoAs with severe depletion of free CoA has been found in patients with
Reye’s Syndrome and impaired fatty acid oxidation (236). Although the exact cause of
Reye’s Syndrome is unknown, the disease has been associated with aspirin consumption
in children with viral illness, and might be linked to: (i) accumulation of acyl-CoA esters
which cause mitochondrial damage, (ii) inhibition of mitochondrial pathways for
ureagenesis, gluconeogenesis, and fatty acid oxidation, and (iii) transitory
hyperammonemia (237-241).
52
In some inborn errors of metabolism, the accumulation of CoA esters has been implicated
in the physiopathology of the diseases. Mitchell et al. reviewed diseases of acyl-CoA
metabolism and the pathological situations that occur when one or more acyl-CoA
species accumulate to high levels (136). They refer to acute CoA sequestration, toxicity,
or redistribution (CASTOR) as a state in which many basic cell processes are
compromised by acyl-CoA accumulation, or by free CoA and acetyl-CoA depletion
(136). The latter can lead to the inhibition of some major pathways like gluconeogenesis,
oxidative phosphorylation, and ureagenesis (242-245).
Since free CoA and acetyl-CoA participate in a large number of chemical reactions of
intermediary metabolism, changes of free CoA and/or acetyl-CoA levels can disrupt multiple pathways of cell metabolism (246). For example, the oxidation of glucose and fatty acids through the CAC could decrease if there is less free CoA available for their conversion to acetyl-CoA (47). In addition, a decrease in free CoA activates (allosteric) pyruvate dehydrogenase kinase, which inhibits pyruvate dehydrogenase activity (247).
The two latter points could result in the following: (i) accumulation of pyruvate and therefore plasma lactate, potentially leading to lactic acidosis, (ii) inhibition of endogenous ketogenesis secondary to the inhibition of endogenous fatty acid oxidation,
(iii) overconsumption of glucose (secondary to the inhibition in the formation of acetyl-
CoA from glucose and fatty acids, resulting in decreased plasma glucose concentrations), and/or (iv) inhibition of gluconeogenesis via the absence of the activation of pyruvate carboxylase (by acetyl-CoA), which could contribute to hypoglycemia (4; 32; 81; 248-
252).
53
In regard to the drug of abuse GHP, the trapping of CoA in the metabolites of GHP might
interfere with a number of CoA-dependent reactions of intermediary metabolism and
contribute to the toxicity of this drug.
54
Figure 2.6. Evidence of CoA Trapping in CoA Esters Derived from 4-Hydroxy-n-acids.
Evidence for CoA trapping in livers perfused with increasing concentrations of 4- hydroxyacids with 4 to 8 carbons. Concentrations of free CoA (A), acetyl-CoA (B), and malonyl-CoA (C). Zhang, G.F., J. Biol. Chem. 284: 33521-33534, 2009. (151)
55
CHAPTER 3
LEVULINIC ACID
3.1. Overview
Levulinic acid (= 4-ketopentanoic acid) is a member of a rare class of organic compounds
known as the gamma-keto acids. Levulinic acid has two functional groups (i.e. a carbonyl
and a carboxyl), making it a versatile building block for the synthesis of various organic
chemicals (including acrylic acid, 2-methyltetrahydrofuran, 1,4-pentanediol, succinic acid,
and 4,4-bisphenolvaleric acid) (253; 254). In addition, levulinic acid is a food additive
(255). As a calcium salt, it is used as an oral and intravenous form of calcium
supplementation (256-262). Since levulinic acid has a carbonyl and carboxyl group,
physiologically it can react as a ketone and as a fatty acid. Although there is no
information on the catabolism of levulinic acid in mammals, it is thought to undergo
complete or partial β-oxidation to yield metabolites, which are eliminated in the urine (263;
264).
Note: The metabolism of 5-aminolevulinate, the first intermediate of the porphyrin pathway and a precursor of heme, has been extensively studied (265-267). However, it is a field that is unrelated to the present work.
3.2. Production and Industrial Applications of Levulinic Acid
An extensive amount of research has been done worldwide on the conversion of biomass waste products to biofuels and intermediates for products of industrial use. Levulinic acid
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has recently received a lot of attention and has been identified as a promising, green,
biomass-derived platform chemical with numerous industrial uses (268). Levulinic acid
can be produced by high temperature acid hydrolysis of carbohydrates (i.e. glucose,
sucrose, galactose, fructose) and from materials such as raw wood and biomass waste
products (e.g. waste feedstock, agricultural residues) (253; 269-273). Considering its
inexpensive production, levulinic acid has a variety of industrial applications. It is used in
the manufacture of resins, nylons, synthetic rubbers, plasticizers, textiles and coatings,
adhesives, lubricants, paints, printing ink and polymers (253; 254; 269; 274; 275). Various
derivatives of levulinic acid, such as 2-methyltetrahydrofuran and levulinate esters may be
used as gasoline and biodiesel additives (i.e. fuel extenders), respectively (275; 276), and
are proposed to aid in generating cleaner-burning fuels for cars than petroleum-based
gasoline. The δ-aminolevulinate derivative is a broad spectrum herbicide and insecticide
(277). In addition, the bisphenol derivative (i.e. 4,4-bisphenolvaleric acid) has been
proposed as a substitute for bisphenol A (i.e. used in the synthesis of plastics) (270). There
are a multitude of patents for the use of levulinic acid in specific industrial applications (see
Table 1 and 3 of (270)).
Levulinic acid is also a food additive allowed by the Food and Drug Administration (255).
It is listed under “synthetic flavoring substances and adjuncts” that can be added directly to
food for human consumption (21 CFR, 172.515) (255). For example, levulinic acid, ethyl levulinate and butyl levulinate are used as a food flavoring agents (278). Levulinic acid is
also used as an acidulant in carbonated beverages, fruit juices, jams, jellies and mayonnaise
(261). In addition, levulinic acid may have activity as a general preservative similar to the
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salts of other short-chain organic acids (279; 280). Indeed there is a patent relating to a
method for inhibiting the growth and proliferation of bacterial pathogens (specifically
Listeria monocytogenes) by addition of levulinic acid in ready-to-eat foods (281). The
World Health Organization (WHO) also lists levulinic acid as a safe food additive in
quality control documents. The International Program on Chemical Safety (WHO
committee) reports that there are no safety concerns at the current level of intake (< 1.8 mg
per day) (264). Interestingly, levulinic acid is used as an additive in cigarettes because (i)
it is a flavorant through its sweet, caramel taste, (ii) it decreases the smoke pH, thus
enhancing the sensory smoothness of smoke and increasing the nicotine delivery in smoke,
and (iii) it enhances the binding of nicotine to neural receptors that would not ordinarily be
responsive to the nicotine molecule (282).
3.3. Levulinic Acid as a Calcium Salt
In the pharmaceutical field, the calcium salt of levulinic acid (i.e. calcium levulinate) is
commercially available and employed as a source of calcium in both oral and intravenous
calcium supplements. For example, Bulgarian Pharmaceutical Group Ltd. and R.P. Traders
(India) manufactures a calcium gluconate/calcium levulinate solution for intravenous
injection, which provides 21 mg elemental calcium and 124 mg levulinate/10 ml (283).
Master Pharxm™ (New York) also manufactures calcium levulinate available for
intravenous injection (284). Global Calcium Ltd. (India) manufactures an oral grade of
calcium levulinate, (i.e. calcium levulinate dihydrate [Ca(levulinate)2•2H2O]), which provides ~6 g of levulinate per 1000 mg of elemental calcium (285). One can even find calcium levulinate listed as “a calcium replenisher to be used in hypocalcemic states” in
58
reference guides for professionals that contain information on chemicals, drugs, and
biologicals (286). Listings are included in sources such as the Merck Index, and the U.S.,
European, Indian, Chinese and Japanese pharmacopeias.
Most of the research on the use of levulinic acid as a calcium salt dates back to the 1930s -
1940s (287). At that time researchers were looking for an organic salt that would (i) increase the solubility and stability of calcium in a water solution, (ii) allow for high calcium content, and (iii) have low toxicity and no irritating effects. The therapeutic use of organic salts of metals depends considerably on the organic vehicle, or the carrier of the metal ion. This is the part that affects the solubility of the salt, the pH and stability of its solutions, and the reaction of the attached metal on normal tissues (particularly when the salt is given by intramuscular or intravenous injections) (260). Early researchers concluded that the calcium salt of levulinic acid possesses the desired properties for injection: (i) the salt contains 13.1% calcium, (ii) it is very soluble in water and is stable in 25-30% solutions, (iii) the pH of a 10% solution lies between pH 7 and 8, and (iv) skin ulceration does not seem to follow extravasation when given intravenously (256; 258; 288). In addition, on a weight basis, calcium levulinate contains 40% more calcium than calcium gluconate, and about the same amount of calcium as calcium lactate, providing another reason to consider trials of calcium levulinate in humans (257).
In 1942, a complete bibliography of 423 references covering levulinic acid and its salts was compiled in a book by the Division of Research Development of the A.E. Staley
Manufacturing Company (287). This bibliography and my own literature search revealed
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only a few references of physiological nature. Proskouriakoff et al. reported that calcium
levulinate was suitable for oral (650 mg/day X 15 days), subcutaneous (3 ml of a 10%
solution every 5 days X 20 days), and intravenous (3 ml of a 5% solution every other day X
10 days) administration in rats and humans, and did not produce any irritating side effects
(257). However, “side effects” were determined on an observation basis (i.e. diarrhea, weight loss, signs of weakness). Tischer et al. reported that ingestion of levulinic acid (3
ml of pure levulinic acid diluted in 150 ml of fruit juice per day X 30 days) in healthy male
adults did not affect the (i) sugar or non-protein nitrogen content of the blood, (ii) color,
pH, or specific gravity of the urine, or (iii) the general physical well-being of the men
(261). In addition, Greville et al. reported that intravenous injection of calcium levulinate
(3 ml of a 10% solution) to (i) two healthy patients, (ii) four tetany patients, and (iii) five
jaundiced patients did not produce any “unpleasant results” (undefined) (258; 259).
Although the previous studies concluded that levulinic acid is “non-toxic and safe to use as
a calcium salt”, biochemical parameters were usually not measured and/or discussed. In
addition, there are a few examples in the literature of “negative side-effects” following
administration of calcium levulinate. For example, in 1933, Gorden et al. reported that
some tuberculosis patients treated orally with calcium levulinate (1 to 4 g/day) experienced
loss of appetite and nausea (257). In 1937, Gisselsson and Sylvan, in self experiments
using calcium levulinate, caused a slight acidosis by administration of higher doses (doses
undefined) (289). In 1947, Berlin et al. reported a rare case of calcinosis of the skin in a
tuberculosis patient given an intravenous injection of calcium levulinate (dose undefined)
(290). In addition, in 2007, Williams et al. reported that lactic acidosis developed in
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premature babies following oral administration of calcium levulinate (0.2 – 0.4 g kg−1 day−1) to treat hypocalcemia (see below) (262). Although it is possible that there are
individual variations in the reaction to levulinate, it appears that calcium levulinate may be
linked to some metabolic perturbations.
To the best of the review of the literature, there is only one study that examined the
absorption of calcium levulinate in mammals. In 1931, Wokes published data comparing
the absorption of calcium chloride, calcium lactate, calcium gluconate, and a mixture of
calcium oleate/calcium levulinate (0.7% to 5%) from solutions given orally to mice (291).
The data suggested that the most readily absorbed calcium salt was the chloride and the mixture of oleate/levulinate was absorbed at “six-tenths” the value of the chloride (291).
3.4. Past Observations on Levulinate Metabolism
Although calcium levulinate has been administered to humans for more than a century, it is remarkable that the metabolism of levulinate in mammalian cells has not been investigated. One could then reasonably assume that, at the doses used for dietary calcium supplementation or for the occasional intravenous treatment of hypocalcemia,
calcium levulinate is probably a safe compound (257). However, there are two reports
that provide some insight on the metabolism of levulinate, via clinical observations
following administration of calcium levulinate. In 1977, Kolvraa et al. found excretion in
the urine of the 4-hydroxy analog of levulinate, or 4-hydroxypentanoate (GHP), following intravenous administration of calcium levulinate (~0.1 g/day) to children (219). The latter was reported as an incidental analytical finding without firm biochemical explanation
61
(219). Still, this finding hinted at the existence of a mechanism that reduces levulinate to
GHP.
The first clear report of a levulinate-associated pathology was published in 2007. In
premature babies treated with oral calcium levulinate for hypocalcemia, serious lactic
acidosis developed during the 20 day treatment period (262). As shown in Figure 3.1, (i)
the average blood pH was < 7.22 and the average blood lactate was > 3.3 mM in all
patients (panel A), and (ii) the plasma lactate concentration started to increase above the
reference range after 4 days of treatment with calcium levulinate (panel B), with the
highest concentration reaching 15 mM in one of the patients (262). This unexplained
metabolic perturbation vanished when calcium levulinate was replaced by calcium
gluconate.
Very recently, Zhang et al. found that levulinate is released by rat livers perfused with GHP
(151). Thus, there is evidence of a mechanism in the human body and in rat liver that
interconverts levulinate and GHP, neither of which are physiological compounds.
Considering that levulinate is converted to GHP in vivo, there is some potential that
calcium levulinate could be used as a legal precursor to a drug of abuse (i.e. GHP).
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Figure 3.1. Biochemical Parameters in the Blood of Premature Babies Following Treatment with Calcium Levulinate.
Evidence for lactic acidosis in premature babies treated for hypocalcemia with oral calcium levulinate. Biochemical parameters measured in the blood, presented as averages over 20 day treatment period (panel A). *Highest value of lactate observed in patient A (= 15 mM). Profile in the concentration of lactate in the blood of one patient treated with calcium levulinate (panel B). Modified from: Williams, M., Ned. Tijdschr. Geneeskd. 151: 1191-1196, 2007. (262)
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CHAPTER 4
HEXANE TOXICITY
4.1. Overview
Some of the findings presented in this thesis hinted at an analogy between a side pathway
of levulinate metabolism and a pathway that contributes to the neurotoxicity of hexane.
For this reason, the mechanism of hexane toxicity is briefly reviewed here.
Chronic exposure of workers to industrial solvents such as n-hexane and methyl n-butyl
ketone resulted in outbreaks of neuropathy in these workers (292). These solvents are
metabolized to 2,5-hexanedione (2,5-diketohexane), a γ-diketone that forms pyrrole
adducts with neurofilament proteins causing specific neurotoxic effects (292). The
relevance of hexane toxicity to the metabolism of levulinate centers around the
mechanism by which pyrrole adducts formed from the 2,5-hexanedione metabolite of
hexane contribute to its toxicity. The mechanism of hexane toxicity is described in the
following section. The formation of pyrrole adducts derived from levulinate and their
potential link to the toxicity of levulinate is discussed in Chapter 7 (i.e. Discussion). In
addition, in our findings (described in Chapter 7), a γ-diketo-acyl-CoA derived from the
elongation of levulinyl-CoA, may react with lysine in a way similar to 2,5-diketohexane.
4.2. Metabolism of n-Hexane
Various studies have demonstrated that n-hexane is absorbed following inhalation of vapors, ingestion of the solvent, or via topical application to the skin (293). However, the
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most frequent exposure is via inhalation. Approximately 10-20% of n-hexane absorbed
by inhalation is excreted unchanged in exhaled air; the remainder is metabolized (294).
Metabolism takes place in the liver via mixed-function oxidase reactions. The initial
reaction is the oxidation of n-hexane to hexanols (1-hexanol, 3-hexanol and
predominately 2-hexanol) by cytochrome P-450 enzymes. Hydroxylation of n-hexane at
the 1 and 3 positions (forming 1-hexanol and 3-hexanol) are considered detoxification
pathways. 1-Hexanol is converted to hexanoic acid which undergoes β-oxidation. 3-
Hexanol is probably partly oxidized to 3-hexanone which is excreted. Further reactions
(presumably catalyzed by oxidases) convert 2-hexanol to 2-hexanone, 2,5-hexanediol, 5- hydroxy-2-hexanone, and 2,5-hexanedione (293; 295-297). In vitro and in vivo studies have revealed that the neurotoxicity of n-hexane is associated with the metabolite 2,5- hexanedione (= 2,5-diketohexane) (292).
4.3. Hexane Toxicity via the Formation of Pyrrole Adducts
Pyrrole adduct formation in neurofilament proteins has been implicated as the mechanism of induction of neuropathy by the γ-diketones (298-300). See Figure 4.1. Katritzky et al. confirmed the reaction mechanism for the pyrrolation of proteins by 2,5-hexanedione
(301). 2,5-Hexanedione is a symmetrical compound with 2 gamma carbonyl groups.
The lone electron pair of an amino group on a protein can attack either of the electron deficient carbonyl carbons to form an enamine (with loss of hydrogen). Subsequently, the lone electron pair of the enamine nitrogen attacks the second carbonyl carbon, joining the chain (with loss of water) to form the 2,5-dimethylpyrolle adduct (301). Pyrrole formation is unique to γ-diketones that have a 2 carbon spacing between the carbonyl
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functions (302). Pyrrolation requires an amino group at the end of a carbon chain, not an
alpha-amino group of aminoacids. The reactions of γ-diketones with protein ε-amino moieties of lysine to yield pyrrole adducts has been demonstrated in many in vitro (303;
304) and in vivo systems (305; 306). Pyrrole formation explains γ-diketone neuropathy
(302).
In the case of n-hexane, the 2,5-hexanedione metabolite induces modification of the primary amine side chain of critical lysine residues of neurofilament or related cytoskeletal proteins (307-309) to form pyrroles (299; 300; 308; 310-312). Both in vitro and in vivo studies have demonstrated that 2,5-hexanedione reacts covalently with lysine
ε-amine groups to form 2,5-dimethylpyrrole adducts on neurofilaments and other proteins
(299; 300; 308). The formation of pyrrole adducts results in changes in neurofilament physiological characteristics including solubility, electrostatic potential, and 3- dimensional structure (300; 312-314). If these chemically modified neurofilament proteins cannot interact with the cytoskeletal network appropriately, then axon atrophy can develop (302).
For example, pyrrolation of neurofilament and cytoskeletal proteins in the peripheral and central nervous systems induces alterations in axon diameter (via swelling or atrophy). In addition, pyrrole adducts can undergo secondary oxidative reactions that yield cross- linked proteins that also contribute to alterations in the physical integrity of axons (302;
315-318). This results in axon degeneration and nerve fiber damage (via protein modification) in both the peripheral nervous system (i.e. tibial and sciatic nerves) and the
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central nervous system (i.e. optic nerve and spinal cord). Nerve fiber damage is further
linked to neurophysiologic dysfunction (i.e. muscular weakness, postural and gait
abnormalities) (302; 315-317; 319; 320). Thus, 2,5-hexanedione induces neuropathy as a
consequence of pyrrolation of lysine ε-amino groups on neurofilament and cytoskeletal
proteins. This explains the neurotoxicity of hexane (321).
Note that 2,5-hexanedione may form pyrrole adducts with non-neuronal proteins because
virtually all proteins contain one or more lysine ε-amine side chains that are potential sites of adduction of 2,5-hexanedione (302).
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Figure 4.1. Formation of a Dimethylpyrrole Adduct from 2,5-Diketohexane and an Amine.
The proposed mechanism for the synthesis of dialkylpyrroles from γ-diketones and primary amines. R = the protein of which the amino group is attached. DeCaprio, A. P., Mol. Pharmacol. 32: 542-548, 1987. (304)
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CHAPTER 5
RESEARCH PLAN
5.1. Overview
The following research plan builds upon a couple of findings in the literature: (i)
identification of GHP in children having taken calcium levulinate (219), (ii) induction of
lactic acidosis in premature babies having taken calcium levulinate (262), and (iii) a
general mechanism on the metabolism of 4-hydroxyacids recently described by our lab
(151). The finding of release of levulinate by livers perfused with GHP and the
unexplored formation of GHP from levulinate in humans suggest the existence of at least
one oxido-reduction mechanism that interconverts levulinate and GHP. This mechanism
may be strongly influenced by variations in [NADH]/[NAD+] ratios induced by ethanol
oxidation in the liver. Therefore, the combination of levulinate and ethanol ingestion
may result in accumulation of toxic levels of GHP in body fluids. The finding of lactic
acidosis in premature babies treated for hypocalcemia with calcium levulinate suggests
that the trapping of free CoA by derivatives of levulinate may perturb a number of CoA-
dependent reactions in the liver. The previous finding that GHP metabolism in the liver
leads to very high concentrations of 4-phosphopentanoyl-CoA (151) suggests that
ingestion of levulinate (especially with ethanol) could lead to very high concentrations of
4-phosphopentanoyl-CoA in the liver and possibly other organs (i.e. brain). The presence
of a keto group on C-4 of levulinate suggests that its catabolism may be more
complicated than what has been suggested previously. The catabolism of levulinate may
involve steps in which the carbonyl on C-4 is reduced to a hydroxyl group.
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The investigation of the above questions required experiments to be conducted in vivo
(intravenous infusions or gavages) and in perfused rat livers. Last, the use of multiple
13 [ Cn]levulinate substrates allows to follow the fates of the carbons of levulinate through
the hypothesized pathways of catabolism, using metabolomics and mass isotopomer
distribution of the expected metabolites of levulinate.
5.2. Part A: Metabolism of Levulinate ± Ethanol in Perfused Rat Livers and in Live
Rats
5.2.1. Mechanisms for the Conversion of Levulinate to 4-Hydroxypentanoate
Our initial interests in levulinate arose from the recent study of the metabolism of 4-
hydroxyacids in our lab. First of all, it was found that 4-hydroxynonanoate (a metabolite
of the lipid peroxidation product 4-hydroxynonenal) reversibly interconverts with 4-
ketononanoate in perfused rat livers (151). Secondly, in an orientation liver perfusion
with 4-hydroxypentanoate (GHP), its oxidized form 4-ketopentanoate (i.e. levulinate) was identified in the perfusate (151). In addition, there was the clinical observation of
GHP found in the urine of children treated with calcium-levulinate (219). All of the latter suggested that there is a mechanism in the body that interconverts levulinate and GHP, which led to the conception of this project. The recent use of GHP as a drug of abuse
(more toxic than 4-hydroxybutyrate (GHB)) (197), led us to hypothesize that levulinate could be used as a pro-drug (i.e. precursor of GHP).
The reduction of levulinate to GHP could occur in the liver and other organs, possibly catalyzed by NADH- and/or NADPH- dependent dehydrogenase(s). Known cases of
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hydroxy and keto acid interconversion occur via dehydrogenases specific to 2- and 3-
hydroxy-n-acids (i.e. lactate and β-hydroxybutyrate dehydrogenases) (227). However,
there is no evidence in the literature of a dehydrogenase or enzyme system that
interconverts 4-ketoacids and 4-hydroxyacids longer than 4 carbons in mammalian cells.
Kaufman’s group reported that there are two enzymes that interconvert GHB and succinic
semialdehyde: (i) a cytosolic NADP+ dehydrogenase, and (ii) a mitochondrial
transhydrogenase catalyzing the reaction GHB + α-ketoglutarate ↔ succinic semialdehyde
+ α-hydroxyglutarate (181-183; 322; 323). The transhydrogenase has an absolute requirement for α-ketoglutarate (322). However, whether the latter two enzymes would work on levulinate and GHP is unknown. Therefore, we hypothesized that the interconversion of levulinate and GHP is likely catalyzed by a dehydrogenase or a more complex enzyme system that is not yet described.
GHP, as a racemic mixture, is a new drug of abuse used in lieu of GHB (149; 197; 324-
326). So we also considered that the metabolism of levulinate could generate both enantiomers of GHP. Many enzymes have chiral substrate specificity (i.e. β- hydroxybutyrate dehydrogenase is specific to (R)-β-hydroxybutyrate) (227). So a dehydrogenase(s) catalyzing the interconversion of levulinate and GHP might be influenced by the chirality of GHP, as GHP has two optically active enantiomers (i.e. (R)- and (S)-GHP) (325). In addition, previous work in our lab demonstrated (i) the production of (R)- and (S)-GHP from levulinate, (ii) production of levulinate from (R,S)-GHP, and (iii) that the total concentration of GHP decreases by more than half in livers perfused with
(R,S)-GHP, showing that both enantiomers are used by the liver. So we considered the
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possibility that a racemase or racemase system exists that would allow for (R)- and (S)-
GHP to be used via the same route of degradation (i.e. pathways A and B in Figure 5.1).
For example, a racemase could interconvert (R)- and (S)-GHP, or (R)- and (S)-GHP-CoA.
On the other hand, a racemase system could exist if there are two enzymes present that act on levulinate to form (R)- and (S)-GHP. In addition, an alternate mechanism may interconvert the CoA esters of of levulinate and GHP. Although there is no information on an enzyme that would interconvert levulinyl-CoA and GHP-CoA, it may be similar (or identical) to the S-3-hydroxyacyl-CoA dehydrogenase involved in fatty acid β-oxidation. If a new type of acyl-CoA dehydrogenase interconverts levulinyl-CoA and GHP-CoA, it may have optical specificity for the GHP-CoA (as 3-hydroxyacyl-CoA dehydrogenase has specificity for the (S)-3-hydroxyacyl-CoAs) (47). Based on all of the latter, we hypothesized that the potential interconversion mechanisms are as follows:
1. NAD+-linked dehydrogenase in the cytosol or mitochondria.
2. NADP+-linked dehydrogenase in the cytosol or mitochondria.
3. Levulinate → Levulinyl-CoA ↔ (S)-GHP-CoA → (S)-GHP, where the
interconversion of levulinyl-CoA and GHP-CoA is catalyzed by a new type
of acyl-CoA dehydrogenase, probably located in the mitochondria.
Based on the effects of ethanol on liver metabolism, we further hypothesized that ethanol
would stimulate (i.e. accelerate) the reduction of levulinate to GHP. The oxidation of
ethanol to acetaldehyde and then to acetate in the liver results in an increase in cytosolic and mitochondrial [NADH]/[NAD+] ratios (128; 327). An increase in [NADH]/[NAD+]
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ratios secondary to the oxidation of ethanol, could accelerate the reduction of levulinate to
GHP, if this reduction is linked to a hepatic NADH dehydrogenase.
5.2.2. Pathways of Levulinate Catabolism
To the best of our review of the literature, the metabolism of levulinate has not been investigated in mammalian cells. The work completed by Zhang et al. on the metabolism
of 4-hydroxyacids with five or more carbons demonstrated that they are metabolized by
two parallel pathways (i.e. pathways A and B, shown in Figure 2.2) (151). However, this
work did not consider the possibility of two forms of pathway B, depending on the
presence of a keto or hydroxy group on C-4 of the 4-hydroxyacid. We hypothesized that
the metabolism of levulinate and GHP would follow a scheme similar to that presented
by Zhang et al., but would include an additional pathway (considering that levulinate has
a keto group on C-4, but is also reduced to GHP which has a hydroxy group on C-4). As
shown in Figure 5.1, levulinate and GHP can be interconverted and activated to their
respective CoAs, and then degraded via three pathways. The hypothesized scheme for
the metabolism of levulinate has features similar to Zhang’s scheme: the isomerization
of 4-hydroxypentanoyl-CoA to 3-hydroxypentanoyl-CoA via 4-phosphopentanoyl-CoA
followed by β-oxidation to propionyl-CoA and acetyl-CoA (pathway A) (151). In
addition, we hypothesized that 4-hydroxypentanoyl-CoA and levulinyl-CoA are degraded
by two parallel β-oxidation processes in pathways B and B’ that converge at 3-keto-4- hydroxypentanoyl-CoA. The latter would undergo thiolytic cleavage to acetyl-CoA and
the putative lactyl-CoA (which has never been identified in mammalian cells). Finally, lactyl-CoA could be hydrolyzed to lactate and/or α-oxidized to acetyl-CoA and formate
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(223; 224). Note: the only difference between pathways B and B’ is the presence of a keto or hydroxy group on C-4 of the five carbon intermediates.
13 To test these hypotheses we tried to design a [ Cn]levulinate substrate that would (i) yield different mass isotopomers of the two acetyl-CoAs presumably derived from C-1+2 of levulinate (via pathways A or B + B’) and C-3+4 of levulinate (via the α-oxidation of
lactyl-CoA from pathways B + B’), and (ii) allow for tracing the production of formate
13 from C-3 of levulinate. However, we could not design a single [ Cn]levulinate substrate
that would allow for tracing of all the processes outlined in Figure 5.1. In addition, the
labeling of levulinate with deuterium (2H) is not an option because of the loss of 2H via exchange with water in (i) keto-enol tautomerization, (ii) hydroxy-ketoacid interconversion, and (iii) β-oxidation. The latter makes it difficult to predict which 2H
atoms would be lost from levulinate, making the interpretation of data next to impossible.
13 Thus we settled on [ C5]levulinate (M5 levulinate) for most of the planned experiments.
Among livers perfused with C4 to C11 4-hydroxyacids, the highest concentrations of 4-
phosphoacyl-CoA and related acyl-CoAs were found in livers perfused with GHP (see
Figure 2 and supplemental Figure 3S of (151)). In addition, premature babies treated for
hypocalcemia with calcium levulinate developed serious lactic acidosis (262). This led
us to hypothesize that the metabolism of levulinate and GHP might result in the
accumulation of CoA esters that trap free CoA. CoA trapping in the metabolites of
levulinate might (i) induce perturbations in the concentration profiles of other
physiological CoAs (i.e. free CoA and acetyl-CoA), and (ii) cause perturbations of
74
intermediary metabolism such as hypoglycemia, hypoketonemia, and lactic acidosis
(136). As discussed in Chapter 2 (section 2.2.4), (i) a decrease in free CoA activates pyruvate dehydrogenase kinase, which inhibits pyruvate dehydrogenase activity (247),
(ii) a decrease in free CoA can decrease the oxidation of glucose and fatty acids through the CAC (since free CoA is required for their conversion to acetyl-CoA) (47), and (iii) a decrease in acetyl-CoA can prevent the activation of pyruvate carboxylase (249). Thus decreases in the concentrations of acetyl-CoA and free CoA could affect the pyruvate dehydrogenase system and could result in: (i) accumulation of pyruvate and therefore lactate, (ii) inhibition of endogenous ketogenesis secondary to the inhibition of endogenous fatty acid oxidation, (iii) overconsumption of glucose secondary to the inhibition in the formation of acetyl-CoA from glucose and fatty acids, and/or (iv) inhibition of gluconeogenesis via the absence of the activation of pyruvate carboxylase by acetyl-CoA (4; 32; 81; 248-252). The latter effects could lead to lactic acidosis, hypoketonemia, hypoglycemia and potentially other perturbations of cellular metabolism.
The accumulation of 4-phosphopentanoyl-CoA, might have toxic effects in its own right.
Mice unable to dispose of endogenous GHB have high concentrations of 4- phosphobutyryl-CoA in both liver and brain (151). The accumulation of 4- phosphobutyryl-CoA may be related to the severe epileptic seizures suffered by these
mice and to the inability to reason in people intoxicated with GHB (186; 188). The
accumulation of 4-phosphopentanoyl-CoA might also be linked to the acute mental
dysfunction in persons ingesting GHP. Thus 4-phosphopentanoyl-CoA could perturb
some brain functions by acting as a neuromodulator.
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Last, since drugs of abuse are often taken with alcoholic beverages, there is a potential link
between ethanol consumption and levulinate toxicity (i.e. for addicts who ingest levulinate
as a precursor to GHP in addition to alcohol to get high). Aside from the potential
stimulation of the reduction of levulinate to GHP, we hypothesized that ethanol might
inhibit the catabolism of levulinate. First, an increase in the mitochondrial
[NADH]/[NAD+] ratio resulting from the oxidation of ethanol in the liver, inhibits the 3-
hydroxyacyl-CoA dehydrogenase reaction of β-oxidation (128; 129). The latter would
inhibit β-oxidation in all three pathways of levulinate catabolism, thereby decreasing the
catabolism of levulinate and GHP (see Figure 5.1, reaction 6, in pathways B and B’, as well
as in the β-oxidation steps in pathway A). In addition, an increase in [NADH]/[NAD+]
ratios resulting from liver ethanol oxidation inhibits reactions of the CAC (130).
Therefore, ethanol oxidation probably inhibits the oxidation of acetyl-CoA derived from levulinate. Second, the carbon from liver ethanol oxidation is released as acetate, which is rapidly taken up and used by peripheral tissues (328). The peripheral oxidation of acetate, involves activation to acetyl-CoA (328). Since levulinate catabolism also yields acetyl-CoA, its catabolism might be inhibited by another precursor of acetyl-CoA (i.e. the acetate derived from liver ethanol oxidation) via competition for free CoA and the enzymes of β-oxidation (329). The result could be substantial accumulation of levulinate-derived intermediates (i.e. GHP and levulinate-derived CoA esters) in the presence of ethanol. This might exacerbate the potential negative “side effects” previously mentioned (i.e. toxic accumulation of 4-phosphopentanoyl-CoA, and significant CoA trapping potentially resulting in hypoglycemia, lactic acidosis, hypoketonemia).
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5.2.3. Public Heath Relevance
The two clinical observations on levulinate metabolism (i.e. (i) GHP found in the urine of
children given calcium levulinate (219), and (ii) the development of lactic acidosis in
premature babies treated for hypocalcemia with calcium levulinate (262)) provide
examples of the potential link of calcium levulinate to a drug of abuse and to
perturbations of intermediary metabolism. The use of calcium levulinate is relevant to
two main target populations: (i) addicts who ingest large amounts of calcium levulinate
as a precursor of GHP (with and without ethanol) to get high, and (ii) the general
population using calcium levulinate as a dietary calcium supplement or for the
intravenous treatment of acute hypocalcemia. Considering that little is known about the
conversion of levulinate to GHP or about the safety of calcium levulinate in the treatment
of hypocalcemia, the metabolism of levulinate and GHP merits research. In addition,
since calcium levulinate is freely available to the public for purchase, the potential use of
calcium levulinate as a precursor of GHP by drug addicts may become a serious public
health problem.
5.2.4. Strategy
The aforementioned problems lie within the levulinate moiety of calcium levulinate, rather
than the calcium moiety. Therefore, our initial experiments focused on levulinate. We
tested the above hypotheses in live rats, in perfused rat livers, and in subcellular fractions
of liver tissue using a combination of metabolomics (221; 222) and mass isotopomer
analysis (220), as outlined below. Overall, our data confirm that (i) levulinate is
converted to GHP, (ii) ethanol stimulates this conversion in the perfused rat liver and in
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live rats, (iii) levulinate is metabolized by the three pathways outlined in Figure 5.1
yielding acetyl-CoA, propionyl-CoA and lactate, and (iv) ethanol inhibits the catabolism of levulinate and GHP.
5.2.4.1. In Vivo Experiments
To test the hypotheses that levulinate is converted to GHP and that ethanol accelerates
this conversion, overnight fasted male Sprague-Dawley rats weighing 200-300 g were
anesthetized with 2% isoflurane and fitted with carotid and jugular catheters. After a 20
min equilibration, sodium levulinate was infused intravenously, as a 150 mM solution, at
2, 4, 6, 8, 10, or 12 μmol∙min-1∙kg-1 for 2 hours. At infusion rates < 6 μmol min−1 kg−1, a plateau in the concentration of levulinate was observed (at up to 0.8 mM). At higher rates of infusion (i.e. 8, 10, and 12 μmol min−1 kg−1), continuous levulinate accumulation in the plasma was observed (almost linearly). These experiments provided an idea of the maximum
capacity of the rat to use or dispose of levulinate, 6 - 8 μmol min−1 kg−1. At all rates of
levulinate infusion, GHP accumulation was observed, which increased linearly without
plateauing, reaching 0.47 mM after 2 hours of levulinate infusion at 12 μmol min−1 kg−1.
Overall, these orientation in vivo experiments demonstrated that an infusion rate of 12
μmol min−1 kg−1 levulinate (i) exceeds the capacity of the rat to metabolize levulinate, and
(ii) produces a plasma GHP concentration that is in the range measured in humans
intoxicated with GHP (197; 326), a concentration that might be toxic (toxic levels of GHB
= 0.5 – 1 mM). Thus, we settled on a levulinate infusion rate of 12 μmol min−1 kg−1 for our rat in vivo experiments.
78
Twelve rats were intravenously infused with 12 μmol∙min-1∙kg-1 levulinate for 2 hours. In
half of the rats, a bolus of 10% ethanol (1.7 M) in saline was injected intraperitoneally at
-15 min in an amount calculated to achieve 10 mM in total body water. This was followed by a constant intravenous infusion of ethanol at 40 μmol·min-1·kg-1 for the 2 hour protocol to compensate for ethanol metabolism. The other rats, used as controls
(infused with levulinate but no ethanol), were injected and infused with saline. Arterial blood was sampled every 20 min for 2 hours. Note: The rate of ethanol infusion was based on the rate of ethanol utilization in the rat liver (~ 1-2 μmol min−1 g liver−1) (330).
Since the weight of the liver is approximately 4% of a rat’s body weight (40 g/kg), the
infusion rate of ethanol was calculated as (40 g liver/kg) x 1 μmol min−1 g liver−1.
5.2.4.2. Perfused Liver Experiments
To test the scheme of levulinate catabolism (shown in Figure 5.1), 4 groups of livers from
overnight fasted male Sprague-Dawley rats (200-250 g) were perfused (331) with
recirculating bicarbonate buffer containing 4% dialyzed, fatty acid-free bovine serum
13 albumin, 4 mM glucose and the following: (i) 2 mM [ C5]levulinate (M5 levulinate); (ii)
2 mM M5 levulinate + 20 mM ethanol; (iii) 20 mM ethanol; and (iv) nothing (control).
The perfusate was sampled every 20 min and livers were quick frozen at the end of the 2
hour protocol.
5.2.4.3. Pilot Enzymatic Assays
To test for mitochondrial and/or cytosolic NADH- and NADPH-dependent
dehydrogenases that reduce levulinate to GHP, series of incubations of levulinate with
79
subcellular fractions of the liver were conducted. Rat liver mitochondria and cytosolic
extracts (100,000 X g supernatants) were prepared as described in (332). Mitochondria
(2.5 or 6 mg) were incubated in 1.2 ml of buffer (100 mM KCl, 50 mM MOPS, 5 mM K-
phosphate, 1 mM EGTA, pH 7.4) containing 4.2 mM glutamate and 6 μM rotenone at
30°C in Mitocell MT200 incubation chambers. The mix of substrates is an NADH generating system. The kinetics were started by adding 5 mM acetoacetate (controls) or levulinate. Incubations were conducted for 30 min (acetoacetate) or 120 min (levulinate) with multiple sampling during the incubations. Cytosolic extracts (2.4 mg of protein)
were incubated for up to 2 hours with 0.3 mM NADPH and 2.8 mM levulinate in a total volume of 3.5 ml of 50 mM K-phosphate buffer, pH 7.4, at 30°C. Samples were taken every 30 min. Note: GHP accumulation was linear with time and protein concentration in all assays.
5.2.4.4. Analytical Procedures
The concentrations and mass isotopomer distributions of various acids, keto acids, and hydroxyacids in rat plasma and liver perfusate were assayed by GC-MS of trimethylsilyl or pentafluorobenzyl derivatives using analog labeled or unlabeled compounds as internal standards. The concentration and labeling of acetate and formate were assayed using negative chemical ionization of the pentafluorobenzyl derivatives (333). The concentration of ethanol in rat plasma was assayed by head space GC-MS with an internal standard of 1-propanol (334). The concentrations and mass isotopomer distributions of acyl-CoAs and levulinate-derived CoA esters were assayed by modification of the LC-MS procedure described by Zhang et al. (151). The
80
chromatographic conditions were optimized to separate the C5 acyl-CoAs derived from levulinate. To assay the distribution of chiral enantiomers of GHP in liver perfusate, and in rat liver mitochondrial and cytosolic extracts, we prepared the (R)-2-butyl-O-acetyl
derivative by modification of the procedure of Struys et al. (335).
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Figure 5.1. Proposed Scheme for the Catabolism of Levulinate.
The hypothetical enzyme activities, designated by numbers in italics are: 1, acid-CoA ligase; 2, hydroxyacid dehydrogenase; 3, 4-hydroxyacyl-CoA dehydrogenase; 4, acyl- CoA dehydrogenase; 5, enoyl-CoA hydratase; 6, 3-hydroxyacyl-CoA dehydrogenase; 7, 3-ketoacyl-CoA thiolase; 8, 4-hydroxyacyl-CoA kinase; 9, acyl-CoA hydrolase; 10, α- oxidation enzymes. The “multiple reactions” mentioned between 4-phosphopentanoyl- CoA and 3-hydroxypentanoyl-CoA result in the isomerization of 4-hydroxypentanoyl- CoA to 3-hydroxypentanoyl-CoA.
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5.3. Part B: Metabolism of Calcium Levulinate
5.3.1. Metabolism of Calcium Levulinate Given Enterally ± Ethanol
Our concerns about (i) CoA trapping in the metabolites of levulinate and GHP and their potential link to perturbations in the concentrations of other physiological acyl-CoAs, and
(ii) the accumulation of 4-phosphopentanoyl-CoA, were supported by the concentration
profiles of acyl-CoAs derived from levulinate in the perfused liver experiments. In addition, (i) the stimulation by ethanol on the conversion of levulinate to GHP, and (ii)
the inhibition by ethanol on the catabolism of levulinate and GHP, were confirmed in
both liver perfusion and in vivo experiments. Therefore, the use of calcium levulinate as a dietary supplement is potentially dangerous, especially for populations at risk (i.e. addicts).
In the experiments from the first phase of the project (i.e. described in part A of the research plan), we confirmed that levulinate is converted to GHP in vivo, and that ethanol stimulates this conversion. So we hypothesized that (i) calcium levulinate could be used
as a legal precursor to the drug of abuse (GHP), and (ii) addicts could take large doses of
calcium levulinate in addition to alcohol to stimulate the conversion of levulinate to GHP
(to produce the desired drug effects). The liver perfusion experiments also confirmed
that the metabolism of levulinate results in the accumulation of levulinate-derived CoA
esters that trap free CoA. This resulted in large decreases in the concentrations of acetyl-
CoA, propionyl-CoA, succinyl-CoA, methylmalonyl-CoA and malonyl-CoA when
compared with control livers. So we hypothesized that substantial CoA trapping in the
metabolites of levulinate might interfere with number of CoA-dependent reactions of
83
intermediary metabolism and may explain the lactic acidosis that was observed in the
premature babies treated for hypocalcemia with calcium levulinate (262). Considering
that ethanol inhibited levulinate catabolism, while enhancing the production of GHP from
levulinate, we further hypothesized that the potentially harmful effects of levulinate
metabolism (i.e. CoA trapping, accumulation of 4-phosphopentanoyl-CoA, and the
potential lactic acidosis and/or hypoglycemia) could be amplified by ethanol.
All of the latter called for an investigation of the effects of oral calcium levulinate (with
and without ethanol) on liver and brain metabolism. After ingestion of calcium levulinate, one can expect a high concentration of levulinate in the portal vein. The latter might result in the release of high concentrations of GHP into the peripheral blood
(especially if ingested with ethanol). High concentrations of GHP reaching the brain
might induce high concentrations of 4-phosphopentanoyl-CoA in the brain. This could
perturb some brain functions by acting as a neuromodulator. In addition, the metabolic
consequences of levulinate catabolism, resulting from GHP accumulation and CoA
trapping in the metabolites of levulinate, are unknown.
The fact that calcium levulinate is freely available for purchase could have direct public
health implications because it represents a legal form of a drug of abuse. Therefore, this
aspect of the project focused on testing whether oral ingestion of calcium levulinate ±
ethanol would (i) induce the accumulation of levulinate-derived CoA esters in the liver
and brain, and (ii) result in changes in plasma glucose and lactate concentrations. To test
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the latter, experiments were originally outlined as described below (using the calcium
salt, not the sodium salt).
5.3.2. Strategy
Overnight fasted male Sprague-Dawley rats weighing 200-250 g were divided into 4
groups (9 rats per group) and given an oral gavage of one of the following: (i) 2 mmol/kg
calcium levulinate; (ii) 2 mmol/kg calcium chloride (control for (i)); (iii) 2 mmol/kg
calcium levulinate + 13.4 mmol/kg ethanol, and (iv) 2 mmol/kg calcium chloride + 13.4
mmol/kg ethanol (control for (iii)). The dose of calcium levulinate provided about 2X
the RDA for calcium for adults (RDA = 1000 mg/day for adults 19-50 years) (336). The
dose of ethanol was calculated to induce 20 mM in total body water, assuming
instantaneous diffusion. To put this concentration range in perspective, 17 mM ethanol
(0.08%) is the legal limit for driving a vehicle, and alcoholic coma occurs between 30-50
mM (337). Rats were given the gavage at 0 min, and were killed at 7 min, followed by
15 min intervals up to 120 min. At the time of kill the following samples were taken: (i)
portal and peripheral plasma, (ii) urine, (iii) a lobe of the liver, and (iv) the whole brain.
5.3.2.1. Analytical Procedures
The concentrations of various acids, keto acids, and hydroxyacids in the plasma were
assayed by GC-MS of pentafluorobenzyl derivatives using analog labeled compounds as
internal standards. The concentration of plasma glucose was assayed on the penta-acetate
derivative, a method modified from (338). The concentration of plasma ethanol was
85
assayed enzymatically. The concentrations of acyl-CoAs and levulinate-derived CoA
esters in the liver and brain were assayed by LC-MS as described in (151).
Our data confirm that following an oral dose of calcium levulinate given to rats: (i)
levulinate derived CoA esters accumulate in both the liver and brain, (ii) levulinate-
derived CoA esters remain longer in the brain than in the liver, (iii) ethanol increases the
concentrations of levulinate-derived CoA esters in both the brain and liver, and (iv)
ethanol increases the concentration of plasma GHP. However, a single oral dose of
calcium levulinate (~2X the RDA for calcium in adult humans) ± ethanol did not increase
plasma lactate or decrease plasma glucose concentrations in the two hours following the
oral dose.
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CHAPTER 6
PUBLICATIONS
6.1. Overview
The hypotheses, observations, and conclusions presented in Chapter 5 are reported in the
following two papers, which are included in this thesis.
6.1.1. Harris, S.R., Zhang, G.F., Sadhukhan, S., Murphy, A.M., Tomcik, K.A., Vazquez, E.J., Anderson, V.E., Tochtrop, G.P., Brunengraber, H. Metabolism of levulinate in perfused rat livers and in live rats: Conversion to the drug of abuse 4- hydroxypentanoate. J. Biol. Chem. 286: 5895-5904, 2011.
6.1.2 Harris, S.R., Zhang, G.F., Sadhukhan, S., Puchowicz, M.A., Anderson, V.E., Tochtrop, G.P., Brunengraber, H. Cyclical CoA esters derived from levulinate. (To be submitted to J. Biol. Chem July 2011)
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6.1.1.
METABOLISM OF LEVULINATE IN PERFUSED RAT LIVERS AND LIVE
RATS:
CONVERSION TO THE DRUG OF ABUSE 4-HYDROXYPENTANOATE
Stephanie R. Harris1, Guo-Fang Zhang1, Sushabhan Sadhukhan2, Anne M.
Murphy1, Kristyen A. Tomcik1, Edwin P. Vazquez1, Vernon E. Anderson3,
Gregory P. Tochtrop2, and Henri Brunengraber1
From Departments of Nutrition1, Chemistry2, and Biochemistry3
Case Western Reserve University, Cleveland Ohio 44106.
Running head: Levulinate metabolism in liver
Address correspondence to: Henri Brunengraber, Department of Nutrition Case Western
Reserve University, School of Medicine - 10900 Euclid Avenue, WG-48;
Cleveland OH 44106-4954. Fax: (216) 368-6560. Email: [email protected]
This research was originally published in The Journal of Biological Chemistry.
Metabolism of levulinate in perfused rat livers and live rats: Conversion to the drug of abuse 4-hydroxypentanoate. The Journal of Biological Chemistry. 2011; (286): 5895-
5904. © The American Society for Biochemistry and Molecular Biology.
88
ABBREVIATIONS
GC-MS, gas chromatography-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry
FOOTNOTE
1. Mass isotopomers are designated as M, M1, M2,...Mn where n is the number of heavy atoms in the molecule. The isotopic enrichment of each mass isotopomer is expressed as mol percent.
ACKNOWLEDGMENTS
This work was supported by the NIH (Roadmap grant R33DK070291 and grant
R01ES013925 to H.B.; Grant RO1HL053315 to G.P.T.). We thank Dr. Janos Kerner for advice on mitochondrial incubations. We also thank the Case Mouse Metabolic and
Phenotyping Center (www.mmpc.orgwww.mmpc.org) for help with the in vivo
experiments.
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ABSTRACT
Calcium levulinate (4-ketopentanoate) is used as an oral and parenteral source of
calcium. We hypothesized that levulinate is converted in the liver to 4- hydroxypentanoate, a new drug of abuse, and that this conversion is accelerated by
ethanol oxidation. We confirmed these hypotheses in live rats, perfused rat livers and
liver subcellular preparations. Levulinate is reduced to (R)-4-hydroxypentanoate by a
cytosolic and a mitochondrial dehydrogenase which are NADPH and NADH-dependent,
respectively. A mitochondrial dehydrogenase or racemase system also forms (S)-4-
13 hydroxypentanoate. In livers perfused with [ C5]levulinate, there was substantial CoA
trapping in levulinyl-CoA, 4-hydroxypentanoyl-CoA and 4-phosphopentanoyl-CoA.
This CoA trapping was increased by ethanol, with a six-fold increase in the concentration
of 4-phosphopentanoyl-CoA. Levulinate is catabolized by 3 parallel pathways to
propionyl-CoA, acetyl-CoA and lactate. Most intermediates of the 3 pathways were
identified by mass isotopomer analysis and metabolomics. The production of 4-
hydroxypentanoate from levulinate and its stimulation by ethanol is a potential public
health concern.
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INTRODUCTION
Levulinate (4-ketopentanoate) is a food additive allowed by the Food and Drug
Administration. As a calcium salt, it has been used for many years as an oral and
intravenous form of calcium administration (1,2). It is listed in the pharmacopeias of a
number of countries (USA, Europe, India, China, Japan, etc). It is also listed in quality
control documents of the World Health Organization. The dry salt is sold on the internet
as a non-prescription dietary supplement. To the best of our review of the literature, the
catabolism of levulinate has not been investigated in mammalian cells. We found one
report on the identification of the reduced form of levulinate, 4-hydroxypentanoate, in the
urine of children with β-ketothiolase deficiency treated with intravenous calcium
levulinate (3). We also found an unexplained report of induction of lactic acidosis in
premature babies treated with oral calcium levulinate for hypocalcemia (4).
Our interest in levulinate arose from our recent study of the metabolism of 4-
hydroxyacids which are products of lipid peroxidation (C9, C6) or drugs of abuse (C4, C5)
(5,6). We showed that 4-hydroxyacids with 5 or more carbons are metabolized by two
new pathways. The first pathway involves the isomerization of 4-hydroxyacyl-CoAs to 3-
hydroxyacyl-CoAs via 4-phosphoacyl-CoAs, a new class of acyl-CoA esters. After
isomerization, the 3-hydroxyacyl-CoAs are catabolized via classical β-oxidation to acetyl-CoA and, in the case of odd-chain 4-hydroxyacids, to propionyl-CoA. The second pathway involves a sequence of beta, alpha and beta-oxidation steps leading to acetyl-
CoA, formate and, in the case of even-chain 4-hydroxyacids, to propionyl-CoA.
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The five-carbon 4-hydroxypentanoate, as a racemic mixture, is a new drug of abuse used in lieu of 4-hydroxybutyrate, which is now a controlled substance in the US (7-11).
Based on our previous work, we hypothesized that calcium levulinate could be used as a precursor of 4-hydroxypentanoate, i.e., as a pro-drug of abuse. The reduction of levulinate to 4-hydroxypentanoate would occur in the liver and other organs, possibly catalyzed by NADH- and/or NADPH- dependent dehydrogenase(s) (Fig. 6.1, Reaction
2). We further hypothesized that the reduction of levulinate to 4-hydroxypentanoate could be stimulated by ethanol ingestion. The oxidation of ethanol in liver increases the
[NADH]/[NAD+] ratios in cytosol and mitochondria (12), and would stimulate the
reduction of levulinate by a hepatic NADH-dehydrogenase. We also considered that the
metabolism of levulinate in cells could generate both enantiomers of 4-
hydroxypentanoate. Lastly, we hypothesized that the metabolism of levulinate and 4-
hydroxypentanoate would follow the reactions of the scheme outlined in Figure 6.1. This
scheme has features demonstrated previously, i.e., the isomerization of 4-
hydroxypentanoyl-CoA to 3-hydroxypentanoyl-CoA via 4-phosphopentanoyl-CoA
followed by β-oxidation to propionyl-CoA + acetyl-CoA (Pathway A). In addition, we
hypothesized that 4-hydroxypentanoyl-CoA and levulinyl-CoA are degraded by two
parallel β-oxidation processes: pathways B (previously described for 4-hydroxynonanoate
(5)) and B’ (hypothetical) that converge at 3-keto-4-hydroxypentanoyl-CoA (Fig. 6.1).
The latter would undergo thiolytic cleavage to acetyl-CoA + the putative lactyl-CoA
which has not been identified in mammalian cells. Lactyl-CoA could be hydrolyzed to
lactate, and/or α-oxidized to acetyl-CoA + formate.
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13 To test these hypotheses, we tried to design a [ Cn]levulinate substrate which would yield (i) different mass isotopomers1 of the two acetyl-CoAs presumably derived from C-
1+2 and C-4+5 of levulinate, and (ii) allow tracing the production of formate from C-3 of
13 levulinate. In our previous study, we had designed 4-hydroxy-[3,4- C2]nonanoate which yielded M2 and M1 acetyl-CoA via Pathways A and B, respectively. However, we could
13 not design a single [ Cn]levulinate which would allow tracing of all the processes
13 outlined in Fig. 6.1. We settled on M5 [ C5]levulinate for most of the planned
13 13 experiments. We also synthesized [3- C]levulinate and [1,2,4,5- C4]levulinate to test
for the production of formate from C-3 of levulinate.
We previously showed that, among livers perfused with one of the C4 to C11 4-
hydroxyacids, the highest concentrations of 4-phosphoacyl-CoA and related acyl-CoAs
were found in livers perfused with 4-hydroxypentanoate (see Figs 2 and 3S of (5)). This
raised the question of whether the metabolism of levulinate ± ethanol in the liver would
lead to substantial CoA trapping, a process linked to a number of perturbations of
intermediary metabolism (13). We tested the above hypotheses in live rats and in
perfused rat livers using a combination of metabolomics (14,15) and mass isotopomer
analysis (16). Our data confirm that levulinate is converted to 4-hydroxypentanoate, and
that it is metabolized by the three pathways outlined in Figure 6.1.
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EXPERIMENTAL PROCEDURES
Materials.
Sigma-Aldrich-Isotec supplied most chemicals and the following isotopically labeled
2 13 2 compounds: [ H7]butyric acid, [ C3]propionic acid, [ H9]pentanoic acid, sodium
13 13 2 [ C2]acetate and sodium [ C]formate. [3,3,5,5,5- H5]Levulinate was prepared by
2 2 isotopic exchange between unlabeled levulinate, H2O and NaO H (17). 4-Hydroxy-
2 2 [3,3,4,5,5,5- H6]pentanoate was prepared by reducing [3,3,5,5,5- H5]levulinate with
2 2 NaB H4. The same procedure was used to prepare (R,S)-3-hydroxy-[ H5]pentanoate from
13 13 13 3-ketopentanoate ethyl ester. Variously C-labeled levulinates ([ C5], [3- C] and
13 [1,2,4,5- C4]) were prepared as in ref (18). The purity of synthesized compounds was
verified by gas chromatography-mass spectrometry (GC-MS) and NMR. The lactones of
all 4-hydroxypentanoates were hydrolyzed with 10% excess NaOH at 60°C for 1 h.
2 2 [ H5]Propionyl-CoA and [ H9]pentanoyl-CoA (internal standards for acyl-CoA profiles)
were prepared from the acids as described in ref (19).
In vivo experiments.
Overnight-fasted male Sprague-Dawley rats were anesthetized with 2% isoflurane and
fitted with carotid and jugular catheters. After 20 min equilibration, Na-levulinate was
infused intravenously, as a 150 mM solution, at 2, 4, 6, 8, 10 or 12 μmol min−1 kg−1 for 2
h. In half of the rats, a bolus of 10% ethanol (1.7 M) in saline was injected intraperitoneally at -15 min in an amount calculated to achieve 10 mM in total body
water. This was followed by a constant intravenous ethanol infusion at 40
μmol min−1 kg−1. Arterial blood was sampled every 20 min for 2 h.
94
Perfused liver experiments.
Four groups of livers from male rats (200-250 g) were perfused (20) with recirculating
bicarbonate buffer containing 4% dialyzed, fatty acid-free, bovine serum albumin, 4 mM
glucose, ± 2 mM M5 levulinate ± 20 mM ethanol (or 20 mM ethanol without levulinate).
Perfusate was sampled every 20 min. Livers were quick-frozen at 2 h.
Analytical Procedures.
The concentrations and mass isotopomer distributions of the various acids, ketoacids and
hydroxyacids were assayed by GC-MS of trimethylsilyl or pentafluorobenzyl derivatives,
using analog unlabeled or labeled compounds as internal standards. The concentration
and labeling of acetate and formate were assayed using negative chemical ionization of
the pentafluorobenzyl derivatives. The concentrations and mass isotopomer distributions
of acyl-CoA esters were assayed as in ref (5). The chromatographic conditions optimize
the separation of short- and medium-chain acyl-CoAs. Since the peaks of levulinyl-CoA and 4-hydroxypentanoyl-CoA partially overlapped (retention times: 14.3 and 14.7 min, respectively), peak integrations were conducted on the front 50% of the levulinyl-CoA peak and the rear 50% of the 4-hydroxypentanoyl-CoA peak. For the assay of the concentrations of new acyl-CoA esters for which unlabeled and labeled standards are not
available, we used a calibration curve of acetyl-CoA concentration with an internal
2 standard of [ H9]pentanoyl-CoA prepared from the acid (19).
To assay the distribution of chiral enantiomers of 4-hydroxypentanoate and lactate, we
prepared the (R)-2-butyl-O-acetyl derivatives by a modification of Struys et al.’s
95
procedure (21). This procedure involves reacting the samples with 2(R)-butanol + HCl, extracting the 2(R)-butyl hydroxyester, and acetylating the latter with acetic anhydride.
We achieved the double derivatization in one step by reacting the samples with 2(R)-
butanol + acetyl chloride. The derivatives were assayed by electron ionization GC-MS.
The concentration of ethanol in rat plasma was assayed by head-space GC-MS with an
internal standard of 1-propanol (22).
Pilot enzymatic assays.
Rat liver mitochondria and cytosolic extracts (100,000 x g supernatants) were prepared as in (23). Mitochondria (2.5 or 6 mg) were incubated in 1.2 ml buffer (100 mM KCl, 50
mM MOPS, 5 mM K-phosphate, 1 mM EGTA, pH 7.4) containing 4.2 mM glutamate,
and 6 μM rotenone at 30°C in Mitocell MT200 incubation chambers. The kinetics were started by adding 5 mM acetoacetate (controls) or levulinate. Incubations were conducted for 30 min (acetoacetate) or 120 min (levulinate) with multiple sampling.
Cytosolic extracts (2.4 mg protein) were incubated for up to 2 h with 0.3 mM NADPH and 2.8 mM levulinate in a total volume of 3.5 ml 50 mM potassium phosphate buffer pH
7.4 at 30°C. The production of 4-hydroxypentanoate was assayed as the trimethylsilyl derivative and as the chiral 2(R)-butyl-O-acetyl derivative.
Calculations.
Correction of measured mass isotopomer distributions for natural enrichment was performed using the CORMAT software (24). The data points shown in the figures represent means of duplicate GC-MS or LC-MS/MS injections, which differed by <2%.
96
The statistical differences between some profiles were tested using an unpaired t test
(Graph Pad Prism Software, version 3).
97
RESULTS
Reduction of levulinate to 4-hydroxypentanoate in vivo and in isolated livers; stimulation
by ethanol.
In orientation in vivo experiments, we infused anesthetized rats with Na-levulinate at
rates ranging from 2 to 12 μmol min−1 kg−1. At infusion rates of levulinate from 2 to 6
μmol min−1 kg−1, plasma levulinate concentrations plateaued at up to 0.8 mM (not
shown). At higher rates of levulinate infusions (6 to 12 μmol min−1 kg−1), the levulinate
concentration kept increasing almost linearly. In all cases, we observed the accumulation
of 4-hydroxypentanoate which increased linearly without plateauing at all rates of
levulinate infusion, reaching 0.47 mM after 2 h of levulinate infusion at 12
μmol min−1 kg−1 (not shown). Because this plasma 4-hydroxypentanoate concentration is
in the range measured in humans addicted to 4-hydroxypentanoate (7,11), we settled on a
levulinate infusion rate of 12 μmol min−1 kg−1 for our rat in vivo experiments. We infused 12 overnight-fasted anesthetized rats with 12 μmol min−1 kg−1 levulinate for 2 h.
At -15 min, 6 rats received an intraperitoneal injection of 10% ethanol in saline. The dose was calculated to induce ethanol concentrations of about 10 mM in total body water.
The same 6 rats were infused intravenously with 10% ethanol in saline at 40
μmol min−1 kg−1 from 0 to 120 min to compensate for ethanol metabolism. This rate of
infusion corresponds to the reported activity of alcohol dehydrogenase in the whole liver
(12). The plasma ethanol concentration at 120 min was 3.7 ± 1 mM (n = 6). Because the
half-maximal ethanol uptake by perfused rat livers was observed at a perfusate
concentration of 0.25 mM (25), ethanol oxidation was operating at maximal capacity
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throughout the experiment. The other 6 rats, used as controls (infused with levulinate but no ethanol), were injected and infused with saline.
Figure 6.2 shows the profiles of plasma levulinate and 4-hydroxypentanoate concentrations in the two groups of rats. Compared to the levulinate controls, ethanol administration resulted in higher plasma levulinate and 4-hydroxypentanoate concentrations, and significantly increased [lactate]/[pyruvate] ratios (not shown). These experiments demonstrated that (i) levulinate is reduced to 4-hydroxypentanoate in vivo,
(ii) ethanol stimulates the reduction of levulinate to 4-hydroxypentanoate, and (iii) ethanol decreases total levulinate metabolism in vivo. However, because we do not know the volumes of distribution of levulinate and 4-hydroxypentanoate in rats, we could not calculate metabolic rates from the data of Figure 6.2. This is why we turned to perfused rat liver experiments.
We perfused 4 groups of isolated rat livers for 2 h with recirculating buffer containing initially 4 mM glucose and either (i) nothing (controls), (ii) 2 mM M5 levulinate, (iii) 2 mM M5 levulinate + 20 mM ethanol, or (iv) 20 mM ethanol. Figure 6.3 shows for groups (ii) and (iii), which contained M5 levulinate, the total levulinate uptake and 4- hydroxypentanoate production over 2 h. Contrary to what we observed in vivo, ethanol almost doubled the uptake of levulinate by the liver. Also, ethanol tripled the production of 4-hydroxypentanoate from levulinate by the isolated liver. Ethanol shifted the distribution of levulinate metabolism between 4-hydroxypentanoate production and catabolism from 1/1 to 4/1. Chiral assay of 4-hydroxypentanoate in the final perfusates
99
of groups (ii) (levulinate) and (iii) (levulinate + ethanol) revealed that the proportions of
the (R)- enantiomer were 84 ± 0.3% and 79 ± 1.4%, respectively (p = 0.007).
Evidence that levulinate and 4-hydroxypentanoate are catabolized as outlined in Figure
6.1.
The putative pathways of levulinate metabolism were confirmed by the identification of a
number of acyl-CoAs, as well as by the assays of their concentrations and their mass
isotopomer distributions (Tables 6.1 and 6.2). Further confirmation was obtained from the mass isotopomer distribution of some carboxylic acids (Table 6.2). In livers perfused
with an initial 2 mM of M5 levulinate ± 20 mM ethanol, the three C5 acyl-CoAs initially
derived from the substrate (levulinyl-CoA, 4-hydroxypentanoyl-CoA and 4-
phosphopentanoyl-CoA, Fig 6.1) accumulated in large amounts (Table 6.1). These three
acyl-CoAs, which are the initial intermediates of Pathways B’, B and A, respectively
(Fig. 6.1), were only M5 labeled as expected. Consistent with Pathway A, propionyl-
CoA was 24% and 47% M3-labeled in livers perfused with M5 levulinate or M5
levulinate + ethanol, respectively (Table 6.2). We did not identify the products of 4-
hydroxypentanoyl-CoA isomerization, i.e., 3-hydroxypentanoyl-CoA and the latter’s
oxidized form, i.e., 3-ketopentanoyl-CoA in liver tissue. However, we identified the corresponding 3-hydroxypentanoate in the perfusate. Unexpectedly, the mass isotopomer
distribution of the latter was not only M5, but was mostly M3 with a small component of
M5 (Table 6.2). The only possible explanation of the isotopomer distribution of 3-
hydroxypentanoate is the combined reversibility of the reactions catalyzed by 3-
hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase (Pathway A) in intact
100 liver cells. This combined reversibility results in the exchanges of (i) the C-1+2 moiety of 3-hydroxypentanoyl-CoA with free acetyl-CoA, and (ii) the C-3+4+5 moiety of 3- hydroxypentanoyl-CoA with propionyl-CoA. However, the propionyl moiety exchanges much more slowly than the acetyl moiety (26). This explains the much lower M2 than
M3 enrichment of 3-hydroxypentanoate (Table 6.2). The very different rates of exchange by thiolase of the moieties of 3-ketoacyl-CoAs had been originally reported by Hüth et al. for acetoacetyl-CoA (27).
Consistent with Pathways B and B’, we identified the six C5 acyl-CoAs which we had hypothesized to derive from 4-hydroxypentanoyl-CoA and levulinyl-CoA (Fig. 6.1). The identifications were based on LC-MS/MS with specific mother/daughter ion relationships and on the mass isotopomer distributions of the compounds. The mass isotopomer distribution was instrumental at differentiating the isomeric compounds 4-keto-3- hydroxypentanoyl-CoA and 3-keto-4-hydroxypentanoyl-CoA (Fig. 6.1) which have the same mother/daughter ion pairs. We had anticipated that 3-keto-4-hydroxypentanoyl-
CoA, a presumed substrate of a reversible thiolase, would have a component of M3 labeling as a result of isotopic exchange with acetyl-CoA. This was the case: 56% M5 and 44% M3. In contrast, we anticipated that 4-keto-3-hydroxypentanoyl-CoA, which would not be a substrate for a thiolase, would be mostly M5 labeled. This was the case:
94% M5.
3,4-Dihydroxypentanoyl-CoA (Fig. 6.1, Pathway B) yielded two peaks, in relative abundances of about 40:60, because it is a mixture of diastereomers. The OH on C-3 is
101
(S)- because of the specificity of enoyl-CoA hydratase. The OH on C-4 is either (R)- or
(S)- because 4-hydroxypentanoate derived from levulinate is 85% (R)- and 15% (S)-
(more on this in the Discussion). The two diastereomers of 3,4-dihydroxypentanoyl-CoA
had similar mass isotopomer distributions: 83% M5 and 15% M3, vs 87% M5 and 11%
M3 (Table 6.2). The presence of M3 isotopomers of the two diastereomers of 3,4-
dihydroxypentanoyl-CoA shows that the acetyl moiety of each diastereomer exchanges
with acetyl-CoA via 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase.
We did not identify lactyl-CoA which would be formed, after the convergence of
Pathways B and B’, via thiolytic cleavage of 3-keto-4-hydroxypentanoyl-CoA. However,
we found that perfusate lactate was mostly M3 labeled, and was more M3 labeled than
pyruvate (Table 6.2). This showed a precursor to product relationship of lactate to
pyruvate supporting the formation of M3 lactate by hydrolysis of the putative lactyl-CoA.
Our hypothetical scheme of levulinate catabolism involved the α-oxidation of lactyl-CoA
which would yield formate. As expected, M1 formate accumulated in perfusions with
M5 levulinate (Fig. 6.4). To check whether the production of M1 formate derived indeed
from C-3 of levulinate, we perfused 2 livers with [3-13C]levulinate and 2 livers with
13 13 [1,2,4,5- C4]levulinate. As expected M1 formate was generated from [3- C]levulinate.
13 To our surprise, M1 formate was also generated from [1,2,4,5- C4]levulinate. Fig. 6.4
shows the concentrations of M1 formate at 120 min in the perfusates of the experiments
13 13 13 with [ C5]levulinate (± ethanol), [3- C]levulinate and [1,2,4,5- C4]levulinate. The data
are presented as M1 formate concentrations because of the ubiquitous contamination of
102
reagents with traces of unlabeled formate. Formyl-CoA, a very unstable intermediate of
fatty acid α-oxidation (28), was not detected in any of the livers. We suspected that M1
formate was formed, not from C-3 of levulinate via α-oxidation, but by an indirect route:
levulinate ➔➔ propionyl-CoA (via Pathway A) ➔ anaplerosis ➔ citric acid cycle
intermediates ➔ PEP ➔➔ 3-phosphoglycerate ➔➔ serine ➔ glycine ➔ formate.
Consistent with this long process, we found in the final perfusates of experiments with
13 13 13 [ C5]levulinate, [3- C]levulinate or [1,2,4,5- C4]levulinate, that tissue glycine was M1 labeled in the range of 0.6 to 2.1% without significant difference between the labels.
13 Thus, the production of M1 formate from [ Cn]levulinate does not reflect an α-oxidation
process generating formate from C-3 of levulinate. However, we cannot exclude that a
very small amount of formate is formed by α-oxidation of lactyl-CoA.
13 Last, in perfusions with [ C5]levulinate ± ethanol, the M2 enrichment of acetyl-CoA was
4.2 and 8.0%, respectively. Based on the Scheme of Fig. 6.1, the catabolism of 1 molecule of levulinate yields 1 acetyl-CoA via Pathway A or 1 to 2 acetyl-CoA via
Pathways B + B’. Thiolytic cleavage of 3-keto-4-hydroxypentanoyl-CoA yields 1 acetyl-
CoA. Decarboxylation of pyruvate derived from lactate can generate up to 1 more acetyl-CoA. However, there is very little pyruvate decarboxylation in livers from overnight-fasted rats, as demonstrated by Koeppe et al. from the labeling pattern of liver glutamate after injection of [2-14C]pyruvate (29). The decarboxylation of pyruvate is
further decreased by ethanol oxidation which inhibits citric acid cycle activity (12).
Therefore, only 1 acetyl-CoA is formed per molecule of levulinate. Thus, the M2
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enrichment of acetyl-CoA represents the contribution of levulinate to acetyl-CoA
production.
Pilot enzymatic assays.
To test for the existence of a mitochondrial dehydrogenase that would reduce levulinate, we incubated intact liver mitochondria with glutamate + rotenone (NADH-generating system) + 5 mM levulinate at 30°C. We observed the production of 4-hydroxypentanoate which was assayed by GC-MS of the trimethylsilyl derivative. The production of 4- hydroxypentanoate was linear with time for 120 min, and with protein concentration (for
0.25 to 0.75 mg protein per incubation). The apparent activity at 5 mM levulinate and
30°C was 0.48 ± 0.03 nmol min−1 (mg protein)−1 or 12.0 ± 0.8 nmol min−1 (g liver)−1 (SE, n = 7) based on 25 mg mitochondrial protein/g liver. In parallel control incubations of mitochondria with 5 mM acetoacetate instead of levulinate, the production of 3- hydroxybutyrate was 32 ± 1.3 nmol min−1 (mg protein)−1 or 789 ± 32 nmol min−1 (g liver)−1 (n = 7). The rates of acetoacetate reduction are similar to published values.
Chiral GC-MS assays showed that 85 ± 0.5% of the 4-hydroxypentanoate formed from levulinate was the (R)-enantiomer (n = 7 different livers). In the same chiral assay, a pure
standard of (R)-4-hydroxypentanoate (Sigma) yielded an apparent 97% of the (R)- enantiomer.
To test for a cytosolic dehydrogenase that would reduce levulinate, we incubated a
100,000 x g supernatant of liver homogenate with levulinate + NADPH. We observed a
linear production of 4-hydroxypentanoate. The apparent activity at 30°C was 0.043 ±
104
0.003 nmol min−1 (mg protein)−1 or 4.6 ± 0.36 nmol min−1 (g liver)−1 (SE, n = 6). When the assay was conducted in the presence of NADH, the activity was less than 20% of the activity measured with NADPH. Chiral GC-MS assay of 4-hydroxypentanoate formed in one cytosolic incubation showed 94% of the (R)-enantiomer. Because of the low sensitivity of the chiral assay, this assay was conducted on the pooled cytosolic extracts
of 6 livers.
The total apparent activity (mitochondrial + cytosolic) of enzymes that reduce levulinate
to 4-hydroxypentanoate in vitro, is about 16.5 ± 0.95 nmol min−1 (g liver wet wt)−1 at
30°C. This rate is equivalent to 82.5 nmol min−1 (g liver dry wt)−1 at 37°C, assuming a
(37°C)/(30°C) activity ratio of 1.5 and a (dry weight)/(wet weight) of 0.31 for in vivo liver (16.5 x 1.5 ÷ 0.31 = 82.5). This rate, measured in subcellular fractions of non- perfused livers, accounts for 62% and 21% of the rates of production of 4- hydroxypentanoate measured in rat livers perfused with 2 mM levulinate ± ethanol (134 and 390 nmol min−1 (g liver dry wt)−1, respectively at 37°C (see Fig. 6.3, upper sections
of bars)). The above pilot enzymatic assays which identify activities that convert
levulinate to 4-hydroxypentanoate are the basis of future studies which will characterize
the kinetics of the uncovered activities.
105
DISCUSSION
Interconversion of levulinate and 4-hydroxypentanoate.
In live rats and in perfused rat livers, we demonstrated the reduction of levulinate to 4- hydroxypentanoate (Figs 6.2, 6.3). Also, in livers perfused with (R,S)- or (R)-4- hydroxypentanoate we observed the accumulation of levulinate (not shown). Thus, there is at least one dehydrogenase system that interconverts levulinate and 4- hydroxypentanoate in liver. We could not find any report on an enzyme system that interconverts 4-ketoacids and 4-hydroxyacids longer than 4 carbons in mammalian cells.
Kaufman’s group (30-32) reported on two enzymes that interconvert succinic semialdehyde and 4-hydroxybutyrate: a cytosolic NADP+-dehydrogenase and a
mitochondrial transhydrogenase catalyzing the reaction 4-hydroxybutyrate + α- ketoglutarate ↔ succinate semialdehyde + α-hydroxyglutarate. The transhydrogenase
has an absolute requirement for α-ketoglutarate.
In our pilot enzymatic studies, the slow cytosolic NADPH-dependent reduction of
levulinate we detected may be catalyzed by the cytosolic 4-hydroxybutyrate
dehydrogenase described by Kaufman (30). The more rapid reduction of levulinate in
mitochondria incubated with glutamate and rotenone must be catalyzed by one or more
enzymes different from Kaufman’s transhydrogenase. The reduction of levulinate by the
4-hydroxybutyrate transhydrogenase would be presumably inhibited by α-ketoglutarate
derived from glutamate. Since the addition of glutamate to mitochondria forms a NADH
generating system, the reduction of levulinate to 4-hydroxypentanoate is most likely
catalyzed by at least one NADH-dehydrogenase. This conclusion is supported by the
106
stimulation of levulinate reduction by ethanol in perfused livers (Fig. 6.3). The oxidation
of ethanol leads to marked increases in the cytosolic and mitochondrial [NADH]/[NAD+] ratios (12). Both enantiomers of 4-hydroxypentanoate were generated in mitochondria incubated with levulinate (85% (R)) and in livers perfused with levulinate alone (84%
(R)), or with levulinate + ethanol (79% (R)).
The data suggest that the reduction of levulinate in liver mitochondria is catalyzed by either (i) one (R)- and one (S)-NADH-dehydrogenase, (ii) one (R)-NADH-dehydrogenase and a racemase, or (iii) one (R)-NADH-dehydrogenase and a racemase system. The (R)- dehydrogenase could be (R)-3-hydroxybutyrate dehydrogenase, the enzyme that interconverts the physiological ketone bodies, acetoacetate and (R)-3-hydroxybutyrate
(33). In our mitochondria incubations, the rate of levulinate reduction to 4- hydroxypentanoate was 1.6% of the rate of acetoacetate reduction to 3-hydroxybutyrate.
Thus, the reduction of levulinate may be a side reaction of (R)-3-hydroxybutyrate dehydrogenase which also interconverts (R)-3-hydroxypentanoate and 3-ketopentanoate
(26,34).
A possible racemase system would follow the sequence: levulinate ➔ levulinyl-CoA ➔
(S)-4-hydroxypentanoyl-CoA ➔ (S)-4-hydroxypentanoate (Fig. 6.1., reactions 1, 3 and reversal of reaction 1). Although there is no information on the enzyme that interconverts levulinyl-CoA and 4-hydroxypentanoyl-CoA, it may be similar (or identical) to the (S)-3-hydroxyacyl-CoA dehydrogenase involved in fatty acid β-
107
oxidation. A fraction of the (S)-4-hydroxypentanoyl-CoA would be hydrolyzed to (S)-4-
hydroxypentanoate.
If the above interpretation is correct, (S)-4-hydroxypentanoyl-CoA is formed from
levulinyl-CoA via (S)-3-hydroxyacyl-CoA dehydrogenase or a similar enzyme. (R)-4- hydroxypentanoyl-CoA is formed via the activation of (R)-4-hydroxypentanoate derived from the reduction of levulinate by (R)-3-hydroxybutyrate dehydrogenase. The 15% (S)- component of 4-hydroxypentanoate derived from levulinate represents a minimal (S)- component of 4-hydroxypentanoyl-CoA.
Ethanol decreases slightly but significantly the fraction of (R)-4-hydroxypentanoate derived from levulinate in perfused livers (from 84 to 79%). This effect results probably from the inhibition of the three oxidation reactions catalyzed by 3-hydroxyacyl-CoA dehydrogenase (Fig. 6.1, reactions 6) by the increase in the [NADH]/[NAD+] ratio
resulting from ethanol oxidation. The decrease in the catabolism of (S)-4- hydroxypentanoyl-CoA probably increases its hydrolysis.
Pathways of levulinate catabolism.
In our previous study (5), we showed that 4-hydroxyacids with at least 5 carbons are metabolized by two pathways listed in Fig 6.1 as A and B. Pathway A involves the isomerization of 4-hydroxyacyl-CoAs to 3-hydroxyacyl-CoAs via 4-phosphoacyl-CoAs.
Pathway B involves a sequence of β-oxidation and α-oxidation steps. In the present study, we demonstrated that an additional β-oxidation process (pathway B’) proceeds
108
from levulinyl-CoA in parallel to pathway B, linking with the latter at 3-keto-4-hydroxy-
pentanoyl-CoA. Pathways B and B’ differ by the presence of a hydroxy or a keto group
on carbon 4 of the five-carbon intermediates. The identification of acyl-CoAs which are intermediates in pathways B and B’ (Fig. 6.1) demonstrates that the presence of a keto or a hydroxy group on C-4 of a fatty acid does not prevent any of the reactions of β- oxidation to proceed. In perfusions with (R,S)-4-hydroxypentanoate, the two enantiomers are taken up at the same rate (not shown). One can thus conclude that the configuration of the hydroxyl on C-4 of some acyl-CoA metabolites of levulinate does not affect their flux rates through Pathways A and B (in Pathway B’, C-4 is a carbonyl). Further information on the enantiomeric and diastereomeric distribution of levulinate metabolites will require the development of mass spectrometry techniques to measure the chiral composition of hydroxy- and dihydroxyacyl-CoAs.
In the presence of ethanol, we observed marked increases in the uptake of levulinate and in its conversion to 4-hydroxypentanoate by perfused livers. The increase in the
[NADH]/[NAD+] induced by ethanol oxidation (12) explains the increase in the reduction
of levulinate to 4-hydroxypentanoate. The subsequent decrease in intracellular levulinate
concentration stimulates levulinate uptake. The small but significant decrease in the
catabolism of levulinate (Fig. 6.3, lower bars) results from the inhibition of the three
reactions catalyzed by 3-hydroxycyacyl-CoA dehydrogenase in Pathways A, B and B’
13 (Fig. 6.1., reactions 6). In an apparent contradiction, the inhibition of [ C5]levulinate catabolism by ethanol was accompanied by marked increases in the M2 enrichment of acetyl-CoA (from 4.2 to 8%, Table 6.2) and in the M3 enrichment of propionyl-CoA
109
(from 24 to 47%). The most likely explanation of these shifts in metabolite enrichments
is that the production of unlabeled acetyl-CoA and propionyl-CoA from endogenous
long-chain fatty acids and amino acids was more inhibited by ethanol oxidation than the
13 production of labeled acetyl-CoA and propionyl-CoA from [ C5]levulinate. When
ethanol is oxidized, the total acetyl-CoA production by the liver is decreased because the
respiratory chain is fueled mostly by reducing equivalents formed in the oxidation of
ethanol to acetate (12).
13 In livers perfused with [ C5]levulinate ± ethanol, the mass isotopomer distribution of lactate and pyruvate (Table 6.2) is compatible with labeled lactate being formed directly from levulinate rather than from pyruvate. In most cases, pyruvate is the precursor of lactate, except when lactate is generated from the oxidation of 1,2-propanediol (35). In
13 the presence of [ C5]levulinate, the higher M3 enrichment of lactate compared to
pyruvate demonstrates a precursor to product relationship where lactate is the precursor
of pyruvate. This occurred in spite of the production of unlabeled pyruvate from glucose
and aminoacids. The enrichment ratio (M3 lactate)/(M3 pyruvate) increased from 1.6 to
5.1 in the presence of ethanol. This reflects the decreased isotopic equilibration between
lactate and pyruvate as a result of the increase in the [NADH]/[NAD+].
13 Although the M3 isotopomer of lactate is formed from [ C5]levulinate, M2 and M1
isotopomers of lactate were also detected. This reflects the redistribution and loss of
label in the pyruvate ➔ oxaloacetate ➔ PEP ➔ pyruvate cycle with continuous partial
isotopic equilibration between lactate and pyruvate. Although some (S)-lactate should be
110
formed from (S)-4-hydroxypentanoyl-CoA via Pathway B (Fig. 6.1), no (S)-lactate was
detected in the perfusate. This results from the abundant formation of (R)-lactate (L-
lactate) via glycolysis and other reactions of intermediary metabolism.
Ethanol increased levulinate uptake in isolated livers, but it decreased the disposal of levulinate in vivo (compare Figs 6.2 and 6.3). Since peripheral tissues do not oxidize ethanol, one cannot consider an inhibition of levulinate catabolism by an increase in the
[NADH]/[NAD+] ratio. A more likely explanation involves the peripheral oxidation of
acetate derived from ethanol oxidation in liver. The group of Hellerstein has documented
a major decrease (73%) in whole-body lipid oxidation in humans after ethanol ingestion
(36). Because levulinate catabolism yields acetyl-CoA, its catabolism is inhibited by
another precursor of acetyl-CoA, i.e., acetate. The latter is used rapidly by peripheral
tissues.
Scope and public health relevance.
Although calcium levulinate has been administered to humans for more than a century, it
is remarkable that the metabolism of levulinate in mammalian cells has not been
investigated. One could then reasonably assume that, at the doses used for dietary
calcium supplementation or for the occasional intravenous treatment of hypocalcemia,
calcium levulinate is probably a safe compound (2). However, some tuberculosis patients
treated orally with calcium levulinate (1 to 4 grams per day) experienced loss of appetite
and nausea (2). Also, in premature babies treated with oral calcium levulinate for
hypocalcemia, serious lactic acidosis developed (4). This unexplained metabolic
111
perturbation vanished when calcium levulinate was replaced by calcium gluconate. In
other children treated with calcium levulinate, the presence of 4-hydroxypentanoate and
levulinate in urine was reported as an incidental analytical finding without firm
biochemical explanation (3). While we recognize that there are differences in the
metabolism of xenobiotics between humans and rats, we strongly feel that our data are
relevant to human health.
The recent use of 4-hydroxypentanoate as a drug of abuse (more toxic than 4-
hydroxybutyrate (11)) led us to hypothesize that levulinate could be used as a pro-drug.
This hypothesis was prompted by our finding that 4-hydroxynonanoate (a metabolite of
the lipid peroxidation product 4-hydroxynonenal) reversibly interconverts with 4-
ketononanoate in perfused rat livers (5). The reduction of levulinate to 4-
hydroxypentanoate in liver might be stimulated by ethanol oxidation as a result of the
increase in cytosolic and mitochondrial [NADH]/[NAD+] ratios (12). Two other public
health concerns resulted from the very high accumulation, in livers perfused with 4-
hydroxypentanoate, of 4-phosphopentanoyl-CoA and a number of related acyl-CoAs
((5)). First, in mice with 4-hydroxybutyrate aciduria resulting from a deficiency in
succinic semialdehyde dehydrogenase (37), the concentration of 4-phosphobutyryl-CoA
in liver and brain was 40 times greater than in wild type mice (5). The accumulation of
4-phosphobutyryl-CoA may be related to the severe epileptic seizures suffered by these mice, and to the inability to reason in persons intoxicated with 4-hydroxybutyrate.
Second, substantial CoA trapping by metabolites of levulinate might interfere with a
112
number of CoA-dependent reactions of intermediary metabolism (13), and might explain
the lactic acidosis observed in premature babies treated with calcium levulinate (4).
Our concerns about CoA trapping and the accumulation of 4-phosphoacyl-CoA are
supported by the concentration profiles of acyl-CoAs derived from levulinate in livers
perfused with 2 mM levulinate ± 20 mM ethanol (Table 6.1). The first three acyl-CoAs
derived from levulinate, i.e., levulinyl-CoA, 4-hydroxypentanoyl-CoA and 4-
phosphopentanoyl-CoA accumulated to high levels. This resulted in large decreases in
the concentrations of acetyl-CoA, propionyl-CoA, methylmalonyl-CoA, succinyl-CoA
and malonyl-CoA, compared to controls. The large decrease in acetyl-CoA concentration
induced by levulinate is somewhat blunted by the addition of ethanol, presumably
because ethanol oxidation generates acetate which is activated to acetyl-CoA. Of
particular concern is the very high concentration of 4-phosphopentanoyl-CoA in livers
perfused with levulinate + ethanol. After ingestion of calcium levulinate, one can expect
a high concentration of levulinate in the portal vein. The stimulation by ethanol of
levulinate uptake and conversion to 4-hydroxypentanoate (Fig. 6.3) should result in the release of high concentrations of 4-hydroxypentanoate into peripheral blood (probably in higher concentrations than after the intravenous administration of levulinate (Fig. 6.2)).
High concentrations of 4-hydroxypentanoate reaching the brain may induce high concentrations of 4-phosphopentanoyl-CoA which could perturb some brain functions by acting as a neuromodulator. The drug effects of 4-hydroxypentanoate are less potent than those of 4-hydroxybutyrate (11). Addicts may ingest large doses of 4-hydroxypentanoate
(or levulinate + ethanol) to produce the desired effects. This is a potentially serious
113 public health concern which calls for an investigation of the effects of the oral ingestion of calcium levulinate + ethanol on brain metabolism and on behavior in live rodents.
114
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FIGURE LEGENDS
FIGURE 6.1. Proposed scheme for the metabolism of levulinate and 4- hydroxypentanoate. The hypothetical enzyme activities, designated by numbers in italics are: 1. acid-CoA ligase; 2. hydroxyacid dehydrogenase; 3. 4-hydroxyacyl-CoA dehydrogenase; 4. acyl-CoA dehydrogenase; 5. enoyl-CoA hydratase; 6. 3- hydroxyacyl-CoA dehydrogenase; 7. 3-ketoacyl-CoA thiolase; 8. 4-hydroxyacyl-CoA kinase; 9. acyl-CoA hydrolase; 10. alpha-oxidation enzymes. The “multiple reactions” mentioned between 4-phospho-pentanoyl-CoA and 3-hydroxypentanoyl-CoA result in the isomerization of 4-hydroxypentanoyl-CoA to 3-hydroxypentanoyl-CoA (5).
FIGURE 6.2. Profiles of plasma concentrations of levulinate (A) and 4- hydroxypentanoate (B) in rats infused intravenously with sodium levulinate ± ethanol. Data are presented as mean ± SEM (n = 6).
FIGURE 6.3. Effect of ethanol on levulinate uptake and metabolism in perfused rat livers. The lower section of each bar, labeled “To catabolism” was calculated as the difference between the uptake of levulinate and the release of 4-hydroxypentanoate. It corresponds to the disposal of levulinate via Pathways A, B and B’. Data are presented as mean ± SEM (n = 6).
13 13 FIGURE 6.4. Release of [ C]formate by rat livers perfused with [ Cn]levulinate. Shown are concentrations of [13C]formate at the end of 120 min perfusions with 13 13 13 [ C5]levulinate (n = 6), [ C5]levulinate + 20 mM ethanol (n = 6), [3- C]levulinate (n = 13 2) or [1,2,4,5- C5]levulinate (n = 2).
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TABLE LEGENDS
TABLE 6.1. Concentrations of acyl-CoAs in perfused rat livers. Livers were perfused for 2 h with buffer containing 4 mM glucose and either nothing (control), 2 mM 13 13 [ C5]levulinate (M5 LEV), 2 mM [ C5]levulinate + 20 mM ethanol (M5 LEV + ETOH) or 20 mM ethanol (ETOH). Concentrations of acyl-CoAs are expressed as nanomole/g dry weight (mean ± S.E.; n = 6). Statistics: *, significantly different from controls; †, significantly different from M5 LEV; ‡, significantly different from M5 LEV + ETOH (p < 0.05); ¢, p = 0.06 compared to M5 LEV.
13 TABLE 6.2. Mass isotopomer distribution of [ C5]levulinate metabolism in perfused rat livers. Livers were perfused for 2 h with buffer containing 4 mM glucose 13 13 and either 2 mM [ C5]levulinate or 2 mM [ C5]levulinate + 20 mM ethanol. Data are presented as percent distributions of mass isotopomers (mean ± S.E.; n = 6). The absence or presence of ethanol in the perfusate is indicated by a – or + sign in the third column. 13 Data from perfusions with [ C5]levulinate + ethanol are shown in italics. Statistics: †, significantly different from M5 LEV (p < 0.05).
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Figure 6.1.
120
Figure 6.2.
121
Figure 6.3.
122
Figure 6.4.
123
Table 6.1.
124
Table 6.2.
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6.1.2.
CYLICAL COA ESTERS DERIVED FROM LEVULINATE
Stephanie R. Harris1, Guo-Fang Zhang1, Sushabhan Sadhukhan2, Michelle A.
Puchowicz1, Vernon E. Anderson3, Gregory P. Tochtrop2, and
Henri Brunengraber1
From Departments of Nutrition1, Chemistry2, and Biochemistry3
Case Western Reserve University, Cleveland Ohio 44106.
Running head: Cyclical CoA esters derived from levulinate
Address correspondence to: Henri Brunengraber, Department of Nutrition Case Western
Reserve University, School of Medicine - 10900 Euclid Avenue, WG-48;
Cleveland OH 44106-4954. Fax: (216) 368-6560. Email: [email protected]
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ABBREVIATIONS
GC-MS, gas chromatography-mass spectrometry; LC-MS, liquid chromatography-mass spectrometry
FOOTNOTE
1. Mass isotopomers are designated as M, M1, M2,...Mn where n is the number of heavy atoms in the molecule. The isotopic enrichment of each mass isotopomer is expressed as mol percent.
ACKNOWLEDGMENTS
This work was supported by the NIH (Roadmap grant R33DK070291 and grant
R01ES013925 to H.B.; Grant RO1HL053315 to G.P.T.). We thank the Case Mouse
Metabolic and Phenotyping Center for help with the in vivo experiments.
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ABSTRACT
We recently reported that levulinate (4-ketopentanoate) is converted in the liver to
4-hydroxypentanoate, a drug of abuse, and that the formation of 4-hydroxypentanoate is stimulated by ethanol oxidation. We also identified 3 parallel β-oxidation pathways by which levulinate and 4-hydroxypentanoate are catabolized to propionyl-CoA and acetyl-
CoA. We now report that levulinate forms three seven-carbon CoA esters by processes starting with the elongation of levulinyl-CoA by acetyl-CoA to 3,6-diketoheptanoyl-CoA.
The latter gamma-diketo ester undergoes two parallel cyclization processes. One process yields a mixture of acyl-CoA tautomers, cyclopentenyl/cyclopentadienyl-acyl-CoAs. The second cyclization process yields a methyl-pyrrolyl-acetyl-CoA containing a nitrogen atom derived from the epsilon nitrogen of lysine, but without carbons from lysine. These cyclical CoA esters were identified in rat livers perfused with levulinate, and in livers and brains from rats gavaged with calcium levulinate ± ethanol. Our data suggest that 3,6- diketoheptanoyl-CoA, like 2,5-diketohexane may pyrrolate lysine residues from proteins.
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INTRODUCTION
We recently reported on new pathways of levulinate (4-ketopentanoate) metabolism (1).
This compound, as a calcium salt, is used as an oral or intravenous source of calcium. We
found that levulinate is converted in the liver to 4-hydroxypentanoate, a drug of abuse, and that the formation of 4-hydroxypentanoate is stimulated by ethanol oxidation. We
also identified 3 parallel β-oxidation pathways by which levulinate and
4-hydroxypentanoate are catabolized to propionyl-CoA and acetyl-CoA. One of the three
β-oxidation pathways involves the formation of 4-phosphopentanoyl-CoA, an
intermediate in the isomerization of 4-hydroxypentanoyl-CoA to 3-hydroxypentanoyl-
CoA. Using a combination of metabolomics and mass isotopomer analysis, we identified
10 C5 acyl-CoA esters involved in the catabolism of levulinate and 4-hydroxypentanoate.
Of these 10 acyl-CoAs derived from levulinate, 3 accumulate in large concentrations:
levulinyl-CoA, 4-hydroxypentanoyl-CoA and 4-phosphopentanoyl-CoA, resulting in
substantial CoA trapping.
In this follow-up study, we identified three C7 CoA esters derived from levulinyl-CoA.
The first ester derives from the elongation of levulinyl-CoA to 3,6-diketoheptanoyl-CoA.
The latter undergoes two parallel cyclization processes. One process yields a mixture of
acyl-CoA tautomers, cyclopentenyl/cyclopentadienyl-acyl-CoAs. The second cyclization
process yields a methyl-pyrrolyl-acetyl-CoA containing a nitrogen atom derived from the
epsilon nitrogen of lysine. These cyclical CoA esters were identified in rat livers
perfused with levulinate, and in livers and brains from rats gavaged with calcium
levulinate ± ethanol.
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EXPERIMENTAL PROCEDURES
Materials.
Sigma-Aldrich-Isotec supplied most chemicals and the following isotopically labeled
13 15 15 13 15 2 compounds: [ C6]glucose, NH4Cl, [6- N]lysine, [ C6, N2]lysine, [ H7]butyric acid,
2 13 2 [ H9]pentanoic acid, sodium [ C2]acetate. 4-Hydroxy-[3,3,4,5,5,5- H6]pentanoate was
2 2 13 prepared by reducing [3,3,5,5,5- H5]levulinate with NaB H4. [ C5]Levulinate was
prepared as in Ref. (2). 3,6-Diketoheptanoate ethyl ester was synthesized as in Ref. (3).
The purity of synthesized compounds was verified by gas chromatography-mass
2 spectrometry (GC-MS) and NMR. [ H9]Pentanoyl-CoA (internal standard for acyl-CoA
profiles) was prepared from the acid as described in Ref. (4).
In vivo experiments.
Overnight fasted male Sprague-Dawley rats weighing 200-250 g were divided into 4
groups (9 rats per group) and given an oral gavage of one of the following: (i) 2 mmol/kg
calcium levulinate; (ii) 2 mmol/kg calcium chloride (control for (i)); (iii) 2 mmol/kg
calcium levulinate + 13.4 mmol/kg ethanol, and (iv) 2 mmol/kg calcium chloride + 13.4
mmol/kg ethanol (control for (iii)). The dose of calcium levulinate per kg is equivalent to
twice the recommended daily allowance of calcium for adult humans. The dose of
ethanol was calculated to induce 20 mM in total body water, assuming instantaneous
diffusion. Rats were given the gavage at 0 min, and were killed at 7 min, followed by 15
min intervals up to 120 min. At the time of kill the following samples were taken: (i)
portal and peripheral plasma, (ii) a lobe of the liver (quick-frozen), and (iii) the whole
brain (quick-frozen).
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Perfused liver experiments.
Additional data were obtained from 2 groups of 6 rat livers perfused for 2 hr with
recirculating bicarbonate buffer containing 4% dialyzed, fatty acid-free, bovine serum albumin, 4 mM glucose, 2 mM M5 levulinate ± 20 mM ethanol (1). In addition, rat livers
13 were perfused (5) with 2 mM unlabeled levulinate and either (i) 4 mM [ C6]glucose, (ii)
13 4 mM unlabeled glucose + 10 mM [ C2]acetate, (iii) 4 mM unlabeled glucose + 5 mM
15 15 NH4Cl, (iv) 4 mM unlabeled glucose + 0.4 mM [6- N]lysine (1 h), (v) 4 mM glucose +
13 15 1 mM [ C6, N2]lysine, or (vi) 4 mM glucose + unlabeled lysine. Additional perfusions
were conducted with 4 mM unlabeled glucose + 1 mM 3,6-diketoheptanoate ethyl ester +
15 13 15 0.4 mM [6- N]lysine (1 h) or 1 mM [ C6, N2]lysine. Perfusate was sampled every 20
min. Livers were quick-frozen at the end of the experiment.
Analytical Procedures.
The concentrations and mass isotopomer distributions of the various acids, ketoacids and
hydroxyacids were assayed by GC-MS of pentafluorobenzyl derivatives, using analog
unlabeled or labeled compounds as internal standards. The concentrations and mass
isotopomer distributions of acyl-CoA esters were assayed as in Ref. (6), except that the
mass spectrometer was set to monitor masses up to 1050. The chromatographic
conditions optimize the separation of short- and medium-chain acyl-CoAs. Since the
peaks of levulinyl-CoA and 4-hydroxypentanoyl-CoA partially overlapped (retention
times: 14.3 and 14.7 min, respectively), peak integrations were conducted on the front
50% of the levulinyl-CoA peak and the rear 50% of the 4-hydroxypentanoyl-CoA peak.
For the assay of the concentrations of new acyl-CoA esters for which unlabeled and
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labeled standards are not available, we used a calibration curve of acetyl-CoA
2 concentration with an internal standard of [ H9]pentanoyl-CoA prepared from the acid
(1).
Calculations.
Correction of measured mass isotopomer distributions for natural enrichment was
performed using the CORMAT software (7). The data points shown in the figures
represent means of duplicate GC-MS or LC-MS/MS injections, which differed by <2%.
The statistical differences between some profiles were tested using an unpaired t test
(Graph Pad Prism Software, version 3).
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RESULTS
Perfused liver experiments.
In reviewing the multiple reaction monitoring raw data of the assays of acyl-CoA profiles in livers perfused with M or M5 levulinate (6), we noticed two peaks (A and B) for which
the masses of the mother/daughter pairs (i) differed by 507 amu (as in all acyl-CoAs and
free CoA (8)), (ii) were heavier than all previously detected CoA esters derived from
levulinate (24 and 23 amu heavier than levulinyl-CoA, respectively), (iii) increased by 5 or 7 amu when unlabeled levulinate was replaced by M5 levulinate, and (iv) were not
present in control livers perfused without levulinate. Product ion scan analyses
confirmed that the two compounds were CoA esters because of the presence of fragments
that are typical of CoA and all CoA esters (m/z 428, 261 and 160, (8)).
We hypothesized that the two unknown CoA esters contained more than the five carbons
of levulinate. In support of this hypothesis, in livers perfused with M5 levulinate, the two
compounds were M5 and M7 labeled (Table 6.3, rows c and d). This suggested that CoA
esters A and B contained the 5 carbons of levulinate, and 2 carbons derived from the
metabolism of M5 levulinate, probably a C2 group. We had previously shown that M5
levulinate is catabolized to M2 acetyl-CoA (1). In livers perfused with M5 levulinate, the
M5 enrichment of levulinyl-CoA and the M2 enrichment of acetyl-CoA were 95% (1)
and 4.2%, respectively (Table 6.3, row c). One would predict based on probability
analysis that, if unknowns A and B derive from the elongation of levulinyl-CoA by
acetyl-CoA, their M5 and M7 enrichments should be 91% and 4% in livers perfused with
M5 levulinate. In fact, the M5 and M7 enrichments of unknowns A and B were 78-80%
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and 10%, respectively (Table 6.3, row c). A likely explanation for the discrepancy is that,
at the site of thiolase, the acetyl-CoA derived from M5 levulinate (via thiolase) has not
fully been diluted by unlabeled acetyl-CoA derived from unlabeled substrates. Thus, the
M2 enrichment of acetyl-CoA used to elongate levulinyl-CoA is greater than the 4%
measured in the liver extract.
Similar calculations apply to the labeling of the unknown CoA esters in livers perfused
with M5 levulinate + ethanol (Table 6.3, row d). There, the M5 enrichment of levulinyl-
CoA and the M2 enrichment of acetyl-CoA were 95% and 8.0%, respectively. The predicted M5 and M7 enrichments of unknowns A and B would be 87.4% and 7.6% (vs the measured 72-75 and 14-15% (Table 6.3, row d)).
The m/z of unknown A (890/383) is 24 amu more than levulinyl-CoA (866/359). So we considered what process(es) could add 24 amu to levulinyl-CoA. Addition of one acetyl
unit (+42) followed by dehydration (-18) would yield the observed 24 mass unit
difference. This suggested that unknown A is formed via the following sequence. First,
levulinyl-CoA is elongated to a seven carbon intermediate, presumably 3,6-
diketoheptanoyl-CoA (Fig. 6.5, compound (2)). The elongation could be catalyzed by an elongase using malonyl-CoA or by the reverse reaction of 3-ketoacyl-CoA thiolase using acetyl-CoA. Second, 3,6-diketoheptanoyl-CoA could, as a gamma-diketo compound, undergo spontaneous cyclization to a hydroxy-cyclopentanone intermediate (Fig. 6.5, compound (3)). Third, compound (3) could undergo dehydration, presumably via the
134
reversal of an enoyl-CoA hydratase. Fourth, the product of compound (3) dehydration
(unknown A) would be a mixture of tautomers (Fig. 6.5, compounds (4) and (5)).
The m/z of unknown B (889/382) is 23 amu more than levulinyl-CoA (866/359). There is no clear combination of addition and subtraction of Cx, Hy and Oz that would account for
the addition of 23 amu to levulinyl-CoA by standard reactions starting with elongation (+
42 amu). This suggested that nitrogen (14 amu) is part of the 23 increase in amu of
unknown B versus levulinyl-CoA. Because nitrogen is trivalent or pentavalent, it would
allow for the addition of an odd-number of mass units, such as 23, in the conversion of
levulinyl-CoA to unknown B. The suggestion that unknown B (i) is formed via the
gamma-diketo compound (2), prone to cyclization, and (ii) contains a nitrogen atom in its
acyl moiety, evoked the formation of pyrrolic compounds from the reaction between 2,5-
diketohexane (a product of hexane oxidation) and either lysine or lysine residues of
proteins (9-12). We thus hypothesized that unknown B is a pyrrolated acyl-CoA, i.e.,
compound (6) of Fig. 6.5.
To gather more information on the structures of unknowns A and B, we conducted
additional experiments with unlabeled levulinate and substrates labeled with 13C, 15N or
13 both (Table 6.3). In livers perfused with unlabeled levulinate + either [ C6]glucose (row
13 e) or [ C2]acetate (Table 6.3, row f) , unknowns A and B were only M2 labeled to a
degree similar to the corresponding M2 enrichments of acetyl-CoA. Because
13 13 [ C6]glucose and [ C2]acetate are both precursors of M2 acetyl-CoA, the data of rows e
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and f of Table 6.3 confirm that unknowns A and B contain 2 carbons derived from acetyl-
CoA.
To check whether unknown B contains a N atom, we first perfused livers with unlabeled
15 glucose + unlabeled levulinate + 5 mM NH4Cl (Table 6.3, row g). No label was
15 detected in compounds A or B. However, as expected in livers perfused with NH4Cl
(13), perfusate glutamate was 35% M1 labeled at 60 min. Therefore, if unknown B contains a N atom, this atom does not derive from non-essential aminoacid(s) which
15 become rapidly labeled from NH4Cl. To test whether unknown B contains a N atom
derived from the essential aminoacid lysine (which is involved in pyrrolation reactions),
we perfused livers with unlabeled glucose + unlabeled levulinate + either [6-15N]lysine or
13 15 15 [ C6, N2]lysine (Table 6.3, rows i and j). In the presence of [6- N]lysine, unknown B
13 15 was about 6% M1 labeled. In the presence of [ C6, N2]lysine, unknown B was about
7% M1 labeled, and no heavier mass isotopomers of B were detected. Thus, compound B
contains a N atom derived from the ε-position of lysine, but no carbon from lysine.
Lastly, we perfused livers with unlabeled glucose + unlabeled 3,6-diketoheptanoate ethyl
15 13 15 ester + [6- N]lysine or [ C6, N2]lysine. We detected unlabeled 3,6-diketoheptanoyl-CoA
(Fig. 6.5, compound (2)), unlabeled levulinyl-CoA (Fig. 6.5, compound (1)) and M1
labeled unknown B (Table 6.3, rows k and l ). Unknown A was detected but was
unlabeled. In livers perfused with 3,6-diketoheptanoate ethyl ester (and no levulinate), the
simultaneous presence of levulinyl-CoA and 3,6-diketoheptanoyl-CoA strongly supports
the hypothesis that levulinyl-CoA is elongated via a thiolase.
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In vivo experiments.
Because levulinate is ingested by humans as a calcium salt, we investigated the impact of
gavaging rats with calcium levulinate ± ethanol on the acyl-CoA profile in liver and
brain. The protocol involved an oral gavage of either: (i) 2 mmol/kg calcium levulinate;
(ii) 2 mmol/kg calcium chloride (control for (i)); (iii) 2 mmol/kg calcium levulinate +
13.4 mmol/kg ethanol, and (iv) 2 mmol/kg calcium chloride + 13.4 mmol/kg ethanol
(control for (iii)). In the 2 groups gavaged with calcium levulinate, the plasma
concentration of levulinate followed similar profiles in the absence or presence of ethanol
(Fig. 6.6A). The plasma concentration of 4-hydroxypentanoate, derived from reduction of
levulinate (Fig. 6.6B), increased faster in the presence of ethanol, based on linear
regression of the data over 80 min: calcium levulinate experiments: slope = 0.0026; R2 =
0.91; calcium levulinate + ethanol experiments: slope = 0.0044, R2 = 0.97; p < 0.002 . In
previous perfused rat liver experiments with initial 2 mM levulinate concentrations, the
reduction of levulinate to 4-hydroxypentanoate was also stimulated by ethanol (1). In
previous in vivo experiments where levulinate was infused intravenously, the co-
administration of ethanol also significantly increased the accumulation of
4-hydroxypentanoate (1). The plasma lactate and glucose concentrations in rats gavaged with levulinate ± ethanol were not different from control values (not shown).
In the livers of rats gavaged with calcium levulinate, the concentrations of the 3 most abundant CoA esters derived from levulinate (levulinyl-CoA, 4-hydroxypentanoyl-CoA
and 4-phosphopentanoyl-CoA) peaked at 45 min (Fig. 6.7A). A similar profile was
observed in livers of rats gavaged with calcium levulinate + ethanol (Fig. 6.7B). The sum
137
of the 3 concentrations peaked at 45 min at levels of 645 and 755 nmol/g dry wt in the
absence and presence of ethanol, respectively (Fig. 6.7C). This represents major trapping
of CoA. Figs 6.8A and 6.8B show the concentrations of unknowns A and B in livers of
rats gavaged with calcium levulinate ± ethanol. These C7 compounds are present at much
lower concentrations than the 3 main C5 acyl-CoAs derived from levulinate. Note that the
concentrations of the C7 acyl-CoAs did not return to the zero baseline after 120 min.
In the brains of rats gavaged with calcium levulinate ± ethanol, the concentrations of the
3 most abundant CoA esters derived from levulinate peaked at 45 - 75 min (Figs 6.9A
and 6.9B). The sum of the 3 concentrations peaked at 60 - 75 min at levels of 3.1 and 4.1
nmol/(g wet wt) in the absence and presence of ethanol, respectively (Fig. 6.9C).
Although the total concentrations of the esters were 20 to 60% higher in the presence
than in the absence of ethanol, statistical analysis could not be conducted because we had
only one rat per time point. Traces of the C7 acyl-CoAs were detected in the brains of
both groups of rats, but their concentrations could not be measured accurately (range
0.004 to 0.01 nmol/g wet weight).
138
DISCUSSION
Our initial study of levulinate metabolism in liver had characterized its catabolism via three parallel β-oxidation pathways (1). We had identified the 10 C5 acyl-CoAs which are
intermediates in the 3 pathways. One of these intermediates, 4-phosphopentanoyl-CoA is
part of a new class of acyl-CoAs derived from 4-hydroxyacids (6). In this follow-up
study, we detected additional CoA esters derived from levulinate. Our mass isotopomer
studies (Table 6.3) revealed that these esters contain the 5 carbons of levulinate + 2
carbons provided by acetyl-CoA. The latter is formed both from levulinate catabolism
and from the metabolism of glucose, fatty acids and a number of endogenous compounds.
We thus hypothesized that the first step in the formation of the C7 CoA esters is the
elongation of levulinyl-CoA to 3,6-diketoheptanoyl-CoA (Fig. 6.5, compound (2)). Very
low concentrations of this intermediate were detected in all livers perfused with 2 mM
levulinate: 0.6 to 1.3 nmol/(g dry wt). Because of the very low concentrations, the
labeling pattern of 3,6-diketoheptanoyl-CoA could not be calculated. In perfusions with
unlabeled 3,6-diketoheptanoate ethyl ester (no levulinate), the concentration of 3,6-
diketoheptanoyl-CoA was about 51 to 53 nmol⋅(g dry wt-1). In these perfusions,
levulinyl-CoA was also identified. This confirmed the existence of a process by which
levulinyl-CoA and acetyl-CoA are reversibly converted to 3,6-diketoheptanoyl-CoA.
This reaction is most likely catalyzed by a 3-ketoacyl-CoA thiolase.
3,6-Diketoheptanoyl-CoA is a gamma diketo compound. Such compounds are known to
easily undergo spontaneous cyclization without involvement of another compound. This
is because of the acidity of the -CH2- group between the two carbonyls on carbons 1 and
139
3. The initial putative product of the cyclization (Fig. 6.5, compound (3)) was not detected. However, we detected a CoA ester (compound A), the mass of which is compatible with it being formed by dehydration of compound (3). The putative formula of this CoA ester (Fig. 6.5, compound (4)) is typical of compounds that exist in two tautomeric forms (Fig. 6.5, compounds (4) and (5)). The dehydration of compound (3) is presumably catalyzed by enoyl-CoA hydratase. Walsh’s group had described the formation of a similar cyclopentenone compound (termed C5N) from the cyclization of 5- aminolevulinyl-CoA (a gamma-dicarbonyl compound) in microorganisms (14).
The mass of another C7 CoA ester (compound B) derived from levulinate (23 amu above that of levulinyl-CoA) suggested the presence of a nitrogen atom in its non-CoA component. Gamma-diketo compounds, such as 2,5-diketohexane, are known to form pyrrolic compounds by binding with the ε-N of lysine (9-12). This is why we tried to
15 label compound B by perfusing livers with unlabeled levulinate + either NH4Cl, [6-
15 13 15 15 N]lysine or [ C6, N2]lysine. Compound B was not labeled from NH4Cl, a substrate which labels the α-amino nitrogen of non-essential aminoacids such as glutamate (13).
However, compound B became similarly M1 labeled from [6-15N]lysine and from
13 15 [ C6, N2]lysine (Table 6.3, rows i and j). Thus, this unknown C7 CoA ester contains a N atom derived from the ε-amino group of lysine, but does not contain carbon from lysine.
The 7 carbons of the non-CoA moiety of this compound are derived from levulinate (5 carbons) and from acetyl-CoA (2 carbons). Based on all the data, we hypothesize that compound B has a pyrrolic structure (Fig. 6.5, compound (6) = B).
140
The presence in compound (6) of the ε-nitrogen but not the carbon of lysine evokes the
first step of the saccharopine pathway of lysine catabolism. In the formation of
saccharopine, the ε-nitrogen of lysine is transferred to α-ketoglutarate forming glutamate
(15,16). Conceivably, 3,6-diketoheptanoyl-CoA could play the role of α-ketoglutarate in
a “saccharopine-like” reaction. 3,6-Diketoheptanoyl-CoA would react with lysine,
abstracting its ε-nitrogen, and would undergo a re-arrangement forming compound (6)
which would play the role of glutamate in the “saccharopine-like” reaction.
We had previously reported that levulinate is converted in the liver to the drug of abuse
4-hydroxypentanoate, and that the conversion is accelerated by ethanol oxidation (1).
4-Hydroxypentanoate has become a popular substitute for 4-hydroxybutyrate which is
now a controlled substance in the US (17). However, 4-hydroxypentanoate has a weaker
drug effect and is more toxic than 4-hydroxybutyrate (18). Thus, addicts may use very
toxic doses of 4-hydroxypentanoate to achieve the desired levels of drug effect.
Alternatively, addicts may ingest large doses of calcium levulinate (which is freely
available) with an alcoholic beverage to stimulate the conversion of levulinate to
4-hydroxypentanoate.
To test whether the ingestion of calcium levulinate ± ethanol would induce the
accumulation of levulinate-derived CoA esters in the brain, we gavaged rats with calcium
levulinate ± ethanol. In the liver and brain of these rats, we observed the accumulation of
the main C5 CoA esters derived from the initial metabolism of levulinate, i.e., levulinyl-
CoA, 4-hydroxypentanoyl-CoA and 4-phosphopentanoyl-CoA. The accumulation of 4-
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phosphopentanoyl-CoA is of concern for the following reasons. In mice and in patients
deficient in succinic semialdehyde dehydrogenase, body fluids contain mM
concentrations of 4-hydroxybutyrate (19,20). We previously reported (6) that, in the
deficient mice, the brain concentration of 4-phosphobutyryl-CoA is 50 times higher than
in control mice (from 0.0005 to 0.025 nmol/g). This suggested that 4-phosphobutyryl-
CoA may contribute to the perturbation of brain metabolism in these mice who
experience severe epileptic seizures (21). Also 4-phosphobutyryl-CoA may contribute to
the mental retardation of patients with 4-hydroxybutyric aciduria (20). Lastly, 4-
phosphobutyryl-CoA may be implicated in the acute mental dysfunction of subjects who
ingested 4-hydroxybutyrate. By analogy, mental dysfunction may be caused by 4-
phosphopentanoyl-CoA in the brain of subjects ingesting either 4-hydroxypentanoate or
calcium levulinate + ethanol. This concern is heightened by the fact that, after gavage
with calcium levulinate ± ethanol, the peak concentration of 4-phosphopentanoyl-CoA in
rat brain (0.8 to 1.4 nmol/g, Figs 6.9A, 6.9B) is up to 56 times higher than the brain
concentration of 4-phosphobutyryl-CoA in mice deficient in succinic semialdehyde dehydrogenase (0.025 nmol/g (6)).
The assay of the liver CoA ester profile in in vivo gavage experiments confirmed the trapping of CoA in levulinate-derived CoA esters. The total liver concentration of levulinyl-CoA + 4-hydroxypentanoyl-CoA + 4-phosphopentanoyl-CoA peaked at 755 nmol/(g dry wt) in rats gavaged with calcium levulinate + ethanol (Fig. 6.7C). This amounts to about 60% of the total CoA pool of the liver (1,200 to 1,300 nmol/(g dry wt)
(22)). In liver, one can expect that such trapping of CoA would impact on CoA-
142
dependent reactions (23). Williams et al reported that in premature babies treated with
oral calcium levulinate, lactic acidosis developed after 4 days of treatment (24). This may result from inhibition of pyruvate dehydrogenase flux by a chronically low CoA content of the liver. In our acute in vivo experiment, we did not observe increases in plasma
lactate during the 2 hr after gavage with calcium levulinate ± ethanol. We plan to
investigate the effects of long-term administration of calcium levulinate on plasma lactate
concentration and metabolomic profiles in liver and brain.
The corresponding total concentration of levulinyl-CoA + 4-hydroxypentanoyl-CoA + 4-
phosphopentanoyl-CoA in brain was much lower than in liver: 4 nmol/(g wet wt) (Fig.
6.9C). Note that the total CoA content of brain is 3 to 10 times lower than in liver (25). It
is not clear whether CoA trapping contributes to the brain toxicity of
4-hydroxypentanoate. As mentioned above, 4-phosphopentanoyl-CoA may alter brain
function.
The C7 cyclical CoA esters derived from levulinate (or their corresponding free acids)
may also interfere with metabolic processes in liver and brain. Lastly, 3,6-
diketoheptanoyl-CoA (or its free acid equivalent) may like 2,5-diketohexane form pyrrole
adducts with lysine residues of proteins (9-12). The formation of pyrrole-lysine adducts
in neurofilaments is a major contributor to the toxicity of 2,5-diketohexane derived from
hexane (26).
143
In conclusion, our new findings suggest additional potential deleterious metabolic effects of levulinate over those previously reported (1). Investigations of these effects will require the development of techniques to synthesize the CoA esters we have identified.
Also, in vitro studies should be conducted with 3,6-diketoheptanoyl-CoA to elucidate the formation of the cyclical CoA esters (Fig. 6.5, compounds (4/5) and (6)), and to test for the formation of pyrrole-lysine adducts. Lastly, proteomic studies should be conducted on brains of rats treated with levulinate ± ethanol to test for the presence of pyrrole adducts on lysine residues. The data of the present report emphasize the public health relevance of further mechanistic investigations on the metabolic perturbations induced by levulinate.
144
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17. Anderson, I. B., Kim, S. Y., Dyer, J. E., Burkhardt, C. B., Iknoian, J. C., Walsh, M. J., and Blanc, P. D. (2006) Trends in gamma-hydroxybutyrate (GHB) and related drug intoxication: 1999 to 2003.
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26. DeCaprio, A. P. (1987) Neurotoxicology 8, 199-210
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FIGURE LEGENDS
FIGURE 6.5. Proposed scheme for the formation of cyclical CoA esters from levulinate. Compound names are: 1 = levulinyl-CoA (4-ketopentanoyl-CoA); 2 = 3,6- diketoheptanoyl-CoA; 3 = 2-hydroxy-2-methyl-5-oxocyclopentanecarboxylic acid-CoA ester; 4 = 2-methyl-5-oxocyclopent-1-enecarboxylic acid-CoA ester; 5 = 5-hydroxy-2- methylcyclopenta-1,4-dienecarboxylic acid-CoA ester; 6 = 2-(5-methyl-1H-pyrrol-2- yl)acetic acid-CoA ester or 2-(5-methyl-1H-pyrrol-2-yl)acetyl CoA.
FIGURE 6.6. Concentrations of levulinate (panel A) and 4-hydroxypentanoate (panel B) in plasma of rats gavaged with calcium levulinate (open squares) or calcium levulinate + ethanol (closed diamonds).
FIGURE 6.7. Liver concentrations of the main C5 acyl-CoAs derived from levulinate in rats gavaged with calcium levulinate ± ethanol. Panel A: concentrations of levulinyl-CoA (blue), 4-hydroxypentanoyl-CoA (red) and 4-phosphopentanoyl-CoA (green) after gavage with calcium levulinate. Panel B: concentrations of the same acyl- CoAs after gavage with calcium levulinate + ethanol. Panel C: sum of the concentrations of the 3 acyl-CoAs after gavage with calcium levulinate (open squares) or calcium levulinate + ethanol (closed diamonds).
FIGURE 6.8. Liver concentrations of the C7 cylical CoA esters derived from levulinate in rats gavaged with calcium levulinate (panel A) or calcium levulinate + ethanol (panel B). The concentrations of unknowns A and B are presented as solid and open circles, respectively.
FIGURE 6.9. Brain concentrations of the main C5 acyl-CoAs derived from levulinate in rats gavaged with calcium levulinate ± ethanol. Panel A: concentrations of levulinyl-CoA (blue), 4-hydroxypentanoyl-CoA (red) and 4-phosphopentanoyl-CoA (green) after gavage with calcium levulinate. Panel B: concentrations of the same acyl- CoAs after gavage with calcium levulinate + ethanol. Panel C: sum of the concentrations
147 of the 3 acyl-CoAs after gavage with calcium levulinate (open squares) or calcium levulinate + ethanol (closed diamonds).
148
TABLE LEGEND
TABLE 6.3. Distrbution of labeled mass isotopomers of C7 CoA esters and acetyl- CoA in rat livers perfused with levulinate (LEV) and substrates labeled with 13C, 15N, or both. Enrichments of compounds are expressed as molar percent entrichment ± S.E. Data from individual livers are shown in rows e and f. In row d, values different from the corresponding values in row c are indicated with an * (p < 0.02).
149
Figure 6.5.
150
Figure 6.6.
151
Figure 6.7.
152
Figure 6.8.
153
Figure 6.9.
154
Table 6.3.
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CHAPTER 7
DISCUSSION, IMPLICATIONS AND FUTURE DIRECTIONS
7.1. Reduction of Levulinate to 4-Hydroxypentanoate In Vivo and in Isolated Livers
7.1.1. Discussion and Conclusions
In rats intravenously infused with levulinate ± ethanol (Figure 6.2), ethanol
administration resulted in significantly higher concentrations of levulinate and 4-
hydroxypentanoate (GHP) in the plasma and significantly increased [lactate]/[pyruvate]
ratios. These experiments demonstrated that (i) levulinate is reduced to GHP in vivo, (ii)
ethanol stimulates the reduction of levulinate to GHP, and (iii) ethanol decreases the
whole-body disposal of levulinate. The increase in the [NADH]/[NAD+] ratios resulting
from liver ethanol oxidation (128) explains the increase in the reduction of levulinate to
GHP. However, because peripheral tissues do not oxidize ethanol, we cannot consider an
inhibition on total levulinate metabolism in vivo by increases in the [NADH]/[NAD+]
ratio in peripheral organs. A more likely explanation centers around the fact that
levulinate catabolism yield acetyl-CoA. At the same time, there is rapid uptake and use
of acetate in peripheral tissues (328). Acetate is the product of liver ethanol oxidation, and its metabolism involves activation to acetyl-CoA. Because levulinate catabolism yields acetyl-CoA, its catabolism is inhibited by another source of acetyl-CoA (i.e. the
acetate derived from ethanol) via competition for free CoA and the enzymes of β-
oxidation (329). The latter competition inhibits the metabolism of levulinate in
peripheral tissues. However, because the volumes of distribution of levulinate and GHP
156
are unknown in rats, metabolic rates could not be calculated from the data of this
experiment.
To gain more insight on the effects of ethanol on levulinate metabolism, we turned to
13 liver perfusion experiments. In livers perfused with [ C5]levulinate (M5) ± ethanol
(Figure 6.3), ethanol (i) almost doubled the uptake of levulinate, (ii) tripled the
production of GHP from levulinate, and (iii) inhibited levulinate catabolism. Although
ethanol stimulated the uptake of levulinate in the liver, it appears that the carbons of
levulinate are preferably sent to GHP and out of the liver, rather than to catabolism. The
increase in [NADH]/[NAD+] induced by liver ethanol oxidation (128) clearly explains the increase in the reduction of levulinate to GHP. Subsequently, the decrease in the intracellular concentration of levulinate stimulated levulinate uptake in the liver. The decrease in the catabolism of levulinate by ethanol can be explained by the redox inhibition of the three reactions catalyzed by 3-hydroxyacyl-CoA dehydrogenase in pathways A, B, and B’ (Figure 6.1, reactions 6) (128; 129).
7.1.2. Future Directions
We have demonstrated that the reduction of levulinate to GHP occurs in the liver and also in the heart (orientation experiments). It is possible that this conversion occurs in other organs, such as the muscle, kidney, or brain. It is also possible that other organs use
GHP. In order to investigate the latter one could measure arterial-venous concentration differences across organs.
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7.2. Mechanisms for the Conversion of Levulinate to 4-Hydroxypentanoate
7.2.1. Discussion and Conclusions
In live rats and in perfused rat livers, we demonstrated the reduction of levulinate to GHP
(Figures 6.2 and 6.3). Also, in livers perfused with (R,S)- or (R)-GHP, the accumulation of levulinate was observed. Therefore, there is at least one dehydrogenase system that interconverts levulinate and GHP in the liver. Based on the stimulation in the reduction of levulinate to GHP by ethanol, it is likely that this reduction is linked to a NADH- dependent dehydrogenase(s). This is supported by our pilot enzymatic studies in intact liver mitochondria incubated with levulinate plus glutamate and rotenone (an NADH generating system). The apparent activity at 5 mM levulinate, 30°C, and pH 7.4 was 0.48 nmol∙min-1∙(mg of protein)-1. Chiral assay revealed that both enantiomers of GHP are formed from levulinate in the mitochondria; 85% is the (R)-enantiomer.
We also tested for a cytosolic dehydrogenase that would reduce levulinate. In incubations of 100,000 X g supernatant of liver homogenate with levulinate and NADPH, the production of GHP was linear with time. The apparent activity at 30°C and pH 7.4 was 0.043 nmol∙min-1∙(mg of protein)-1. Chiral assay of the GHP formed from levulinate revealed that 94% was the (R)-enantiomer.
There is no information on an enzyme system that interconverts 4-keto acids and 4- hydroxyacids longer than four carbons in mammalian cells. Because the addition of glutamate to intact mitochondria forms an NADH generating system, the reduction of levulinate to GHP is most likely catalyzed by at least one NADH-dehydrogenase. This
158
conclusion is supported by the stimulation of levulinate reduction by ethanol in our
perfused liver experiments (i.e. the oxidation of ethanol increases mitochondrial
[NADH]/[NAD+] ratios (128)). In addition, both enantiomers of GHP were generated in mitochondria incubated with levulinate (85% (R)), and in livers perfused with levulinate
alone (84% (R)), or levulinate + ethanol (79% (R)). We cannot exclude the possibility
that a mitochondrial NADPH-dehydrogenase exists that can reduce levulinate to GHP,
but there are no known conditions under which intact mitochondria can generate NADPH
(but no NADH) to test this.
Overall, the data suggest that the reduction of levulinate in liver mitochondria is
catalyzed by: (i) one (R)- and one (S)- NADH-dehydrogenase; (ii) one (R)-NADH-
dehydrogenase and a racemase; or (iii) one (R)-NADH-dehydrogenase and a racemase
system. The (R)-dehydrogenase could be the (R)-3-hydroxybutyrate dehydrogenase that
interconverts acetoacetate and (R)-3-hydroxybutyrate (i.e. ketone bodies) (339). Since
the rate of levulinate reduction to GHP in the mitochondrial incubations was only 1.6%
of the rate of acetoacetate reduction to 3-hydroxybutryate, the reduction of levulinate
may be a side reaction of (R)-3-hydroxybutyrate dehydrogenase. A possible
mitochondrial racemase system that could generate (S)-GHP would follow the sequence
(Figure 6.1, reactions 1, 3, and reversal of 1):
Levulinate → levulinyl-CoA → (S)-4-hydroxypentanoyl-CoA → (S)-GHP
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Although there is no information on the enzyme that interconverts levulinyl-CoA and 4- hydroxypentanoyl-CoA, it might be similar (or identical) to the (S)-3-hydroxyacyl-CoA dehydrogenase involved in fatty acid β-oxidation. If the latter is correct, then (S)-4- hydroxypentanoyl-CoA is formed from levulinyl-CoA via (S)-3-hydroxyacyl-CoA dehydrogenase or a similar enzyme. A fraction of the (S)-4-hydroxypentanoyl-CoA would be hydrolyzed to form the small fraction of (S)-GHP detected in the mitochondria.
In the cytosol, there might be one (R)- and one (S)-NADPH-dehydrogenase that could form the (R)- and (S)-GHP detected. However, there is also the possibility that some (S)-
GHP is formed from leakage of a racemase system from broken mitochondria contaminating the cytosolic extract. It appears that the cytosolic activity for reduction of levulinate is much lower than the mitochondrial activity.
7.2.2. Future Directions
The total apparent activity of enzymes (mitochondrial NAD-dehydrogenase + cytosolic
NADP-dehydrogenase) that reduce levulinate to GHP in vitro accounts for 62% of the rate of GHP production measured in rat livers perfused with 2 mM levulinate. Thus, our initial assays of the dehydrogenase activities need to be optimized.
The formation of mostly (R)-GHP from levulinate in vitro and in vivo suggests that the enzymes involved in the conversion of levulinate to GHP favor of formation of the (R)- enantiomer. However, both enantiomers of GHP are used as the same rate in livers perfused with (R,S)-GHP. Although we identified a mitochondrial NADH- dehydrogenase and a cytosolic NADPH-dehydrogenase, the chiral specificity of the
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dehydrogenases that interconvert levulinate and GHP still needs to be determined. Future
enzyme characterization should include (i) measuring the activity of the enzyme(s) in both directions under optimal conditions (once determined), and (ii) partial purification of the
enzyme(s) to determine its kinetic parameters. Partial purification will increase the specific activity of the enzyme(s) by removing potential inhibitors or interferences. It can be achieved by using classical protein purification techniques: ammonium sulfate fractionation, calcium phosphate gel absorption, and/or diethylamino-cellulose chromatography. To determine enzyme kinetics (i.e. Vmax and KM) of the semi-purified
enzyme(s), a series of experiments can be done with various concentrations of levulinate or
GHP. Protocols should include incubating increasing concentrations of levulinate (R)-
GHP, or (S)-GHP with (i) liver tissue extract, (ii) K-phosphate buffer at the pH that is optimal for enzyme activity, and (iii) the reduced or oxidized co-enzyme(s) that are linked to the conversion.
To explore the possibility that (S)-3-hydroxyacyl-CoA dehydrogenase (or a new type of acyl-CoA dehydrogenase) catalyzes the conversion of levulinyl-CoA to (S)-4- hydroxypentanoyl-CoA in the mitochondria, levulinyl-CoA can be synthesized and incubated with intact liver mitochondria. Levulinyl-CoA can be synthesized by (i) making levulinic-anhydride (340), and (ii) reacting levulinic anhydride with CoA (341). To test for the formation of (R)- or (S)-4-hydroxypentanoyl-CoA, the 4-hydroxypentanoyl-CoA can be submitted to aminolysis with chiral sec. butylamine ((R)- or (S)-) (342). The latter removes the CoA and forms an amide bond with GHP. The diastereomers of 4- hydroxypentanoate-N(sec. butylamide) should separate on GC-MS. If the resulting
161
butylamide is not sufficiently volatile for GC-MS, then derivatization of the 4-OH group with TMS can follow aminolysis. This assay is also applicable to levulinyl-CoA for testing conversion in the opposite direction (i.e. 4-hydroxypentanoyl-CoA to levulinyl-CoA).
Note: The synthesis of 4-hydroxyacyl-CoAs is difficult because of the propensity of all 4- hydroxyacids for lactonization. However, previous work has demonstrated that incubation of acetyl-CoA and GHP with 4-hydroxybutyryl-CoA transferase (isolated from Clostridium aminobutyricum) yields acetate and 4-hydroxypentanoyl-CoA (343). This enzyme transfers CoA from acetyl-CoA to GHP, and works only with 4-hydroxyacids with 4 or 5 carbons, but not with longer chain substrates. Thus, 4-hydroxypentanoyl-CoA can also be synthesized following the latter techniques and used to test for enzymatic activities.
7.3. Pathways of Levulinate Catabolism
7.3.1. Discussion and Conclusions
The proposed scheme for the three pathways of levulinate metabolism (Figure 6.1) was
13 confirmed by perfusing livers with [ C5]levulinate (M5 levulinate), and by the
identification and mass isotopomer distribution of (i) most of the acyl-CoAs and end
products of pathways A, B, and B’ in the liver tissue, or (ii) their corresponding acids in
the liver perfusate (Tables 6.1, 6.2). The three initial intermediates of pathways A, B, and B’ (i.e. the three initial C5 acyl-CoAs derived from levulinate), which are levulinyl-
CoA, 4-hydroxypentanoyl-CoA, and 4-phosphopentanoyl-CoA, were all M5 labeled as
expected. Consistent with catabolism of levulinate via pathway A, we identified M3
propionyl-CoA in liver tissue. We did not identify the products of 4-phosphopentanoyl-
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CoA isomerization (i.e. 3-hydroxypentanoyl-CoA or 3-ketopentanoyl-CoA) in liver
tissue, but we identified the corresponding 3-hydroxypentanoate as a free acid in the
perfusate. To our surprise, the latter was mostly M3 labeled with a small component of
M5. The only possible explanation for this isotopomer distribution of 3-
hydroxypentanoate is the combined reversibility of the two reactions catalyzed by 3- hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase (pathway A, reactions 6 and 7). The latter results in the exchange of (i) the C-1+2 moiety of 3-hydroxypentanoyl-
CoA with free acetyl-CoA, and (ii) the C-3+4+5 moiety of 3-hydroxypentanoyl-CoA with free propionyl-CoA. However, the propionyl moiety exchanges much more slowly with thiolase than the acetyl moiety (56), as originally reported by Huth et al. using acetoacetyl-CoA (55). This explains the much lower M2 than M3 enrichment of 3- hydroxypentanoate.
Consistent with catabolism of levulinate via pathways B and B’, we identified the six C5
acyl-CoAs that derive from levulinyl-CoA and 4-hydroxypentanyl-CoA. The mass
isotopomer distributions allowed for differentiating between two isomeric compounds in
pathways B and B’ (i.e. 4-keto-3-hydroxypentanoyl-CoA and 3-keto-4-
hydroxypentanoyl-CoA), which both have the same mother/daughter ion pair on LC-MS,
making their individual identification difficult. The 3-keto-4-hydroxypentanoyl-CoA
was predicted to be a substrate of the reversible thiolase, resulting in a component of M3
labeling. We confirmed the latter, as it was 56% M5, and 44% M3. The 4-keto-3-
hydroxypentanoyl-CoA, which would not be a substrate of the reversible thiolase, would
be mostly M5 labeled. This was also confirmed, as it was 94% M5. In addition, we
163 identified two peaks for 3,4-dihdyroxypentanoyl-CoA, because it is a mixture of diastereomers: (i) the OH on C-3 is (S)-, because of the specificity of enoyl-CoA hydratase, and (ii) the OH on C-4 is (R)- or (S)-, because the GHP derived from levulinate is 85% (R)- and 15% (S)-. The presence of M3 isotopomers for the two diastereomers of 3,4-dihdyroxypentanoyl-CoA (pathway B’) demonstrates that the acetyl moiety of each diastereomer exchanges with free acetyl-CoA via 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase.
Our proposed scheme for the catabolism of levulinate also involved the potential α- oxidation of lactyl-CoA, which would yield formate. As expected, we identified M1 formate in the perfusions with M5 levulinate (as C-3 of levulinate was hypothesized to end up in formate). However, in order to confirm the α-oxidation of lactyl-CoA, we would need to demonstrate a clear precursor to product relationship between the substrate
(lactyl-CoA) and the product (either acetyl-CoA, formyl-CoA or formate) of its α- oxidation. The latter was impossible because (i) lactyl-CoA has never been identified in mammalian cells, (ii) we could not develop a labeling pattern for levulinate that would allow us to distinguish between the acetyl-CoA formed in pathway A versus that formed in pathways B + B’, and (iii) we could not identify formyl-CoA (because it is a very unstable intermediate of fatty acid α-oxidation (344)). So to check whether the production of M1 formate is specific to the α-oxidation of lactyl-CoA (and that M1 formate is derived from C-3 of levulinate), we synthesized and perfused livers with [3-
13 13 C]levulinate and [1,2,4,5- C4]levulinate. As expected, M1 formate was found in the perfusions with [3-13C]levulinate. However, M1 formate was also found in perfusions
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13 with [1,2,4,5- C4]levulinate (Figure 6.4). Therefore, M1 formate production from
13 [ Cn]levulinate does not reflect the α-oxidation of lactyl-CoA, generating formate from
13 C-3 of levulinate. Instead, the formation of M1 formate from [ Cn]levulinate is likely formed via an indirect route:
Levulinate →→ Propionyl-CoA (pathway A) → Anaplerosis → CAC Intermediates →
PEP →→ 3-Phosphoglycerate →→ Serine → Glycine → Formate
Consistent with the latter process, we found liver tissue glycine similarly M1 labeled
13 from the three [ Cn]levulinate labels used in perfusions. However, we cannot exclude that a small amount of formate is formed by the α-oxidation of lactyl-CoA.
In summary, we have confirmed that levulinate is catabolized to propionyl-CoA, acetyl-
CoA and lactate via the three pathways outlined in Figure 6.1. The identification of the six acyl-CoAs, which are intermediates in pathways B and B’ demonstrates that the presence of a keto or a hydroxy group on C-4 of a fatty acid, does not prevent any of the normal reactions of β-oxidation to proceed. In addition, in livers perfused with (R,S)-
GHP, both enantiomers are used at the same rate. So the chirality of the hydroxy group on C-4 of some of the acyl-CoAs derived from levulinate, likely does not affect their flux through pathways A and B’ (note that in pathway B, the C-4 is a keto group).
The prediction of CoA trapping in esters derived from levulinate is supported by the concentration profiles of acyl-CoAs (shown in Table 6.1). In livers perfused with M5
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levulinate, the first three acyl-CoAs derived from levulinate (i.e. levulinyl-CoA, 4-
hydroxypentanoyl-CoA, and 4-phosphopentanoyl-CoA) accumulated to high levels in the
liver. This resulted in the perturbation of the concentration profiles of other physiological
CoAs (i.e. large decreases in the concentrations of acetyl-CoA, propionyl-CoA,
methylmalonyl-CoA, succinyl-CoA, and malonyl-CoA when compared to control livers).
Substantial CoA trapping in the metabolites of levulinate might interfere with a number
of CoA-dependent reactions of intermediary metabolism (136), and might explain the lactic acidosis that was observed in premature babies treated with calcium levulinate
(262) (discussed in next section, 7.4). In addition, in an orientation heart perfusion with levulinate, we observed accumulation of the three main levulinate-derived CoA esters in the heart at concentrations similar to those observed in the perfused rat liver (see Figure
7.1). Thus, it is likely that substantial CoA trapping occurs in different organs as a result
of levulinate metabolism.
Ethanol increases the accumulation of all C5 acyl-CoAs derived from levulinate (i.e. sum
of their concentrations) in livers perfused with M5 levulinate. This occurs because an
increase in the [NADH]/[NAD+] ratio resulting from liver ethanol oxidation inhibits the
3-hydroxyacyl-CoA dehydrogenase reaction of β-oxidation (in pathways A, B, and B’)
(128; 129). The inhibition of this reaction prevents the catabolism of levulinate, causing accumulation of levulinate-derived CoA esters. Of particular concern, is the very high concentration of 4-phosphopentanoyl-CoA induced by ethanol. In mice unable to dispose of endogenous 4-hydroxybutyrate (GHB) who suffer from severe epileptic seizures (188), the concentration of 4-phosphobutyryl-CoA was 50 times higher in the liver and the brain
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when compared to wildtype mice (151). Thus the accumulation of 4-phosphobutyryl-
CoA might be related to perturbations in brain metabolism in these mice, and in the
inability to reason in persons intoxicated with GHB. Whether the accumulation of 4-
phosphopentanoyl-CoA (derived from levulinate and GHP) causes similar perturbations
in brain metabolism is unknown. However, if the latter does occur, it is likely that
ethanol will exacerbate these effects.
7.3.2. Future Directions
Our previous experiments with M5 levulinate did not allow for the calculation of the
distribution of levulinate catabolism between pathway A and pathways B + B’. This is
because (i) both pathways of catabolism generate M2 acetyl-CoA, and (ii) we could not
devise a labeling strategy for levulinate where one of the pathways would generate M2
acetyl-CoA and the other M1 acetyl-CoA. However, a potential strategy that might
allow for this calculation involves measuring the turnover of total propionyl-CoA in
livers perfused with M5 levulinate. Note that propionyl-CoA is formed from levulinate
only in pathway A. The strategy is outlined in Figure 7.2., and involves infusing a low
concentration of [3-13C]propionate into the recirculating perfusate that contains M5 levulinate and then measuring (i) the uptake of [3-13C]propionate, (ii) the M1 and M3
enrichments of propionyl-CoA in the liver tissue, and (iii) the uptake of levulinate and the
release of GHP. In this system, M3 propionyl-CoA is derived from M5 levulinate, M
propionyl-CoA is derived from endogenous unlabeled amino acids, and M1 propionyl-
CoA is derived from the infused [3-13C]propionate tracer. The following calculations can
167 then be made to determine the absolute flux through pathways A and B + B’, and thus the distribution of levulinate catabolism through these pathways.
(A) Total catabolism of LEV = (uptake of LEV) – (release of GHP)
(B) Total turnover of propionyl-CoA in the liver = (uptake of M1 propionate) / (M1
enrichment of propionyl-CoA)
(C) Production of propionyl-CoA from LEV = (B) x (M3 enrichment of propionyl-CoA)
*This is the absolute flux through pathway A
(D) Absolute flux through pathways B + B’ = (A) – (C)
Although we observed the formation of mostly (R)-GHP from levulinate in vivo, we also found that both enantiomers of GHP are used in the liver at the same rate. In addition,
GHP, as a drug of abuse, is ingested as the racemic mixture. Whether the (R)-enantiomer has specific effects on intermediary metabolism and brain function when compared to the
(S)-enantiomer has yet to be investigated. Thus, the metabolism of both (R)- and (S)-
GHP merits research.
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Figure 7.1. Trapping of Heart CoA by the Metabolites of Levulinate.
The accumulation of the three main levulinate-derived CoA esters (i.e. levulinyl-CoA, 4- hydroxypentanoyl-CoA, and 4-phosphopentanoyl-CoA) in the perfusate of a heart perfused with 2 mM levulinate (n = 1).
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Figure 7.2. Tentative Strategy for Measuring the Distribution of Levulinate Catabolism.
Tentative strategy for measuring the flux of levulinate through Pathway A and the turnover of propionyl-CoA. M3 propionyl-CoA is derived from M5 levulinate; M propionyl-CoA is derived from endogenous unlabeled amino acids; M1 propionyl-CoA is derived from the infused [3-13C]propionate tracer.
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7.4. Metabolism of Oral Calcium Levulinate ± Ethanol
7.4.1. Discussion and Conclusions
In rats gavaged with calcium levulinate, we observed a strong wave in the concentration of levulinate in the portal vein plasma over the two hour protocol. The high plasma concentration of levulinate (~3.5 mM), reflects the rapid absorption of levulinate from the stomach. In addition, the concentration of levulinate decreased almost linearly over the two hour protocol, demonstrating that the time table of the experiment was optimal. The wave in the concentration of GHP in the portal vein was low, but peaked around 75 minutes (at 0.28 mM). The concentration profiles of levulinate and GHP in the portal vein did not change much in the presence of ethanol. In the livers of rats gavaged with calcium levulinate, we observed a strong wave in the concentrations of the three most abundant CoA esters derived from levulinate (i.e. levulinyl-CoA, 4-hydroxypentanoyl-
CoA, and 4-phosphopentanoyl-CoA) (Figure 6.7A). Their concentrations were high and peaked around 45 minutes (with the sum of the three concentrations at 645 nmol/g dry weight, Figure 6.7C). This represents major CoA trapping and amounts to about 50-60% of the total CoA pool of the liver (1,200 to 1,300 nmol/g dry weight) (345). Ethanol only slightly increased the wave of CoA trapping in the three most abundant CoA esters derived from levulinate in the liver (Figure 6.7B). The sum of the three concentrations peaked around 45 minutes at 755 nmol/g dry weight (Figure 6.7C). This is in contrast with the effect of ethanol on the perfused liver, where the concentrations of C5 acyl-CoAs
derived from levulinate dramatically increased. We do not have an explanation for the
different degrees of action of ethanol in the two models.
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We also measured the same metabolites of levulinate in the peripheral plasma and in the brain. The peripheral plasma concentration of levulinate followed similar profiles in the absence or presence of ethanol (Figure 6.6A). However, ethanol almost doubled the peak concentration of GHP derived from levulinate in peripheral plasma (0.45 mM versus 0.25 mM without ethanol (Figure 6.6B). In addition, based on linear regression of the data over 80 minutes, the plasma concentration of GHP increased faster in the presence of ethanol (calcium levulinate experiments: slope = 0.0026, R2 = 0.92 vs. calcium levulinate
+ ethanol experiments: 0.0044, R2 = 0.97; p < 0.002). In rats gavaged with calcium
levulinate, we also observed a strong wave in the concentrations of brain levulinyl-CoA,
4-hydroxypentanoyl-CoA and 4-phosphopentanoyl-CoA (Figure 6.9A), but at much lower concentrations than in the liver. This is typical of physiological acyl-CoAs when comparing their concentrations in the brain to the liver (i.e. 3-10 times lower in the brain)
(346; 347). However, these three most abundant levulinate-derived CoA esters were found in higher concentrations than some of the physiological acyl-CoAs found in the brain (such as malonyl-CoA, methylmalonyl-CoA, propionyl-CoA, heptanoyl-CoA and octanoyl-CoA). As found in the liver, ethanol also only slightly increased the wave of
CoA trapping in the three main levulinate-derived CoA esters in the brain (Figure 6.9B).
However, the wave of CoA trapping in levulinate-derived CoA esters seems to last longer in the brain (Figure 6.9), than in the liver (Figure 6.7). This could mean that the potential toxic effects of CoA ester accumulation are longer-lived in the brain.
In addition to levulinyl-CoA, 4-hydroxypentanoyl-CoA, and 4-phosphopentanoyl-CoA, the other seven C5 acyl-CoAs derived from levulinate (identified in liver perfusion
172 experiments as previously discussed and shown in Table 6.1) were identified in the livers of rats gavaged with calcium levulinate ± ethanol. A few of the latter were also identified in the brains of these rats. Note: Considering that levulinate-derived CoA esters were found in the brain, there must be a mechanism that allows for levulinate and/or GHP to cross into the brain, probably via the monocarboxylic acid transporter that is used by ketone bodies (348).
Although we considered that CoA trapping in the metabolites of levulinate might result in hypoglycemia and lactic acidosis, this was not observed in the two hours following a gavage of calcium levulinate ± ethanol. The lack of effect of calcium levulinate on lactate and glucose concentrations might be due to (i) the dose used, (ii) or the short time frame in which metabolites were measured during this acute study. High plasma lactate concentrations were observed after three to four days in premature babies who were treated for hypocalcemia with calcium levulinate (262). Therefore, this specific metabolic perturbation might be a consequence of long-term CoA trapping and a decrease in pyruvate dehydrogenase (PDH) activity (i.e. a decrease in free CoA activates PDH kinase, which inhibits PDH activation (247)). This leaves open the possibility that lactic acidosis could develop with chronic treatment and/or supplementation of calcium levulinate. This would not have been demonstrated in our acute study. Hypoglycemia was not observed in the premature babies treated with calcium levulinate, but this was unlikely to occur considering these infants were fed every couple of hours. In our experiments, the stress of the gavage could have increased plasma glucose levels, counterbalancing any potential decrease in glucose by levulinate. Although the rats were
173
fasted overnight, a small amount of liver glycogen can substantially increase plasma
glucose levels. For example, in overnight fasted rats, with a liver glycogen content of
0.3% (versus 5% in fed rats), hydrolysis of the remaining glycogen woud increase
glucose concentration by ~3.5 – 4 mM. Thus, glycogenolysis could have prevented the
development of hypoglycemia and may account for the lack of effect of levulinate on
glucose metabolism observed in this acute study.
The accumulation of 4-phosphopentanoyl-CoA in the brains of rats gavaged with calcium
levulinate ± ethanol is of particular concern. Brain concentrations of 4-phosphobutyryl-
CoA (derived from endogenous GHB) are 50 times higher in mice deficient in succinic
semialdehyde dehydrogenase than in control mice (0.0005 to 0.025 nmol/g wet weight)
(151). The latter suggests that 4-phosphobutyryl-CoA may contribute to the perturbation of brain metabolism in these deficient mice who experience severe epileptic seizures
(229). 4-Phosphobutyryl-CoA may also be implicated in the acute mental dysfunction of subjects who ingest GHB. Thus, by analogy, mental dysfunction may be caused by accumulation of 4-phosphopentanoyl-CoA in the brain of subjects ingesting GHP or calcium levulinate + ethanol (as a pro-drug). This concern is highlighted by the data shown in Figures 6.9 A and B. After gavage with calcium levulinate ± ethanol, the peak concentration of 4-phosphopentanoyl-CoA in rat brain (0.8 to 1.4 nmol/g wet weight) is up to 56 times higher than the brain concentration of 4-phosphobutyryl-CoA in mice deficient in succinic semialdehyde dehydrogenase (0.025 nmol/g) (151). Whether or not
4-phosphopentanoyl-CoA acts a neuromodulator is still unknown and will require future studies.
174
Overall, the gavage experiment simulated what might occur in a drug addict taking a
single dose of calcium levulinate with or without ethanol. It is clear that (i) levulinate-
derived CoA esters accumulate in both the brain and liver following a gavage with
calcium levulinate, (ii) these CoA esters remain longer in the brain than in the liver, and
(iii) ethanol increases the plasma concentration of GHP. Note that the dose of calcium
levulinate used in this study only provided twice the RDA for calcium (for an adult
human). However, addicts may ingest much larger doses of calcium levulinate ± ethanol to produce the desired effects.
7.4.2. Future Directions
It will be important to conduct experiments using a range of high doses of calcium levulinate (± ethanol) that might be used by a drug addict. In order to determine the metabolic effects of chronic calcium levulinate ingestion, a feeding study could be conducted in which rodent diets are spiked with various amounts of calcium levulinate.
This would allow for the investigation of the long-term effects of CoA trapping on pyruvate dehydrogenase activity (and on other CoA-requiring enzymes). In addition, the effect of calcium levulinate on glucose turnover, plasma fatty acid turnover and lipolysis,
2 2 could be investigated with isotopic tracers such as [6,6- H2]glucose, [ H5]glycerol, and
13 [ C18]oleate (349).
We identified levulinyl-carnitine and 4-hydroxypentanoyl-carnitine (which correspond to
two of the most abundant acyl-CoAs derived from levulinate) in a liver perfused with levulinate. Thus, it may be possible to alleviate CoA trapping in the metabolites of
175 levulinate by administration of carnitine or glycine (350-352). Experiments with acute loading of levulinate ± carnitine or glycine could be used to test whether carnitine or glycine may be used as part of the future treatment of intoxicated addicts. The acylcarnitine or acylglycine profile in plasma, urine or organ perfusate would reflect the removal of CoA trapping acyl groups from cells (350). However, clinical improvement could occur with carnitine or glycine supplementation even if this is not demonstrated biochemically. For example, the concentrations of acylcarnitines in the urine of some patients with fatty acid oxidation disorders treated with carnitine, is small. Yet the treatment with carnitine has beneficial clinical effects in some fatty acid oxidation disorders and in other CASTOR diseases (352). In addition, in some CASTOR diseases acyl-CoA conjugation with glycine is more efficient than conjugation with carnitine
(351). Note that glycine administration also loads the patient with nitrogen.
Last, it will be important to assess the behavioral changes induced by calcium levulinate, likely resulting from accumulation of GHP in the plasma and 4-phosphopentanoyl-CoA in the brain. Behavioral studies could be conducted in rodents injected or fed with levulinate and/or GHP ± ethanol. Following administration of the latter, behavioral tests, such as the Rotarod test or the open field locomotor test (353) (and many others), could be used to assess changes in coordination and overall locomotor function. These tests would allow for the comparison of behavioral side effects caused by levulinate/GHP, with what is known for other drugs of abuse such as GHB. We hope that neuroscientists will investigate the effect of 4-phosphopentanoyl-CoA (or the corresponding 4-
176
phosphopentanoate) on brain receptors that may be involved in the toxicity of GHP (such as the GHB or GABAB receptors).
7.5. New Cyclical CoA Esters Derived from Levulinate
Note: In this section, bold numbers in parentheses and in italics refer to compound
numbers in Figure 7.3.
7.5.1. Discussion and Conclusions
After identifying many C5 acyl-CoAs derived from levulinate, we found additional CoA
esters with 7 carbons which are derived from levulinate by the hypothetical scheme shown in Figure 7.3. This is supported by additional experiments with levulinate or 3,6-
13 15 15 13 15 diketoheptanoate + different C-substrates, NH4Cl, [6- N]lysine, or [ C6, N2]lysine
(data in Table 6.3). The scheme starts with the elongation of levulinyl-CoA (1) by
acetyl-CoA to form 3,6-diketoheptanoyl-CoA (2), followed by the cyclization of the γ-
diketo acyl-CoA (2) to a cyclopentanone acyl-CoA (3) and then dehydration to the
cyclopentenyl-acyl-CoA (4) and cyclopentadienyl-acyl-CoA (5) tautomers (i.e compound
A). In parallel, compound (2) appears to form a pyrrolated acyl-CoA (i.e. a methyl-
pyrrolyl-acetyl-CoA) that includes a nitrogen atom derived from the ε-amino of lysine,
but without the carbons of lysine. The pyrrolated acyl-CoA (6) (i.e. compound B) is
reminiscent of pyrrole adducts formed in brain and central nervous system proteins by γ-
diketo-hexane (i.e. 2,5-diketohexane) derived from n-hexane (303-306; 314). Thus, 3.6-
diketoheptanoyl-CoA, like 2,5-diketohexane, may pyrrolate lysine residues from proteins.
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3,6-Diketoheptanoyl-CoA (2) and the two C7 cyclical CoA esters (i.e. compounds A (4/5)
and B (6)) derived from levulinate were identified in our perfused liver experiments with
levulinate ± ethanol and in the livers of rats gavaged with calcium levulinate ± ethanol
(Figure 6.8). Although these C7 compounds are present at much lower concentrations in the liver than the three main C5 acyl-CoAs derived from levulinate (i.e. levulinyl-CoA, 4-
hydroxypentanoyl-CoA, and 4-phosphopentanoyl-CoA), they appear to remain longer in
the liver than the C5 CoA esters (Figure 6.8 versus Figure 6.7). In addition, traces of
compounds A (4/5) and B (6) were detected in the brains of rats gavaged with calcium levulinate ± ethanol (range 0.004 to 0.01 nmol/g wet weight). The data suggest that formation of cyclical CoA esters from levulinate may result in toxic effects on the brain and/or liver. For example, in liver, pyrrolation of enzymes such as carbamoyl phosphate synthetase might explain the inhibition of urea synthesis recently observed by Dr. Nissim in rat hepatocytes (unpublished). Also, 3,6-diketoheptanoyl-CoA may form pyrrole adducts with lysine groups of proteins in various tissues.
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Figure 7.3. Scheme for the Formation of New Cyclical CoA Esters Derived from Levulinate.
Note that compounds (4) and (5) are the two tautomers of Compound A, and compound (6) is the pyrrolated acyl-CoA (Compound B), as referred to in the text. Note that compound (2) can undergo two types of pyrrolation reactions: (i) using only the ε- nitrogen of lysine to form compound (6), or (ii) binding to free lysine or lysine residues of proteins.
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7.5.2. Future Directions
Although we have the tentative identification of new C7 CoA esters derived from
levulinate, more experiments are required to confirm these findings and to identify other
cyclical CoA esters or acids that may be derived from levulinate. Future work should
attempt to identify the intermediates in the pyrrolation reaction forming the pyrrolated
acyl-CoA (compound B) (6) from 3,6-diketoheptanoyl-CoA (2). The latter will help
explain how the nitrogen of lysine is incorporated into compound B (6), while the carbon
skeleton of lysine is lost. Note that the presence in compound B (6) of the ε-nitrogen of
lysine (but not the carbon skeleton of lysine) evokes the first step of mammalian lysine catabolism via the saccharopine pathway. In the formation of saccharopine, the ε-
nitrogen of lysine is transferred to α-ketoglutarate forming glutamate (354; 355).
Perhaps, in a “saccharopine-like” reaction, 3,6-diketoheptanoyl-CoA (2) could (i) play
the role of α-ketoglutarate (abstracting the ε-nitrogen of lysine), and (ii) undergo a
rearrangement forming compound B (6), which would be the counterpart of glutamate.
In vitro experiments aimed at generating these cyclical CoA esters derived from
levulinate can also be used to gather more information on (i) the enzymes catalyzing the
elongation of levulinyl-CoA (1) with acetyl-CoA to form 3,6-diketoheptanoyl-CoA (2),
followed by dehydration of (2) to form compound A (4/5), and (ii) to confirm that (2)
undergoes pyrrolation with free lysine to form compound B (6) and possibly an adduct of
(2) and lysine that includes the carbon skeleton of lysine. A potential strategy for the
latter would involve infusion of the reaction mixtures described below into the mass
spectrometer and observing the formation of expected compounds in real time (356).
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13 13 1) If M or M5 ([ C5]) levulinyl-CoA (1) is incubated with M or M2 ([ C2]) acetyl-
CoA + thiolase (in a buffer), then one should observe 3,6-diketoheptanoyl-CoA
(2).
2) If (2) undergoes spontaneous cyclization, then one should observe (3). However,
if (2) does not spontaneously cyclize, it may require a cyclase.
3) If enoyl-CoA hydratase is added to the above incubation contents, then one
should observe (4) and (5), if no cyclase is needed to make (3).
13 13 4) If M or M5 ([ C5]) levulinyl-CoA (1) is incubated with M or M2 ([ C2]) acetyl-
15 13 15 CoA + thiolase + lysine (M, M1 ([ε- N]), or M8 ([ C6, N2])), then one should
observe a pyrrolated lysine + (6).
Absolute proof for the identification of compounds A (4/5) and B (6) would require isolating them from perfused livers. Compounds (4/5) and (6) could also be isolated from the in vitro experiments described above. Then the compounds could be used as standards to confirm retention time, mass, exact mass, and fragmentation patterns on LC-
MS. Their structures could be confirmed using NMR.
Last, proteomics studies could be conducted to detect pyrrolated proteins in the brain, liver, and central nervous system tissue of rats treated with levulinate ± ethanol.
Proteomics studies of brain and liver tissue will probe for proteins that are specifically modified at lysine sites with pyrrole adducts (357).
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7.6. Overall Summary and Implications
A number of public health issues arise from our studies on levulinate metabolism. First, levulinate, a freely available nutritional supplement, is converted in the liver to a toxic drug
of abuse GHP. Second, the formation of GHP from levulinate is stimulated by ethanol oxidation. This raises the possibility that drug addicts will switch from GHB or GHP
(controlled substances) to levulinate + an alcoholic beverage. Third, chronic exposure to
levulinate may induce CoA trapping in various organs with perturbations of intermediary
metabolism, leading possibly to lactic acidosis. The latter could be compounded by ethanol
ingestion. Fourth, 4-phosphopentanoyl-CoA, which accumulates in the liver and brain
following levulinate ingestion (especially + ethanol), may contribute to the toxicity of GHP
derived from levulinate on the brain. Fifth, the C7 cyclic CoA esters derived from
levulinate, as well as their related free acids, may exert toxic effects on the brain. Sixth,
pyrrole adducts of lysine residues of brain proteins may be formed from 3,6- diketoheptanoyl-CoA.
The above six mechanisms that we have identified may result in serious deleterious effects of levulinate on various aspects of organ metabolism. The Drug Enforcement Agency should take a hard look at the potential consequences of keeping calcium levulinate or levulinic acid as a freely available pro-drug of abuse.
182
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