University of Florida Thesis Or Dissertation Formatting

HYDROGEN SULFIDE BIOMARKERS AND VITAMIN B-6 STATUS

BARBARA NEAL DERATT

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2016

To all my family and friends

ACKNOWLEDGMENTS

I have been so lucky to have such a great support system during my time as a doctoral student. I would like to thank my mentor, Dr. Jess Gregory for guiding me in my research project. I am forever thankful for all you taught me and I aspire to be a scientist of your caliber one day. I would also like to thank my committee members, Dr.

Harry Sitren, Dr. Susan Percival and Dr. Yueh-Yun Chi for their input on my project and support through the many meetings and exams. I would also like to thank my lab members and friends, Dr. Luisa Rios-Avila, Dr. Lili Huang, and Maria Ralat, for their positive influence and advice.

My family and friends have also been a blessing through these past 5 years. I would like to thank my parents and my sister Jamie for always listening to my problems and pushing me to finish. I would also like to thank my sister Lindsey for being the best roommate/ chemistry advisor I know. To all my friends, who have listening to me complain and helped with any of my scientific questions or to give me emotional support, I am so indebted to you. And finally, I have to thank Duke Lemmens for being an amazing boyfriend during the hardest part of my doctoral degree. You always supported me and wanted to listen to everything I was doing even though you had no idea what I was talking about.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

LIST OF ABBREVIATIONS ...... 10

ABSTRACT ...... 14

CHAPTER

1 LITERATURE REVIEW ...... 16

Vitamin B-6 ...... 16 Hydrogen Sulfide ...... 22 One Carbon Metabolism ...... 26 Transsulfuration Pathway ...... 27 Cardiovascular Disease ...... 29 Study Aims and Objectives ...... 30

2 KINETIC STUDIES OF LANTHIONINE AND HOMOLANTHIONINE IN CELL CULTURE ...... 35

Introduction ...... 35 Materials and Methods...... 36 Materials ...... 36 Primary Human Hepatocytes ...... 36 HepG2 Cell Culture ...... 37 Analytical Methods ...... 37 Isotopically Labelled H2S Biomarker Synthesis and Purification ...... 39 In-Vivo Kinetics of the Transsulfuration Pathway ...... 39 Statistical Analysis ...... 40 Results ...... 41 Vitamin B-6 Analysis in Cell Culture Experiments ...... 41 PAG Inhibition Affects In-Vivo Kinetics and Concentrations Similarly in Primary Human Hepatocytes and HepG2 Cells ...... 41 HepG2 Amino-thiol Concentrations Differ Slightly by Treatment Group After Incubation with Labeled Lanthionine and Homolanthionine ...... 45 Discussion ...... 46

3 EFFECT OF SHORT-TERM, MARGINAL VITAMIN B-6 DEFICIENCY ON THE CONCENTRATION OF H2S BIOMARKERS, LANTHIONINE AND HOMOLANTIHONINE, IN HEALTHY HUMAN SUBJECTS ...... 57

Abstract ...... 57 Introduction ...... 58 Material and Methods ...... 60 Human Vitamin B-6 Restriction Protocols...... 60 Analytical Methods ...... 61 Statistical Analyses ...... 62 Results ...... 62 Effect of Vitamin B-6 Restriction Protocol ...... 62 H2S Biomarkers in Healthy Human Subjects ...... 63 Relationships Among Lanthionine, Homolanthionine, Total Homocysteine, Total Cysteine and Functional Indicators of Vitamin B-6 Deficiency ...... 64 Discussion ...... 65

4 METABOLIC CONSEQUENCES OF PLASMA CYSTATHIONINE ELEVATION IN SUBJECTS WITH SUSPECTED CORONARY ARTERY DISEASE ...... 72

Introduction ...... 72 Materials and Methods...... 73 Human Subjects ...... 73 Analytical Methods ...... 74 Statistical Analyses ...... 75 Results ...... 75 Targeted Metabolomics ...... 75 LCMS Analysis ...... 77 NMR Analysis ...... 78 Discussion ...... 79

5 CONCLUSIONS AND FUTURE DIRECTIONS ...... 106

APPENDIX

A ISOTOPICALLY LABELED LANTHIONINE AND HOMOLANTHIONINE SYNTHESIS ...... 111

B PAG CONCENTRATION STUDY ...... 113

REFERENCES ...... 116

BIOGRAPHICAL SKETCH ...... 130

LIST OF TABLES

Table page

2-1 Isotopic enrichments of methionine and cysteine in media in primary human hepatocytes and HepG2 cells...... 47

2-2 Molar equivalent concentrations of H2S from reactions in the transsulfuration pathway of primary human hepatocytes...... 48

2-3 Molar equivalent concentrations of H2S from reactions in the transsulfuration pathway of HepG2 cells ...... 48

3-1 Concentrations of amino acid and vitamin B-6 functional biomarkers in preprandial human plasma before and after 28-d dietary vitamin B-6 restriction ...... 68

4-1 Baseline characteristics of 80 subjects from previously conducted Norwegian trials ...... 82

4-2 Concentrations of selected plasma variables in 80 subjects from previously conducted Norwegian trials (NORCAD) ...... 83

4-3 Peak areas of identified metabolites by LCMS analysis in both positive and negative ion mode that had a VIP score of greater than 2...... 85

4-4 Peak areas of unidentified metabolites by LCMS analysis in both positive and negative ion mode that had a VIP score of greater than 2...... 87

4-5 Peak areas of the top 35 contributors to differences seen in the LC/MS model...... 95

4-6 Identified peaks from NMR spectra that were significant contributors to difference seen in the model...... 97

A-1 Experimental conditions for isotopically labeled lanthionine and homolanthionine synthesis reactions...... 111

A-2 Concentration of amino-thiols before and after synthesis reactions determined by DTNB assay...... 111

LIST OF FIGURES

Figure page

1-1 The structures the B-6 vitamers and their phosphorylated forms...... 32

1-2 Depiction of all the possible reactions producing H2S in vivo ...... 33

1-3 One carbon metabolism and transsulfuration pathway...... 34

2-1 Enrichments over 6-h time course experiments of the labeled precursors 13 [ C5] L-methionine and [D2] L-cysteine with and without the addition of PAG. .. 49

2-2 Appearance of labeled of cystathionine is elevated in cells with PAG intracellularly and extracellularly...... 50

2-3 Enrichment of homolanthionine over the 6-h time course in primary human hepatocytes and HepG2 cells...... 51

2-4 Appearance of labeled lanthionine in Subject 1 primary human hepatocytes, Subject 2 primary human hepatocytes, and HepG2 cells with and without the addition of PAG...... 52

2-5 Labeled product appearance as determined from total cystathionine concentration derived from serine/cysteine intracellular competition...... 53

2-6 Total intracellular glutathione and cysteinylglycine concentrations in the presence or absence of PAG...... 54

2-7 Aminothiol concentrations intracellularly and extracellular over 12-h time course after the addition of labeled lanthionine and homolanthionine ...... 55

3-1 Human plasma concentrations of homolanthionine and lanthionine before and after 28-d dietary vitamin B-6 restriction ...... 69

3-2 Relationships between lanthionine and its precursor cysteine and between homolanthionine and its precursor homocysteine in human plasma before and after 28-d dietary vitamin B-6 restriction ...... 69

3-3 Relationship between lanthionine and homolanthionine in human plasma before and after 28-d dietary vitamin B-6 restriction ...... 70

3-4 Relationship between homolanthionine with 3-hydroxykynurenine, the ratio of 3-hydroxykynurenine:xanthurenic acid, and cystathionine in human plasma before and after 28-d dietary vitamin B-6 restriction ...... 71

4-1 Score plot from partial least squares-discriminant analysis (PLS-DA) of overall targeted metabolite concentrations in subjects with high or low plasma cystathionine ...... 99

4-2 Loadings plot shows targeted metabolite variable contributions to the differences seen in the PLS-DA scores plot model...... 100

4-3 Score plot from partial least squares-discriminant analysis (PLS-DA) of LCMS peak areas in subjects with high or low plasma cystathionine...... 101

4-4 Score plot from partial least squares-discriminant analysis (PLS-DA) of 1H- NMR spectra in subjects with high or low plasma cystathionine...... 102

4-5 Representative spectra of each cystathionine group...... 103

4-6 Variable importance to the projection (VIP) scores versus chemical shift (PPM) from NMR spectra...... 104

4-7 Spectral representation of the differences between the low and high cystathionine group...... 105

5-1 Multivariate PLS-DA scores plot of subjects according to acute myocardial outcomes...... 110

A-1 Representative GC/MS chromatograms of purified biomarkers...... 112

B-1 Human liver lysate CSE activity was measured by colorimetric assay under various concentrations of PAG...... 114

B-2 Mouse liver lysate CSE activity was measured by colorimetric assay under various concentrations of PAG...... 114

B-3 Cysteine and glutathione concentrations in human hepatocytes with various concentrations of PAG added after 6-h incubation...... 115

LIST OF ABBREVIATIONS

µL microliter

µM Micomolar

AA Anthranilic Acid

ADP Adenosine Diphosphate

ATP Adenosine Triphosphate

B-6 Vitamin B-6

CBS Cystathionine β-synthase

CDC Centers for Disease Control

CO2 Carbon dioxide

CoA Coenzyme A

CSE Cystathionine γ-lyase

Csn Cystathionine

CVD Cardiovascular disease

Cys Cysteine

DHA Docosahexanoic Acid

DHF Dihydrofolate

DHFR Dihydrofolate reductase

DNA Deoxyribonucleic acid

EAR Estimated Average Requirement

EDTA Ethylenediaminetetraacetic acid

Ep Enrichment plateau

EPA Eicosapentaenoic Acid

FSR Fractional synthesis rate

GABA Gamma-aminobutyric acid

GC/MS Gas chromatography/mass spectroscopy

Gly Glycine

GSH Glutathione

H2S Hydrogen sulfide

HAA Hydroxyanthranilic Acid

HBSS Hank’s Balanced Salt Solution

HCl Hydrochloride

Hcy Homocysteine

HEPES 4-(2-hydroxyethyl)-1-piperarzineethanesulfonic acid

HFBA heptafluorobutyric anhydride

HK 3-Hydroxykynurenine

HPLC High performance liquid chromatography I Initial rate

IDO Indoleamine 2,3-dioxygenase

IOM Institute of Medicine

KA Kynurenic Acid

LCMS Liquid Chromatography/ Mass Spectroscopy

LDL Low Density Lipoprotein

LPH Lactase-phlorizin hydrolase

MEM/EBSS Minimum Essential Medium with Earle’s Balanced Salts

Met Methionine mg milligram mM Millimolar

NAD Nicotinamide adenine dinucleotide

NH4OH Ammonium hydroxide

NHANES National Health and Nutrition Examination Survey nM Nanomolar

NMR Nuclear Magnetic Resonance

PL Pyridoxal

PLP Pyridoxal phosphate

PM Pyridoxamine

PMP Pyridoxamine phosphate

PN Pyridoxine

PNG Pyridoxine 5’-β-D-glucoside

PNGH Pyridoxine 5’-β-D-glucoside hydrolase

PNP Pyridoxine phosphate

RDA Recommended dietary allowance

RM Remethylation

RT Retention Time

SAH S-adenosylhomocysteine

SAM S-adenosylmethionine

Ser Serine

SHMT Serine hydroxylmethyl transferase

SHMT Serine hydroxymethyltransferase

TCA Trichloroacetic acid

TCEP Tris (2-carboxyethyl)phosphine

TDO tryptophan 2,3-dioxygenase

THF Tetrahydrofolate

TM Transmethylation

TMAO Trimethylamine N-oxide

TS Transsulfuration pathway

UL Upper limit

XA Xanthurenic acid

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

HYDROGEN SULFIDE BIOMARKERS AND VITAMIN B-6 STATUS

Barbara DeRatt

December 2016

Chair: Jess Gregory Major: Nutritional Sciences

Hydrogen sulfide (H2S) is an endogenous gasotransmitter involved in many facets of human metabolism, including cardio-protection and inflammation. H2S is produced primarily in the transsulfuration pathway by two PLP-dependent enzymes, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). Lanthionine and homolanthionine are produced concurrently with H2S in specific non-canonical reactions, although little is known about their metabolism. This dissertation focused on the study of lanthionine and homolanthionine concentrations and metabolism in health and disease. Specifically, I determined a normal range of concentrations of lanthionine

(89.0 to 372 nmol/L) and homolanthionine (5.75 to 32.3 nmol/L) in healthy adults.

These ranges were not affected by short-term vitamin B-6 deficiency. To examine the effect of disease on lanthionine and homolanthionine concentrations, plasma samples from subjects with suspected coronary artery disease were separated by high or low plasma cystathionine. Lanthionine concentrations were 75% higher in subjects with elevated plasma cystathionine compared to subjects with low cystathionine (P<0.001), while homolanthionine concentrations were not different. Homolanthionine concentrations in these older diseased subjects were 10 times higher than the normal

range seen in healthy young adults. To study the mechanisms of production and catabolism of lanthionine and homolanthionine, I utilized primary human hepatocytes

13 and HeG2 cell cultures. Using stable isotope tracers, [U- C5] L-methionine and (3, 3

D2) L-cysteine, I determined the metabolic fluxes through the transsulfuration pathway.

Vitamin B-6 deficiency was simulated using a CSE specific inhibitor, propargylglycine

(PAG). In both cellular models, I confirmed serine is the preferred substrate for cystathionine synthesis by CBS and cysteine only accounts for <10% of total cystathionine produced. Lanthionine concentrations (P<0.001), but not enrichments, were higher in PAG-treated cells than controls, suggesting CSE does not contribute to lanthionine synthesis but CSE may catalyze some lanthionine cleavage.

Homolanthionine production in control cells greatly exceeded that observed in PAG- treated cells (P<0.001), which confirmed that CSE is essential in homolanthionine synthesis. HepG2 cell experiments supported results seen in primary human hepatocyte studies which showed that CSE is the primary source of homolanthionine production while CBS is solely responsible for lanthionine production. These data illustrate how this HepG2 model is useful for mechanistic investigation of H2S production. This dissertation is a compilation of novel experiments which have shown alterations in H2S synthesis is disease and under specific stresses to the transsulfuration pathway.

CHAPTER 1 LITERATURE REVIEW

Vitamin B-6

Vitamin B-6 is the generic term for 3-hydroxy-2-methylpyridine derivatives containing the metabolic activity of pyridoxine. The subclasses have a common base structure, 2-methyl, 3-hydroxy, 5-hydroxymethyl pyridine with different substituents at the fourth position of the ring. Pyridoxal (PL) contains an aldehyde, pyridoxine (PN) an alcohol, and pyridoxamine (PM) an amino group at the 4’-carbon position. In addition, each group can be esterified by a phosphate group at the 5’-hydroxymethyl position to form 3 additional vitamers (Figure 1-1) (1). These structural differences yield functional differences; PN has a hydroxymethyl function, PM has an aminomethyl function, and PL has a formyl function (2). The presence of several ionic sites causing complex ionization on the molecule explain the vitamin’s reactivity in many enzymatic processes

(3). Most coenzymatic properties of vitamin B-6 involve carbonyl-amine condensation.

The carbonyl group of PLP forms a Schiff base with the primary amine of the amino acid substrate, which then undergoes tautomerization and hydrolysis to yield PMP and an α- keto acid. The amine group transferred to PMP can then be transferred to another α- keto acid by the regeneration of the active transaminase-PLP Schiff base to release a new amino acid (4).

Vitamin B-6 exists in many dietary sources. B-6 vitamers are naturally interspersed throughout food products. Plant products such as whole-grains and vegetables contain PN or pyridoxine-5’-β-D-glucoside (PNG), while PLP and PMP make up >80% of vitamin B-6 found in organ meats and muscle (5, 6). Additionally, many breakfast cereal products and energy drinks are fortified with the vitamin. PNG and PN

are much more stable than PL and PM, suggesting cooking of vegetables causes minimal losses of the vitamin while thermal processing of meats causes substantial vitamin loss (7). Due to the stability of PN, most supplements and food fortification use pyridoxine hydrochloride (8). Vitamin B-6 is light and heat sensitive and unstable in neutral or alkaline conditions (9).

To become nutritionally active, plant sources require β-glucosides in the intestine to hydrolyze the glucoside. PNG is taken up by diffusion and converted to PN by cytosolic PNG hydrolase or brush border lactase-phlorizin hydrolase (LPH) and later oxidized to PLP (10, 11). Hydrolysis of PNG by LPH is competitively inhibited by lactose in vitro (10). The co-ingestion of PNG with free PN reduces the utilization of PN (12).

PNG provides 15% of total vitamin B-6 intake in a typical mixed diet (13). Relative to

PN, PNG is only 50% as bioavailable in humans (5, 14). Generally, animal products are

10% more digestible than plant sources. Based on plasma PLP levels in male human subjects, bioavailability in an average American diet is between 61% and 81% (15).

The phosphorylated vitamers and PLP-protein adducts are dephosphorylated by alkaline phosphatases in the small intestine, and then passively diffuse through the brush border membrane into circulation. The only active co-enzyme form of the vitamin is PLP, so after absorption PN and PM are carried to the liver (the primary site of vitamin B-6 metabolism) to be converted to PLP by PNP/PMP oxidase. Circulating PL can be taken up by a number of tissues and trapped in the cytosol after becoming rephosphorylated by pyridoxal kinase (16-19). The majority of total vitamin B-6, mainly as PLP, is stored in the muscle bound to glycogen phosphorylase (20-22). Muscle concentrations have been found to be stable during dietary depletion and

supplementation, containing approximately 170 mg of total vitamin B-6 (21, 23).

Therefore, muscle vitamin B-6 concentrations serve as a measure of long term stores.

Only a small portion of total body vitamin B-6 is present in blood, usually as PLP bound to albumin by Schiff base linkages. Plasma B-6 shows significant changes in restriction and supplementation, thereby reflecting short-term stores (23). PLP is catabolized by aldehyde oxidase in the liver to form 4-pyridoxic acid which is then excreted in the urine.

Vitamin B-6, in the form of PLP, is a cofactor in over 160 enzymatic reactions, many of which are associated with amino acid metabolism, organic acid production, heme synthesis, one-carbon metabolism, neurotransmitter production, sphingolipid metabolism and glycogenolysis. Transaminases are vitamin B-6-dependent enzymes responsible for the catabolism of most amino acids (24). The sensitivity of aminotransferases to B-6 deficiency has been known for many years, and their activities and coenzyme saturation in red blood cells are traditional biomarkers of B-6 nutritional status (1). One such amino acid sensitive to vitamin B-6 deficiency and inflammation is tryptophan catabolism (25). The tryptophan catabolic pathway begins with the oxidation of tryptophan to form N-formylkynurenine catalyzed by tryptophan 2,3-dioxygenase

(TDO) in the liver (26). Indoleamine 2,3-dioxygenase (IDO) is responsible for this reaction in extra hepatic tissues (27). N-formylkynurenine is converted to kynurenine by kynurenine formamidase (28). Kynurenine then has 3 potential fates: conversion to anthranilic acid (AA) by kynureninase, conversion to kynurenic acid (KA) by kynurenine aminotransferase, or conversion to 3-hydroxykynurenine (HK) by kynurenine 3- hydroxylase (29). Kynurenine aminotransferase then can catalyze the formation of xanthurenic acid (XA) from HK. Alternatively, HK can be converted to 3-

hydroxyanthranilic acid (HAA) by kynureninase (29). HAA is used to form quinolinic acid and later nicotinamide adenine dinucleotide (NAD), the active form of niacin in metabolism (27, 30). Kynureninase and kynurenine aminotransferase are PLP- dependent enzymes (29).

In addition to amino acid metabolism, vitamin B-6 is also essential in the production of many other compounds. The synthesis of hemoglobin requires PLP while additional binding of PLP and PL to sites on the β-chains enhances the O2-binding capacity and inhibits the deformation of sickle-cell hemoglobin (31). Serotonin, epinephrine, norepinephrine, and γ-aminobutyric acid all require vitamin B-6 in their biosynthesis. PLP is necessary for the release of glucose from glycogen by glycogen phosphorylase (2). The transsulfuration pathway also contains two PLP-dependent enzymes responsible for the catabolism of homocysteine and the production of cysteine and glutathione (Figure 1-3). While these and many other enzymes are B-6-dependent, their susceptibility to deficiency varies greatly. Specifically in 1-carbon metabolism, cystathionine β-synthase (CBS) is refractory and cystathionine γ-lyase (CSE) is very sensitive to vitamin B-6 insufficiency (32). The numerous functions of this vitamin demonstrate its importance in-vivo and indicate the many pathways that may potentially be affected in deficiency.

Adequate dietary intake of vitamin B-6 (RDA 1.3 mg/d) is needed for sufficient cellular production of plasma PLP greater than 20 nM. This dietary requirement is increased in the elderly and during pregnancy and lactation. Plasma PLP is currently the primary indicator of B-6 status, however, a need exists for better functional indicators. According to the CDC’s most recent National Health and Nutrition

Evaluation Survey (NHANES), 10% of Americans have plasma PLP below the 20 nmol/L criterion of deficiency (33, 34), and at least another 10% fall in the 20-30nmol/L range considered to indicate insufficient or marginal B-6 status. The prevalence of deficiency is greater in women and adults over 60 years old, as well as alcoholics.

Some studies suggest the current RDA is not optimal for plasma PLP concentrations greater than 30nmol/L and should be increased (35, 36). Wide variation in B-6 status is due to variable food selection, lack of systemic fortification and bioavailability.

Inflammatory conditions accentuate B-6 insufficiency but the mechanism remains unclear (37, 38). Recent metabolomic and targeted metabolite profiling studies have shown that experimental dietary B-6 restriction resulting in plasma PLP <30nmol/L in healthy young adults induces a wide variety of metabolic changes, including evidence of aberrant amino acid and organic acid metabolism (39-41). Chronic B-6 insufficiency

(<30 nmol/L PLP) has been associated with altered biochemical markers (39, 42). The combination of chronic and severe deficiency causes clinical manifestations such as cheilosis and glossitis and if untreated can result in neurological dysfunction and seizures. However, this is not prevalent in the American population (2, 43). Although toxicity is also uncommon, the current upper limit is 100 mg/day due to reports of sensory neuropathy caused by the use of 500 mg pyridoxine supplements for an extended period of time (44). Most daily multi-vitamins contain approximately 2 mg of vitamin B-6, and toxicity has never been reported through dietary intake alone.

Metabolic consequences of mild-to-moderate vitamin B-6 deficiency in healthy adults yielded changes in amino acid patterns of 1C metabolism, transsulfuration pathway and tryptophan catabolism as well as in amino acid ratios (39, 40, 45-48).

Targeted metabolomic panels of one-carbon metabolites and tryptophan catabolic pathway revealed increases in cystathionine, serine, and 3-hydroxykynurenine and decreases in creatine, creatinine, dimethylglycine, and kynurenic acid after 28-day dietary B-6 restriction (39). Untargeted analysis of the same samples showed significant changes in glutamine/glutamate and 2-oxoglutarate/glutamate ratios and increases in concentrations of acetate, pyruvate, and TMAO (40). A recent analysis of low and high B-6 status in oral contraceptive users showed significant negative nonlinear associations between plasma PLP and metabolite concentrations of homocysteine, glutathione, and ratios of asymmetric dimethylarginine/arginine, 3- hydroxykynurenine/ 3-hydroxyanthranilic acid and 3-hydroxykynurenine/ kynurenic acid

(41).

The association between B-6 status and CVD development was first reported over 50 years ago with the observation that B-6 deficiency caused atherosclerotic lesions in Rhesus monkeys (49, 50). Epidemiological studies have shown that low dietary intake of B-6 and low plasma PLP concentrations are independent risk factors for CVD, stroke and venous thrombosis (51-57). Low PLP concentration is a risk factor of coronary artery disease independent of homocysteine concentration, suggesting a potential protective effect of B-6 through mechanisms unrelated to homocysteine metabolism (56, 58). Additional mechanistic explanations for the association between

B-6 and CVD are the role of B-6 in coagulation by inhibiting ADP receptors and prolonging bleeding by occupying the glycoprotein IIb/IIIa receptor or down-regulating its synthesis (57, 59-64), the possible induction of hypercholesterolemia by inhibition of advanced glycation and lipooxidation of end-products (65-67). Omega-3 fatty acids

(EPA and DHA) have also been shown to be decreased in vitamin B-6 deficiency, thus preventing their cardioprotective effects (68-71). Impaired immunological function has also been suggested due to aberrant T lymphocyte, macrophage differentiation, and interleukin production in vitamin B-6 deficiency (72-74). Systemic inflammation is associated with lower plasma PLP and may reflect altered B-6 homeostasis and function associated with CVD development (37, 38). C-reactive protein is also a predictor of cardiovascular disease risk and is inversely related to plasma vitamin B-6 concentrations (75-77). Atherosclerosis is a complex multi-factorial disease, and currently B-6-related atherogenesis is poorly understood. There is a distinct need for more in-depth metabolomic analysis to further discern the metabolic effects of B-6 insufficiency and its association with CVD risk.

Hydrogen Sulfide

Hydrogen sulfide (H2S) is a naturally occurring gas, most commonly associated with the smell of rotting eggs. A few breaths of air containing high levels of the gas can cause death. It can be found in crude petroleum, natural gas, volcanic gas, and in sulfur hot springs (78). The history of hydrogen sulfide begins much like the story of nitric oxide; a once toxic chemical now found to be endogenously produced and necessary for physiological functions. Nitric oxide has been studied for over 50 years now but only in the past twenty years was H2S discovered in-vivo.

As seen in Figure 1-2, the majority of H2S is synthesized endogenously by cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). These two transsulfuration enzymes require vitamin B-6 in the form of PLP; however CSE has been shown to be more susceptible to vitamin B-6 deficiency than CBS (32).

Homocysteine enters the transsulfuration pathway from methionine in the one carbon

cycle. The purpose of the transsulfuration pathway is to catabolize homocysteine to maintain safe levels in the blood and to also provide the necessary components for glutathione synthesis. While the normal progression of the transsulfuration pathway does not result in H2S formation, the side reactions produce significant amounts. Of particular interest are amino acids, lanthionine and homolanthionine, which are produced concurrently with H2S. Currently their utilization in-vivo or their breakdown and excretion is not understood. By quantifying the metabolism of these amino acids, indirect comparisons can be made about H2S status, thereby categorizing them as biomarkers. In patients with hyperhomocysteinemia, homolanthionine has been found in urine (79). There is another mechanism of H2S production, although it is thought to be minor in-vivo (80). The combined action of 3-mercaptopyruvate sulfurtransferase and cysteine-aminotransferase, involved in the cysteine catabolic pathway, can produce

H2S but only in the presence of a reductant (Figure 1-2). Metabolic removal of H2S occurs in the mitochondrion where it is oxidized to thiosulfate. This reaction provides electrons to the electron-transport chain, thus generating energy. Thiosulfate is further metabolized into sulfate which is then excreted in urine (81).

Kinetic simulations in the Banerjee lab predict at physiological substrate concentrations the α, β elimination of cysteine to form serine and one molecule of H2S is the preferred route of H2S generation by CSE (82). Additionally, the generation of homolanthionine (Vmax = 6.6±0.47 units/mg with 1 unit corresponding to 1 µmol of product formed per minute; Km = 5.9±1.2 mM) is greatly favored over lanthionine generation (Vmax = 1.2±0.3 units/mg; Km = 33±8 mM) catalyzed by CSE. CSE is sensitive to homocysteine concentrations; under conditions of hyperhomocsyteinemia,

the activity of CSE shifts to the condensation of homocysteine to form homolanthionine.

Similarly, the β-replacement of cysteine by homocysteine is thought to be the primary mechanism for H2S generation by CBS at maximal velocity and physiologic substrate concentrations (83). Although the condensation of cysteine and homocysteine may constitute the majority of H2S produced (Vmax = 18.7±2.6 units/mg; Km = 6.8±1.7 mM), the condensation of serine and homocysteine is still the favored reaction for the production of cystathionine (Vmax = 5.05±0.3 units/mg; Km = 2.76±0.5 mM) according to kinetic parameters. The simulations predict that a) if CBS and CSE are present at equimolar concentrations and b) if CBS is fully activated by adenosylmethionine (SAM), then CBS compared to CSE (7:3 ratio of contribution to H2S production) is the primary source of H2S generation under physiological conditions (83). However, the relative contributions of CBS and CSE to total H2S production vary by tissue enzyme concentration. In the brain, CBS expression is greater than CSE and therefore assumed to be the major contributor to H2S production (84). This was confirmed in CSE knockout mice, where brain H2S concentrations were unchanged. In contrast, H2S levels in serum and in heart were significantly reduced, suggesting CSE is the primary contributor of H2S generation in peripheral tissues (85). When compared to liver, the kidney and brain have lower CBS and CSE activities (86-88). Contributions of each enzyme to H2S generation are tissue specific and comparisons between activities are limited because of differences in substrate concentrations used for activity measurements.

Endogenous concentrations of H2S have been reported to range 30-50 µM in the peripheral system and 50-160 µM in the brain, but these seem to be grossly

overestimated (89-93). Recent publications have reported significantly lower H2S concentrations of 14 and 17 nM in liver and brain, respectively (93). Quantifying H2S is difficult due to due to rapid oxidation and volatility. Since H2S is weakly acidic (pKa=

6.76 at 37°C), at physiological pH of 7.4 18.5% of total sulfide exists as the undissociated from and 81.5% exists as the HS- anion (94). Therefore, an indirect method for quantifying total H2S is needed and proposed to be possible through the biomarkers, lanthionine and homolanthionine (82, 95).

As a gasotransmitter, H2S is involved in many mechanisms essential for life (96-

99). Current research suggests H2S is involved in the regulation of nitric oxide mediated signaling events and/or vice versa (100). Specifically, H2S has been shown to downregulate inducible NO synthase (iNOS) thereby controlling the production of another gasotransmitter (101). H2S also functions as a neuromodulator by enhancing

N-methyl-D aspartate receptor-mediated responses and facilitating the induction of long term potentiation in the hippocampus (84). The antioxidant activity of H2S is seen through its interaction with several cytotoxic oxidant species (eg. hypochlorite, peroxynitrite, hydrogen peroxide, and nitric oxide), thus preventing cell death (102-105).

In inflammation, H2S can induce the upregulations of anti-inflammatory and cytoprotective genes such as heme oxygenase (HO1) in smooth muscle cells (106). By upregulating HO1, H2S induces the production of CO, another gasotransmitter with known cyotprotective and anti-inflammatory effects (107).

Since H2S has many functions involved with vasodilation, studies began looking toward H2S as a possible modulator in cardiovascular disease. In addition to inducing vasodilation of smooth muscle cells by opening KATP channels, H2S can also activate

adenyl cyclase and cAMP-mediated vasodilation(108, 109) (89, 94, 110-112). H2S can attenuate myocardial ischemia-reperfusion injury by protecting mitochondrial function

(108). Mice with CSE deleted have reduced concentrations of H2S in serum, heart, aorta, and other tissues, along with hypertension and reduced vaso-relaxation (85).

Due to its ability to modulate vasodilation, H2S has been identified as a cardioprotective agent. Human patients with coronary heart disease had significantly lower levels of H2S compared to angiographically normal controls. Specifically, acute myocardial infarction patients had 60% less H2S than normal controls (113). Low plasma H2S even been suggested to reflect cardiovascular disease severity in humans. The conclusions related to the directionality of H2S change in heart disease have been contradictory in the literature. One such study found subjects with coronary artery disease, peripheral arterial disease, or any vascular disease had higher plasma-free H2S compared to subjects without vascular disease (114). These studies express the need for additional research in the field of H2S and cardiovascular disease.

One Carbon Metabolism

The one-carbon metabolism pathway provides one-carbon units essential for nucleotide biosynthesis and methylation reactions (115). All aspects of this pathway are essential in humans, such as the production of s-adenosylmethionine (SAM), the major methyl donor in-vivo (116). DNA and purine synthesis stem from the exchange of one- carbon units from the folate cycle. Briefly, dietary folate or folic acid is converted to tetrahydrofolate (THF) by dihydrofolate reductase (DHFR). Once THF is formed, it can redistribute a one carbon unit from serine to THF to yield glycine and 5,10- methyleneTHF in a reversible reaction. This reaction is catalyzed by the PLP- dependent enzyme serine hydroxymethyltransferase (SHMT). The formation of 5,10

methyleneTHF from THF can also occur from the mitochondrial glycine-cleavage system (GCS), which decarboxylates glycine to produce the required 1C unit concurrent with the production of CO2 and ammonia (117). This reaction also requires PLP as a cofactor for glycine decarboxylase. 5,10 methyleneTHF has many possible fates; such as a one-carbon unit donor for the synthesis of thymidylate from deoxyuridylate, an initial and rate-limiting step in DNA synthesis or it can be converted to 10-formylTHF which later is used in purine synthesis. If 5,10 methyleneTHF remains in the folate cycle it is irreversibly reduced to form 5-methylTHF, catalyzed by methylene tetrahydrofolate reductase (MTHFR). Riboflavin, as FAD, is a cofactor for MTHFR but folate status can also affect functionality in people with the common genetic polymorphism (MTHFR677C→T) (118). 5-methylTHF will then donate its methyl group to remethylate homocysteine, forming methionine and later SAM. This remethylation reaction is catalyzed by methionine synthase which is vitamin B12 dependent.

Methionine is activated by ATP to form S-adenosylmethionine (SAM). SAM, as the universal methyl donor in over 100 methyltransferase reactions such as in DNA methylation, neurotransmitter synthesis, and phospholipid synthesis, can then be demethylated to form SAH. The loss of adenosine from SAH yields homocysteine. If homocysteine is not remethylated, it will be catabolized in the transsulfuration pathway(Figure 1-3).

Transsulfuration Pathway

In addition to the transsulfuration pathways’ function in the production of hydrogen sulfide, it also maintains a safe concentration of homocysteine in-vivo, and produces cysteine. Cysteine can then serve as a precursor for glutathione production.

Homocysteine, derived from methionine in the methylation reaction of one-carbon

metabolism, has two possible fates. It can be remethylated by methionine synthase into methionine, which in turn leads to the recycling of intracellular folates, or it can enter the transsulfuration pathway. Metabolic regulation of homocysteine is tissue dependent, with all tissues possessing the methionine cycle. The transsulfuration pathway is limited to the liver, kidney, small intestine, and pancreas (119). The Km for homocysteine remethylation is estimated to be approximately 0.06 mM while the same value for CBS ranges from 1-25 mM. Therefore, at lower concentrations of homocysteine, remethylation is favored. When homocysteine concentration increases, so does the redirection to the transsulfuration pathway. Homocysteine catabolism is essential.

Hyperhomocysteinema is associated with greater risk of all-cause cardiovascular disease mortality (120). In the first enzymatic step of the transsulfuration pathway, cystathionine β-synthase catalyzes the condensation of cysteine or serine with homocysteine to form cystathionine. Serine, Kd of 56 ± 6 µM, will compete with cysteine

(Kd of 400 ± 27 µM) to bind to the PLP site according to enzymatic experiments (83). At intracellular levels of cysteine and serine, serine is expected to be the preferred substrate for CBS. S-adenosylmethionine is an allosteric activator of CBS.

Cystathionine is then cleaved by cystathionine γ-lyase to form cysteine and α- ketobutyrate. Cystathionine elevation in plasma is an indicator of vitamin B-6 deficiency due to the susceptibility of CSE to inadequate vitamin B-6 status. Glutathione is formed in an additional 2-step reaction requiring glutamate and glycine and 2 moles of ATP.

The production of this antioxidant is therefore dependent upon this pathway and thus vitamin B-6. This progression is depicted in Figure 1-3. The transsulfuration pathway is irreversible in humans.

Cardiovascular Disease

Cardiovascular disease is the leading cause of death in the world, with coronary artery disease being the most common. CVD is often associated with perturbed energy homeostasis and metabolism such as obesity, insulin resistance, and diabetes. While diagnostic markers of cardiovascular disease are well established in literature, there is still poor understanding of perturbations in metabolic pathways prior to clinical events, or prognostic markers (121). Prognostic markers of CVD outcomes are LDL cholesterol but nutritional correlates of disease remain unclear (122). Diagnostic biomarkers of acute changes, such as troponin I and troponin T for myocardial infarction and B-type naturiuretic peptide for congestive heart failure are the current measures used to diagnose specific events. However, there lacks a valid general profile for heart disease. Previously conducted metabolomic studies seeking to evaluate the severity of coronary artery disease were later found to be confounded by cholesterol-lowering therapies (123, 124). A recent metabolic study in patients undergoing planned myocardial infarction identified changes in circulating concentrations of metabolites in pyrimidine metabolism, the tricarboxylic cycle and the pentose phosphate pathway. The metabolic signature consisted of aconitic acid, hypoxanthine, trimethylamine N-oxide

(TMAO), and threonine (125). Targeted metabolic profile analysis has shown changes in amino acid and acylcarnitine patterns associated with CVD development (126).

Interestingly, many of these biomarkers involve PLP-dependent pathways. The use of high-throughput metabolomic research is growing, and being able to understand and quantify numerous metabolic changes in cardiovascular disease could aid in better and faster diagnoses.

Study Aims and Objectives

There are currently many gaps in knowledge related to the study of H2S due to its recent discovery as an endogenous gaso-signaling molecule. Possibly related to the varied physiological effects attributed to H2S, little is known about H2S concentrations and production capacity in tissues. Furthermore, mechanisms of regulation of H2S production and clearance are not known (127). Even less is known about H2S biomarkers, lanthionine and homolanthionine. Previous research in this lab has shown

H2S production is proportional to the production of these biomarkers and both are dependent upon vitamin B-6 status (95). This dissertation is focused on elucidating kinetic information about lanthionine and homolanthionine (Chapter 2), as an indirect measurement of H2S, as it relates to vitamin B-6 deficiency (Chapter 3) and cardiovascular disease in humans (Chapter 4). This dissertation consists of a set of novel experiments seeking to fill in gaps in knowledge about H2S and its biomarkers.

Chapter 2: Kinetic studies of lanthionine and homolanthionine in cell culture

 Aim 1: Determine the role of CSE and CBS on the production of homolanthionine and lanthionine in primary human hepatocytes and in HepG2 cells. o Hypothesis: CSE produces all of homolanthionine found yet only contributes minimally to the production of lanthionine. CBS produces the majority of lanthionine.  Aim 2: Compare the substrates, cysteine and serine, in the production of cystathionine in the primary human hepatocyte study and HepG2 cell culture model. o Hypothesis: Serine is the favored substrate in the transsulfuration pathway resulting in the majority of cystathionine formation.  Aim 3: Determine the rate of uptake, catabolism, and turnover of lanthionine and homolanthionine in a HepG2 cell culture model in sufficient and deficient vitamin B-6 concentration.

o Hypothesis: Both biomarkers will be taken up by the cells but at a slower rate than other amino acids (e.g. methionine and cysteine). The rate of uptake will not be affected by vitamin B-6 status. Lanthionine catabolism will be impaired in vitamin B-6 deficiency but homolanthionine catabolism will not be affected. Homolanthionine will have a higher turnover rate than lanthionine.  Aim 4: Identify any effects in the transsulfuration pathway from the addition of lanthionine and homolanthionine. o Hypothesis: There will likely be no changes in transsulfuration metabolite concentrations from the addition of labeled lanthionine and homolanthionine to media.

Chapter 3: Effect of short-term, marginal vitamin B-6 deficiency on the concentration of

H2S biomarkers, lanthionine and homolanthionine, in healthy human subjects

 Aim 1: Determine the impact of a short-term 28-d dietary vitamin B-6 restriction in healthy humans on the concentrations of lanthionine and homolanthionine in plasma. o Hypothesis: Vitamin B-6 deficiency will significantly reduce the concentration of lanthionine and homolanthionine in plasma.

 Aim 2: Determine the relationship between precursors of H2S biomarkers and their respective products in subjects before and after dietary vitamin B-6 restriction. o Hypothesis: Cysteine and homocysteine will be positively correlated with lanthionine and homolanthionine concentrations, respectively, both before and after vitamin B-6 restriction.

 Aim 3: Determine the relationship between H2S biomarkers and other functional biomarkers of vitamin B-6 deficiency in healthy human subjects before and after 28-d dietary vitamin B-6 restriction. o Hypothesis: Cystathionine, 3-hydroxykynurenine, and 3- hydroxykynurenine:kynurenic acid will be inversely correlated with lanthionine and homolanthionine concentrations after the 28-d dietary vitamin B-6 restriction.

Chapter 4: Metabolic consequences of plasma cystathionine elevation in subjects with suspected coronary artery disease

 Aim 1: Determine lanthionine and homolanthionine concentrations in subjects with high and low plasma cystathionine. o Hypothesis: Lanthionine and homolanthionine will be significantly lower in subjects with high cystathionine.  Aim 2: Explore differences between subjects with high and low plasma cystathionine by untargeted metabolomics. o Hypothesis: By separating subjects according to plasma cystathionine, there will also be clear differences in LC/MS and NMR spectra.

Figure 1-1. The structures the B-6 vitamers and their phosphorylated forms. (1) Pyridoxine and pyridoxine 5’-phosphate; (2) pyridoxamine and pyridoxamine 5’-phosphate; and (3) pyridoxal and pyridoxal 5’-phosphate form. ©Barbara Deratt.

Figure 1-2. Depiction of all the possible reactions producing H2S in vivo (128).

Figure 1-3. One carbon metabolism and transsulfuration pathway. Abbreviations: DHF, dihydrofolate; THF, tetrahydrofolate; SHMT, serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; MS, methionine synthase; DMG, dimethylglycine; SAM, s-adenosylmethionine; SAH, s- adenosylhomocysteine; CBS, cystathionine β-synthase; CSE, cystathionine γ- lyase. ©Barbara DeRatt

CHAPTER 2 KINETIC STUDIES OF LANTHIONINE AND HOMOLANTHIONINE IN CELL CULTURE

Introduction

Pathway kinetics of the traditional transsulfuration products have been previously reported in relation to vitamin B-6 status in humans (45, 46, 129). Short-term deficiency induced by a 28-day dietary restriction lead to elevated plasma glycine concentration, cystathionine concentration and synthesis, and altered proportions of other one-carbon metabolites. The rates of homocysteine synthesis and remethylation as well as the total flux of the transsulfuration pathway were not affected by this short-term marginal deficiency in humans (42, 45, 47). Severe deficiency in rats affected the concentrations of homocysteine, S-adenosylmethionine, and glutathione (32, 130-132). The variations of transsulfuration metabolites under different severities of vitamin B-6 deficiency confirm the susceptibility of CSE to vitamin B-6 deficiency compared to CBS. In-vitro and cell lysate studies show that both enzymes contribute substantially to H2S production and cysteine desulfhydration (127, 133), and recently I have shown in

HepG2 cells that vitamin B-6 status affects the synthesis of H2S and its biomarkers (95).

The impact of each enzyme on the production of H2S is still in disagreement and additional research is needed to understand the metabolism of lanthionine and homolanthionine biomarkers.

This chapter will investigate the impact of individual enzymes in the transsulfuration pathway on the production of H2S biomarkers in primary human hepatocytes and HepG2 cells. To evaluate individual enzyme contributions, I will inhibit

CSE in a subset of cells, resulting in only CBS functionality. This experiment will use the inhibitor, propargylglycine (PAG), which is a suicide inhibitor of CSE. I postulate

that enzyme activities between primary human hepatocyte subjects will vary and the inhibition of CSE will decrease the production of homolanthionine and lanthionine. This study provides an opportunity to evaluate the impact of each transsulfuration enzyme

(CBS and CSE) in the formation of H2S biomarkers in two live cell models. This chapter will also investigate the metabolism of lanthionine and homolanthionine after formation and provide new information about the cycling of lanthionine and homolanthionine in the

HepG2 cell model under conditions of vitamin B-6 deficiency and adequacy.

Materials and Methods

Materials

13 Isotopically labeled amino acids, [U- C5] L-methionine (97-98% purity), [3,3,4,4-

D4] DL-Homocysteine (98% purity), and [3,3-D2] L-cysteine (98% purity), were obtained from Cambridge Isotope Laboratories. All other HPLC-grade chemicals and solvents were purchased from Fisher Scientific or Sigma Aldrich. The HepG2 human hepatoma cell line was obtained from American Type Culture Collection (Manassas, VA). All cell culture media and media supplements were purchased from HyClone, Cellgro, or BD

Biosciences.

Primary Human Hepatocytes

Stable isotope studies in primary human hepatocytes were completed in collaboration with Dr. Robin da Silva at the University of Alberta (Edmonton, Canada).

Pre-prandial primary human hepatocytes were isolated using collagenase-based perfusion of liver fragments obtained by resection of specimens far away from the tumor margin during biopsy as previously described (134). Human liver samples used for hepatocyte isolation were obtained from patients undergoing operations for therapeutic purposes at the Service of Digestive Tract Surgery, University of Alberta. Ethical

approval was obtained from the University of Alberta's Faculty of Medicine Research

Ethics Board and all patients consented to participate in the study. Isolated primary hepatocytes were plated in 60mm collagen-coated dishes (BD Biosciences) at a concentration of 1.5 million cells per dish containing 2.5 mL of cell culture medium. The cells were incubated in RPMI-1640 culture medium (GIBCO) containing 10% fetal bovine serum (GIBCO) for 2 h to allow the cells to adhere to the plate. All incubations were conducted at 37°C in humidified air containing 5% CO2 and viability was assessed using trypan blue exclusion on parallel incubations. The final volume of medium was 2 mL per dish; with 3 independent flasks per time point per subject.

HepG2 Cell Culture

HepG2 cells were grown to confluency in modified essential Eagles medium with

Earle’s Balanced Salts (MEM/EBSS) obtained as the standard formulation containing approximately 1800 nmol/L pyridoxal (PL) and in custom form (without PL) from

HyClone (Logan, UT). All media were supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, antibiotic/antimycotic solution (1x), and 10% FBS, the later which contributes 14.8 nmol/L PL and 0.807 nmol/L PLP to the total media concentration. Thus, cells grown in custom media had 15 nmol/L PL added and were considered vitamin B-6 deficient as previously reported (95). Cells grown with the standard formulation were considered controls. All incubations were conducted at

37°C in humidified air containing 5% CO2. The final volume of medium was 8 mL per

75 cm2 flask; with 4 independent flasks per time point.

Analytical Methods

Intracellular PLP was determined in primary hepatocytes and HepG2 cells.

Briefly, cell lysates (500 µL) were combined with 500 µL 10% (w/v) trichloroacetic acid

and centrifuged at 10,600 g to deproteinize cells. Semicarbazide derivatives were then determined by HPLC (Dionex) with fluorescence detection (135). Protein concentration was determined by Bradford assay using a 50 µL aliquot of cell lysate diluted 2-fold

(without TCA) (136). The values of PLP and other metabolites were expressed per milligram protein.

Amino-thiol concentrations (i.e. total homocysteine, cysteine, glutathione, and cysteinylglycine) were quantified as 7-fluorescence-2-oxa-1,3-diazole-4-sulfonate derivatives by reverse-phase HPLC with fluorescence detection (137). Cell lysates and media were treated with tris(2-carboxyethyl)phosphine to reduce disulfide bonds, allowing for determination of total concentration for each amino-thiol.

Isotopic enrichment and concentrations of methionine, cystathionine, lanthionine, and homolanthionine were determined by gas chromatography mass spectroscopy

(GCMS) (Thermo DSQII) as N-propyl ester, heptafluorobutyramide derivatives in negative chemical ionization mode with selected-ion monitoring (129, 138). Methionine

13 was quantified relative to [ C5]-methionine internal standard. Cystathionine, lanthionine

13 and homolanthionine were quantified relative to [ C1] L-phenylalanine and norleucine internal standards. Homolanthionine reference standard was synthesized enzymatically using recombinant CSE and quantified relative to the cystathionine response curve, since it is not commercially available. Enrichment was determined as a ratio of labeled to total (unlabeled + labeled) species of methionine, cysteine, homocysteine, lanthionine, and homolanthionine. For cysteine and homocysteine, this analysis allowed for the determination of enrichment in the reduced form only, which was assumed to be representative of enrichment of total pools.

Isotopically Labelled H2S Biomarker Synthesis and Purification

To synthesize isotopically labeled lanthionine and homolanthionine, commercially available precursors [3,3-D2] L-cysteine, (98% purity) and [3, 3, 4, 4-D4] DL- homocysteine (98% purity) were purchased from Cambridge Isotope Laboratories.

Emzymatic reactions were executed in 100 mM Hepes buffer containing TCEP, PLP, and BSA at pH 8.5. All reactions were incubated at 37°C for the duration of the synthesis. Briefly, 40 mM cysteine wasconverted to lanthionine by human recombinant

CBS (3.0 mg/ mL; donated by Dr. Jan Kraus, University of Colorado) after a 24-h reaction (82% yield). Similarly, 8 mM Hcy was converted to homolanthionine after 6-h reaction catalyzed by recombinant CSE (2.5 mg/mL; provided by Dr. Ruma Banerjee,

University of Michigan) (97% yield). Lanthionine and homolanthionine were purified by ion exchange chromatography using DOWEX® 50WX8-200 resin by linear gradient elution from 0.0125 M ammonium formate pH 3.0 to 0.15 M ammonium formate pH 8.0.

After HPLC identification as dansyl-chloride derivatives, elution vials containing purified biomarkers were dried by speed-vac. Pure isotopically labeled lanthionine and homolanthionine were reconstituted in water prior to dilution into media.

Concentrations of purified stable isotopes were quantified by previously described

GCMS methods. Additional data from purification methods can be found in Appendix A.

In-Vivo Kinetics of the Transsulfuration Pathway

In all samples, fresh media was added prior to initiation of the stable isotope protocol. Cells were washed with PBS and a subset of human hepatocytes and HepG2 cells were treated for 3 minutes with media containing 1000 µM propargylglycine (PAG).

The concentration of PAG reduced CSE enzyme activity to <5% of normal activity

(Appendix B). For the first experimental protocol, the addition of fresh media containing

13 [U- C5] L-Methionine and [3, 3 D2] L-Cysteine was considered time point 0. Cell and media samples were collected at 0-, 0.5-, 1-, 2-, 4-, and 6-h time points. In the second experimental protocol, the addition of media containing synthesized [D4]-lanthionine and [D8]-homolanthionine was considered time point 0. Subsequent time points were

0.5-, 1-, 3-, 6-, 9-, and 12-h. All cells were lysed immediately by the addition of TCA to a final concentration of 10% (w/v), followed by 30 second ultrasonication (Fisher, Sonic

Dimembrator Model 100) continuous mode setting 4. Supernatants and media were stored at -80°C until analysis. Each time point contained 4 flask replicates.

All enrichment values were corrected for natural abundance and expressed as the molar ratio of labeled amino acid divided by the sum of the total forms (labeled + unlabeled). Data are presented as per milligram protein, as determined by Bradford assay (136).

Dr. Robin da Silva completed the stable isotope protocol in primary human hepatocytes at the University of Alberta, Canada using my methodology. Cell and media samples were shipped to the University of Florida by overnight shipping with dry- ice. All samples were frozen upon arrival. I completed the analyses for enrichment and concentrations on all samples. The enrichment protocol for HepG2 cells was done at the University of Florida by me.

Statistical Analysis

All data were analyzed in the statistical software package SigmaPlot 12.5.

Overall significant differences among metabolite concentrations and enrichments in human hepatocytes and HepG2 cells with added isotopically labeled cysteine and methionine were determined by two-way ANOVA (Factor A: Time, Factor B: Inhibition) with significance determined by Holm-Sidak test for pairwise comparisons. All data

were logarithm (base 10) transformed to meet the Gaussian assumption. Statistical significance was determined at P<0.05.

Results

Vitamin B-6 Analysis in Cell Culture Experiments

PLP and PL concentrations were determined in cell and media samples of controls and treatment experiments. In the media exposed to primary hepatocytes, PLP was 12.1 ± 2.95 nM while PL was 111 ± 15.8 nM. HepG2 cell commercial media contained approximately 1800 nM PL. All medium contained 10% FBS which has been shown to contribute 14.8 nM PL and 0.807 nM PLP to complete media. In media devoid of PL, FBS was the sole source of PLP and PL.

The concentration of PL and PLP showed great inter-subject variability. The concentration of PLP in Subject 1 was 303 ± 32.7 pmol/mg cell protein while PL concentration was 51.4 ± 9.70 pmol/mg cell protein. PLP and PL concentrations were

195 ± 9.72 and 82.8 ± 38.8 pmol/mg cell protein respectively, in Subject 2. The concentrations of PLP and PL in HepG2 cells grown in commercial media were 205 ±

39.5 pmol/mg cell protein and 4.45 ± 0.723 pmol/mg cell protein, respectively. The addition of PAG to any cells did not affect the concentrations of PLP or PL.

PAG Inhibition Affects In-Vivo Kinetics and Concentrations Similarly in Primary Human Hepatocytes and HepG2 Cells

13 In studies of cells incubated with [U- C5] L-methionine and [D2] L-cysteine, time

13 course plots of intracellular [U- C5]-methionine and [D2]-cysteine reached an intracellular enrichment plateau instantaneously and were maintained throughout the time course (Figure 2-1). In primary human hepatocytes, methionine enrichment was approximately 20% in both subjects and methionine concentrations were equivalent.

Cysteine concentrations varied by subject due to differences in the addition of labeled cysteine. Total cysteine concentration in media of subject 1 was 362±7.63 µM. Subject

2 total media concentration was 643±14.7 µM. Therefore, cysteine enrichment was

30% higher in subject 2 compared to subject 1 (P<0.001; Table 2-1). In HepG2 cells, methionine and cysteine enrichments were 14% and 13% respectively. Intracellular methionine enrichment was significantly higher in control cells compared to those treated with PAG (Figure 2-1D).

13 13 13 The appearance of [ C4]-homocysteine, [ C4]-cystathionine, and [ C4]-

13 homolanthionine derived from [ C5]-methionine was readily detected. Intracellular homocysteine concentrations were not detected but extracellular homocysteine enrichment was not affected by PAG addition (data not shown). The concentration of labeled cystathionine in cells and media was more than 6 times higher in primary human hepatocytes and HepG2 cells when PAG was present (P<0.001), suggesting the level of inhibition was sufficient to inhibit catabolism of cystathionine by CSE (Figure 2-

2). Synthesis rates of cystathionine were not affected by inhibition since initial rates of enrichment were not different. When CSE was inhibited, there was a 76% reduction of homolanthionine production in primary human hepatocytes (P<0.041; Figure 2-3A). In

HepG2 cells, homolanthionine was also significantly reduced by PAG, however these changes were not as profound (Figure 2-3B). The appearance of labeled lanthionine over the 6h time course for primary human hepatocytes subject 1 showed no differences in the presence or absence of PAG inhibition (P=0.198). Subject 2, which had higher cysteine enrichment and total concentration, concentrations of lanthionine were significantly higher when CSE was inhibited (P=0.029; Figure 2-4B). HepG2 cell

showed similar results compared to subject 2, such that labeled lanthionine was significantly higher in control cells compared to PAG inhibition at the 6-h time point

(P=0.001; Figure 2-4C). These results suggest a possible role of CSE in lanthionine catabolism.

The use of cysteine and methionine as labeled precursors allowed for the determination of the competition between serine and cysteine in the formation of cystathionine catalyzed by CBS. [D2]-cystathionine was easily detected in the time course study in both culture models. To ensure that only CBS-derived cystathionine was considered, only cells treated with PAG were used. Cysteine only accounted for 0.35% of total cystathionine production in primary human hepatocytes subject 1. When cysteine enrichment elevated in subject 2, cysteine accounted for 18.8% of cystathionine production (Figure 2-5). The competition between serine and cysteine in the formation of cystathionine is affected by cysteine concentration. However, even at elevated cysteine concentrations, serine is the favored substrate of CBS catalyzed production of cystathionine. These results were confirmed in the HepG2 study (Figure

2-5).

In addition to the amino-thiols that were quantified and data reported as appearance of labeled products, cysteinylglycine and glutathione concentrations were also determined intracellularly. Glutathione was significantly lower in HepG2 cells and primary human hepatocytes exposed to PAG compared to control cells (P<0.01; Figure

2-6 C and 2-6D). In HepG2 cells, cysteinylglycine concentrations were greater in control cells at some time points (P<0.05; Figure 2-6B). There were no differences between cysteinylglycine concentrations in primary human hepatocytes (Figure 2-6A).

Since H2S is produced concurrently with lanthionine and homolanthionine in a

1:1 ratio, the concentration of lanthionine or homolanthionine is a molar equivalent to

H2S produced in that specific reaction. Taken together, I calculated the concentration of

H2S produced in some of the non-canonical transsulfuration reactions. For instance, at

20% [3,3-D2] cysteine enrichment with no inhibition (total cysteine concentration in media was 342 µM), [D2]-cysteine only accounted for 0.35% of total cystathionine production. The formation of cystathionine from cysteine also produces one molecule of

H2S. By multiplying the percentage of cystathionine derived from cysteine by the steady state intracellular concentration of cystathionine, I concluded 13.6 pmol/mg cell protein

H2S was formed by this reaction. The total intracellular concentration of lanthionine is equivalent to the concentration of H2S derived in that reaction. After adding lanthionine concentration and cystathionine derived from cysteine, H2S concentration from CBS reactions was 90.1 pmol/mg cell protein in hepatocytes with low cysteine enrichment.

Homolanthionine concentration is also a molar equivalent to H2S concentration from this synthesis reaction. Therefore, 277 pmol/mg cell protein H2S was formed by this reaction. From the above mentioned reactions, I calculated CSE accounted for approximately 70% of total H2S production while CBS only accounted for 30% in primary human hepatocytes (Table 2-2). Using the same calculation in HepG2 cells, I determined CSE accounted for 41% and CBS for 59% of H2S produced in these reactions (Table 2-3). Therefore, I can conclude in these listed reactions that CSE has a greater role in the formation of H2S then previously reported (83). Due to higher the cysteine concentration in subject 2, I could evaluate the impact of cysteine on enzyme contributions to the formation of H2S. At high cysteine concentrations, 18.8% of

cystathionine was derived from cysteine (determined by the appearance of [D2]- cystathionine). Therefore, 727 pmol/mg cell protein H2S was formed in this reaction

(determined by molar equivalents). Lanthionine production was also increased, but there was no effect on homolanthionine production at high cysteine concentrations.

Therefore, at higher cysteine concentrations, CBS accounted for 78% of H2S derived from the reactions listed while CSE (which was not sensitive to elevated cysteine) accounted for 22% (Table 2-2).

HepG2 Amino-thiol Concentrations Differ Slightly by Treatment Group After Incubation with Labeled Lanthionine and Homolanthionine

Total amino-thiol concentrations in cells and media from the labeled lanthionine and homolanthionine time course experiment were measured. Total cysteine concentrations were similar for all three groups (control, B-6-def, and PAG inhibited) intracellularly and extracellularly (Figure 2-7A and 2-7B). In media, total cysteine concentrations decreased after the addition of labeled tracers. Intracellular total homocysteine concentrations remained steady throughout the 12-h time course and were not different between groups (Figure 2-7C and 2-7D). Extracellular total homocysteine was higher in

B-6-deficient cells compared to controls and PAG treated cells. While extracellular total glutathione was undetectable, intracellular concentrations were lower in cells treated with PAG compared to controls and B-6 deficient cells (Figure 2-7G). Total cysteinylglycine concentration in cells was highest in cells treated with PAG, suggesting the lower glutathione concentrations were due to increased catabolism. B-6-deficient cells had the highest concentration on total cysteinylglycine in the media followed by

PAG-treated cells. Control media had the lowest concentration of total cysteinylglycine

(Figure 2-7E and 2-7F).

Discussion

This tracer study sought to expand the understanding of the metabolism of the transsulfuration pathway, specifically in its production of H2S measured indirectly by biomarkers, lanthionine and homolanthionine. Previous studies have shown the sensitivity of CSE to vitamin B-6 insufficiency, such that marginal deficiency can significantly decrease enzyme activity (131). To simulate severe deficiency in primary human hepatocytes, CSE was inhibited using the enzyme specific inhibitor, PAG. The use of a CSE-suicide inhibitor allowed for the determination of the role of each transsulfuration enzyme on the production of the H2S biomarkers and thus the molar equivalents of H2S produced. Over 76% of homolanthionine was produced by CSE.

The additional 24% could have been produced through unknown reactions or nonenzymatic formation, since homolanthionine has been found in commercial media and small quantities are produced during derivatization. While CSE has the ability to catalyze lanthionine formation, CBS is thought to produce 99.9% of lanthionine due to v/[E] values estimated to be 2.9 X 10-8 s-1 for CSE and 3.9 X 10-4 s-1 for fully SAM- activated CBS (83). No detectable amount of lanthionine was produced by CSE in this study. Interestingly, Singh et. al stated significant cleavage activity of CSE for lanthionine with Vmax of 1.03±0.05 unit/mg and a Km,lan= 0.75 ± 0.17 mM (Km/Vmax ratio=

0.728) compared to with Vmax of 3.1±0.1 unit/mg and a Km,lan= 0.28 ± 0.03 mM (Km/Vmax ratio= 0.090) for cystathionine (83). In this study, CSE cleaved 67% of lanthionine in cells when cysteine enrichment was high. HepG2 cells also showed some cleavage of lanthionine by CSE, although not as apparent. This was the first study to show CSE- catalyzed cleavage of lanthionine in live cells. Overall, this study provided additional

insights on H2S production in the transsulfuration pathway but further studies need to be conducted to confirm and strengthen these findings.

The use primary human hepatocytes and HepG2 cells allowed for comparison of culture models in this experiment. In the canonical reactions of the transsulfuration pathways, both models behaved similarly. In the production of cystathionine from cysteine, CBS contributed to approximately 85% of production while CSE only contributed 15%. These percentages were the same in both models. Homolanthionine production rates were also agreeable. The only difference between the models was the impact of CBS on H2S formation (by previously described calculations). In primary human hepatocytes, CSE had a greater impact on H2S formation compared to in

HepG2 cells. Overall, this study showed the use of HepG2 cell, which are more readily available, are comparable to healthy primary human hepatocytes for study of the transsulfuration pathway.

Table 2-1. Isotopic enrichments of methionine and cysteine in media in primary human hepatocytes and HepG2 cells1,2. Methionine Cysteine Enrichment (%) Enrichment (%) Subject 1 20.1±0.537 21.3±1.21 Primary Human Hepatocytes Subject 2 22.2±0.386 56.4±1.31 HepG2 cells 13.9±2.02 12.9±0.542 1All data are presented as means ± SD. Averages taken over 6h time course with and without PAG.

2Enrichment=tracer/(tracer + tracee)

Table 2-2. Molar equivalent concentrations of H2S from reactions in the transsulfuration pathway of primary human hepatocytes1. Low Cysteine High Cysteine Reaction H2S Conc. % H2S Conc. % Contribution Contribution pmol/mg cell protein 2Cys→Lan + H S 90.1 27 263 78 CBS 2 Hcy + Cys→Csn + H2S 13.6 727 CSE 2Hcy→Hlan + H2S 277 73 277 22 1Concentrations of lanthionine, cystathionine, and homolanthionine used in calculations were averages of 4-6 hr total concentrations (n=1 subject; 3 independent cell flasks). During these time points, concentrations reached a plateau. H2S has an equimolar relationship to each amino acid (ex. 1 mol H2S per 1 mol lan).

Table 2-3. Molar equivalent concentrations of H2S from reactions in the transsulfuration pathway of HepG2 cells.1 Reactions H2S Conc. % Contribution pmol/mg cell protein 2Cys→Lan + H S 63.6 41.2 CBS 2 Hcy + Cys→Csn + H2S 130 CSE 2Hcy→Hlan + H2S 139 58.2 1Concentrations of lanthionine, cystathionine, and homolanthionine used in calculations were averages of 4-6 hr total concentrations (n=4). During these time points, concentrations reached a plateau. H2S has an equimolar relationship to each amino acid (ex. 1 mol H2S per 1 mol lan).

Figure 2-1. Enrichments over 6-h time course experiments of the labeled precursors (A 13 and B) [ C5] L-methionine and (C and D) [D2] L-cysteine with and without the addition of PAG. (▲) represents primary human hepatocytes and (●) represents HepG2 cells. All data are shown as means ± SD, n=2 in primary human hepatocytes and n=4 in HepG2 cells. *Designates significance by Holm-Sidak method for multiple comparisons at P<0.05.

Figure 2-2. Appearance of labeled of cystathionine is elevated in cells with PAG intracellularly and extracellularly. (▲) represents primary human hepatocytes and (●) represents HepG2 cells. Data are shown as means ± SD, n=2 in primary human hepatocytes and n=4 in HepG2 cells.

Figure 2-3. Enrichment of homolanthionine over the 6-h time course in primary human hepatocytes and HepG2 cells. (▲) represents primary human hepatocytes and (●) represents HepG2 cells. Data are shown as means ± SD, n=2 in primary human hepatocytes and n=4 in HepG2 cells. *Designates significance by Holm-Sidak method for multiple comparisons at P<0.05.

Figure 2-4. Appearance of labeled lanthionine in A) Subject 1 primary human hepatocytes, B) Subject 2 primary human hepatocytes, and C) HepG2 cells with and without the addition of PAG. Data are shown as means ± SD, n=3 flasks per subject in primary human hepatocytes and n=4 in HepG2 cells. *Designates significance by Holm-Sidak method for multiple comparisons at P<0.05.

Figure 2-5. Labeled product appearance as determined from total cystathionine concentration derived from serine/cysteine intracellular competition. *Designates significant difference (P<0.05) between concentrations in each time point, determined by ANOVA with multiple pairwise comparisons based on Holm-Sidak method. Data are means ± SD, n=2 in primary human hepatocytes and n=4 in HepG2 cells.

Figure 2-6. Total intracellular glutathione and cysteinylglycine concentrations in the presence or absence of PAG; A) Hepatocyte total cysteinylglycine; B) HepG2 total cysteinylglycine; C) Hepatocyte total glutathione; D) HepG2 total glutathione. (▲) represents primary human hepatocytes and (●) represents HepG2 cells. All data expressed as means ± SD, n=2 in primary human hepatocytes and n=4 in HepG2 cells. *Denotes significance determined by Holm-Sidak method for multiple comparisons at P<0.05.

Figure 2-7. Aminothiol concentrations intracellularly and extracellular over 12-h time course after the addition of labeled lanthionine and homolanthionine; A)

Intracellular cysteine; B)Extracellular cysteine; C) Intracellular homocysteine; D) Extracellular homocysteine; E) Intracellular cysteinylglycine; F) Extracellular cysteinylglycine; G) Intracellular glutathione.

CHAPTER 3 EFFECT OF SHORT-TERM, MARGINAL VITAMIN B-6 DEFICIENCY ON THE CONCENTRATION OF H2S BIOMARKERS, LANTHIONINE AND HOMOLANTIHONINE, IN HEALTHY HUMAN SUBJECTS1

Abstract

Background: Suboptimal vitamin B-6 status is associated with increased cardiovascular disease risk, although the mechanism is unknown. The synthesis of the vasodilator hydrogen sulfide (H2S) occurs through side reactions of the transsulfuration enzymes, cystathionine β-synthase (CBS) and cystathionine -lyase (CSE), with pyridoxal 5’-phosphate (PLP) as coenzyme. Two proposed H2S biomarkers, lanthionine and homolanthionine, are produced concurrently.

Objective: To determine whether H2S production is reduced by vitamin B-6 deficiency, we examined the relationships among plasma concentrations of lanthionine and homolanthionine, along with other components of the transsulfuration pathway

(homocysteine, cystathionine, serine and cysteine) in a secondary analysis of samples from two vitamin B-6 restriction studies in healthy men and women.

Methods: Metabolite concentrations were measured in plasma from 23 subjects before and after 28-d controlled dietary vitamin B-6 restriction (0.37±0.04 mg/d). Vitamin B-6 restriction effects on lanthionine and homolanthionine concentrations were assessed.

Associations among H2S biomarkers, transsulfuration metabolites and functional indicators of vitamin B-6 deficiency were analyzed by linear regression.

1 Reprinted in full with the exception of reference list with permission from Deratt B, Ralat M, Gregory J. Short-term vitamin B-6 restriction does not affect plasma concentrations of H2S biomarkers, lanthionine and homolanthionine, in healthy men and women. J Nutr. 2016. pii: jn227819.

Results: Mean lanthionine and homolanthionine concentrations were not affected by

28-d dietary vitamin B-6 restriction (P<0.66), with marked heterogeneity of individual responses. Plasma homolanthionine and lanthionine were not associated with the vitamin B-6 status markers cystathionine, 3-hydroxykynurenine, or 3- hydroxykynurenine:xanthurenic acid in adequate vitamin B-6 status. However, homolanthionine was positively associated with 3-hydroxykynurenine and 3- hydroxykynurenine:xanthurenic acid in B-6 deficiency. Plasma lanthionine was positively correlated with cysteine concentration before and after dietary restriction

(P=0.002). Homolanthionine concentration was positively correlated with homocysteine concentration only in vitamin B-6 adequacy (P=0.001). Certain individuals exhibited marked increases or decreases in lanthionine and homolanthionine concentrations suggestive of increases and decreases in H2S after restriction.

Conclusions: In spite of a lack of effect of vitamin B-6 restriction on mean concentrations of H2S biomarkers, correlations that suggest homolanthionine production is dependent on adequate vitamin B-6 status.

Introduction

Hydrogen sulfide (H2S) is produced in mammalian cells by the pyridoxal 5’- phosphate (PLP)-dependent transsulfuration enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). As a regulatory molecule, H2S promotes vasodilation through the opening of KATP channels (89, 94, 110-112), which attenuates myocardial ischemia-reperfusion injury and hypertension (85, 108). Accurate measurement of H2S is difficult and prone to inaccuracy due to its volatility and susceptibility to oxidation (93).

Lanthionine and homolanthionine, which have been previously described as markers of

H2S synthesizing reactions, are formed concurrently with H2S in side reactions of CBS and CSE from cysteine and homocysteine, respectively (82, 95, 133, 139).

Previous research has shown that vitamin B-6 insufficiency causes a differential effect on the two enzymes of the transsulfuration pathway. Although PLP is well established as a coenzyme of CBS (140), vitamin B-6 deficiency in rats yields only modest losses of CBS activity (32). In contrast, CSE exhibits substantial losses of activity consistent with the extent of lowering of liver PLP in rats (32). The increase in plasma and urinary cystathionine in humans along with elevations of liver cystathionine in rats during moderate vitamin B-6 deficiency documents the existence of a metabolic bottleneck created by reduced cellular PLP supply and the resulting reduction of CSE activity (32, 45, 48, 132, 141). In spite of the sensitivity of CSE, vitamin B-6 restriction in humans does not reduce overall flux of the transsulfuration pathway (47) nor does it alter overall cysteine flux (45). The elevation of cystathionine concentration is postulated to provide a driving force (i.e., increased ratio of cystathionine concentration to its Km) that compensates for the reduced CSE activity (i.e., lower Vmax), thus maintaining transsulfuration flux and cysteine production at most degrees of vitamin B-6 insufficiency.

In spite of this resilience and adaptability of the canonical reactions of transsulfuration pathway, the effects of vitamin B-6 deficiency on H2S production in humans or a mammalian animal model are unclear. Recent cell culture studies have shown that the adequacy of vitamin B-6 supply to cells influences their capacity to form

H2S and governs the rate and extent of lanthionine and homolanthionine synthesis (95), suggesting H2S production may be reduced by vitamin B-6 insufficiency in vivo. We

hypothesize that the condensation of two molecules of cysteine by CBS to form lanthionine would be minimally affected by vitamin B-6 restriction in humans.

Homolanthionine formation by CSE catalyzed condensation of 2 homocysteine molecules is postulated to be lowered after vitamin B-6 restriction due to the reduction of CSE activity and the potential competitive inhibition by the alternate substrate, cystathionine. However, the in vivo nutritional regulation of H2S production by transsulfuration enzymes, including the influence of vitamin B-6 status, has not been fully determined.

We report here quantitative analysis of the surrogate markers of H2S production, lanthionine and homolanthionine, in healthy men and women before and after vitamin B-

6 restriction in two previously reported dietary protocols (47, 48). This secondary analysis from the same plasma samples allows assessment of lanthionine and homolanthionine, within the context of previous kinetic, metabolite profiling and metabolomic analyses (39, 40) to provide initial information regarding the influence of short-term vitamin B-6 insufficiency on H2S production.

Material and Methods

Human Vitamin B-6 Restriction Protocols

Plasma samples analyzed in this study were obtained from 23 healthy participants from 2 identical dietary vitamin B-6 restriction studies that were previously reported (47, 142). In these studies, health was determined through routine tests of hepatic, renal, thyroid and hematological function as well as a physical examination (47,

142). Further exclusions were no history of gastrointestinal surgeries, chronic disease, chronic smoking, alcohol or drug use, and body mass index less than 28 kg/m2.

Additional exclusion criteria included consumption of supplements containing vitamins,

amino acids, or proteins. Preprandial assessment determined nutritional adequacy of serum folate (>7 nmol/L), serum vitamin B12 (>200 pmol/L), plasma PLP (>30 nmol/L), and plasma total homocysteine (<12 µmol/L). The baseline blood sample was taken after a 2-d standardized diet that was nutritionally adequate (total vitamin B-6=1.02 ±

0.11 mg/d). After the completion of the 28-d restriction diet (total vitamin B-6=0.37 ±

0.04 mg/d), a second preprandial plasma sample was collected. Samples were collected in EDTA tubes and separated by centrifugation of whole blood at 1650 X g for

15 minutes at 4°C. All samples were stored at 80°C with minimal freeze-thaws. These protocols and subsequent metabolite analyses were approved by the University of

Florida Institutional Review Board and the University of Florida Clinical Research Center

Scientific Advisory Committee and registered at clinicaltrials.gov as NCT00877812.

Analytical Methods

Plasma PLP concentrations were determined by reverse phase HPLC (Dionex) with fluorescence detection (95, 135). Aminothiol concentrations (i.e. total homocysteine, cysteine, glutathione, and cysteinylglycine) were quantified as 7- fluorescence-2-oxa-1,3-diazole-4-sulfonate derivatives by reverse-phase HPLC with fluorescence detection (137). Concentrations of methionine and the transsulfuration metabolites lanthionine, homolanthionine and cystathionine were determined by gas chromatography mass spectroscopy (GCMS) (Thermo DSQII) as N-propyl ester, heptafluorobutyramide derivatives in negative chemical ionization mode with selected- ion monitoring (129, 138). Lanthionine and homolanthionine were measured relative to a norleucine internal standard, while cystathionine and methionine were quantified

13 relative to [ C5]-methionine and [D4]-cystathionine internal standards (Cambridge

Isotopes). As described previously (82), recombinant CSE (provided by Dr. Ruma

Banerjee, University of Michigan) was employed for enzymatic synthesis of a homolanthionine reference standard. Homolanthionine was quantified relative to the cystathionine response curve. Previously reported concentrations of homocysteine, cysteine, 3-hydroxykynurenine and xanthurenic acid were utilized for vitamin B-6 restriction study data comparison (39, 40, 143).

Statistical Analyses

All data are presented as mean ± SD. The changes in lanthionine and homolanthionine concentrations, pre-restriction versus post-restriction, were analyzed by Student’s paired t-test after log transformation. Linear regression analysis was performed to compare functional indicators of B-6 deficiency (i.e. cystathionine, 3- hydroxykynurenine, and 3-hydroxykynurenine:xanthurenic acid) and transsulfuration amino acids to H2S biomarkers before and after dietary restriction using SigmaPlot 12.5.

Shapiro-Wilk normality test and constant variance test were performed before modeling.

Statistical significance was determined at the 0.05 level for all procedures used.

Results

Effect of Vitamin B-6 Restriction Protocol

As reported previously, all participants had serum folate, vitamin B-12 and total homocysteine in the normal range at the beginning and end of the study (47, 48). The

28-d dietary vitamin B-6 restriction protocol lowered plasma PLP from 52.6 ± 2.93 to

21.5 ± 0.95 nmol/L (P<0.001). After B-6 restriction, 15 of the participants were considered to be in the marginally deficient range (plasma PLP 20-30 nmol/L) and 8 were considered to be vitamin B-6 deficient (plasma PLP<20 nmol/L) after 28-d dietary vitamin B-6 restriction. A 2-sample t test after restriction revealed no significant difference between male and female participants (P=0.10; data not shown). Descriptive

data regarding the 23 participants in this protocol have been reported previously (39).

Additionally, the elevations of cystathionine, 3-hydroxykynurenine and the ratio of 3- hydroxykynurenine:xanthurenic acid constituted functional evidence of vitamin B-6 insufficiency (P<0.001; Table 3-1), as reported previously. At this extent of deficiency, the concentrations of cysteine and homocysteine, which are precursors of lanthionine, homolanthionine and H2S, were not altered (Table 3-1).

H2S Biomarkers in Healthy Human Subjects

Lanthionine and homolanthionine concentrations ranged from 89.0 to 372 nmol/L and 5.75 to 32.3 nmol/L in plasma during normal vitamin B-6 status, respectively. There was no significant effect of the vitamin B-6 restriction on mean lanthionine and homolanthionine concentrations (Table 3-1). Plasma lanthionine exceeded homolanthionine by approximately a factor of 10 both before and after vitamin B-6 restriction. Lanthionine and homolanthionine concentrations before and after dietary restriction did not differ between male and female participants (P=0.35). The sum of lanthionine and homolanthionine concentrations also did not change after dietary restriction (P=0.75).

Despite the lack of significant effect of vitamin B-6 restriction on mean concentrations of plasma lanthionine and homolanthionine, we observed substantial variability among individuals in their response of both lanthionine and homolanthionine to vitamin B-6 restriction (Figure 3-1A and 3-1B). Of the 23 total subjects, 12 (52% of total) had higher lanthionine concentrations after the 28-d B-6 restriction diet. More females had higher lanthionine concentrations after restriction, while the majority of the males exhibited lower lanthionine concentrations after restriction. The mean percent increase was 22% in plasma lanthionine for all subjects that had higher concentrations

after restriction, while the mean percent decrease was 19% in plasma lanthionine for all subjects who had lower concentrations after restriction. The heterogeneity in lanthionine concentrations was not related to the concurrent changes in cysteine concentration.

The majority of subjects (61%) had lower homolanthionine concentrations after restriction. Fifty five percent of females and 67% of males had lower homolanthionine concentrations after the 28-d B-6 restriction. The mean percent changes for homolanthionine for those that increased versus those that decreased were 43% and

44%, respectively. Increases or decreases in homolanthionine concentration after restriction were not related to changes in homocysteine concentrations.

Relationships Among Lanthionine, Homolanthionine, Total Homocysteine, Total Cysteine and Functional Indicators of Vitamin B-6 Deficiency

Previously reported plasma metabolite concentrations from the 23 subjects were compared with newly determined concentrations of lanthionine and homolanthionine.

Linear regression analysis indicated no significant relationship between the concentration of lanthionine and the functional indicators of vitamin B-6 deficiency (i.e. cystathionine, 3-hydroxykynurenine, and 3-hydroxykynurenine:xanthurenic acid) before dietary restriction. Homolanthionine was not associated with 3-hydroxykynurenine, or 3- hydroxykynurenine:xanthurenic acid before dietary restriction but was positively associated with cystathionine (R2=0.38, P=0.002; Fig. 3-4). After 28-d restriction, homolanthionine was positively correlated with 3-hydroxykynurenine (R2=0.22,

P=0.025) and the ratio of 3-hydroxykynurenine:xanthurenic acid (R2=0.39, P=0.033; Fig.

3-4). Lanthionine concentrations were significantly correlated with cysteine concentrations (the precursor of lanthionine) in both adequate (R2=0.36, P=0.002) and

B-6-restricted (R2=0.37, P=0.002) states (Figure 3-2A and 3-2B). Before dietary

restriction of vitamin B-6, homolanthionine was positively correlated with its precursor, homocysteine (R2=0.41, P<0.001) even though all participants exhibited plasma total homocysteine concentration within the normal range (<12 µmol/L). The positive correlation between homocysteine and homolanthionine concentrations disappeared after vitamin B-6 restriction (Figure 3-2C and 3-2D). Homolanthionine and lanthionine were positively correlated when vitamin B-6 status was adequate (R2=0.36, P=0.002) vitamin B-6 restriction caused the relationship to disappear after (R2=0.062, P=0.25)

(Figure 3-3). Cystathionine and homocysteine concentrations were positively correlated in B-6 adequate (R2=0.23, P=0.021) and restricted (R2=0.18, P=0.047) states, suggesting the precursor concentration determines product concentration (data not shown). However, only in B-6 adequacy was cystathionine positively correlated with cysteine production (R2=0.17, P=0.049). Homocysteine was positively correlated to cysteine concentration before (R2=0.76, P<0.001) and after (R2=0.51, P<0.001) dietary restriction (data not shown).

Discussion

This study quantified lanthionine and homolanthionine plasma concentrations in healthy adults before and after a 28-d dietary vitamin B-6 restriction. We have previously reported regarding these vitamin B-6 restriction studies the elevation of cystathionine, disruptions of the tryptophan pathway, and other consequences (39, 40,

47, 48). In the present study, we employed several functional biomarkers of vitamin B-6 insufficiency that reflect metabolic effects: cystathionine, 3-hydroxykynurenine and the ratio 3-hydoxykynurenine:xanthurenic acid (144), all of which are increased during

deficiency. In this manner, we could evaluate the relationship of the lanthionine and homolanthionine relative to functional indicators of the deficiency state.

Although vitamin B-6 restriction did not alter the mean concentrations of lanthionine and homolanthionine or their precursors, linear regression analysis showed significant associations among the transsulfuration metabolites. The precursor-product relationship between cysteine and lanthionine existed before and after vitamin B-6 restriction, which suggests that lanthionine production by CBS is not affected by short- term vitamin B-6 insufficiency. There was, however, an apparent relationship between homolanthionine and vitamin B-6 status. The precursor-product relationship of homocysteine and homolanthionine was diminished after the vitamin B-6 restriction, likely due to the reduction of CSE activity caused by vitamin B-6 insufficiency. The positive correlation between homolanthionine and the functional biomarkers, 3- hydroxykynurenine and 3-hydroxykynurenine:xanthurenic acid, after restriction was unexpected since we predicted lower homolanthionine concentrations with vitamin B-6 deficiency. These findings suggest homolanthionine production is associated with vitamin B-6 status in humans, although a moderate short-term deficiency did not significantly change plasma homolanthionine concentrations.

Kinetic simulations by Singh predicted that, at normal cellular homocysteine concentrations, CBS would account for 70% of transsulfuration-derived H2S production, with the relative contribution from CBS declining in conditions of elevated homocysteine

(83). From these simulations, the CBS catalyzed β-replacement reaction of homocysteine and cysteine to form cystathionine produced the majority of H2S, with significantly less H2S from the condensation of 2 molecules of cysteine to form

lanthionine (83, 145). In our study, mean total plasma cysteine was 37 times higher than total plasma homocysteine irrespective of vitamin B-6 status, which corresponded to lanthionine concentrations that were 10 times greater than homolanthionine concentrations. Since essentially all lanthionine is produced by CBS and homolanthionine by CSE (95), our results provide in vivo evidence that CBS-catalyzed

H2S-generating reactions greatly exceeded those of CSE and were dependent on the concentration of the respective substrates, cysteine and homocysteine.

Interestingly, the concentration of plasma lanthionine was equivalent to that of cystathionine in this study. The canonical and predominant reaction producing cystathionine is the CBS-catalyzed condensation of serine and homocysteine which does not result in H2S formation. Since only a small portion of cystathionine is derived from the condensation of homocysteine with cysteine (145), which produces 1 molecule of H2S concurrently, this suggests more H2S is produced by the CBS-catalyzed formation of lanthionine than previously expected (83). The limitation of our current findings is that we report only plasma concentrations, which are governed by rates of appearance of cysteine, homocysteine, cystathionine, lanthionine and homolanthionine from cells, in addition to their disposal rates by metabolism or excretion. Absolute quantitative inferences regarding the functions of CBS and CSE in H2S production will require additional information about in-vivo rates of synthesis and disposal of the biomarkers lanthionine and homolanthionine.

Homolanthionine has been found in urine of patients with homocystinuria, thereby defining the relationship between precursor and product (79). Furthermore,

Chiku et al. suggested homolanthionine could serve as a biomarker of H2S production in

elevated homocysteine conditions (82). Our results show that homolanthionine is also correlated with homocysteine under normal conditions, extending its application as an

H2S biomarker in normal physiological conditions.

In summary, the major implication of this study was that moderate, short-term vitamin B-6 insufficiency may not substantially reduce the production of H2S. In cell culture studies, a severe level of vitamin B-6 deficiency yielded reduced cellular concentrations of lanthionine and homolanthionine and the H2S production capacity

(95). This suggests that more severe deficiency in humans, potentially accentuated by inflammatory conditions or vitamin B-6 antagonists, could have more extensive effects on H2S production by transsulfuration enzymes.

Table 3-1. Concentrations of amino acid and vitamin B-6 functional biomarkers in preprandial human plasma before and after 28-d dietary vitamin B-6 restriction.1

Metabolite Pre-Restriction Post-Restriction P Value

Lanthionine, nmol/L 188 ± 18 193 ± 16 0.659

Homolanthionine, nmol/L 14.9 ± 1.7 13.5 ± 1.9 0.460

Total Cysteine, µmol/L2 256 ± 39 253 ± 36 0.577

Total Homocysteine, µmol/L2 6.97 ± 1.26 6.97 ± 1.33 0.925

Cystathionine, nmol/L 145 ± 60.4 232 ± 79.3 <0.001

3-hydroxykynurenine, nmol/L2 24.5 ± 9.46 32.4 ± 11.0 <0.001

3-hydroxykynurenine:xanthurenic acid 3.36 ± 1.92 5.20 ± 3.10 <0.001

Plasma PLP, nmol/L2 52.6 ± 2.93 21.5 ± 0.95 <0.001

1Mean ± SD, n=23.

2Previously reported concentrations (39).

Figure 3-1. Human plasma concentrations of A) homolanthionine and B) lanthionine before and after 28-d dietary vitamin B-6 restriction. Dashed lines are female subjects and solid lines are male subjects, n=23.

Figure 3-2. Relationships between lanthionine and its precursor cysteine (A and B) and between homolanthionine and its precursor homocysteine (C and D) in human plasma before and after 28-d dietary vitamin B-6 restriction (n=23).

Figure 3-3. Relationship between lanthionine and homolanthionine in human plasma A) before and B) after 28-d dietary vitamin B-6 restriction (n=23).

Figure 3-4. Relationship between homolanthionine with 3-hydroxykynurenine (A and B), the ratio of 3-hydroxykynurenine:xanthurenic acid (C and D), and cystathionine (E and F) in human plasma before and after 28-d dietary vitamin B-6 restriction (n=23). This figure was Supplemental Figure 1 in the original publication.

CHAPTER 4 METABOLIC CONSEQUENCES OF PLASMA CYSTATHIONINE ELEVATION IN SUBJECTS WITH SUSPECTED CORONARY ARTERY DISEASE

Introduction

Metabolomics, the systematic analysis of low molecular weight biochemical compounds in a biological specimen, has been increasingly applied to biomarker discovery for human disease due to the ability to highlight the breadth of metabolic disturbances. The concept of metabolic profiling uses a simple sample preparation to quantify multitudes of metabolites in a single run. The most common analyses are nuclear magnetic resonance (NMR) and liquid chromatography mass spectroscopy (LC-

MS). NMR analysis of plasma samples has the potential to detect perturbations in profiles of constituents reflecting the status of intermediary metabolism (e.g., lipids, amino acids and organic acids) along with other relevant compounds. LC-MS metabolomics will allow further evaluation with detection of a broader array of constituents, including those below detection thresholds of HPLC and NMR that may have importance as discriminating biomarkers of cardiovascular disease (CVD).

CVDs are the leading cause of deaths world-wide but the molecular etiology is still unknown. This multifactorial disease has genetic and environmental components causing its complexity. Therefore, measuring metabolic profiles provides a more proximal disease metabolism than genetic markers alone. These studies can provide both novel biomarkers with clinical potential associated with disease type and information about the mechanisms of disease development (146). This study will attempt to understand the role of cystathionine in distinguishing metabolite profiles in subjects with suspected heart disease.

CVD is often associated with perturbed energy homeostasis and metabolism such as obesity, insulin resistance, and diabetes. Current cardiology practice includes diagnostic biomarkers of acute changes, such as troponin I and troponin T for myocardial infarction and B-type naturiuretic peptide for congestive heart failure yet there lacks a general profile for heart disease (147, 148). Furthermore, prognostic markers of CVD outcomes such as LDL cholesterol are disputative and nutritional correlations of disease remain unclear (122). There is a distinct need for more in-depth metabolomic analysis to further discern the metabolic effects of B-6 insufficiency and its association with CVD risk. This study sought to determine the relationship between cystathionine and CVD and to explore lanthionine and homolanthionine concentrations in subjects with suspected coronary artery disease. This is a secondary analysis of plasma from NORCAD trials.

Materials and Methods

Human Subjects

This study used a subset of 80 plasma samples from previously conducted

Norwegian trials (WENBIT, NORVIT, BECAC) which aimed to study the prognostic markers of incident lifestyle disease among patients with suspected coronary artery disease. Initial subject recruitment occurred at Haukeland University Hospital of patients undergoing cardiac catheterization for suspected coronary artery disease. The majority of patients had stable angina pectoris and the NORVIT study included subjects with acute coronary syndrome. Patient lifestyle and medical history was obtained from self- administered questionnaires and verified by hospital records. Statin use was 74% of all subjects. Blood sampling, blood pressure and assessment of anthropometric data were performed by trained nurses. The subset of samples was randomly selected

based on plasma cystathionine concentrations: with a high cystathionine group

(average=1.7±0.76 µmol/L; n=40) and low cystathionine group (average=0.081±0.0099

µmol/L; n=40). My analyses were conducted only on plasma samples. Previously collected information was provided by collaborators as de-identified data corresponding to subject number. This metabolomic study was approved by the Norwegian Regional

Ethics committee and the University of Florida Institutional Review Board.

Analytical Methods

Routine blood analyses, such as hematologic parameters, renal function markers, and lipid-related factors were analyzed in fresh samples at the Laboratory of

Clinical Biochemistry, Haukeland University Hospital, using standard methods. Total plasma homocysteine, cystathionine and B vitamins were analyzed at the laboratory of

Bevital AS, Bergen, Norway, using previously described methods and have been previously reported (149). I measured total cysteine, glutathione, and cysteinylglycine as SBD-F derivatives using previously described methods. Lanthionine and homolanthionine plasma concentrations were determined by gas chromatography-mass spectroscopy (Thermo DSQII) in house as previously described (150).

Untargeted metabolomic analyses were performed by the Southeast Center for

Integrated Metabolomics. Briefly, LCMS analysis used Dionex UHPLC and Thermo Q-

Exactive Oribtrap mass spectrometer which measured samples in positive and negative heated electrospray ionization with mass resolution of 35,00 m/z 200 in separate injections. For LCMS, MZmine (freeware) was used to identify features, deisotope, align features, and perform gap filling to correct features that may have been missed in the first alignment algorithm. 1H-NMR spectra were obtained on a Varian INOVA 600 spectrometer with water presaturation at 25°C as previously described (143).

Statistical Analyses

All data are presented as means ± SDs. Differences in plasma metabolite and vitamin concentrations by cystathionine groupings were analyzed by Student’s t-test with significance determined by Bonferroni correction for multiple comparisons

(P<0.002). LC/MS peak areas were analyzed by Student’s t-test with adjustments for multiple comparisons (P<0.0002). Metabolomic analyses were performed by SIMCA

13.0. Linear regression analysis was performed by SigmaPlot 12.5.

Results

Targeted Metabolomics

Our targeted metabolomic analyses focused on vitamin and metabolite concentrations related to the one-carbon and transsulfuration pathways. In addition, we included measures of kidney function and inflammation. Study characteristics (Table 4-

1) showed approximately 80% of subjects were male with no significant gender differences between cystathionine groups. There were no significant differences between specific heart conditions or current heart medications between subjects according to cystathionine group. Subjects with high cystathionine were significantly older (66 ± 9.3 y) compared to subjects with low plasma cystathionine (57 ± 7.7 y;

P<0.001). Subjects in the lower cystathionine group were more likely to be fasted

(P<0.001) and had a significantly longer time since the last meal (P<0.001) compared to the high cystathionine group.

One carbon metabolism requires vitamin B-6, B12, and riboflavin as cofactors to different enzymes throughout the pathway. Riboflavin, folate, and B-6 vitamers (PLP,

PL, and 4-PA) in plasma were not different between groups. Although plasma PLP was not significantly different between groups (P<0.03), 35% of subjects in the high

cystathionine group had plasma PLP less than 30 nmol/L indicating marginal deficiency, compared to only 15% in the low cystathionine group. Serum cobalamin was not different between cystathionine groups but MMA, a functional indicator of vitamin B12 status, was significantly higher in subjects with high cystathionine (P<0.001; Table 4-2).

In the remethylation pathway, methionine was significantly higher (P<0.001) in the high cystathionine group which lead to higher homocysteine (P<0.001; Table 4-2). Plasma choline concentrations were greater in subjects with high cystathionine (P<0.001) but the conversion to betaine and ultimately DMG by methionine synthase was not affected.

All of the tryptophan catabolism metabolites were significantly higher in subjects with high plasma cystathionine (Table 4-2), which suggests some level of inflammation or vitamin B-6 insufficiency. While CRP levels were not significantly different, PAr index was significantly higher in subjects with high cystathionine (P<0.001), indicating increased vitamin B-6 catabolism due to inflammation. In addition to higher inflammation, subjects with higher cystathionine were more likely to have reduced kidney function, as measured by GFR (P<0.002).

The purpose of the transsulfuration pathway is to remove homocysteine and produce glutathione, an important antioxidant. The transsulfuration is also the primary location for H2S production and biomarkers lanthionine and homolanthionine. Plasma lanthionine concentrations were 75% higher in subjects with high cystathionine

(0.12±0.044 µmol/L) compared to subjects with low cystathionine (0.032±0.013 µmol/L;

P<0.001). These differences in lanthionine concentrations were not a result of higher precursor concentrations, as cysteine concentrations were not different between groups

(P=0.02; Table 4-2). While plasma homolanthionine was notably higher than

lanthionine in all subjects, homolanthionine concentrations were not different between the groups (P=0.18). The end-product of the transsulfuration pathway, glutathione, was significantly lower in the high cystathionine group (P<0.001; Table 4-2).

Multivariate PLS-DA analysis revealed a significant separation by cystathionine class (Figure 4-1). This model had a goodness of fit of R2=0.83 and a predictive power of Q2=0.71. Loadings plot analysis (Figure 4-2), determined which compounds had the greatest impact on the class differences seen in the scores plot model at the 99% confidence interval. From this analysis, the tryptophan metabolites (Trp, Kyn, HK, KA,

XA, HKA), PAr index, MMA, choline, lanthionine, total homocysteine and glutathione, methionine, cysteine, diabetes, fasting and age were significant contributors to differences seen the PLS-DA scores plot model. These findings agreed with statistical differences in concentrations between cystathionine groups.

LCMS Analysis

Untargeted LCMS analysis in positive ion mode detected a total of 5061 features while negative ion mode detected 1259 features. Using metabolite identification software, 310 metabolites were identified in both ion modes. In SIMCA, all features were analyzed according to cystathionine group (Figure 4-4). The clear separation between the high and low cystathionine group confirms a pattern of discriminating features by LC/MS analysis. Significant features were determined by a VIP score greater than 2. From this criterion, 36 known metabolites (Table 4-3) and 215 unknown features (Table 4-4) were found. Average peak areas were compared for all significant features. Uridine in both positive and negative ion mode, phenethylamine and pyridine-

2,3-dicarboxylate were significantly higher in the low cystathionine group (P<0.0002).

Compounds that were higher in subjects with high plasma cystathionine were amino

acids (phenylalanine, cysteine, proline, glutamic acid, tyrosine, isoleucine, tryptophan, valine, leucine and alanine/sarcosine), acylcarnitines, and tryptophan catabolism metabolites (kynurenine, kynurenic acid), creatinine, aldopentose, cysteinylglycine, malate, choline, and homovanillate. Phenethylamine, proprionylcarnitine, phenylalanine, L-kynurenine, n-acetyl-l-alanine, and indole were in the top 35 most significant metabolites according to SIMCA multivariate statistical software (Table 4-4).

Many of these metabolites were also significant contributors to differences seen in the

NMR spectra, which are reported below.

NMR Analysis

Untargeted proton NMR analysis revealed different spectra according to groupings of plasma cystathionine. Representative spectra from one subject in the low cystathionine group (Figure 4-1 A) and one subject the high cystathionine group (Figure

3-1 B) provide an indication of spectral differences. Further analysis of spectra peaks by multivariate PLS-DA analysis revealed significant differences between subjects with low or high plasma cystathionine (Figure 4-6). VIP values and chemical shift (PPM) were plotted to determine which peaks on the spectra were significant contributors to differences seen in the PLS-DA model (Figure 4-2). Once contributing peaks were marked on the spectra (VIP value >2), previous publications and the Human

Metabolome Database were used to determine their identities (Table 4-1) (151, 152).

Many of the metabolites that contributed to significant differences in the model were amino acids and glucose. Signals representing creatine, acetylcarnitine, 3- hydroxybutyrate, phenylacetic acid, acetate, betaine and choline were also significantly different. To examine the direction of changes, we subtracted the average spectra of high versus low cystathionine groups. From this we determined, glucose peaks tended

to be higher in subjects with high cystathionine compared to the low cystathionine group. Signals representing alanine, choline, and lactate were all higher in subjects with high plasma cystathionine. Signals representing acetate, 3-hydroxybutrate, acetyl carnitine, taurine, proline, and valine were higher in the low cystathionine group.

Results from NMR analysis agreed with metabolite differences found in other metabolomic analyses.

Discussion

To my knowledge, this is the first study to examine metabolomic effects of elevated plasma cystathionine in subjects with pre-existing heart conditions. A recent metabolic study in patients undergoing planned myocardial infarction identified changes in circulating concentrations of metabolites in pyrimidine metabolism, the tricarboxylic cycle and the pentose phosphate pathway. The metabolic signature consisted of alanine, isoleucine/leucine, hypoxanthine, trimethylamine N-oxide, threonine, serine, inosine, choline, proline and malonic acid 60 minutes after AMI (125). Similar studies showed changes in amino acid patterns and acylcarnitine patterns associated with CVD development (126). In the current study, acylcarnitines were disrupted as well as

TMAO, choline, branched chain amino acids, alanine and proline. These results, stratified by cystathionine class, were comparable to the above-mentioned studies which did not measure plasma cystathionine. Therefore, cystathionine status may be an important factor to consider in CVD metabolomic analyses since subjects had significantly different metabolite profiles according to cystathionine concentration.

Cystathionine elevation is apparent in folate, B12 and B-6 deficiencies.

Cobalamin and folate deficiencies also have elevated plasma homocysteine (153). In the current study, serum folate was not different in cystathionine groups, suggesting

adequate folate status in all subjects. Although serum cobalamin was not different,

MMA was higher in subjects with high cystathionine, however still within the normal range. Most likely, subjects with high cystathionine also had impaired B-6 status.

Although PLP concentrations were not significantly different (P=0.02), the elevation of cystathionine and the PAr index suggest suboptimal B-6 status likely due to increased catabolism. Metabolic consequences of mild-to moderate vitamin B-6 deficiency in healthy adults yielded changes in amino acid patterns of 1C metabolism, transsulfuration pathway and tryptophan catabolism as well as in amino acid ratios. (39,

40, 45-48). Many of these same changes were seen in my study population. The current study provides evidence of impaired B-6 status (as determined by functional indicator cystathionine) and its effect on metabolomic profiles in diseased subjects. Many of the above mentioned metabolites affected in cardiovascular disease are associated with

PLP-dependent pathways, such as the production of lanthionine and homolanthionine.

H2S is known to be related to cardiovascular disease, although the exact mechanism is still not understood. Since the measurement of H2S is difficult, we propose indirect measurement of H2S produced in specific reactions by quantifying lanthionine and homolanthionine (82, 95). Both sulfur compounds have no known function in-vivo and are only produced in H2S-generating reactions. H2S is believed to be depressed in CVD disease, and many patients had lower circulating levels with AMI

(113). However, homolanthionine is significantly higher in both subject groups compared to healthy young adults (150). Lanthionine concentration was much lower than normal adults in subjects with low plasma cystathionine (150). When plasma cystathionine was high, lanthionine concentrations were equivalent to those in healthy

young adults. This elevation in lanthionine leads to the conclusion that maybe excretory pathways of lanthionine and homolanthionine were impaired in older, diseased patients.

Supporting this claim, the high cystathionine group had significantly lower GFR indicative of a reduction in kidney function. Since we know homolanthionine has been excreted in urine of hyperhomocysteinemia patients, we suspect lanthionine would be cleared by the body in the same manner (79). The elevation of homolanthionine in both groups is more difficult to interpret. Clearance maybe inhibited due to reduced kidney function, or CSE activity maybe upregulated to produce H2S as a compensatory mechanism of protection. These relationships are still unclear and additional research is needed to elucidate the mechanisms behind these findings.

Table 4-1. Baseline characteristics of 80 subjects from previously conducted Norwegian trials (NORCAD).1 Low High Variable Cystathionine Cystathionine P-Value2 Group (n=40) Group (n=40) Men 31 (78%) 33 (83%) 0.58 Age (y) 57 ± 7.7 66 ± 9.3* <0.001 Fasting 31 (78%) 6 (15%)* <0.001 Time Since last meal (h) 8.6 ± 4.4 3.2 ± 2.9* <0.001 Treatment 28 (70%) 25 (63%) 0.48 Previous or Current Heart Conditions Coronary Artery Bypass 5 (13%) 3 (8%) 0.46 Acute Mydocardial Infarction 15 (38%) 23 (58%) 0.07 Angiographic Coronary Stenosis 10 (25%) 16 (40%) 0.16 Stroke 0 (0%) 4 (10%) 0.04 Atherothrombosis 21 (53) 29 (73%) 0.07 Heart Failure 1 (3%) 5 (13%) 0.09 Percutaneous Coronary Intervention 6 (15%) 12 (30%) 0.11 Peripherial Artery Disease 3 (8%) 4 (10%) 0.70 Diabetes 6 (15%) 24 (60%) 0.01 Other Illness 4.8 ± 7.9 5.2 ± 7.5 0.83 Statin Use3 Lovastatin Simvastatin 0.39 Statin Dose3 Moderate Moderate 0.39 1 Data are presented as mean ± SD (n=80) for continuous variables or number

(%) for categorical variables.

2Overall significant differences among variable concentrations were determined by Student’s t-test with Bonferroni correction for multiple comparisons. * Denotes statistical significance at P<0.002.

3Statin use is most common drug. Statin dose is based on 0-6 by increasing dosage.

Table 4-2. Concentrations of selected plasma variables in 80 subjects from previously conducted Norwegian trials (NORCAD).1 Low Cystathionine High Cystathionine Variable P-Value2 Group (n=40) Group (n=40)

Methylmalonic Acid, µmol/L 0.142 ± 0.0516 0.211 ± 0.0957* <0.001 Pyridoxal 5’ phosphate, µmol/L 60.1 ± 56.1 39.0 ± 20.9 0.03 Pyridoxal, µmol/L 12.5 ± 10.7 9.97 ± 3.36 0.17 Pyridoxic acid, µmol/L 26.0 ± 14.3 29.8 ± 13.4 0.22 Total Homocysteine, µmol/L 9.32 ± 2.22 13.1 ± 4.54* <0.001 Methionine, µmol/L 15.9 ± 3.65 21.2 ± 5.78* <0.001 Lanthionine, µmol/L 0.0317 ± 0.0133 0.121 ± 0.0437* <0.001 Homolanthionine, µmol/L 0.243 ± 0.116 0.257 ± 0.0892 0.37 Serine, µmol/L 125 ± 19.2 117 ± 30.6 0.37 Cysteine, µmol/L 248 ± 34.4 267 ± 39.1 0.02 Glutathione, µmol/L 5.37± 2.21 3.49 ± 1.68* <0.001 Glycine, µmol/L 214 ± 66.0 202 ± 50.1 0.35 Riboflavin, nmol/L 14.7 ± 17.7 14.9 ± 11.0 0.94 Serum Folate, nmol/L 16.8 ± 10.9 12.0 ± 6.46 0.02 Serum Cobalamin, pmol/L 349 ± 144 329 ± 154 0.73 Palmitoylcarnitine, pmol/L 80.8 ± 26.3 85.8 ± 40.1 0.51 Choline, µmol/L 8.48 ± 1.85 10.8 ± 2.45* <0.001 Betaine, µmol/L 46.0 ± 30.4 42.0 ± 18.0 0.47 Dimethylglycine, µmol/L 3.86 ± 1.61 5.10 ± 2.01 0.003 Tryptophan, µmol/L 54.3 ± 10.5 72.4 ± 16.9* <0.001 Kynurenine, µmol/L 1.04 ± 0.236 1.92 ± 0.574* <0.001 Kynurenine: Tryptophan 1.97 ± 0.551 2.76 ± 1.05* <0.001 Hydroxykynurenine, nmol/L 18.3 ± 6.33 48.0 ± 31.8* <0.001 Kynurenic Acid, nmol/L 32.1 ± 9.97 64.3 ± 23.4* <0.001 Xanthurenic Acid, nmol/L 8.45 ± 2.90 19.0 ± 8.51* <0.001 Anthranilic Acid, nmol/L 11.9 ± 4.30 16.1 ± 6.73* 0.001 3-hydroxyanthranilic Acid, nmol/L 19.3 ± 7.61 44.0 ± 17.9* <0.001 Trimethylamine N-oxide, µmol/L 5.22 ± 6.58 11.5 ± 8.41* <0.001 C-reactive Protein, mg/L 3.45 ± 5.80 7.30 ±13.8 0.11 Hemoglobin A1C, % 6.27 ± 1.62 5.98 ± 1.31 0.07 Glomerular Filtration Rate, mL/min 104 ± 10.1 79.8 ± 19.0* <0.001 PAr Index 0.448 ± 0.205 0.692 ± 0.343* <0.001 1 Data are presented as mean ± SD (n=80).

Table 4-3. Peak areas of identified metabolites by LCMS analysis in both positive and negative ion mode that had a VIP score of greater than 2. These were considered to be significant contributors to differences seen in the PLS-DA model.1 Row High Row VIP Low Cystathionine Metabolite Ion Mode Retention Cystathionine P-value m/z Score Group Peak Area time Group Peak Area Phenethylamine Positive 122.10 6.849 3.03 2.32E+07 7.34E+06 6.63E-14 Proprionylcarnitine Positive 218.14 6.694 2.94 2.15E+07 4.21E+07 1.61E-14 Phenylalanine Positive 166.09 6.215 2.91 1.18E+09 1.62E+09 3.54E-14 L-Kynurenine Positive 209.09 6.473 2.79 1.72E+07 3.07E+07 8.86E-13 N-acetyl-L-alanine Negative 154.05 2.150 2.77 4.85E+06 6.95E+06 1.90E-12 Pyridine-2,3-dicarboxylate Positive 168.03 1.600 2.73 1.65E+06 5.04E+05 2.34E-10 L-Cysteine Negative 120.01 0.760 2.66 1.59E+06 3.22E+06 4.46E-11 L-Proline Positive 116.07 0.835 2.61 1.74E+09 2.57E+09 8.39E-11 Kynurenic Acid Negative 188.03 7.650 2.59 1.12E+06 2.25E+06 1.10E-10 3-sulfino-L-alanine Negative 152.00 0.760 2.56 8.60E+05 1.68E+06 2.71E-10 Creatinine Positive 114.07 0.916 2.55 6.91E+08 9.98E+08 2.64E-10 Aldopentose Negative 149.05 0.830 2.50 8.41E+06 1.46E+07 2.37E-09 2-Hydroxyphenylalanine Positive 182.08 3.339 2.50 5.05E+08 7.53E+08 8.65E-10 N-acetyl-L-alanine Positive 154.05 2.149 2.49 3.98E+06 5.46E+06 1.23E-09 Cys-Gly Positive 179.05 0.917 2.46 3.36E+06 8.12E+06 1.57E-09 L-Glutamic Acid Negative 146.05 1.050 2.40 2.32E+06 3.51E+06 9.09E-09 Isovalerylcarnitine Positive 246.17 8.072 2.38 2.57E+06 5.90E+06 7.53E-09 L-Cysteine Positive 122.03 0.752 2.37 2.66E+07 5.08E+07 3.54E-08 L-Tyrosine Negative 180.07 3.350 2.37 1.42E+07 2.09E+07 1.01E-08 Kynurenic Acid Positive 190.05 7.652 2.36 3.01E+05 9.02E+05 9.66E-09 4-Acetamidobutanoate Positive 168.06 3.363 2.35 2.12E+06 4.29E+06 4.98E-08 Malate Negative 133.01 1.080 2.30 5.02E+07 8.40E+07 8.87E-08 Choline Positive 104.11 0.809 2.26 3.28E+08 3.99E+08 5.36E-08 N-acetyl-L-aspartic acid Negative 174.04 1.390 2.23 9.79E+05 1.45E+06 9.60E-08 L-Isoleucine Negative 130.09 2.190 2.21 4.81E+05 7.83E+05 1.84E-07

Table 4-3. Continued. Row High Row VIP Low Cystathionine Metabolite Ion Mode Retention Cystathionine P-value m/z Score Group Peak Area time Group Peak Area Uridine Positive 267.06 2.721 2.18 3.11E+06 2.22E+06 6.94E-07 Uridine Negative 243.06 2.710 2.18 1.03E+07 7.51E+06 7.48E-07 Isovalerylcarnitine Positive 246.17 8.176 2.15 4.22E+06 1.04E+07 2.97E-07 Deoxycarnitine Positive 146.12 1.192 2.15 7.45E+07 9.55E+07 6.03E-07 Homovanillate Negative 181.05 7.470 2.12 9.07E+06 1.53E+07 1.31E-06 Creatine-D3 Positive 135.10 0.884 2.12 2.74E+07 2.45E+07 1.01E-06 Tryptophan-NH3 Positive 188.07 7.655 2.06 2.97E+08 3.86E+08 1.19E-06 L-Valine Negative 116.07 1.100 2.06 3.25E+06 4.42E+06 2.20E-06 Leucine Positive 132.10 2.435 2.05 2.92E+09 4.16E+09 1.50E-06 Alanine/Sacrcosine Positive 90.05 0.719 2.04 5.51E+07 7.46E+07 2.06E-06 L-Leucine-D10 Positive 142.16 2.413 2.04 6.13E+07 5.84E+07 1.71E-06 Acyl-Carnitine(5-OH) Positive 262.16 6.682 2.03 9.03E+05 1.36E+06 1.84E-06 L-Isoleucine Negative 130.09 2.490 2.03 1.09E+06 1.62E+06 1.91E-06 1Significance was determined from average peak area between the low and high cystathionine group by student’s t-test after Bonferonni correction (P<0.001).

Table 4-4. Peak areas of unidentified metabolites by LCMS analysis in both positive and negative ion mode that had a VIP score of greater than 2. These were considered to be significant contributors to differences seen in the PLS-DA model.1 Row Low High Ion Row VIP Metabolite Retention Cystathionine Cystathionine P-value Mode m/z Score Time Group Peak Area Group Peak Area p-unknown-2168 Positive 97.06 10.076 3.93 3.84E+05 1.04E+05 3.55E-31 p-unknown-2135 Positive 137.10 10.041 3.79 3.76E+05 9.83E+04 2.85E-28 p-unknown-2313 Positive 137.10 10.326 3.61 3.35E+05 1.18E+05 1.07E-22 p-unknown-1699 Positive 126.04 7.067 3.55 6.18E+05 2.79E+05 8.12E-22 p-unknown-534 Positive 169.09 8.144 3.54 4.93E+06 2.21E+06 2.96E-21 p-unknown-1070 Positive 153.09 8.859 3.51 3.10E+06 3.78E+05 1.26E-22 p-unknown-3570 Positive 125.06 10.046 3.49 1.12E+05 2.33E+04 4.08E-20 p-unknown-2898 Positive 207.14 10.980 3.47 2.44E+05 7.28E+04 1.51E-20 p-unknown-2029 Positive 185.15 10.919 3.41 6.08E+05 1.96E+05 5.47E-19 p-unknown-1609 Positive 109.06 8.729 3.37 6.50E+05 3.33E+05 1.30E-17 p-unknown-2496 Positive 251.09 9.676 3.33 2.29E+05 8.43E+04 7.31E-16 p-unknown-968 Positive 171.10 8.991 3.29 2.15E+06 8.60E+05 2.89E-17 p-unknown-1675 Positive 211.09 8.215 3.20 5.30E+05 2.58E+05 9.85E-15 p-unknown-1656 Positive 215.13 10.023 3.06 5.78E+05 3.06E+05 1.60E-11 p-unknown-280 Positive 131.05 6.215 3.02 8.06E+06 1.13E+07 1.95E-15 n-unknown-197 Negative 250.15 15.230 2.97 1.42E+06 1.72E+06 8.73E-13 p-unknown-1498 Positive 185.15 12.949 2.91 1.89E+06 5.36E+05 2.81E-11 p-unknown-827 Positive 250.04 6.216 2.88 1.58E+06 2.06E+06 8.92E-14 p-unknown-39 Positive 167.09 6.215 2.87 1.18E+08 1.61E+08 1.34E-13 p-unknown-311 Positive 267.06 1.378 2.85 6.96E+06 1.05E+07 3.46E-13 n-unknown-44 Negative 164.07 6.260 2.85 1.28E+07 1.78E+07 2.43E-13 p-unknown-860 Positive 77.04 6.216 2.84 1.49E+06 2.14E+06 2.91E-13 p-unknown-1007 Positive 136.08 6.473 2.82 9.67E+05 1.66E+06 4.30E-13

Table 4-4. Continued. Row Low High Ion Row VIP Metabolite Retention Cystathionine Cystathionine P-value Mode m/z Score Time Group Peak Area Group Peak Area p-unknown-1209 Positive 237.11 10.255 2.81 1.55E+06 6.17E+05 1.97E-10 p-unknown-1309 Positive 229.14 8.462 2.80 4.67E+05 1.05E+06 1.50E-11 p-unknown-1066 Positive 169.09 8.729 2.78 1.51E+06 7.92E+05 1.69E-11 p-unknown-1377 Positive 223.02 2.271 2.76 5.36E+05 8.85E+05 5.00E-12 p-unknown-950 Positive 91.05 6.215 2.76 1.21E+06 1.71E+06 1.90E-12 p-unknown-1274 Positive 118.06 6.216 2.75 5.30E+05 9.12E+05 2.44E-12 n-unknown-946 Negative 130.05 1.540 2.75 3.11E+04 1.43E+05 4.70E-12 p-unknown-1469 Positive 169.09 9.662 2.75 8.51E+05 3.29E+05 2.04E-10 p-unknown-412 Positive 282.12 3.654 2.74 4.48E+06 6.58E+06 3.84E-12 p-unknown-1215 Positive 207.16 8.492 2.73 5.88E+05 1.21E+06 4.68E-11 p-unknown-104 Positive 175.03 0.957 2.71 3.91E+07 3.46E+07 1.79E-11 n-unknown-93 Negative 144.05 9.730 2.71 2.45E+06 4.48E+06 9.12E-12 p-unknown-766 Positive 269.07 1.342 2.68 1.77E+06 2.81E+06 3.05E-11 n-unknown-918 Negative 173.06 1.540 2.67 2.03E+05 7.59E+05 3.02E-11 p-unknown-495 Positive 139.08 8.543 2.67 5.07E+06 3.19E+06 9.03E-10 p-unknown-1332 Positive 132.05 6.216 2.66 4.99E+05 8.84E+05 3.39E-11 p-unknown-204 Positive 113.03 2.507 2.65 2.18E+07 8.04E+06 8.75E-11 p-unknown-2564 Positive 169.09 10.369 2.65 4.00E+05 8.60E+04 1.04E-10 n-unknown-801 Negative 294.03 6.260 2.64 1.31E+06 1.65E+06 4.65E-11 p-unknown-481 Positive 107.05 6.216 2.64 3.86E+06 5.08E+06 3.44E-11 n-unknown-1092 Negative 405.18 9.030 2.64 4.61E+04 4.61E+05 8.19E-11 n-unknown-493 Negative 144.05 7.650 2.61 4.72E+05 8.42E+05 7.66E-11 n-unknown-757 Negative 177.04 0.930 2.61 1.30E+07 5.38E+07 1.55E-10 p-unknown-1097 Positive 152.04 7.467 2.58 8.00E+05 1.44E+06 1.38E-10 n-unknown-1102 Negative 217.11 8.730 2.58 2.10E+05 3.38E+05 1.73E-10 p-unknown-2784 Positive 199.13 9.833 2.58 2.10E+05 9.04E+04 7.92E-10 p-unknown-2328 Positive 211.10 6.582 2.58 1.69E+05 2.74E+05 1.54E-10

Table 4-4. Continued. Row Low High Ion Row VIP Metabolite Retention Cystathionine Cystathionine P-value Mode m/z Score Time Group Peak Area Group Peak Area p-unknown-106 Positive 115.07 0.916 2.54 3.16E+07 4.62E+07 3.25E-10 n-unknown-792 Negative 171.04 2.510 2.54 2.68E+05 1.04E+06 3.84E-10 n-unknown-1025 Negative 190.07 2.500 2.54 8.66E+04 1.76E+05 3.62E-10 p-unknown-52 Positive 121.07 0.749 2.53 8.38E+07 1.49E+08 4.05E-10 p-unknown-1759 Positive 181.08 7.242 2.52 5.75E+05 2.84E+05 1.11E-08 p-unknown-1349 Positive 79.05 6.216 2.52 5.44E+05 8.52E+05 4.60E-10 p-unknown-595 Positive 135.10 6.817 2.50 3.69E+06 2.59E+06 5.67E-08 p-unknown-1087 Positive 181.12 9.559 2.49 1.12E+06 6.29E+05 1.91E-09 p-unknown-462 Positive 137.08 3.340 2.49 3.87E+06 5.88E+06 8.68E-10 p-unknown-22 Positive 235.09 0.888 2.49 2.22E+08 1.76E+08 1.27E-09 n-unknown-810 Negative 111.01 1.090 2.47 1.01E+05 1.80E+05 1.61E-09 p-unknown-308 Positive 119.05 3.339 2.47 7.11E+06 9.87E+06 1.47E-09 p-unknown-991 Positive 216.05 2.154 2.47 1.12E+06 1.52E+06 2.45E-09 p-unknown-655 Positive 166.07 2.554 2.46 2.37E+06 3.39E+06 2.40E-09 p-unknown-363 Positive 241.15 6.151 2.44 5.62E+06 9.00E+06 2.67E-09 n-unknown-547 Negative 204.98 6.990 2.43 5.62E+05 3.19E+06 3.12E-09 p-unknown-753 Positive 266.03 3.339 2.43 1.90E+06 2.53E+06 3.05E-09 n-unknown-125 Negative 119.03 0.960 2.42 4.42E+06 7.42E+06 4.38E-09 p-unknown-296 Positive 115.06 0.916 2.42 7.48E+06 1.10E+07 3.82E-09 n-unknown-37 Negative 165.04 0.740 2.41 1.36E+07 2.20E+07 5.02E-09 p-unknown-459 Positive 184.09 3.344 2.41 3.80E+06 5.71E+06 4.71E-09 p-unknown-131 Positive 229.15 6.293 2.40 2.21E+07 4.06E+07 5.83E-09 p-unknown-1133 Positive 244.15 7.893 2.40 6.70E+05 1.53E+06 5.23E-09 p-unknown-154 Positive 148.10 1.721 2.40 1.65E+07 3.42E+07 4.00E-08 p-unknown-2574 Positive 177.08 3.846 2.39 9.41E+04 2.62E+05 6.00E-09 p-unknown-983 Positive 269.12 1.623 2.39 9.46E+05 1.72E+06 1.21E-08 p-unknown-327 Positive 120.09 1.087 2.39 6.94E+06 8.56E+06 1.49E-08

Table 4-4. Continued. Row Low High Ion Row VIP Metabolite Retention Cystathionine Cystathionine P-value Mode m/z Score Time Group Peak Area Group Peak Area n-unknown-1017 Negative 261.01 7.540 2.38 1.04E+05 2.79E+05 1.09E-08 n-unknown-280 Negative 162.04 0.720 2.38 2.74E+05 8.12E+05 1.05E-08 p-unknown-2425 Positive 187.09 6.843 2.38 1.27E+05 3.13E+05 1.46E-08 p-unknown-3386 Positive 162.08 3.418 2.38 4.85E+04 1.23E+05 7.51E-09 p-unknown-2309 Positive 185.15 11.060 2.36 3.08E+05 1.74E+05 4.15E-08 n-unknown-328 Negative 189.03 1.210 2.36 5.53E+05 1.24E+06 1.02E-08 p-unknown-1346 Positive 86.12 2.165 2.36 5.87E+05 8.62E+05 1.15E-08 p-unknown-556 Positive 176.07 3.847 2.36 2.45E+06 4.70E+06 1.58E-08 p-unknown-837 Positive 149.12 7.521 2.36 1.81E+06 1.63E+06 9.37E-08 n-unknown-349 Negative 233.08 0.870 2.36 5.19E+06 2.49E+06 2.02E-08 p-unknown-1960 Positive 150.08 3.656 2.35 2.42E+05 3.83E+05 1.51E-08 n-unknown-820 Negative 242.07 3.350 2.34 1.20E+06 1.88E+06 1.76E-08 n-unknown-873 Negative 168.03 1.490 2.33 9.12E+04 2.44E+05 2.19E-08 n-unknown-196 Negative 333.06 1.380 2.33 1.59E+06 2.34E+06 3.60E-08 n-unknown-281 Negative 383.12 3.350 2.33 1.15E+06 2.10E+06 2.31E-08 p-unknown-2340 Positive 130.07 9.243 2.32 1.55E+05 2.88E+05 2.12E-08 p-unknown-375 Positive 146.08 3.380 2.32 5.34E+06 7.96E+06 3.96E-08 p-unknown-1062 Positive 86.08 2.445 2.32 9.80E+05 1.32E+06 2.44E-08 p-unknown-244 Positive 145.10 0.832 2.31 4.51E+06 1.46E+07 3.32E-08 p-unknown-1994 Positive 183.04 2.690 2.31 3.58E+05 2.26E+05 7.21E-08 n-unknown-95 Negative 171.08 0.800 2.31 1.12E+06 3.47E+06 2.94E-08 n-unknown-500 Negative 155.07 9.650 2.31 5.96E+05 1.44E+06 2.66E-08 n-unknown-984 Negative 174.08 2.190 2.31 2.23E+05 3.64E+05 3.87E-08 n-unknown-709 Negative 171.08 0.920 2.30 2.45E+05 7.37E+05 2.83E-08 p-unknown-36 Positive 173.09 0.793 2.29 5.85E+07 1.73E+08 4.75E-08 n-unknown-977 Negative 196.06 7.830 2.29 5.18E+04 1.95E+05 3.97E-08 p-unknown-1798 Positive 182.07 6.121 2.29 2.84E+05 6.52E+05 6.27E-08

Table 4-4. Continued. Row Low High Ion Row VIP Metabolite Retention Cystathionine Cystathionine P-value Mode m/z Score Time Group Peak Area Group Peak Area p-unknown-4189 Positive 94.06 9.677 2.29 2.63E+04 1.04E+05 4.31E-08 n-unknown-144 Negative 369.01 1.100 2.28 8.28E+06 7.83E+06 6.06E-08 p-unknown-466 Positive 245.08 2.720 2.28 4.95E+06 3.59E+06 1.54E-07 p-unknown-16 Positive 133.10 2.443 2.28 2.16E+08 2.93E+08 1.05E-07 p-unknown-441 Positive 191.05 1.203 2.27 3.25E+06 7.96E+06 6.07E-08 p-unknown-3233 Positive 155.05 2.382 2.27 4.29E+04 4.20E+05 5.85E-08 p-unknown-1342 Positive 86.08 2.165 2.26 5.96E+05 8.50E+05 7.15E-08 p-unknown-243 Positive 146.06 7.655 2.26 9.17E+06 1.25E+07 6.14E-08 p-unknown-126 Positive 148.10 1.167 2.25 2.49E+07 3.32E+07 1.37E-07 n-unknown-780 Negative 133.00 3.350 2.25 5.23E+05 7.25E+05 8.84E-08 n-unknown-133 Negative 333.06 2.710 2.24 3.66E+06 2.63E+06 2.02E-07 p-unknown-555 Positive 166.03 1.240 2.24 3.67E+06 3.25E+06 1.82E-07 n-unknown-128 Negative 87.01 0.950 2.24 5.59E+06 1.27E+07 9.40E-07 p-unknown-480 Positive 181.05 1.126 2.23 3.81E+06 5.73E+06 1.38E-07 p-unknown-3389 Positive 211.12 3.105 2.23 3.77E+04 1.26E+05 9.79E-08 n-unknown-14 Negative 215.07 0.870 2.23 2.69E+07 4.67E+07 9.78E-08 n-unknown-883 Negative 178.04 0.930 2.22 5.57E+05 2.10E+06 8.82E-07 p-unknown-1191 Positive 174.09 0.993 2.22 4.74E+05 1.49E+06 1.02E-07 n-unknown-919 Negative 281.05 1.330 2.22 5.10E+05 7.80E+05 1.48E-07 n-unknown-442 Negative 175.07 0.770 2.22 1.50E+06 2.25E+05 1.20E-07 p-unknown-171 Positive 123.04 3.339 2.22 1.62E+07 2.12E+07 1.10E-07 p-unknown-2173 Positive 169.12 8.490 2.22 2.36E+05 7.47E+05 1.35E-07 n-unknown-774 Negative 258.06 0.750 2.21 7.64E+05 1.19E+06 1.30E-07 p-unknown-2027 Positive 202.98 2.134 2.21 4.69E+05 2.48E+05 1.26E-07 p-unknown-784 Positive 328.25 11.845 2.21 1.60E+06 3.07E+06 1.23E-07 p-unknown-2339 Positive 152.04 9.241 2.21 1.67E+05 2.94E+05 1.34E-07 p-unknown-3980 Positive 386.29 12.638 2.20 3.56E+04 1.36E+05 2.62E-07

Table 4-4. Continued. Row Low High Ion Row VIP Metabolite Retention Cystathionine Cystathionine P-value Mode m/z Score Time Group Peak Area Group Peak Area p-unknown-695 Positive 216.05 2.444 2.20 2.27E+06 2.78E+06 1.72E-07 p-unknown-17 Positive 217.08 0.871 2.20 1.31E+08 2.44E+08 1.79E-07 p-unknown-1943 Positive 130.03 1.691 2.20 2.08E+05 4.03E+05 1.55E-07 n-unknown-871 Negative 167.03 10.120 2.20 9.09E+04 2.34E+05 1.55E-07 p-unknown-1269 Positive 124.05 3.339 2.19 6.19E+05 9.94E+05 1.96E-07 n-unknown-126 Negative 189.04 2.420 2.19 3.57E+06 6.02E+06 1.72E-07 p-unknown-1836 Positive 203.08 3.735 2.19 3.03E+05 5.08E+05 2.79E-07 p-unknown-143 Positive 113.03 2.720 2.19 2.71E+07 2.08E+07 6.75E-07 p-unknown-190 Positive 154.08 2.244 2.18 1.38E+07 2.39E+07 2.59E-07 p-unknown-1939 Positive 262.13 6.084 2.18 2.71E+05 4.26E+05 2.30E-07 p-unknown-117 Positive 229.15 5.699 2.18 2.39E+07 5.65E+07 2.38E-07 p-unknown-954 Positive 143.07 7.654 2.17 1.19E+06 1.64E+06 3.69E-07 p-unknown-831 Positive 150.04 1.259 2.17 1.38E+06 2.14E+06 4.26E-07 p-unknown-1023 Positive 74.02 1.170 2.17 1.03E+06 1.38E+06 4.40E-07 p-unknown-96 Positive 148.04 1.259 2.17 3.12E+07 4.76E+07 5.37E-07 p-unknown-1829 Positive 226.07 1.499 2.17 3.13E+05 4.76E+05 3.56E-07 p-unknown-1600 Positive 87.09 2.273 2.17 3.51E+05 5.76E+05 4.12E-07 p-unknown-98 Positive 76.08 0.884 2.16 2.94E+07 5.87E+07 4.35E-06 p-unknown-115 Positive 229.15 5.469 2.16 2.35E+07 5.41E+07 2.76E-07 p-unknown-870 Positive 163.54 9.243 2.16 1.38E+06 2.09E+06 3.02E-07 p-unknown-1719 Positive 190.09 6.218 2.16 3.55E+05 5.75E+05 4.14E-07 p-unknown-359 Positive 113.03 2.083 2.15 8.50E+06 5.18E+06 3.59E-07 n-unknown-694 Negative 209.05 8.640 2.15 5.96E+05 1.74E+05 3.89E-07 p-unknown-3706 Positive 176.07 6.286 2.15 3.53E+04 1.07E+05 5.79E-07 p-unknown-567 Positive 171.53 1.386 2.14 3.05E+06 4.29E+06 9.17E-07 p-unknown-3445 Positive 367.19 8.411 2.14 1.12E+06 1.48E+05 4.30E-07 p-unknown-373 Positive 117.07 0.835 2.14 5.39E+06 7.64E+06 3.92E-07

Table 4-4. Continued. Row Low High Ion Row VIP Metabolite Retention Cystathionine Cystathionine P-value Mode m/z Score Time Group Peak Area Group Peak Area p-unknown-3810 Positive 195.14 9.683 2.14 3.43E+04 1.19E+05 4.49E-07 n-unknown-351 Negative 215.00 7.990 2.14 4.68E+05 1.07E+06 7.63E-07 n-unknown-122 Negative 336.01 1.070 2.13 1.49E+07 1.41E+07 7.70E-07 p-unknown-2926 Positive 366.86 8.412 2.13 1.94E+06 2.77E+05 4.85E-07 p-unknown-1164 Positive 121.06 3.339 2.13 7.91E+05 1.10E+06 4.67E-07 p-unknown-1254 Positive 300.22 10.534 2.12 5.81E+05 1.26E+06 4.81E-07 p-unknown-3246 Positive 115.52 1.627 2.12 5.11E+05 1.09E+05 5.10E-07 p-unknown-1385 Positive 226.07 1.049 2.10 4.64E+05 1.40E+06 6.35E-07 p-unknown-1186 Positive 199.17 11.690 2.10 1.05E+06 6.51E+05 8.68E-06 p-unknown-1459 Positive 329.03 2.721 2.10 6.54E+05 4.99E+05 2.07E-06 n-unknown-851 Negative 224.06 3.350 2.10 3.09E+05 5.10E+05 7.44E-07 p-unknown-1434 Positive 73.03 2.443 2.09 5.37E+05 7.19E+05 7.24E-07 p-unknown-271 Positive 129.10 0.852 2.09 4.34E+06 1.18E+07 2.67E-06 p-unknown-1842 Positive 112.08 7.155 2.09 8.78E+05 3.01E+05 1.20E-06 p-unknown-2576 Positive 160.03 2.721 2.09 2.20E+05 1.37E+05 2.77E-06 p-unknown-2131 Positive 197.08 8.076 2.09 3.82E+05 1.96E+05 5.05E-05 n-unknown-1010 Negative 258.06 3.350 2.08 4.24E+04 1.02E+05 9.22E-07 n-unknown-380 Negative 117.02 0.970 2.08 6.56E+05 1.51E+06 5.02E-06 n-unknown-619 Negative 547.77 8.350 2.08 5.71E+05 7.89E+04 9.51E-07 n-unknown-455 Negative 245.08 1.330 2.08 1.36E+06 1.93E+06 2.29E-06 p-unknown-1891 Positive 139.02 2.055 2.08 4.11E+05 2.96E+05 1.20E-06 p-unknown-478 Positive 130.06 7.656 2.08 3.92E+06 5.06E+06 9.39E-07 p-unknown-790 Positive 209.05 1.387 2.08 1.78E+06 2.61E+06 2.83E-06 p-unknown-210 Positive 134.03 0.846 2.07 1.05E+07 1.68E+07 5.47E-06 p-unknown-600 Positive 171.53 2.721 2.07 3.52E+06 2.63E+06 2.20E-06 p-unknown-511 Positive 97.99 2.068 2.07 4.18E+06 3.14E+06 1.03E-06 p-unknown-622 Positive 102.09 0.920 2.06 9.77E+06 2.95E+06 2.10E-06

Table 4-4. Continued. Row Low High Ion Row VIP Metabolite Retention Cystathionine Cystathionine P-value Mode m/z Score Time Group Peak Area Group Peak Area p-unknown-376 Positive 158.08 6.624 2.06 5.49E+06 7.62E+06 2.88E-06 p-unknown-1173 Positive 155.04 1.386 2.06 8.12E+05 1.17E+06 3.50E-06 n-unknown-207 Negative 279.04 2.710 2.05 2.07E+06 1.50E+06 2.90E-06 p-unknown-1892 Positive 237.15 10.368 2.05 3.74E+05 2.93E+05 4.07E-06 p-unknown-559 Positive 171.10 7.521 2.05 3.71E+06 3.21E+06 1.36E-06 n-unknown-59 Negative 103.00 0.730 2.05 4.03E+06 6.22E+06 2.81E-06 p-unknown-989 Positive 221.09 3.737 2.05 1.17E+06 1.67E+06 2.10E-06 p-unknown-272 Positive 245.08 1.386 2.04 8.53E+06 1.22E+07 4.11E-06 n-unknown-827 Negative 166.06 8.730 2.04 2.13E+05 4.13E+05 1.53E-06 p-unknown-457 Positive 204.98 2.050 2.03 8.47E+06 3.94E+06 1.68E-06 n-unknown-714 Negative 940.45 8.910 2.03 7.98E+05 2.03E+05 2.51E-06 p-unknown-118 Positive 151.14 0.883 2.03 2.43E+07 6.60E+07 3.18E-05 n-unknown-139 Negative 197.81 0.750 2.03 3.33E+06 2.43E+06 2.85E-06 p-unknown-1700 Positive 184.10 7.168 2.03 3.66E+05 5.39E+05 3.97E-06 p-unknown-2526 Positive 161.09 6.334 2.02 1.38E+05 2.68E+05 2.15E-06 n-unknown-318 Negative 124.99 0.740 2.02 7.92E+06 1.26E+07 6.98E-06 n-unknown-156 Negative 206.08 8.770 2.02 1.67E+06 2.04E+06 5.67E-06 p-unknown-191 Positive 134.08 0.803 2.02 1.14E+07 2.04E+07 2.55E-06 p-unknown-455 Positive 164.09 0.801 2.01 2.57E+06 6.55E+06 3.96E-06 n-unknown-190 Negative 148.94 0.720 2.01 3.15E+06 2.24E+06 8.84E-06 n-unknown-371 Negative 203.07 0.790 2.01 5.99E+06 1.39E+06 2.81E-06 p-unknown-1371 Positive 249.18 14.135 2.01 6.40E+05 6.79E+05 1.50E-05 p-unknown-2299 Positive 227.17 6.571 2.00 3.80E+05 1.64E+05 2.62E-06 1Significance was determined from average peak area between the low and high cystathionine group by student’s t-test after Bonferonni correction (P<0.001).

Table 4-5. Peak areas of the top 35 contributors to differences seen in the LC/MS model. Row Ion Row VIP Low Cystathionine High Cystathionine Number Metabolite Retention P-value Mode m/z Score Group Peak Area Group Peak Area Time 1 p-unknown-2168 Positive 97.06 10.076 3.93 3.84E+05 1.04E+05 3.55E-31 2 p-unknown-2135 Positive 137.10 10.041 3.79 3.76E+05 9.83E+04 2.85E-28 3 p-unknown-2313 Positive 137.10 10.326 3.61 3.35E+05 1.18E+05 1.07E-22 4 p-unknown-1699 Positive 126.04 7.067 3.55 6.18E+05 2.79E+05 8.12E-22 5 p-unknown-534 Positive 169.09 8.144 3.54 4.93E+06 2.21E+06 2.96E-21 6 p-unknown-1070 Positive 153.09 8.859 3.51 3.10E+06 3.78E+05 1.26E-22 7 p-unknown-3570 Positive 125.06 10.046 3.49 1.12E+05 2.33E+04 4.08E-20 8 p-unknown-2898 Positive 207.14 10.980 3.47 2.44E+05 7.28E+04 1.51E-20 9 p-unknown-2029 Positive 185.15 10.919 3.41 6.08E+05 1.96E+05 5.47E-19 10 p-unknown-1609 Positive 109.06 8.729 3.37 6.50E+05 3.33E+05 1.30E-17 11 p-unknown-2496 Positive 251.09 9.676 3.33 2.29E+05 8.43E+04 7.31E-16 12 p-unknown-968 Positive 171.10 8.991 3.29 2.15E+06 8.60E+05 2.89E-17 13 p-unknown-1675 Positive 211.09 8.215 3.20 5.30E+05 2.58E+05 9.85E-15 14 p-unknown-1656 Positive 215.13 10.023 3.06 5.78E+05 3.06E+05 1.60E-11 15 Phenethylamine Positive 122.10 6.849 3.03 2.32E+07 7.34E+06 6.63E-14 16 p-unknown-280 Positive 131.05 6.215 3.02 8.06E+06 1.13E+07 1.95E-15 17 n-unknown-197 Negative 250.15 15.230 2.97 1.42E+06 1.72E+06 8.73E-13 18 Proprionylcarnitine Positive 218.14 6.694 2.94 2.15E+07 4.21E+07 1.61E-14 19 Phenylalanine Positive 166.09 6.215 2.91 1.18E+09 1.62E+09 3.54E-14 20 p-unknown-1498 Positive 185.15 12.949 2.91 1.89E+06 5.36E+05 2.81E-11 21 p-unknown-827 Positive 250.04 6.216 2.88 1.58E+06 2.06E+06 8.92E-14 22 p-unknown-39 Positive 167.09 6.215 2.87 1.18E+08 1.61E+08 1.34E-13 23 p-unknown-311 Positive 267.06 1.378 2.85 6.96E+06 1.05E+07 3.46E-13 24 n-unknown-44 Negative 164.07 6.260 2.85 1.28E+07 1.78E+07 2.43E-13 25 p-unknown-860 Positive 77.04 6.216 2.84 1.49E+06 2.14E+06 2.91E-13 26 p-unknown-1007 Positive 136.08 6.473 2.82 9.67E+05 1.66E+06 4.30E-13 27 p-unknown-1966 Positive 94.06 6.474 2.82 2.32E+05 3.98E+05 4.85E-13

Table 4-5. Continued. Row Ion Row VIP Low Cystathionine High Cystathionine Number Metabolite Retention P-value Mode m/z Score Group Peak Area Group Peak Area Time 29 p-unknown-1309 Positive 229.14 8.462 2.80 4.67E+05 1.05E+06 1.50E-11 30 L-Kynurenine Positive 209.09 6.473 2.79 1.72E+07 3.07E+07 8.86E-13 31 p-unknown-1066 Positive 169.09 8.729 2.78 1.51E+06 7.92E+05 1.69E-11 32 N-acetyl-L-alanine Negative 154.05 2.150 2.77 4.85E+06 6.95E+06 1.90E-12 33 p-unknown-1377 Positive 223.02 2.271 2.76 5.36E+05 8.85E+05 5.00E-12 34 p-unknown-950 Positive 91.05 6.215 2.76 1.21E+06 1.71E+06 1.90E-12 35 p-unknown-1274 Positive 118.06 6.216 2.75 5.30E+05 9.12E+05 2.44E-12

Table 4-6. Identified peaks from NMR spectra that were significant contributors to difference seen in the model. PPM VIP ID 0.95 2.64 Leucine 0.98 3.00 Valine 1.02 3.03 Valine 1.19 5.40 3-hydroxybutyrate 1.31 7.06 Lactate 1.46 4.98 Alanine 1.90 5.20 Acetate 2.22 2.08 2.29 2.68 3-hydroxybutyrate 2.95 2.57 3.03 4.18 Creatine 3.16 3.92 acetylcarnitine 3.21 5.30 Choline 3.23 6.49 Glucose 3.24 5.77 Betaine 3.25 3.16 Taurine 3.32 3.50 Proline 3.35 4.00 3.37 4.34 3.39 6.97 Glucose 3.40 7.05 Glucose 3.41 4.02 Taurine 3.45 4.87 Glucose 3.46 6.83 Glucose 3.48 7.99 Glucose 3.50 5.29 Glucose 3.53 4.55 Phenylacetic acid 3.53 4.91 Glycine 3.61 3.53 Valine 3.68 4.45 3.70 5.70 3.71 6.21 Glucose 3.72 5.84 3.75 4.86 Glucose 3.81 6.54 Glucose 3.83 4.78 3.87 6.11 Glucose 3.89 5.22 Betaine 3.93 4.42 Creatine 4.10 2.84 Lactate

Table 4-6. Continued. PPM VIP ID 4.63 5.18 Glucose 5.22 5.60 Glucose

Figure 4-1. Score plot from partial least squares-discriminant analysis (PLS-DA) of overall targeted metabolite concentrations in subjects with high or low plasma cystathionine. Each datum point represents a function of pooled metabolite profile of each subject, n=80. PLS-DA represented a clear separation between the 2 groups with acceptable goodness of fit (R2=0.83) and predictive power (Q2=0.71), performed by SIMCA multivariate statistical software.

Figure 4-2. Loadings plot (99% confidence interval) shows targeted metabolite variable contributions to the differences seen in the PLS-DA scores plot model. Significant contributors are represented as bars in which the error bars do not cross the x-axis.

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Figure 4-3. Score plot from partial least squares-discriminant analysis (PLS-DA) of LCMS peak areas in subjects with high or low plasma cystathionine. Each datum point represents a function of pooled peak area profile of each subject, n=80. PLS-DA represented a clear separation between the 2 groups with acceptable goodness of fit (R2=0.96) and predictive power (Q2=0.75), performed by SIMCA multivariate statistical software.

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Figure 4-4. Score plot from partial least squares-discriminant analysis (PLS-DA) of 1H- NMR spectra in subjects with high or low plasma cystathionine. Each datum point represents a function of pooled spectral profile of each subject, n=80. PLS-DA represented a clear separation between the 2 groups with acceptable goodness of fit (R2=0.57) and predictive power (Q2=0.46), performed by SIMCA multivariate statistical software.

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Figure 4-5. Representative spectra of each cystathionine group. A) Low cystathionine group; subject 13. B) High cystathionine group; subject 53.

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Figure 4-6. Variable importance to the projection (VIP) scores versus chemical shift (PPM) from NMR spectra. This plot identifies significant peaks contributing to differences seen in the multivariate model.

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Figure 4-7. Spectral representation of the differences between the low and high cystathionine group. Intensity of differences denotes the magnitude of change between groups. Positive direction of peak represents higher concentration in the low cystathionine groups and negative direction of peak represents higher concentration in the high cystathionine group.

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CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS

Vitamin B-6 is essential to amino acid metabolism and to the proper functioning of the transsulfuration, one-carbon, and tryptophan catabolism pathways. Vitamin B-6 also serves as a coenzyme for two enzymes that produce H2S in-vivo. H2S is a gaseous signaling molecule involved with vasodilation, inflammation, antioxidation, and neuromodulation. Vitamin B-6 restriction in cell culture has been shown to impact H2S production as well as H2S biomarkers lanthionine and homolanthionine. Marginal vitamin B-6 deficiency has been associated with increased risk for cardiovascular disease and certain cancers although the mechanism behind these associations in unknown. H2S has similar associations with cardiovascular disease but it remains unknown if these factors are related. This dissertation work sought first to understand the metabolism of H2S biomarkers in live cell culture models under various conditions.

Currently, little is known about lanthionine and homolanthionine in terms of production, stability, breakdown, or recycling. Secondly, this dissertation work characterized these biomarkers in human plasma and their associations with related metabolites and disease outcomes.

Stable isotope studies are unique in that they can provide kinetic measurements about precursors and products in living organisms. Cell models can be further manipulated by inhibiting specific enzymes to explore changes in reaction rates and overall flux. The first set of studies used CSE suicide inhibitor, PAG, to determine the impact of each transsulfuration enzyme on the formation of H2S biomarkers. Labeled precursors, methionine and cysteine, were used in primary human hepatocytes and

HepG2 cells. These studies confirmed the majority of homolanthionine is produced by

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CSE-catalyzed condensation of two homocysteine molecules. Homolanthionine production is significantly decreased when CSE is inhibited. Lanthionine was only produced by CBS and was insensitive to CSE inhibition. Interestingly, lanthionine was shown to be cleaved by CSE in both cell models. The competition of cysteine and serine in the formation of cystathionine was also evaluated. I concluded that serine is the preferred substrate for cystathionine formation in both cell models and that cysteine only contributes minimally to the total cystathionine concentration. This result questions the impact of cysteine-derived cystathionine formation as the major source of H2S in- vivo, although additional research is needed.

After determining kinetic variations of lanthionine and homolanthionine formation in cell culture, I sought to determine concentration changes in 23 subjects after 28-day dietary vitamin B-6 restriction. There was great heterogeneity of individual responses to vitamin B-6 deficiency. The response of plasma lanthionine and homolanthionine concentrations to dietary B-6 restriction was varied (Figure 2-1). Although there were no significant changes in concentration, 28-d dietary B-6 restriction affected the positive association between homocysteine and homolanthionine, which suggested alterations in H2S biomarker metabolism in short-term deficiency. These subjects were fed a controlled diet providing <0.5g/d vitamin B-6 for 28-d. Plasma PLP concentrations reached marginal deficiency in week 3. Therefore, these subjects were deficient for only the last 10 days of the study. It is unknown whether prolonged marginal deficiency or a more severe deficiency would have a more marked effect on lanthionine and homolanthionine concentrations. This study did, however, provide baseline

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concentrations of lanthionine and homolanthionine in plasma of healthy young adults which was lacking in current literature.

The mechanism by which vitamin B-6 status is connected to chronic disease is still unclear. Specifically, impaired vitamin B-6 status is an independent risk factor for coronary artery disease even after accounting for homocysteine risk. This dissertation work aimed to find associations between B-6-related metabolites in subjects with suspected coronary artery disease. Since cystathionine elevation is a functional indicator of vitamin B-6 deficiency, I designed an observational study based on high and low plasma cystathionine concentrations. Multivariate analysis from targeted metabolites, LCMS, and NMR all found significant differences between metabolite profiles in subjects from the high cystathionine group versus the low cystathionine group. Tryptophan catabolism pathway metabolites, lanthionine, branch chain amino acids, and carnitines were significantly higher in subjects with high cystathionine compared to those with low cystathionine. Subjects with higher plasma cystathionine also had higher MMA and homocysteine concentrations and PAr index but lower total glutathione compared to the low cystathionine group. CRP was not significantly different between groups, which suggested the higher inflammation in the high cystathionine group was related to vitamin B-6 catabolism. Lower glutathione in the high cystathionine group could be related to higher oxidative stress. This could be further explored by calculating the ratio between reduced glutathione (GSH) to oxidized glutathione (GSSG); GSH:GSSG. These differences between profiles based only on plasma cystathionine concentration agree with work from Nygård et al. (FASEB

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Meeting, 2016) which suggested that elevated cystathionine could be an apparent risk factor of acute myocardial infarction.

In addition to analyzing these targeted metabolites based on cystathionine concentrations, I also sought to determine if metabolite profiles could indicate risk for future acute myocardial infarction (Figure 5-1). There was separation between metabolite profiles of subjects who went on to have no adverse event and those who had a previous AMI. Subjects who later had an AMI did not have significantly different profiles from those who had no adverse event but the sample size was too small to be conclusive. Subjects who had a previous AMI and went on to have another AMI did have different profiles. Therefore, using targeted metabolomics could help identify subjects who are at risk for another heart attack. These results should be expanded to a larger study subset to confirm these findings.

Overall, this observational study found relationships between the H2S biomarker, lanthionine, and coronary artery disease in subjects with elevated cystathionine. It would be beneficial to have homolanthionine and lanthionine measurements in age- matched controls to compare concentration differences. Furthermore, additional research is needed to evaluate the clinical significance of these results.

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Figure 5-1. Multivariate PLS-DA scores plot of subjects according to acute myocardial outcomes. The four groups represent subjects who have had a previous heart attack (blue), subjects who will later have a heart attack (red), subjects who had had a previous heart attack and will go on to have a later attack (yellow), and subjects who have no documented AMI (green). PLS-DA represented a some separation between the 4 groups with acceptable goodness of fit (R2=0.43) and predictive power (Q2=0.29), performed by SIMCA multivariate statistical software.

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APPENDIX A ISOTOPICALLY LABELED LANTHIONINE AND HOMOLANTHIONINE SYNTHESIS

Table A-1. Experimental conditions for isotopically labeled lanthionine and homolanthionine synthesis reactions. Lanthionine Homolanthionine Substrate Concentration, mM 35.9 8.54 Enzyme Concentration, ug/mL 1.5 200 Time, h 24 6 Final Volume, mL 5 5

Table A-2. Concentration of amino-thiols before and after synthesis reactions (Table A- 1) determined by DTNB assay (154). Reaction Before After % Completion mM Homocysteine 8.54 0.278 96.7 Cysteine 35.9 6.55 81.8

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Figure A-1. Representative GC/MS chromatograms of purified biomarkers. (A) Lanthionine RT=29.5 min and (B) Homolanthionine RT=31.5 min. Internal 5 standard peaks in both chromatograms are [ C13]-methionine; RT=19.4 and [D4]-cystathionine; RT= 29.5.

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APPENDIX B PAG CONCENTRATION STUDY

Previous studies have reported a range of PAG concentrations to completely inhibit CSE, however, at high concentrations the inhibitor can be toxic (127, 155).

Therefore, our preliminary study sought to find the lowest concentration at which CSE activity was completely inhibited. Various concentrations (0, 10, 25, 50, 100, 250, 500,

1000 µM) of PAG were added to a 20% w/v homogenate of human liver cells in 50 mM phosphate buffer at pH 6.8 (Figure B-1). CSE activity was determined using the previously described method (156). This method is based on colorimetry for the determination of pyruvate produced from beta-chloro-L-alanine with the beta-elimination catalyzed by CSE, coupling a color enzymatic reaction with pyruvate oxidase and peroxidase. The absorbance increases with the oxidized color of a leuco dye, N-

(carboxymethylamino)-4,4'-bis (dimethylamino)-diphenylamine at 727 nm is proportional to CSE activity (157). This protocol was repeated with mouse liver as well (Figure B-2).

Downstream metabolites were also measured in cells with different concentrations of

PAG. Cysteine and glutathione in cells had the same curve shape, with PAG concentration of ~250 µM resulting in a significant reduction of total amino-thiol concentrations (P<0.001 and P<0.003; Figure C-A and B). Therefore, I was confident in the use of 1000 µM PAG to inhibit CSE activity to <5% of activity from untreated cells in subsequent tracer experiments.

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CγL Activity in the presence of PAG

0.8

) n

i 0.6

l o

m 0.4

(

i t

c 0.2 A

0.0 0 250 500 750 1000 PAG (mM)

Figure B-1. Human liver lysate CSE activity was measured by colorimetric assay under various concentrations of PAG. Data expressed and means ± SD, n=4.

CgL Activity in the presence of PAG

) n

i 3

l o

m 2

(

i t

c 1 A

0 0 100 200 300 400 500 PAG (mM)

Figure B-2. Mouse liver lysate CSE activity was measured by colorimetric assay under various concentrations of PAG. Data expressed and means ± SD, n=4.

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Figure B-3. (A)Cysteine and (B) glutathione concentrations in human hepatocytes with various concentrations of PAG added after 6-h incubation. Data expressed and means, n=4.

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BIOGRAPHICAL SKETCH

Barbara Neal DeRatt’s undergraduate major was pre-medicine and biology with a double minor in math and chemistry. She later earned her Master of Science from the

University of Florida in food science and human nutrition. Upon completion of her M.S., she pursued her Doctor of Philosophy in nutritional sciences. She graduated from the

Department of Nutritional Sciences at the University of Florida in December of 2016 with her Ph.D. She has accepted a job with Nestle Purina in St. Louis, Missouri.

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