Homocysteine Metabolism and the Characterization of Cystathionine Beta-Synthase, Cystathionine Gamma-Lyase and Methionine Synthase in Piglets From Birth Until Post-Weaning

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Manitoba

by

Desmond Marie Ballance

In partial fulfillment of requirements for the degree of

Masters of Science

July,2004

ODesmond Marie Ballance TIIE TINIVERSITY OF MANITOBA

FACULTY OF' GRADUATE STUDIES ***** COPYRIGHT PERMISSION

Homocysteine Metabolism and the Characterization of Cystathionine Beta-Synthase, Cystathionine Gamma-Lyase and Methionine synthase in Pigtets From Birth until post-weaning

BY

Desmond Marie Ballance

A ThesisÆracticum submitted to the tr'aculty of Graduate Studies of The University of

Manitoba in partial fulfillment of the requirement of the degree of

MASTER OF' SCIENCE

Desmond Marie Ballance @2004

Permission has been granted to the Library of the University of Manitoba to lend or sell copies of this thesis/practicum, to the National Library of Canada to microfilm this thesis and to lend or sell copies of the film, and to University Microfilms Inc. to publish an abstract of this thesis/practicum.

This reproduction or copy of this thesis has been made available by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright owner. I

Acknowledgments

I would like to acknowledge the receipt of the Cyril L. Anderson-Ridley Canada Limited Graduate Fellowship and the National Science and Engineering Council of Canada for their support, without which this thesis would not have been possible.

Though committee meetings were few and far between,I am grateful to my advisory committee members, Dr. Wilhelm Guenter and Dr. Harold Aukema, for their encouragement.

A sincere thank you to Rickie Araneda, my "Spanish mother hen" in the lab. Her technical support and life advice was invaluable --"Control what you can control." I am also extremely grateful to Jody Philippe, Alain Neveux and Ed Devlieger at the Glenlea Swine Research Unit. Not only were they ever-willing to offer their assistance and advice, their jokes and teasing about my "pig in a blanket" were sincerely appreciated!

No one understands the conflicts of a grad student like another grad student. Thank you to all the graduate students for your support, comradery, Boston Pizza lunches and problem- solving office visits. A special thanks to Melanie Shell for her near-speeding ticket support during my animal trial. As well, a big thank you hug to Kat Hebert for tube- labeling, laughing, venting, confiding, advising, procrastinating and "becoming radioactive" with me.

Only two words are needed to describe the support and encouragement I have received from Jen Onyskie, Sandra Kelly Sawatzky, Wendy McClellan, Tricia Schmalenberg and Kari van Middlesworth - girls night!!! Learning to think like a scientist takes time and effort and I am sincerely grateful to Dr. Jim House for taking that time with me. The opporlunities and experiences I have received under his guidance, both as an undergraduate summer student and as a graduate student, are invaluable and will be with me throughout my career-particularly where unit conversions are concerned! Thank you Dr. House.

A big thank you to Chris Bileski whose love, understanding, encouragement and laughter have helped me get to where I am now. You have not only made me want to succeed, but taught me that taking time to relax is part of the process. I promise to get out of school one day but until then, thank you forever.

Finally without my parents, I would be far less of a person than I am proud to say I am today. I am ever grateful to my Mom for her support when I was frustrated, her patience when I was stressed and her hugs when I felt like I just couldn't do it anymore. I also owe a huge thank you to my Dad who has let me pester him with biochemistry questions almost every morning for over two years. Your love, support and advice have been priceless and walking to school/work will never be the same. I love you both! il

Chapter I - Introduction......

Chapter 2 -Literature Review ...... 2

2.1 Cysteine Synthesis and the Methionine Cycle ...... 2 2.2 Essential Functions of Cysteine...... 5 2.2.I Protein Synthesis...... 6 2.2.2 Taurine Synthesis...... 6 2.2.3 Gltttathione Synthesis ...... 8 2.2.4 Inorganic Sulphate Synthesis ...... 11 2.3 Remethylation and Transsulphuration Enzymes: Location and Factors Influencing Changes in Activity ...... 15 2.3.1 Cystathionine Beta-Synthase ...... 16 2.3.2 Cystathionine Gamma-Lyase ...... 22 2.3.3 Methionine Synthase ...... 28 2.4 Sulphur Amino Acids and B-Vitamins: Nutrition and Supply for Prenatal and Post-natal Piglets...... 32 2.4.1 Methionine and Cysteine Availability...... 32 2.4.2 Folate and Vitamin 8,, Availability ...... 37 2.4.3 Yitarnin Bu Availability...... 41 2.5 Summary...... 42

Chapter 3 - Hypotheses and Objectives ...... 44

3.1 Hypotheses...... 44 3.2 Objectives...... 44

Chapter 4 - Materials and Methods...... 45

4.1 4nima1s...... 45 4.2 Diets...... 46 4.3 Study Design...... 46 4.4 Chemicals...... 47 4.5 Analyses of P1asma...... 47 n

4.5.1 Folate and Vitamin 8,, ...... 47 4.5.2 Amino Acids...... 49 4.5.3 Total Homocysteine and Cysteine...... 49 4.6 Hepatic Total Homocysteine and Cysteine...... 51 4.7 Preparation of Tissue Homogenates...... 51 4.8 Measurement of Tissue Protein ..:...... 52 4.9 Analyses of Tissue En4¡me Activity...... 53 4.9.1 Assessment of Protein Concentration and Incubation Length for Optimizing Enzyme Activity...... 53 4.9.2 Cystathionine Beta-Synthase ...... 53 4.9.3 Cystathionine Gamma-Lyase ...... 55 4.9.4 Methionine Synthase ...... 56 4.10 StatisticalAnalyses...... 57

Chapter5-Results ...... 58

5.IGrowth ...... 58 5.2 Plasma Homocysteine and Cysteine...... 58 5.3 Plasma Folate and Vitamin 8r2...... 58 5.4 Plasma Methionine, Serine and Taurine and Other Amino 4cids...... 63 5.5 Hepatic Total Homocysteine and Cysteine...... 63 5.6 Optimization of Enzyme Activity for Enzyme Dilution and Incubation Length...... 67 5.7 Cystathionine Beta-Synthase...... 67 5.8 Cystathionine Gamma-Lyase...... 70 5.9 Methionine Synthase...... 70 5.i0 Significance of Block Effects...... ,73

Chapter6-Discussion...... 74

6.1 Terminal Piglet and Litter Growth Pattems...... 75 6.2 Delayed Development of Enzyme Activity Along the Transsulphuration Pathway...... 76 6.3 Maintenance of Hepatic Cysteine Concentrations...... 80 6.3.1 Cysteine Uptake From Plasma...... 81 6.3.2 Proteolysis...... 82 6.3.3 Cysteine Utilization For Taurine Synthesis...... 84 6.3.4 Speculation on the Involvement of Sulphate and GSH on Cysteine Levels...... 86 6.4 Increased Hepatic Cysteine Concentrations Post-Weaning...... 88 6.5 Delayed Development of MS in Neonatal Pigs...... 89 6.6 Potential Factors Influencing Homocysteine Flux Along the Remetþlation Pathway...... 91 TV

6.6.1 Enryme Characteristics and Factors Potentially Influencing Homocysteine Concentrations...... 92 6.6.2 Folate and Vitamin 8,, Availability For Compiete IviS Activity.....96 6.6.3 Folate Availability for Serine Production ...... 99 6.7 Potential Explanations for the Significant Block Effects Observed...... 101 6.8 Factors Influencing Plasma Homocysteine and Cysteine Pattems in Developing Pigs...... 101 6.9 Similarities Between Piglets and Human Infànts With Regards to Sulphur Amino Acid Metabolism...... 109 6.10 Developing Pigs and Their Potential to Respond to Disease Challenges...1l l 6.11 Summary ...... 113

Chapter 7 - Future Research...... 115

ChapterS-References...... 117

Chapterg-Appendices...... 138 V

List of Tables

Table 5.1 - Plasma Folate and Vitamin 8,, in Developing Pigs From Birth to Post- Weaning...... 62

Table 5.2 - Plasma Methionine, Serine and Taurine in Developing Pigs From Birth to Post-Weaning...... 64

'Weaning...Table 5.3 - Renal, CBS, CGL and MS Activities in Developing Pigs From Birth to Post- ...... 69 VI

List of Figures

Figure 2.1 - Methionine Metabolism Leading to the Formation of Cysteine...... 3

Figure 5. i - Body Weight Averages of Litters and Terminated Pigs from Birth to Post- Weaning...... 59

Figure 5.2 - Plasma Homocysteine in Developing Pigs From Birth to Post- Weaning...... 60

Figure 5.3 - Plasma Cysteine in Developing Pigs From Birth to Post-Weaning...... 6i

Figure 5.4 - Hepatic Homocysteine in Developing Pigs From Birth to Post-Weaning....65

Figure 5.5 - Hepatic Cysteine in Developing Pigs From Birth to Post-V/eaning...... 66

Figure 5.6 - Hepatic CBS Activity in Developing Pigs From Birth to Post- Weaning...... 68

Figure 5.7 - Hepatic CGL Activity in Developing Pigs From Birth to Post-Weaning...... 71

Figure 5.8 - Hepatic MS Activity in Developing Pigs From Birth to Post-'Weaning...... 72

Figure 6.1 - General Pattern of Development Observed for Hepatic CBS Activify...... 77

Figure 6.2 - General Pattern of Development Observed for Hepatic CGL Activity...... 78

Figure 6.3 - General Pattern of Development Observed for Hepatic MS Activity...... 90 VII

List of Appendices

Solution Preparation Methods...... 138

Optimized Enzyme Dilutions and Incubation Lengths For Measuring CBS, CGL and MS...... 144

Plasma Amino Acids in Developing Pigs From Birth to Post- Weaning...... 145 . Hepatic CBS Activity in Developing Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue 8asis...... 146

Renal CBS Activity in Developing Pigs From Birth to Post-'Weaning Reported on a Per Gram of Tissue and Total Tissue 8asis...... 147

Hepatic CGL Activity in Developing Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue 8asis...... 148

Renal CGL Activity in Developing Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue 8asis...... 149

Hepatic MS Activity in Developing Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue 8asis...... 150

Renal MS Activity in Developing Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue Basis...... 151

Summary of Block Effects and Visual Comparison with Litter Size at Day I and Sow Parity...... 152 VIII

Abbreviations

S-MTHF - 5-Methyltetrahydrofolate

5, 1 0-MTHF - 5, 1 0-Methylenetetrahydrofolate ATP - Adenosine triphosphate BCA - Bicinchoninic acid BCS - Bathocuproine disulphonate BHMT - Betaine :homocysteine methyltransferase BSA - Bovine serum albumen CBS - Cystathionine beta-synthase CGL - Cystathionine gamma-lyase DPM - Disintegrations per minute EDTA - Ethylenediaminetetraacetic acid GSH - Glutathione, reduced form GSSG - Glutathione, oxidized form Km - Michaelis-Menton constant KOH - Potassium hydroxide LDH - Lactate dehydrogenase Max - Maximum Min - Minimum mRNA - messenger Ribonucleic Acid MS - Methionine synthase MTHFR - Methylenetetrahydrofolate reductase NADH - Nicotinamide adenine dinucleotide, reduced form NaOH - Sodium hydroxide NRC - National Research Council ODC - Omithine decarboxylase OPA - o-phthaldialdehyde P5 CS - Pyrroline-5 -carboxylate PF - Protein-free PLP - Pyridoxal 5'-phosphate PPG - Propargylglycine P-valuet*t- P-value for treatment effect P-valuebro'k - P-value for block effect RIA - Radioimmunoassay ROS - Reactive oxygen species TpHPLC - reverse-phase High Pressure Liquid Chromatography SAH - S-adenosylhomocysteine SAM - S-adenosylmethionine

S B D F - 7 -flur o -2 - oxa- 1,3 - diazo I e-4 - sulfoni c aci d SHMT - Serine hydroxyrnethyltransferase SSA - Sulphosalicylic acid tBuOOH - Tertiary butyl hydroperoxide TCEP - Tris(2-carboxyethyl)-phosphine hydrochloride IX

THF - Tetrahydrofolate 1

Chapter I - Introduction Cysteine, a sulphur amino acid produced endogenously from methionine, is recognized to be nutritionally dispensable for swine Q'.lational Research Council 1998).

Adequate levels of cysteine are important for optimizingprotein synthesis and the generation of taurine and glutathione. Research indicates that endogenous cysteine production may be limited in the human neonate as a result of developmental delays in cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CGL), two enzymes involved in the transsulphuration pathway directly leading to cysteine synthesis. The developmental pattems of these enzymes have not been previously investigated in pigs.

Methionine synthase, (MS) involved in homocysteine remethylation, may also be involved in the coordination of transsulphuration pathway activity. Establishing the developmental pattern of these enzymes, from birth until shortly after weaning, may offer insight as to whether the early-weaned pig has the capacity to meet its cysteine requirements early in life so as to support taurine and glutathione production as well as complete growth and development. Chapter 2 -Literature Review

2.1 Cysteine Synthesis and the Methionine Cycle

The sulphur amino acid cysteine can be derived for use by animals from the diet, protein breakdown and endogenous cysteine synthesis. Endogenous cysteine proceeds from a series of reactions beginning with the indispensable amino acid methionine

(Figure 2.1). The metabolism of methionine to cysteine occurs via three enrymatic pathways: transmethylation, remethylation and transsulphuration. While transmethylation and transsulphuration are directly involved in cysteine synthesis, remethylation contributes indirectly to its synthesis by regenerating methionine and competing with the transsulphuration pathway for homocysteine. Collectively, these pathways act to maintain a homeostatic balance between the body's requirements for methionine and cysteine.

The transmethylation pathway consists of three steps through which methionine is converted to homocysteine. The first step involves the activation of methionine to a high- energy sulphonium compound, S-adenosylmethionine (SAM) via SAM synthetase. SAM is an active methyl donor in the body (Chiang et al. 1996,Lu 2000) and is involved in transmethylation reactions such as the methylation of glycine to sarcosine and the methylation of DNA and RNA. Various methyltranrf.rur., involved in these reactions also catalyze the transmethylation of SAM (indicated in Figure 1 as X->X-CHr) to form

S-adenosylhomocysteine (SAH). Methyltransferase activity is regulated by the ratio of

SAH to SAM and, as a potent competitor of SAM at several binding sites and a methyltransferase inhibitor, high SAH levels will inhibit methylation (Hoffman et al.

1980, Finkelstein 1990, Molloy et al. 1992). SAH hydrolase further catalyzes the MTHFR 5,10-CH2-THF 5-CH3-THF ild:j CBS (Vitamin B) serine MS (Vitamin B,r) Homocysteine Cystathionine Betaine at 1 Serine THF

Alpha-ketobutyrate SAH Hydrolase + NH¿* CGL (Vitanin B)

Methionine -

ATP Adenosine SAM Synthetase

PPi+Pi

S-Adenosylmethionine S-Adenosyl homocysteine .--* CYsteine 1 Protein /\ \ Grurathione MTs

X x-cH3 Sulphate Taurine

Figure 2.1: Methionine Metabolism Leading to the Formation of cysteine Betaine:Homocysteine Methyltransferase, BHMT; Cystathionine Beta-synthase, CBS; Cystathionine Gamma-Lyase, CGL; Methioníne Synthase, MS; Methylenetetrahydrofolate Reduclase, MTHFR; Methyltransferases, MTs; s-Adenosylméthionine Synthase, sAMs; s-Adenosylhomocysteine, sAHs; Serine Hydroxymethyltransferase, SHMT;

i 4 conversion of SAH to adenosine and homocysteine. When homocysteine concentrations increase, SAH levels increase as a result of the thermodynamically favoured reverse reaction of SAH hydrolase to SAH. To prevent this reaction, homocysteine and adenosine must be further metabolized. Adenosine is converted via adenosine deaminase to inosine for removal and equal amounts of homocysteine (Finkelstein et al' 1984) are diverted through one of two enzymatic pathways, (Green and Jacobsen 1995, Bolander-Gouaille

2002): a) remethylation to methionine or b) transsulphuration to cysteine'

A) Remethylation: The remethylation of homocysteine to methionine occurs via the enzymes methionine synthase (MS) and betaine:homocysteine methyltransferase

(BHMT). Cobalamin-dependent MS catalyzes the transfer of a metþl group from 5- methyltetrahydrofolate (5-MTHF) to homocysteine, forming methionine and the by- product tetrahydrofolate (THF). Resynthesis of S-MTHF requires serine to act as a one-

(5,10- carbon donor for the remethylation of THF to 5,1O-methylenetetrahydrofolate

MTHF), and methylenetetrahydrofolate reductase (MTHFR) to catalyze the f,rnal reaction

of 5,10-MTHF to 5-MTHF (Refsum et al. 1998b). Homocysteine may also be

remetþlated using the choline metabolite betaine as a methyl donor. Remethylation of

homocysteine using betaine and the enzyme BHMT is entirely independent of the folate

pool and results in the production of methionine and the betaine-by-product

dimethylglycine (Finkelstein 1990). Of the 54 per cent of homocysteine that is produced

and remethylated to methionine, MS and BHMT equally.contribute to its methylation in

the liver of rats (Finkelstein and Martin 19S4). However, whereas all cells contain MS

and thus are capable of remethylation, only measurable amounts of BHMT have been reported in the liver and kidney of some species, including rats, monkeys and humans

(Finkelstein et al. 1971, Sturman et al. 1976a, Gaull et al. 1973b).

B)Transsulphuration: The transsulphuration pathway involves the catabolism and oxidation of carbon and sulphur groups on homocysteine resulting in the end-product cysteine (Stipanuk 2004). Homocysteine first condenses with serine to form cystathionine via the vitamin Bu-dependent enzyme cystathionine beta'synthase (CBS). Cystathionine is fuither metabolized by cystathionine gamma-lyase (CGL), also requiring vitamin 86 as a cofactor, to ø-ketobutyrate, NHo* and cysteine. Transsulphuration is the favored reaction when SAM concentrations are high, dietary methionine is adequate or in excess

(Finkelstein and Martin 1986) and cystine is not being provided for methionine-sparing

(Finkelstein and Mudd 1967, Finkelstein et al. 1988). Transsulphuration enzymes are absent in the majority of cells and present primarily in the liver, kidney, pancreas and small intestine.

Cysteine is composed of a thiol group, transferred from the metabolized methionine, and a carbon skeleton donated by serine. Present in one of two forms, cysteine represents the sulphhydral form, found primarily inside the cell while cystine, represents the disulphide form, predominantly located outside the cell.

2.2 Essential Functions of Cysteine

In mature vertebrates, cysteine is considered a dispensable amino acid. However, cysteine is required for various important physiological functions including protein synthesis, glutathione and taurine generation and inorganic sulphate production. 6

2.2.1 Protein Synthesis

Cysteine, either of exogenous or endogenous origin, is required for protein synthesis and has an average occurrence in proteins of 1.9 per cent (Doolittle 1989, as referenced by Nelson and Cox 2000). The oxidation of cysteine's thiol groups to other sulphur compounds in adjoining polypeptide chains for stabilizing protein structure via disulphide linkages is well-documented. Cysteine residues provide the active thiol groups in biological thiol-dependent enzymes such as cysteine proteases (Leung-Toung et al.

2002). For thimet oligopeptidase, a metalloenzyme involved in regulating neuropeptide processing, the location and not just the existence of intermolecular disulphide bonds is important in enzyme activation and regulation (Sigman et al. 2003) As well, glutathione's side chain sulphhydral residue provided by cysteine accounts for most of the antioxidant's physiological properties (Sen 1997).

2.2.2 T aurine Synthesrs

Taurine is a sulphonic acid synthesized as a result of the oxidation of cysteine to cysteinesulphinate, the central intermediate in cysteine catabolism. Cysteinesulphinate undergoes decarboxylation to hypotaurine that is further oxidized to form taurine. In mature, healthy omnivores, taurine is a dispensable nutrient, however it is generally accepted as conditionally-indispensable in developing human infants (Sturman and

Chesney 1995).

Although taurine is not utilized for protein biosynthesis, it is present in relatively high concentrations in most animal tissues. Taurine is regognized as playing an important 7 role in various physiological functions, including membrane stabilization, bile salt formation, antioxidation, calcium homeostasis maintenance, glycolysis and glycogenesis stimulation, growth modulation, osmoregulation, immurie function, neural development and vision (Sturman and Chesney 1995, Redmond et al. 1998). Dietary deficiencies of taurine and physiological aberrations preventing taurine absorption can result in retinal degeneration (Hayes et al. 1975a, Hayes et al. 1975b, as referenced by Sturman and

Chesney 1995), poor reproductive performance (Sturman et al. 1986, Sturman and

Messing 1991), hypernatremic dehydration and seizure activity (Trachtman et al. 1988, as referenced by Sturman and Chesney 1995), and reduced neural development (Tyson et al.

1989, as referenced by Sturman and Chesney 1995).

As with glutathione, taurine synthesis is influenced by factors affecting cysteine availability. In isolated rat hepatocytes, high cysteine availability favored the production of taurine and sulphate whereas low cysteine concentrations favored glutathione synthesis

(Stipanuk et al. I 992). The presence of bathocuprine disulphonate (BCS), a copper chelator that increases the supply of cysteine to the cell by maintaining a high cysteine:cystine ratio in the incubation medium, also resulted in increased cellular taurine synthesis. With increasing cysteine concentrations, the percentage of cysteine metabolism accounted for by taurine increased by 1.3-fold, occurring primarily at the expense of glutathione synthesis (Stipanuk et al. 1992). Cysteine is proposed as a key amino acid in the body during inflammatory conditions (Grimble et al. 1992) with absolute whole body cysteine/cystine being significantly greater and whole body cysteine catabolism being dramatically reduced during infection (Breuillé et al. 1996). Cysteine catabolism to 8 sulphate in septic rats is significantly lower than in pair-fed controls and proposed to be a result of an increased physiological demand for taurine and glutathione (Malmezat et al.

1998). Hepatic taurine and glutathione concentrations were significantly elevated in several organs of septic rats as compared with healtþ controls, suggesting a potential protective mechanism against sepsis-induced oxidative damage. Increased rates of muscle protein wasting concurrent with elevated whole body protein synthesis in septic animals could also contribute to increased cysteine utilization during infection, potentially limiting cysteine availability for glutathione and taurine production (Malmezat et al.

1 ee8).

2.2.3 Glutathione Synthesis

Glutathione (GSH, reduced form) is synthesized as a result of tr¡¡o condensation reactions between cysteine, glutamate and glycine. The y-carboxyl group of glutamate is activated by adenosine triphosphate (ATP) to form an acyl phosphate intermediate that is then joined by the ø-amino group of cysteine. The s-carboxyl group of cysteine undergoes a second condensation reaction to join with glycine, resulting in the tripeptide glutathione (Nelson and Cox 2000). Glutathione may exist in both the thiol-reduced form

(GSH) and the disulphide-oxidized (GSSG) form.

Glutathione participates in a variety of protective.and metabolic functions, including the following: detoxification of xenobiotics, stabilization of thiols in proteins, protection of cells from oxidative stress, serving as a storage and transport form of cysteine, modulation of redox-regulated signal transduction, regulation of cell 9 proliferation, synthesis of DNA, regulation of leukotriene and prostaglandin production and immune response (Meister and Anderson 1983, Sen 1997). Present at quantitatively high levels in mammalian cells (0.5-1OmM; Meister and Anderson 1983), and as a substrate to GSH peroxidase, glutathione serves as the major scavenger of reactive oxygen species (ROS) (Dröge and Breitkreutz 2000). Although most well-recognized for its overall-protective role as an antioxidant, the sensitivity and altered effectiveness of lymphocytes, interleukin-2, lymphokine-activated killer cells and natural killer cells during an immune response to a glutathione deficiency or excess, emphasizes the importance of GSH to whole-body immune response (Ohmori etaL.7982, Yamauchi and

Bloom 1993, Iwata et al. 1994, as referenced by Yamauchi et al. 7999, Dröge and

Breitkreutz 2000).

Glutathione levels are highly influenced by cysteine availability (Gallant and

Cherian 1989, Viña et al. 1995, Lavoie and Chessex 1998). Tateishi etal. (1974) reported that hepatic GSH levels were closely associated with dietary conditions and that GSH concentrations in starved rats decreased to between two-thirds and half the normal level but rapidly returned to normal upon refeeding. Substrate availability, not de novo synthesis of GSH-synthesizing enzymes, was proposed as the primary rate determinant of hepatic GSH synthesis. High levels of hepatic cysteine and glutamate, observed during the initial stage of refeeding, should favor GSH synthesis. However, hepatic cysteine concentrations, as compared to glutamate and glycine, may limit the rate of GSH synthesis since cellular cysteine concentrations were substantially lower than the Km value of gamma-glutamylcysteine synthetase, the first errTpe involved in GSH synthesis 10

(2.5 xlO¡; Tateishi et al. 1974).In agreement, depressed.hepatic GSH levels in rats fed a low protein diet were restored within 24 hours by feeding a diet high in protein, cysteine or methionine (Leaf and Neuberger 1947, Register et al. 1959, as referenced by Tateishi et al. 1974). Tateishi et al. (1981) demonstrated cysteine to be preferentially used over methionine for GSH synthesis. Lower amounts of 3sS from dietary methionine were incorporated into the hepatic GSH pool as compared to equivalent amounts of cysteine.

As well, preformed dietary cysteine was the preferred source for hepatic GSH while methionine-derived cysteine was more often incorporated into liver proteins (Tateishi et al. 1981). Extracellular and intracellular amino acid concentrations also influence cysteine availability for GSH synthesis. Lu (1998) demonstrated the rate of cysteine uptake is approximately 3-fold and 13-fold greater than methionine or cystine respectively, thereby increasing the cellular availability of cysteine for GSH synthesis.

Factors affecting the transsulphuration pathway also influence cysteine availability for GSH synthesis. Exposure of human hepatocytes to oxidants increase the flux of sulphur amino acids through the transsulphuration pathway. In the presence of hydrogen peroxide, a 1.6-fold increase in the flux of homocysteine through the transsulphuration pathway was observed while a 1.5-fold increase in the incorporation of [35S]-methionine into the GSH pool occur¡ed following ins treatment with tertiary butyl hydroperoxide

(tBuOOH; Mosharov et al. 2000). Inhibition of CGL with propargylglycine also significantly decreased intracellular cysteine concentrations with a parallel decline in the

GSH pool and the glutathione to glutathione disulphide (GSH:GSSG) ratio (Gallant and

Cherian 1989, Mosharov et al. 2000). Incubation of human colon carcinoma cells in a 11 cysteine-defrcient medium similarly resulted in a reduced intracellular GSH concentration and redox potential (Miller et aI.2002). Subsequent readdition of cysteine to the deficient cells produced a rapid, near-complete recovery of the GSH pool, demonstrating the importance of cysteine availability to GSH pool maintenance.

Nutritional deficiencies of the B-vitamins involved in methionine metabolism have varied effects on GSH synthesis. Dietary folate depletion in rats had no significant effect on hepatic GSH levels, despite an increase in oxidative stress in the livers (Huang et al. 2001) while Verjee etal. (1975) reported increased concentrations of GSH in vitamin B,r-deficient baboons. Vitamin Bu deflrciency in rats did not influence the level of

GSH in any tissues studied, suggesting cysteine availability was maintained despite a significant reduction in transsulphuration enzyme activity (Takeuchi et al. 1991).

Decreased utilization of cysteine by other metabolic pathways for the maintenance of the cysteine pool is a possibility, increasing the scope of factors that could influence cysteine availability.

2.2.4 Inorganic Sulphate Synthesis

Sulphate is produced following the oxidation of cysteine via the enzyme cysteine desulphhydrase, to pyruvate and inorganic sulphate. This process occrus during protein turnover and following the ingestion of excess protein and/or sulphur compounds present in the diet. Sulphur is required by the body for the production of all but two essential sulphur-containing products (thiamin and biotin), with one of the primary roles for inorgani c sulphate being the synthesis of 3'-phosphoadenosine-5'-phosphosulphate T2

(PAPS). PAPS is required for the synthesis of several important body components, such as the bile salt taurolithocholate sulphate and dermatan sulphate, a glycosaminoglycan found in the skin, blood vessels and heart valves (Instituie of Medicine2004).

Methionine and cysteine are primary organic sulphur compounds and their metabolism is suggested to supply over half of the inorganic sulphate required in humans, with the remainder coming from pre-formed sulphate in water and foods (Institute of

Medicine 2l}4).sulphate deficiency has been observed when dietary protein and sulphur amino acids, specifically cysteine, are restricted in the diet. Glazenberg et al. (1984) demonstrated that rats fed a low-protein diet containing only 8 per cent casein had a serum sulphate concentration 20 per cent that of control rats. Similarly, subjects fed a purified diet with no methionine or cysteine for 8 days had reduced urinary levels of methionine and inorganic sulphate within the first half of the trial period (Lakshmanan et al.l976). Re-addition of sulphate to such diets has resulted in significant growth responses (Byington eT al. 1972, Smith 1973, Sasse and Baker 1974, Anderson et al.

197 5). Sulphate requirements for the growing fetus are also high (Cole et al. 1992 as referenced by Institute of Medicine2004), suggesting sulphur to be important in early growth and development.

The importance of cysteine for protein, GSH, tauline and organic sulphate production is evident. Adequate levels of cysteine particularly at birth are essential to fulfill the rapidly increasing requirements by the newborn for cysteine and its metabolic products. Without suffrcient amounts, growth and development are limited, immunity is jeopardized and the establishment of various physiological functions is suppressed. 13

Although cysteine is considered a dispensable amino acid in adulthood, some researchers suggest cysteine to be an indispensable amino acid in low-birth-weight human infants due to a delay in the development of hepatic transsulphuration enzymes (Sturman et al. i970,

Gaull et al. 1972, Pohlandt 1974). To prevent potential developmental limitations of infants caused by inadequate endogenous cysteine synthesis, cysteine may be included in parenteral feeding solutions to ensure post-natal cysteine requirements are satisfied

(Sturman et al. 1910, Gaull et al. 1972, Pohlandt l974,Yiña et al. 1995, American

Academy of Pediatrics 1998). On the contrary, Zlotkin et al. (1981) reported premature infants on a cysteine-free intravenous diets as having excellent nitrogen retention and growth that was not improved by cysteine inclusion. Zlotkin and Anderson (1982) also demonstrated CGL activity in the kidney, adrenal and pancreas of premature infants to be stable. Given adequate levels of methionine, these authors suggested that endogenous cysteine synthesis via these organs may be sufficient to support neonatal cysteine requirements until the hepatic transsulphuration mechanisms reach an adult activity level.

In pigs, cysteine is classified as a dispensable amino acid, due to its ability to be endogenously synthesized from methionine. A dietary requirement for methionine but not cysteine per se has been established (l.trational Research Council 1998). Since cysteine and cystine can satisff approximately 50 per cent of the pigs' need for total sulphur amino acids (Roth and Kirchgessner 1989, Chung and Baker 1992), dietary inclusion of cysteine can reduce the pigs' methionine requirement. According to the National Research

Council QIIRC)'s Nutrient Requirements for Swine (1998), the estimated amino acid requirement (represented as total amino acids on a percentage of the diet as-fed basis) for 14

methionine plus cysteine for pigs between 5 and 20kg is around 0.5 per cent (Lovett et al.

1986, Chung and Baker 1992, Owen et al. 1995, as referenced by the National Research

Council 1998). Although it is generally assumed that endogenous cysteine synthesis is

adequate to support cysteine requirements in the piglet, unlike for human infants, the

developmental profiles of CBS and CGL in pigs have not been demonstrated, thus weakening these assumptions. If development of these enzymes occurs similarly, cysteine availability to the piglet may be limited until weaning, typically between 14 and 19 days of age (Thacker 1999, 'Whiting 2001). Under normal swine production systems, suckling pigs rely on the sows' milk and as they get older, possibly creep feed until weaning.

However without knowing the developmental patterns of these enzymes, cysteine levels in the piglet may be inadequate throughout suckling, and. creep feed and post-weaning diets, formulated under the assumption that endogenous cysteine mechanisms are at an adult activity level, may in fact contain insufficient levels of dietary cysteine. Although there are numerous dietary factors that may influence cysteine availability via endogenous means, such as methionine (Finkelstein and Mudd 1967, Finkelstein and Martin 1986), folate (Selhub and Miller 1992) and vitamin B', (Doi et al. 1989) availability, understanding the developmental pattems of CBS and CGL is the first step to ensure that these intermediates are provided at sufficient levels to support transsulphuration.

Furthermore characterization of the changes in CBS and.CGL activity with age in pigs will indicate whether these enzymes are capable of supporting pigs' cysteine requirements early in life and if not, at what age an adult level of enzyme activity is achieved. 15

2.3 Remethylation and Transsulphuration Enrymes: Location and Factors Influencing Changes in Activity

Characterization of the developmental pattems of CBS and CGL have received appreciable attention due to their role in endogenous cysteine synthesis via the transsulphuration pathway. The remethylation pathway, specifically involving MS, has also been suggested to indirectly affect cysteine production. Selhub and Miller (1992) proposed a coordinate regulation of the two pathways by SAM, an enzymatic effector that acts as an allosteric inhibitor of MTHFR (Jencks and Matthews 1987) and an activator of

CBS (Finkelstein et al. 1975). When SAM concentrations are low, the synthesis of 5-

MTHF and MS activity is uninhibited whereas cystathionine synthesis and CBS is suppressed. Conversely, a high SAM concentration would suppress 5-MTHF production and MS activity because more homocysteine is diverted inrougtr the transsulphuration pathway due to stimulated CBS. Based on this hypothesis, changes in MS activity with age and its effect on homocysteine levels could impact the development rate of CBS and

CGL by influencing substrate availability for transsulphuration and thus affecting the

SAH:SAM ratio.

Characterization of the developmental patterns of CBS, CGL and MS have been documented in a variety of organs in humans, rats, monkeys and chicks (Finkelstein 1967,

Kashiwamata 1971, Gaull et al. l972,Yolpe and Laster 1972, Gaull et al. 1973b,

Kalnitsky et al. 1982, Zlotkin and Anderson 1 982, VanAerts et al. I 995, Rao et al. 1 998).

Studies on the effects of dietary, hormonal and pharmacological factors on these enzymes 16 have also been performed. The following is a review of the literature relating to the tissue location, enzymatic stimuli and developmental pattems of CBS, CGL and MS, as previously established in various research species. Insight into the characteristics ofthese enzymes may be gained from other animal models, however findings may not be directly applicable to pigs.

2.3.1 Cystathionine Beta-Synthase

Cystathionine beta-synthase has been identified as being catalytically active in a variety of tissues with notably higher activity levels than remethylation enzymes. Mudd et al. (1965) reported substantial CBS activity in liver (184 nmolÆr/mg protein), kidney (84 nmol/hr/mg protein) and pancreas (166 nmol/hrlmg protein) of rats in comparison with lower levels in adipose tissue (38 nmol/hr/mg protein), brain (37 nmol/lr/mg protein) and mucosa of the small intestine (31 nmol/tr/mg protein); the spleen, adrenal, lung, testes, heart and skeletal muscle contained low to no activity luit t.r, than 4 nmollhr/mg protein). Tissue distribution of CBS in rats is similar to that of other experimental species. Hepatic and renal CBS are most commonly analyzed in humans (Sturman et al.

1970), rodents (Finkelstein and Martin 1986, Yamamoto et al. 1995, Yamamoto et al.

1996, Dicker-Brown et al. 2001), broiler chicks (Saunderson and MacKinlay 1990) and primates (Sturman et al. I976b). Several dietary and hormonal factors are known to influence CBS activity. A diet containing excess methionine fed to rats resulted in a significant rise in hepatic CBS activity and coincided with a decline in hepatic serine concentrations (Finkelstein and Martin 1986). Conversely, a low-methionine diet reduced t7 hepatic CBS activity in broiler chicks, likely encouraging remethylation at the expense of transsulphuration. Choline suppiementation of a methioriine-free diet also lowered hepatic CBS activity, suggesting ân adaptive response of the sulphur metabolic pathways to catabolize excess choline and conserve methionine (Saunderson and MacKinlay i990).

In contrast, methionine supplementation of growing steers (Lambert et aL 2002) or sheep

(Radcliffe and Egan lgl8)had no effect on hepatic CBS activity.

The methionine-sparing effect of dietary cystine is effective in lowering hepatic

CBS activity (Finkelstein et al. 1988, Yamamoto et al. 1995). Substituting dietary methionine almost entirely by cystine resulted in a 60 and 66 per cent decrease in CBS activity and SAM concentration, respectively (Finkelstein et al. 1988). Cysteine supplementation of a low-methionine diet reduced rat hepatic CBS activity only slightly more than cysteine supplementation of a methionine-containing diet. Alterations in the

CBS mRNA level caused by dietary manipulation were similar to those observed in

enzyme activity and transcription-level changes were concluded to be the source of variation in CBS activity (Yamamoto et al. 1995).

Aging may also have an effect on the level of repression of CBS activity and gene expression in rats fed a cysteine-free, methionine balanced diet. This diet caused similar reductions in CBS activity in 4- and l2-week old rats whereas a cysteine-containing diet yielded a more significant decline in enzyme activity and gene expression in the younger rats. Results suggest the effect of dietary cysteine on CBS diminishes with aging

(Yamamoto et al. 1995).

Changes in dietary protein are known to affect CBS activity and gene 18 transcription. Hepatic CBS activity of rats fed a protein-free diet, fasted and then fed casein were significantly higher than control rats continuously fed protein-free rations

(Finkelstein 1967). Hepatic CBS activity levels of rats fed a standard or high-protein (62 per cent casein) laboratory diet were respectively 390 and 452 per cent greater than of rats fed the protein-free diet. This supports the enhanced management of a normal or excessive influx of methionine (Finkelstein 1967). Similar trends in CBS activity have been noted Q.takagawa 1971 asreferenced by Yamamoto et al. !996,Yamamoto et al.

1996, Kim et al. 2003). In agreement, Yamamoto et al. (1996) reported a significant increase in hepatic CBS activity in rats fed an 80 per cent casein diet after five days.

Hepatic CBS activity was successively reduced in rats fed intermediate (control) and low levels of dietary casein. As well, the CBS transcription rate of the high-protein diet was approximately 2.6-fold greater than that of the low-protein diet. Results suggest that changes in CBS activity caused by dietary protein were mediated at the level of gene expression (Yamamoto et al. 1996).

The role of vitamin Bu is to serve as the source of the coenzyme pyridoxal 5'- phosphate (PLP) and is required for complete CBS activity. A 50 per cent reduction in hepatic (Kashiwamatal97l, Shannon and Demos 1977) and neural (Kashiwamatal9Tl)

CBS activity in vitamin Bu-def,rcient rats has been previo,usly reported. In contrast,

Mafünez et al. (2000) demonstrated vitamin Bu deficiency did not significantly reduce hepatic CBS activity and that in vivo transsulphuration flux, shown by the hepatic production of [2Hr]-cysteine from [zHr]-serine, increased three-fold. However, the dramatic decline in plasma [2H,]-cysteine concentrations suggests that large reductions in T9 whole body transsulphuration flux occurred during vitamin Bu deficiency. The enzlme

CBS is known to bind four PLP molecules with a tight nonequivalent binding affinity

(Kery et al. l994,Taoka et al. 1999) and in some tissuet, CgS may be less sensitive to the suppressing effects of a vitamin Bu deficiency. In agreement, Sturman et al. (1969) reported that a vitamin Bu deficiency significantly reduced CBS activity only in pancreatic tissue with no changes in the liver or brain and a slight decrease in the kidney.

In addition to the vitamin Bu cofactor, CBS relies on a heme protein for binding

PLP and thus complete enzyme activation (Kery et al. 1994). Unlike other heme-based enzymes whose activity declines when subject to an iron deficiency (Rodvien et al. I974,

Stangl and Kirchgeßner 1998 as referenced by Ballance and House 2001), this was not the case in nutritionally induced, iron-deficient rats (Ballance and House 2001). Iron deficiency in rats was confirmed by low hemoglobin concentrations and high total iron- binding capacity as compared with control and pairfed rats. Hepatic CBS activity in iron- deficient rats was significantly greater, approximately 2.7 nmol/hr/mg protein, as compared with enzyme activity of control and pairfed animals at one-half that amount.

Plasma homocysteine concentrations were significantly higher in iron-deficient animals as compared with pairfed rats (12 versus 9 umol/L, respectively) but were lower than for control rats þermitted ad libitum feed intake), which had unexpectedly high circulating levels of homocysteine (20 umol/L). Plasma folate and vitamin B,, cofactors were also measured and while folate levels were acceptable for all treatment groups, vitamin Bt, concentrations of the iron-deficient rats were significantly lower than either of the other groups. Although the increase in CBS activity in iron-deficient rats could not be 20 definitively established from the present trial, increased CBS activity may be in response to oxidative stress induced during iron-deficient conditions (Yip and Dallman 1996).

Mosharov et al. (2000) noted an increased flux through the transsulphuration pathway in intact human hematoma cells despite no change in CBS gene expression. These authors speculated that increased movement along the transsulphuration pathway may be a means of ensuring increased cysteine synthesis and optimal GSH production. Increased plasma homocysteine in iron-deficient rats was unlikely related to CBS activity and more likely in response to low vitamin 8,, concentrations, known to influence plasma homocysteine concentrations (Lindenbaum et al. 1988, Stabler et al. 1991, Stangl et al. 2000).

Hormonal effects on CBS activity have been investigated. Early studies by

Finkelstein (1967) revealed positive effects of various hormones on liver, kidney, pancreas and brain CBS activity in rats but no treatments resulted in significant, parallel changes in all tissues. Hydrocortisone, alloxan and glucagon significantly increased hepatic CBS while thyroxine and testosterone dramatically lowered renal CBS activity in rats. As well, alloxan significantly reduced CBS activity.in the kidney and pancreas but had no effect on neural activity levels. More recent studies have focused on the effects of insulin in homocysteine metabolism. Increasing concentrations of insulin significantly reduced CBS activity in cultured mouse hepatocytes (Dicker-Brown et al. 200i). Similar effects on CBS activity were observed in vivo for insulin-treated diabetic rats (0.68 nmol/min/mg protein) as compared with untreated diabetic animals (0.98 nmol/min/mg protein) (Jacobs et al. 1998). Ratnam et al. (2002) demonstrated a parallel increase in

CBS activify and its mRNA levels in diabetic rats, both of which were reversible with 2T insulin administration. Glucocorticoids and glucagon, counter-regulatory agents of insulin, also influence CBS activity. Treatment of rat and human hepatomas with glucocorticoids increased the level of CBS protein and mRNA in vitro while insulin inhibited the effect. Changes in CBS activity are confirmed to be a result of stimulation of

CBS gene expression by glucocorticoids and inhibition by insulin at the level of transcription (Ratnam et al.2002). As well, glucagon administration to rats resulted in a

90 per cent increase in CBS activity and mRNA level as compared with control animals

(Jacobs et al. 2001). Flux through CBS increased 5-fold in hepatocytes isolated from glucagon-treated rats with parallel increases in the hepatic concentrations of SAM and

SAH, both allosteric activators of CBS.

In general, CBS activity increases with age in most tissues of species studied.

Volpe and Laster (1972) reported fetal rat hepatic CBS activity as low (< 20 nmol lhrlmg protein) until late gestation (day 20) but rose dramatically (90 to 110 nmol/hr/mg protein) just prior to parturition. Hepatic CBS activity continued to rise gradually, reaching an activity level comparable that of the adult rat by the third post-natal week. Neural CBS activity was undetectable until late in gestation but increased continuously throughout the remaining pre- and post-natal period studied. Adult levels were reached two weeks after birth (Volpe and Laster 1972). CBS activity was not measured in early embryonic or fetal rat tissue but was prominent in surrounding placental tissue early in gestation (VanAerts et al. 1995). Fetal hepatic CBS activity was measurable on day 16 of gestation and progressively increased to 68 per cent of the dams' hepatic CBS activity level near parturition (VanAerts et al. 1995). Finkelstein(1967) and Kashiwamata(1971) reported 22 similar pattems of change for CBS in rat liver and brain. Kashiwamata (1971) also reported no differences between male and female rats in hepatic and neural CBS developmental pattems. Hepatic CBS measured in human fetuses, premature neonates and full-term infants also followed a pattern of increasing enzyme activity with age

(Sturman et al. I970). Sturman et al. (1970) reported hepatic CBS activity of immature humans at birth to be one-quarter to one-third that of CBS activity measured in more mature subjects. Slight differences in the developmental pattem of CBS in fetal, neonate and adult Rhesus monkey tissues have been observed (Sturman et al. 1976b). In contrast to previous trends, hepatic CBS activity peaked at birth (200-350 nmollhr/mg protein) and rapidly declined to adult levels by two weeks of age. While neural CBS activity followed a similar pattem to that previously reported in rat, it did not stabilize at constant levels until two to three months of age.

2.3 .2 Cystathionine Gamma-Lyase

Cystathionine gamma-lyase activity has been reviewed in tissues from numerous animals, including rat, beef cattle, lamb, pig, horse, guinea pig, hamster, monkey and human (Mudd et al. 1965). While the liver, kidney, pancreas and brain contain the highest

CGL activity (Mudd etaI.1965, Finkelstein 1967, Sturman et al. 1969, Heinonen 1973,

Heinonen 1977 , Zlotkin and Anderson 1982), adipose tissue, mucosa of the small intestine, spleen, adrenal, lung and testis also contain measurable levels of enzyme activity (Mudd et al. 1965).

Dietary factors, many of which are known to affect CBS activity, also influence 23

CGL activity. Changes in dietary protein have significant but unique effects on CGL activity in the liver, kidney, pancreas and brain (Finkelstein 1{I67).CGL activity in these tissues was measured and presented by Finkelstein (1967) as the percentage of the specific activity in the tissues of rats maintained on a protein-free (PF) diet. Liver, kidney, pancreas and brain CGL activity of rats fed PF diets andthen fasted, declined to between

53 and 80 per cent while only hepatic CGL activity returned to a PF level of activity upon refeeding casein (Finkelstein 1967). Hepatic CGL activity was nearly four- and three-fold greater in rats fed the standard (protein level not reported by author) and high protein (62 per cent casein) diets while neural CGL activity rose to 27 4 and 171 per cent the level of

PF-fed rats, respectively. Conversely, the standard diet depressed renal and pancreatic

CGL activity to 67 and 46 per cent of the PF activity level. Pancreatic CGL activity was also inhibited in rats fed the high protein diet however renal CGL activity remained unchanged from the PF activity levels (Finkelstein 1967). Heinonen (1973) administered

L-methionine intraperitoneally to one-day old rats and also observed a signif,rcant increase in hepatic CGL activity from 1050 to 1530 nmol cysteine/hrlmg protein. Hepatic CGL activity in rats fed a cysteine-supplemented, low methionine diet was only slightly lower than control rats fed a methionine-adequate diet, with orwithout cysteine supplementation (Finkelstein and Mudd 1967)

Similar to CBS, vitamin Bu as a source of PLP is required for the complete activation of CGL and a deficiency results in a reduced rate of transsulphuration (Sturman et al. 1969, Shannon and Demos 1977). Sturman et al. (1969) reported a significant decline in hepatic, renal and pancreatic CGL activity (31, 25,17 nmol cysteine/hr/mg 24 protein, respectively) in vitamin Bu-deficient rats as compared with pair-fed controls (226,

156,234 nmol cysteine/hr/mg protein) while neural CGL activity remained unchanged.

Similar effects were observed by Shannon and Demos (1g77).The dramatic effects of a vitamin Bu deficiency on CGL suggest that PLP is weakly bound to this enrqe and that a constant source of PLP is required to maintain optimum activity levels (Sturman et al.

1e6e).

The effect of dietary cholic acid and cholesterol on CGL activity was investigated by Loriette et al. (1980) based on the known involvement of cholesterol in atherosclerosis

(Grundy 1999) and the suggestion that sulphur amino acids can also be involved in this disease (McCully 1975, as referenced by Loriette et al. 1980). Hepatic CGL activity in rats fed a cholesterol-free, cholic acid-free diet was 33 per cent less than the activity of rats fed the same diet but supplemented with cholic acid. Rats fed a diet containing cholic acid and supplemented with cholesterol also had significantly lower hepatic CGL activity as compared with rats given a cholic acid-based, cholesterol-free diet. Neither diet combination altered the CGL activity in the kidney. The mechanism of these changes is unknown. Chronic, but not acute, ethanol administration to rats caused a significant increase in hepatic CGL activity (Finkelstein and Kyle 1968). Parenteral supplementation of choline and methionine concurrent with ethanol administration did not prevent the alcohol-induced rise in enzyme activity as had been previously suggested by Lieber ærd

DeCarli (1966).

A variety of hormones influence CGL activity although the response is tissue- specifrc (Finkelstein 1967).Increased hepatic, renal and þancreatic CGL activity with 25 estradiol administration, as observed by Finkelstein (1967), contrasted the decline in hepatic CGL activity in rats observed by Shannon and Demos (1977). Estradiol is known to stimulate several vitamin Bu-dependent enzymes (Rose and Braidman I97I, Braidman and Rose I97I, as referenced by Shannon and Demos 1977), potentially causing a redistribution of vitamin stores and limiting the vitamin availability to other PLP- dependent enzymes such as CGL. Thyroxine treatment signif,rcantly reduced hepatic CGL but increased enzyme activity in the kidney of rats (Finketsteinlg6T).Heinonen (1977) demonstrated that regardless of age, hepatic and pancreatic CGL was more active in hypothyroid animals than normothyroid controls. Administration of L-thyroxine to hypothyroid rats normalized CGL activity levels to control levels. As well, treatment of normothyroid animals with Lthyroxine significantly reduced hepatic CGL activity below that of untreated, normothyroid rats. In contrast, renal CGL activity of hypothyroid

animals was signif,rcantly less than that of normothyroid controls and markedly more

active in thl.roxine-treated animals than in control littermates. The effect of hydrocortisone on CGL activity has also been studied in various tissues of rats

(Finkelstein 1967) and the livers of human infants (Zlotkin and Anderson 1982). Hepatic

CGL activity was not significantly affected by hydrocortisone treatment in either species

(Finkelstein 1967 , Zlotkin and Anderson 1982) as were enzyme activities in the kidney, pancreas and brain ofrats (Finkelstein 1967).

The hormones insulin and glucagon are also recognized effectors of CGL activity and the transsulphuration pathway (Finkelstein 1967, Jacobs et al. 1998). Jacobs et al.

( I 998) demonstrated a two-fold increase in hepatic CGL activity in streptozotocin- 26 induced diabetic rats (1350 nmol/hrlmg protein) while insulin treatment maintained

enzyme activity at control levels (600 nmol/hrimg protein). The significant decrease in plasma homocysteine concentrations in untreated diabetic rats (7uM) versus insulin- treated rats (l1uM) combined with no change measured in the activities of remetþlation enzymes further demonstrated the importance of the transsulphuration pathway. Renal

CGL activity remained unchanged among the three treatments (Jacobs et al. 1998).

Insulin's mode of action on CGL remains to be determined, although enhanced mRNA gene expression, as shown for CBS, is a possibility. Jacobs et al. (2001) also observed a

25 per cent increase in hepatic CGL activity in glucagon-treated rats as compared with controls although no change in CGL mRNA was detected. Similar to results by Jacobs et al. (1998), total plasma homocysteine levels were lowerld UV 30 per cent by glucagon treatment despite no significant change in remethylation enzyme activity.

Characterization of the developmental pattem of CGL has received significant affention due to nutritional concerns for cysteine in low-birth-weight human infants.

Human hepatic CGL activity is demonstrated to increase rapidly shortly after birth and stabilize at an adult level within the first few days of life (Sturman et al. 1970, Gaull et al.

1972, Sturmanetal.l976b,Zlotkin and Anderson 1982). Sturman et al. (1970) reported hepatic CGL activity to be unmeasurable in fetuses or premature infants and only barely detectable in full+erm infants less than 24 hours old (9 nmol cysteinelhr/mg protein).

Significantly higher hepatic CGL activity was measured in mature humans (126 nmol cysteine/hrlmg protein) and corresponded with the virfual absence of cystathionine in the liver (Gaull et al. 1972). Zlotkin and Anderson (1982) also demonstrated a significant 27 difference in hepatic CGL activity in preterm, full-term and control infants who died prior to one year of age. A positive correlation between increasing post-natal age and hepatic

CGL activity (r0.6a) was established. Declining plasma cystathionine and increasing plasma cysteine concentrations measured from human neonates with gestation periods

<32 weeks ,33-36weeks artd>37 weeks, supported the developmental pattem of CGL in humans (Viña et al. 1995).

Species differences in the development of CGL activity have been demonstrated although the general pattem of increasing specific activity with age remains consistent

(Gaull et al. l972,Heinonen 1973, Sturman et al. 1976b, Heinonen 1977).In rats, hepatic

CGL activity increased 3-fold during the last few days prior to term, peaked on the first post-natal day and then declined slightly and stabilized. Mature female rats had the same hepatic CGL activity as at birth while enzyme activity in adult male rats was significantly lower (Heinonen 1973). Gaull etal. (1972) reported hepatic CGL activity from fetalrat, mouse and gerbil late in gestation to be similar to their mother. Hepatic CGL was only measurable in fetal guinea pigs within ten days of term. Enzyme activity in the fetal rabbit was undetectable until seven days prior to term while hepatic CGL in newborn rabbits was measurable but well below adult levels. Hepatic CGL activity was absent in the fetal

Rhesus monkey until late gestation but was measurable immediately prior to and at term in fetal baboons (Gaull et al. 1972). In agreement, Sturman et al. (1976b) reported hepatic

CGL to be undetectable in the fetal Rhesus monkey and demonstrated a gradual increase in CGL activity to adult levels at two to th¡ee months of age.

Organ differences in CGL development have been more actively reported than for 28

other enzymes. Neural CGL was virtually undetectable in the rat until birth, after which

activity levels gradually increased to low adult levels (Heinonen 1973). Similar results

were observed in the brain of the developing Rhesus monkey (Sturman et al. T976b).

Heinonen (1977) reported the liver and pancreas to have the highest levels of CGL

activity in the rat, with renal CGL activity being only half that amount. Similarly, renal

CGL activity in the Rhesus monkey was low and relatively stable throughout the entire

period of development with the exception of a significant decrease in activity

immediately following parturition (Sturman et al. I976b).

2.3.3 Methionine Synthase

Methionine synthase has primarily been studied in the liver, kidney and brain of mammals (Finkelstein etal.197I, Gaull et al. 1973b, Sturman etal.I976a, Kalnitsþ et

al. 1982,Doi et al. 1989, VanAerts et al. 1995,Yamada et al. 2000) but is present at

lower levels in a wide range of tissues. Measurable levels of MS activity have been

documented in the pancreas, lung, heart, spleen, adrenals, testes and adipose tissue of rats

(Finkelstein et al. 1971, Doi et al. 1989, Yamada et al. 2000). Rhesus monkeys and humans were also reported to have some catalytic activity in their lungs (Sternowsþ et

al.1976).

Methionine synthase activity is affected by several dietary factors. Methionine supplementation, considered essential for growing cattle, caused a 35 per cent decline in hepatic MS activity when steers were provided with 5 or 10g/d supplemental methionine

(Lambert el aL.2002). The inclusion of dietary methionine plus betaine or choline in 29 broiler chick diets reduced MS activity to within a similar range (Saunderson and

MacKinlay 1990). Finkelstein and Martin (1986) also demonstrated a 39 per cent decrease in MS specific activity within one day of chærge from a 0.3 per cent to a 1.5 per cent methionine diet. In agreement, substitution of 1.0 per cent methionine with 0.25 per cent methionine and 0.8 per cent cysteine resulted in an 87 per cent increase in activity, supporting the importance of MS in methionine conservation (Finkelstein et al. 1988).

Yamada et al, (2000) induced a severe vitamin 8,, deficiency in rats and reported a significant decline in hepatic holoenzyme and total MS activity as compared with control rats, supporting the strong reliance of MS on its methylcobalamin cofactor. Similarly, Doi et al. (1989) observed hepatic and neural MS activity levels in vitamin B,r-deficient rats as being five and ten per cent that of control animals, respectively. Dietary supplementation of methionine did not influence MS activity in either tissue under 8,, deficient conditions (Doi et al. 1989). Dietary folate deficiencies in micropigs had no effect on in vivo MS activity as measured by Halstead et al. (2002). However Townsend et aL. (2004) reported in vitro MS activity to be significantly reduced as measured in human intestinal cell cultures incubated in a folate-deficient medium. Copper deficiency also reduced hepatic MS activity by 21 per cent as compared with control rats although the physiological mechanism of this change is unknown (Tamura et al. T999).

Studies of the effect of hormones on MS activity is limited. Finkelstein et al.

(1971) reported extrahepatic MS to be more responsive to exogenous hormone treatments than hepatic MS. As compared with untreated, control animals, renal MS activity increased i 51 percent with estrogen administration and decreased to 60 per cent with 30 growth hormone injections. In the pancreas, hydrocortisone treatment resulted in a two- fold increase in MS activity. Sex differences have also been noted with MS activity in female rats as 73 percent that observed in males (Finkelstein et al. I97l). As well, Jacobs et al. (2001) reported no change in hepatic MS activity in hyperglucagonemic rats with similar findings found using insulin on streptozotocin-induced rats (Jacobs et al. 1998).

Oxidation of cob(I)alamin, a cofactor of MS and a highly reactive intermediate involved in the transfer of a methyl groups from 5-methyl-TFlF to homocysteine, is also known to inactivate MS. Reactivation is achieved by a reductive methylation using SAM as a methyl donor (Dixon et al. 1996).

A similar pattern of development for MS has been observed in all species studied to date. Methionine synthase activity in developing human infants, rats and monkeys tends to decline with age. The pattern of development of MS in the human has been characterized by Gaull et al. (1973b), Sternowsky et al. (1976) and Kalnitsþ et al.

(1982). Gaull et al. (1973b) reported MS activity to be significantly greater in a second trimester fetal liver (4.7 nrnollhrlmg protein) as compared with MS activity of neonates (6 to 9 days; 1.8 nmol/lu/mg protein) and adult livers (1.3 nmol/hr/mg protein). A similar downward trend in MS specific activity was noted in kidney and brain tissue also (Gaull et al. 1973b). Stemowsþ et al. (1976) also reported a decline in MS specific activity befween fetal, newborn and mature human lung tissue, with statistical significance

(P<0.05) found only between fetal and newbom values. In agreement, Kalnitsþ et al.

(1982) noted a continuous decrease in hepatic MS activity across preterm infants, full- term infants and control subjects greater than one year of age. Again, specific activity 31 differences for MS between full+erm and control subjects were numerically but not statistically different. These findings support MS activity as reaching an adult level in human by the early neonatal period (Gaull et al. 1973b).-

The patterns of development for MS in the rat and monkey are similar to that of the human infant. VanAerts et al. (1995) reported the highest MS activity in embryonic and fetal tissue on day 12 of gestation and in fetal liver on day 14, after which both declined at arate of approximately 30 pmol/mg protein/min per day. The prenatal decline of MS activity in the liver, brain and kidney continued in the post-natal period as observed in rats raised from body weights of less than ten to more than 250 grams

(Finkelstein et al. l97l).In contrast to the developmental pattem of MS in the liver, kidney and brain, the specific activity of MS in rat pancreas increased with age

(Finkelstein et al. l97l). MS activity in the liver, kidney and brain of the Rhesus monkey was measured pre- and post-natally by Sturman et al. (1976a). Specific activity was higher in all tissues during late gestation and immediately following birth as compared to mature, adult tissues. As well, a significant positive correlation between MS activity and gestational age for liver (r :0.62, P < .001) and brain (r: 0.55, P < .001), not previously observed in human tissue, was noted. A similar decline in MS activity in the lungs of the primate has also been observed. (Sternowsþ et aL.1976).

As mentioned, the developmental profiles from birth to post-weaning for CBS,

CGL and MS have not been previously determined in pigs. Characteizing the developmental patterns of CBS and CGL is important for understanding the kinetics of methionine metabolism as they relate to cysteine synthesis. Similar information for MS 32 would also be useful in clarifying potential changes in homocysteine concentrations.

However, the consideration of substrate (methionine), cofactor (folate and vitamin B,r) and endproduct (cysteine) availability to the piglet is required to provide a more complete picture of the potential for endogenous cysteine synthesis post-natally.

2.4 Sutphur Amino Acids and B-Vitamins: Nutrition and Supply for Prenatal and Post-natal Piglets Growth, development and survival of the piglet depends on whether its nutritional demands are suffrciently met. Prenatally, the fetus relies on nutrient transfer mechanisms from the sow's blood through the placenta while post-natally, nutrients are supplied via colostrum and milk. Since the sow is the primary source of the piglet's nutrition, changes in the sow's nutrition influence pre- and post-natal nutrient availability (Schoknecht

1997). Physiological development of the piglet will also influence its ability to absorb and utilize nutrients (Trugo et al. 1985a). If similar to other species, the pre- and post-natal piglets are likely to rely on the sow for its sulphur amino acids, specifically cysteine, for some period of time prior to the complete development of its own endogenous cysteine mechanisms. The availability of B-vitamin cofactors is also essential to ensure full activity of the remethylation and transsulphuration enzymes during the rapid development of the neonate early in life.

2.4.1 Methionine and Cysteine Availability

The transfer of methionine and cysteine from maternal to fetal plasma in humans has been examined (Gaull et aL t973a). An intravenous infusion of L-methionine to the 33 mother resulted in a transfer of methionine from matemal to fetal plasma against a three- fold difference in concentration; infusions of L-leucine and L-ornithine had similar effects. In contrast, the concentration of L-cysteine in fetal plasma was equal to or lower than maternal plasma cysteine following an L-cysteine load. Fetal plasma cysteine concentrations rose gradually while matemal concentrations rapidly declined, suggesting that cysteine transfer is slower than other amino acids and potentially carrier-mediated.

Similar findings have been observed in the guinea pig and pregnant monkey (Young

1971, as referenced by Gaull etal1973a, Sturman etal.1973). Malloy et al. (1983) further suggested GSH to act as a temporary storage form of cysteine in the placenta and a mechanism for maintaining constant cysteine provision to the fetus without the risk of neurotoxic effects associated with excess cysteine (Karlsen et al. 1981). This was based on observations that in pregnant rats injected with cysteine, the measured increase in placental GSH was similar to the difference in cysteine concentration between the placenta and maternal plasma.

Dietary amino acid deficiency and restricted protein intake by a gestating sow will affect pre- and post-natal growth by limiting indispensable amino acids from the developing offspring (Schoknecht et al. 1993). Sows fed a protein-restricted diet (PR;

0.5% protein) throughout gestation, a protein-restricted diet early in pregnancy followed by a balanced diet (PR-B; 13% protein) or a protein-restricted diet late in pregnancy preceded by a balanced diet (B-PR) had low-birth weight litters. Litter daily gain was significantly lower in PR sows while progeny from B-PR sows had slightly greater rates of gain than other groups between birth and 25 wks of age, suggesting a compensatory 34

growth response to maternal dietary protein restriction in late pregnancy. Jones and Stahly

(1999) demonstrated that despite increased matemal protein mobilization, milk nutrient

output by lactating sows fed low-lysine diets was significantly reduced and reflected in the depressed rate of litter weight gain. Progeny of primiparous so\ils were also suggested

as being more vulnerable to protein-restriction due to nutrient competition between fetal

and continued maternal growth (Schoknecht et al. 1993). If endogenous cysteine

mechanisms are underdeveloped at birth in pigs, maternal protein restriction would

further limit their sources of available cysteine.

Sufficient amino acid uptake by the porcine mammary system is essential to maximize milk protein for the neonate (Wu and Knabe 1994, Trottier et al. 1997, Nielson

et a\.2002). Uptake of some amino acids by the mammary gland is different than the

amino acid profile of the milk, suggesting amino acid retention by the mammary tissue beyond that required for milk synthesis. According to Trottier et al. (1997), methionine and cysteine were not significantly retained by the porcine mammary gland. Guan et al.

(2002) demonstrated that milk protein synthesis was associated with mammary transport of indispensable amino acids. Milk protein synthesis, litter growth rate and net uptake of methionine by the mammary gland was reduced in lactating sows fed a lysine-deficient diet. Although the supply of methionine to the mammary gland was not affected by dietary amino acid availability, the partitioning of plasma methionine to the mammary gland, net uptake and bidirectional transport rates of methionine were reduced (Guan et aL 2002). Litter size and day of lactation also had an effect on porcine maÍìmary amino acid uptake Q.{ielsen et aL.2002). Increased uptake of some amino acids, not including 35 methionine, was observed with increased litter size and proposed to be a function of increased marnmary plasma flow rate. A general increase in the manìmary uptake of indispensable amino acids as lactation advances in the sow was also observed Qllielsen et al.2002). Changes in the concentration of protein-bound and free amino acids in sow's colostrum and milk will affect methionine and cystine availability to the piglet at birth (Wu and Knabe 1994). Milk protein concentrations and protein-bound amino acid levels decreased during the f,rrst eight days of lactation. Protein-bound methionine and cystine concentrations were low in sow's colostrum and milk with cystine being the least abundant amino acid (Wu and Knabe 1994). Trace amounts of free methionine were measured until the eighth day of lactation. Unbound cystine was present at low levels in colostrum with increasing concentrations on the third and twenty-ninth day of lactation.

Taurine was the most abundant free amino acid, potentially due to the de novo synthesis of taurine from cysteine in the lactating mafnmary gland.(Wright et al. 1986). An abundant dietary source of taurine would be valuable to the neonatal piglet if endogenous cysteine synthesis mechanisms were not completely developed at birth. Despite changes in the concentrations of free- and protein-bound amino acids in sow's colostrum and milk, Flynn et al. (2000) reported piglet plasma concentrations of cysteine and taurine as unchanged from day one to twenty-one. Methionine and cysteine availability to the piglet may also be influenced by intestinal catabolism of sulphur amino acids (Wu 1998).

Townsend et al. (2004) studied homocysteine metabolism in human intestinal cells in vitro and suggested that remethylation of homocysteine comprised a significant amount of the free methionine pool (19 per cent), despite adequate methionine availability in the 36 culture medium. These authors considered this reasonable, citing the cells' large methionine and cysteine requirements for protein synthesis and maintenance of methylation reactions due to its large cell tumover rates. Stoll et al. (1998) also noted a significant decrease in the portal appearance of methionine (33 umollkg'hr-r) as compared with methionine intake concentration (69 umol/kg'hr-r), a surprising result considering no significant amount of cystine was observed in portal concentrations. These results suggest that in pigs, circulating cystine is primarily the result of endogenous synthesis and that sulphur amino acids have a specific role in intestinal mucosal cells (Stoll et al. 1998), potentially in GSH synthesis. However, recent work by Stoll et al. (1998) may have underestimated the absorption of cysteine being that a significant proportion of cysteine is protein-bound in portal circulation (Prathapasinghe et al.2004). Cysteine is a component of GSH that is actively synthesized to protect the intestinal mucosa against toxic and peroxide damage (Reeds et al. 1997) to which the piglet is highly susceptible (Clark et al.

1990). An increased requirement for intestinal GSH in the neonate may explain the absence of cystine from portal circulation (Stoll et al. 1998) and further emphasize the importance of sufficient endogenous cysteine synthesis.

Methionine and cysteine are available to the fetus, via matemal-fetal transfer, and to the neonatal pig, via the sow's lactation. Provision of both amino acids to the developing pig is of two-fold importance. The production potential of the enzymes involved in endogenous cysteine synthesis in the neonatal pig are unknown, and an alternate source of cysteine to meet early sulphur amino acid requirements, given the potential for underdeveloped transsulphuration enzyme activity, is essential. Furthermore, 37 sufficient methionine availability is necessary to ensure substrate levels are not inhibiting the activity of developing enzymes involved in endogenous cysteine synthesis.

2.4.2 Folate and Vitamin B' Availability

The transfer of vitamins and minerals from the sow to the fetal and neonatal piglet was reviewed by Mahan and Vallet (1997). Micronutrienl transport mechanisms for the conceptus are established in the f,rrst halfofgestation and increasingly used to transport large quantities of nutrients to fetal and mammary tissue. Active transport mechanisms are likely the primary method of transfer for vitamins and minerals across the maternal- fetal blood barrier (Mahan and Vallet 1997). Folate and vitamin B,, status in piglets and

B-vitamin availability from the sow prior to weaning has been widely studied. However, the adequacy of B-vitamin cofactors in neonatal pigs for supporting the complete development of enzymes involved in sulphur amino acid metabolism has not been reviewed.

Folate is required for thymidine synthesis, the rate-limiting step in DNA synthesis

(Shane and Stokstad 1985) and is essential during the early stages ofrapid fetal development (Mahan and Vallet 1997). Folate deficiencies during early gestation in the rat result in resorption, early fetal death and physical abnormalities (O'Coruror et al.

1989). Folic acid supplementation of gestating sow diets is reported to have a positive effect (10 to 15 per cent increase) on litter prolificacy (Matte et al. 1984, Lindemann and

Kornegay 1989, Thaler et al. 1989, Trembley et al. i989, as referenced by Matte et al.

1993). The remethylation pathway also depends on folatè as a methyl donor. Assuming 38 the remethylation and transsulphuration pathways are coordinated by SAM (Selhub and

Miller 1992), insufficient folate during remethylation could inhibit early endogenous cysteine synthesis. As such, transsulphuration enz¡,rne activity development may be suppressed to a level below that normally reached at adulthood. Adequate folate is also essential for the interconversion of serine and glycine via the enzyme serine

'Work hydroxymethyltransferase (SHMT; Nelson and Cox 2000). by Girgis et al. (1997) and Narkewick et al. (2002) suggest increased MTHF availabilitl leads to an increase in

serine production. If a folate deficiency works in opposition of this, not only would

serine's role in protein, DNA, RNA and phospholipid s¡rrthesis (Sturman et al. 1975, Xu

et al. 1991 , as referenced by Narkewick et al. 2002) be jeopardized, but transsulphuration may be limited due to serine's role in providing the carbon skeleton to which the

homocysteine-sulphur is transferred to for cysteine production.

Large amounts of folate are taken up by the uterine tissue and transferred into the

lumen of the uterus during the first 15 days of gestation, increasing folate availability to

the fetus (Mane etal.1996). The concentration of folate.in seven-week old fetuses also

rose significantly, with the level of supplementary folic acid in the diet of gestating sows

(Matte et al. 1993). These studies corroborate the identification of a folate binding protein

in the intrauterine environment of pigs that increases at approximately the time of

maternal recognition of pregnancy, supporting the transfer of folate from the sow to the

fetus (Vallet et al. 1998, Vallet et al. 1999). Barkow et al. (2001) reported that prior to the

ingestion of colostrum, the serum folate concentration in newborn piglets from folate-

supplemented sows was significantly greater than in piglets from unsupplemented so\ils. 39

Serum folate concentrations in piglets from both folate-supplemented and control sows increased three- to four-fold from birth until day two of lactation and declined slightly by weaning at day 28. Natsuhori et al. (1996) demonstrated a similar pattern, noting that the ratio of 5-MTHF to THF was highest at birth (4.1 :1) and.lowest in adult sows (0.3:1).

This pattern corresponds with changes observed in the sow's colostrum and milk folate concentrations. The folate level in the colostrum was three to four times higher than in the milk at the end of lactation for both control and treated sows, with folate-supplemented animals having significantly greater levels than that of the controls (Barkow et al. 2001).

Matte and Girard (1989) and Matte etaI. (1992) observed similar trends. O'Connor et al.

(1989) reported the most significant reduction in milk folates occurred during the first seven days of lactation. In line with these findings, Natsuhori et al. (1996) reported a rapid decline in neonatal total plasma folate from 102nM at day 2 to less than 20nM by

160 days of age. Colostral and serum folate levels, measured in control animals on day

100 of gestation, were similar (101 nmol/L and 104 nmolll, respectively) as compared to folate-supplemented sows in which colostral folate levels were significantly greater than serum folate levels (Barkow et al. 2001). A preferential route for supplemental folate toward the colostrum pool is supported by several authors (Matte and Girard 1989,

O'Connor et al. 1989, Matte et al. l992,Matfe and Girard 1999, Barkow et al. 2001).

Based on the ratios of serum folate concentrations between sows and piglets, the folate status of a litter is influenced more by the post-natal supply of folate from colostrum and milk than from prenatal sources (Matte et al. 1992, Barkow et al. 2001).

Similar to folate, vitamin 8,, is reported to influence fetal development in humans 40

(Jeffery 1999) although studies on its influence in swine reproduction are limited. Early studies by Frederick and Brisson (1961) indicate that vitamin 8,, influences swine reproduction and neonatal survival. Vitamin 8,, supplementation of nulliparous and multiparous sows increased plasma 8,, levels as well as the amount of vitamin 8,, recovered from the uterine homs, measuring 180 to 300 per cent that found in the plasma.

The large transfer of vitamin B,, to the uterus suggests an important requirement for embryo development and uterine metabolism during the first 15 days of gestation. (Guay et al.2002). The receptor sites for the transcobalamin-vitamin 8,, complex in pigs remains to be characterized although a transcobalamin I receptor has been identified in endometrial tissue (Pearson et al. 1998 as referenced by Guay et aL.2002) and in humans,

a transcobalamin II receptor has been identified in placental tissue (Friedman et al. 1977).

Simard et al. (2002) measured the vitamin B,, concentrations of colostrum and

milk as well as the plasma levels of nursing piglets of sows fed graded levels of vitamin

8,,. Colostral 8,, levels increased with the levels of dietary cobalamin. Milk Br2 levels

decreased by 40 per cent on day I but residual effects ofvitamin supplementation were

observed throughout lactation. Day 0 plasma 8,, concentrations of piglets (592.9 +53.3

pglmL) were similar at all treatment levels while day 1 plasma B,r levels were twice as

high and increased with the level of 8,, in the gestation diet (Simard et al. 2002). A

decrease in plasma homocysteine of piglets, concurtent with the increase in gestational

dietary cobalamin, was also noted. House and Fletcher (2003) reported a similar response

in early-weaned pigs fed graded dietary levels of cobalamin, however an effect was only 4t observed with levels up to 40uglkgBtrsupplementation.

A vitamin B,r-binding protein isolated from so*js milk has been identif,red as the primary mechanism for vitamin 8,, absorption in the neonate (Trugo et al. 1985a,b).

According to Trugo et al. (1985b), absorption and retention of radio-labeled cyanocobalamin was consistently greater in suckling versus early-weaned (2d postpartum) piglets. Animals dosed at 3d postpartum had vitamin 8,, retention values of

83 and 63 per cent while at 7d postpartum, vitamin 8,, retention by the suckling pigs remained high (73 per cent) as compared with the early-weaned group (7 per cent; Trugo et al. 1985b). Ford et al. (1975) reported similar results during the first two weeks of life while noting the absence of intrinsic factor in the gut.A comparison of vitamin B'r-binder and intrinsic factor on vitamin 8,, uptake by intestinal microvilli of 7- and 28-day old piglets revealed that the binder was more effective in promoting uptake in the younger pigs and intrinsic factor in the older animals (Trugo et al. 1985a). As such, this transitory mechanism would ensure vitamin 8,, availability to the neonate prior to the production of intrinsic factor and possibly the complete development of endogenous synthesis mechanisms.

2.4.3 Yitamin Bu Availability

Benedikt et al. (1996) reported that sows' milk is a poor source of vitamin Bu and that based on a factorial calculation (Cobum 1994, as referenced by Matte et al. 2001), is less than half the amount required to sustain the growth rate of piglets. As such, the pyridoxine status of the pig at weaning is likely to be low. Moreover, as shown in rats (Lu 42 and Huang 1997) and humans (Bender 1999), increased protein availability post-weaning as compared to sows' milk, may further increase the vitamin Bu needs of the piglet, because of the increased interconversion and oxidation of amino acids, many of which are pyridoxal-5'-phosphate-dependent. Matte et al. (2001) demonstrated that in early-weaned pigs, plasma levels declined by 20 per cent, suggesting that the long-term balance between the dietary supply of pyridoxine and metabolic utilization was negative.

Furthermore the levels of vitamin Bu provided in the basal diet at the two feeding levels

were five times higher than the recommended supply of Bu (National Research Council

199S) for early-weaned pigs and were still insufficient (Matte et al.2001). Since vitamin

Bu-dependent hepatic CBS and CGL activities are knowr,r to be affected during a vitamin

Bu-deficiency (Takeuchi et al. 1 991), investigation of the PLP status of developing pigs

may help explain any changes or quantitative differences in enzyme activity.

Provision of methionine, cysteine and B-vitamins to the pig are essential for

complete methionine metabolism. Incomplete enzyme activity by CBS, CGL and/or MS

will limit homocysteine remethylation and transsulphuration however, the absence of

adequate substrate precursors (methionine for homocysteine) or enzyme cofactors (folate,

8,, and/or Bu) will also prevent enzymes from achieving adult activity levels. Sufficient

nutrient availability is required to ensure complete enzyrÌle development occurs under

non-limiting conditions.

2.5 Summary

Adequate levels of the sulphur amino acid cysteine are required for complete 43 growth and development in the neonatal pig. In mature animals, suffrcient levels of cysteine are assumed to be produced via endogenous synthesis from methionine. The transsulphuration enzymes CBS and CGL are directly responsible for the endogenous production of cysteine while the remethylation enzyme MS may also be indirectly involved in the regulation of the transsulphuration pathway. Numerous studies have been performed, using various species, that provide insight into the characteristics of CBS,

CGL and MS and their developmental patterns in different organs. Characterization of these patterns have not been performed in the neonatal pig. Although developmental patterns observed for these enzymes in other species suggest CBS and CGL may not have reached an adult specific activity level by birth in the piglet and thus are not fully capable of sufficient endogenous cysteine synthesis, this cannot be confirmed without species specific enzyme characteÅzation. Measurement of the change in the specific activity of these transsulphuration and remethylation enzymes in the developing pig is of three-fold importance. Not only would it provide the first static picture of the developmental pattern of CBS, CGL and MS in swine, but it would also demonstrate whether age-related changes exist and have any influence on the pigs' ability to meet its cysteine requirements early in life. Finally, characterization of these enzymes would set the stage for the measurement of flux in metabolites involved in methionine degradation to cysteine, thus further defining physiological changes that ensure adequate cysteine concentrations in the developing pig. 44

Chapter 3 - Hypotheses and Objectives

3.1 Hypotheses

Alternative Hypothesis.' Transsulphuration enermes will exhibit maturational changes in specific activity in pigs from birth until shortly after weaning at 18 days of age.

Null Hypothes¿s.' Transsulphuration enzymes will not exhibit maturational changes in specific activity in maturing piglets from birth until shortly after weaning at i 8 days of age.

3.2 Objectives

The purpose of this experiment is to create a static picture of the developmental patterns of the transsulphuration enzymes in the pig as a means of assessing the capacity of the transsulphuration pathway to support porcine tissue cysteine requirements.

This will be accomplished through the following objectives: i) To characterize the maturation pattems of hepatic and renal CBS and CGL by

measuring changes in specific activity in develop.ing pigs from birth until shortly

after weaning ii) To assess hepatic and renal MS activity in developing pigs from birth until shortly

after weaning as having a potential influence on the transsulphuration pathway via

coordinate regulation. iii) To determine the concentration and normal pattern of change in tissue and plasma

cysteine concentrations in neonatal pigs from birth until shortly after weaning. 45

Chapter 4 - Materials and Methods

4.1 Animals

Eight pregnant Cotswold gilts and sows, ranging in parities from one to six with an average parity of three litters, were randomly selected. from the gestating herd at the

Glenlea Swine Research Unit (University of Manitoba) to provide litters for the experiment. Animals were housed at the facility in a temperature- and humidity- controlled environment. The dams were moved into farrowing crates one week prior to term and cared for in accordance with standard operating procedures for lactating sows.

Piglets for the present trial were obtained from a production system that was consistent with current commercial practices. Experimental litters were bom within ten days of each other and six piglets from each litter were used in the experiment. All piglets surviving beyond day one were processed (teeth trimming, ear notching, tail docking, castration, iron and Excenelr'injections) according to the standard operating procedure for suckling pigs. Piglets suckled normally from their mother until weaning with creep feed provided ad libitum at l4 days of age. V/eaning occurred on day 18. Litters, both those involved in the present trial and other litters from the barn, were conibined into groups of two or three litters (approximately 20 weanlings per pen) in a temperature- and humidity-controlled room. A standard weanling diet was provided ad libitum to all animals and water was available ab libitum from nipple drinkers. Approval for the study was obtained from the

Protocol Management and Review Committee in accordance with the Canadian Council of Animal Care. 46

4.2 Diets

The diets were purchased from Feed-Rite (Ridley, Inc.). Sows received a nurse sow ration crumbles formulated to contain the following:160/o crude protein, 4.5o/o crude fat, 60/o crude fiber (max) , 0.5Yo salt (actual), 0.2o/o sodium (actual), 1.0% calcium

(actual), 0.8% phosphorous (actual), 25mglkg copper (actual), 2Z}mglkgzinc (actual),

13,200iì/kg vitamin A (min), 1500iulkg vitamin D, (min), l95iuikg vitamin E (min),

30ug/kg vitamin B12, i.0 mg/kgvitamin Bu, l.3mglkg folic acid. Weaned pigs received a medicated (22mglkg Lincomycin and 22mglkg Spectinomycin) AP 25135 creep starter

(mini-pellets) formulated to contain the following:20o/o crude protein (min), 8.1% crude fat (min), 2.2o/o crude fiber (max) , I .2yo salt (actual), 0.5o/o sodium (actual), 0.9% calcium

(actual), 0.8% phosphorous (actual), l25mglkg copper (actual), 500mglkg zinc (actual),

20,000iu/g vitamin A (min), 1500iu/kg vitamin D3, 300iu/kg vitamin E (min), 45uglkg vitamin B,r, 11mg/kg vitamin Bu,2mgkgfolic acid. firis ¿iet was also fed as creep feed to the piglets on day 14.

4.3 Study Design

At parturition (day 0), all piglets were weighed prior to receiving colostrum. The piglet randomly selected for termination received a pre-suckle intramuscular injection of the anaesthetic acepromazine (0.4 mglkg lM)-ketamine (15 mg/kg IM). The piglet was immediately removed from the litter with as little stress as possible. Upon reaching a surgical plane of anaesthesia as assessed by the absence of a withdrawal reflex, the piglet was laid posteriorly in a padded V-trough. Blood was collected by cardiac puncture into 47 sodium heparin vacutainers and stored on ice. Euthanyl, at a dosage of 2 mll4.5kg was immediately administered by cardiac puncture and death was confirmed by the absence of a heart beat for five seconds and observed cyanosis around the snout and mouth. The carcass was promptly dissected and the liver and kidney were recovered, rinsed in a chilled, phosphate-buffered saline solution, weighed and frozen immediately in liquid nitrogen (-70"C). Blood was centrifuged for plasma collection and stored, along with the tissues, at -80'C for future analysis.

Sample collection as described above also was performed on days 1, 9, 18

(immediate pre-weaning period), 19 þost-weaning period) arñ26. Samples representative of the immediate pre-weaning period were collected from piglets terminated just prior to weaning while post-weaning samples were collecled24 hours later. Animals were chosen at random but with consideration of a gender balance at each timepoint. All piglets in each litter were weighed on each designated termination day following the termination of the time-point piglet. All terminations began at approximately the same time each day.

4.4 Chemicals

All chemicals, unless otherwise indicated were obtained from either Sigma

Chemical Co. or Fisher Scientific.

4.5 Analyses of Plasma

4,5.1 Folate and Vitamin 8,, 48

Plasma folate and vitamin B,, were measured using a competitive-binding assay.

Plasma folate was measured using a Quantaphase II Folate Radioassay (Bio-Rad, Catalog no. l9i 1044). All reagents and samples were brought to room temperature prior to beginning the assay. Plasma samples were diluted to a 1 :10 ratio using the zero standard.

Two hundred microlitres of each standard, blank, control and diluted sample was added to labeled reaction tubes (12x75mm) with the standards,.blanks and controls measured in duplicate. All tubes received one milliliter of [r25l]-folate and were vortexed. Total count tubes were prepared by adding one milliliter of [r25l]-folate to the reaction tube and then set aside until the end of the assay. The tubes were covered with aluminum foil and placed in a boiling water bath at 100 'C for at least 20 minutes. Upon removal, the tubes were placed in a cold water bath for approximately 10 minutes to reach room temperature. With the exception of the blanks to which 100uL of the blank reagent

(containing polymer beads in a phosphate buffer with 0.1 per cent sodium azide) was added, all tubes received 100uL of the microbead reagent (which contained an affinity- purified porcine intrinsic factor and folate binding protein bound to polymer beads in a phosphate buffer with 0.1 per cent sodium azide). The tubes were vortexed and incubated at room temperature for one hour. The tubes were then centrifuged at 1500 x g for 10 minutes and the supernatant was immediately and simultaneously decanted from all tubes. The tubes were blotted twice on an absorbent pad to remove any supernatant droplets, and then quickly retumed to their upright position. Tubes were counted for one minute using a gamma counter (LKB Wallace 1282 Compugamma). The diluted sample folate concentrations were determined using the concurrently prepared standard curve and 49 the samples mathematical fit to the curve. Correction for sample dilution gave the folate

concentration of the concentrated plasma sample.

Plasma B,rwas measured using a Quantaphase IIP B,, Radioassay (Bio-Rad,

Catalog no. 191 1046). The procedure was identical to that used to measure plasma folate, except that [57Co]-vitamin 8,, was used and plasma sample dilutions were unnecessary.

4.5.2 Amino Acids

Plasma amino acids were separated by cation-exchange chromatography and quantified by post-column derivatization. Plasma amino acids were measured according to the method of Blom and Huijmans (1985). In brief, 250uL of sample was combined with 250 uL of a five per cent SSA solution containing 0.5 mM norleucine as an intemal standard, in a L5mL microcentrifuge tube. The mixture was vortexed and centrifuged at

14,000 x g for l0 minutes. Supernatant was collected and frozen at -20"C. The supernatant was thawed and recentrifuged prior to analysis. A 50 ul aliquot of supernatant was filtered and injected onto a cation-exchange column contained within the Biochrome

20 Amino Acid Analyzer. Amino acids were separated using the gradient elution system previously described (Blom and Huijmans 1985), reacted with ninhydrin and detected by

2-charnelultraviolet detection (440 nm and 570nm). Peak areas were integrated using theEZ Chrom Integration Software Package (Scientific Software,Inc.).

4.5.3 Total Homocysteine and Cysteine 50

Plasma total homocysteine and cysteine were derivatized with 7-fluro-2-oxa-1,3- diazole-4-sulfonic acid (SBDF) and separated using the reverse-phase high pressure liquid chromatography (rpHPLC) method of Araki and Sako (1987), with modifications as suggested by Gilfix et al. (1997). Thiols were quantified by fluorescence detection. To start, a 0.i mM D, L-homocysteine/L-cysteine was prepared in a 0.1 M potassium borate solution, pH 9.5 (see Solution Preparation Method in the Appendices, pp. 130). Using the

0.1 mM solution as 100 per cent concentration, a standard curve with dilutions of 0,25,

50 and 75 per cent was prepared with the 0.1M potassium borate solution as the diluent.

One hundred and f,rfty microlitres of each prepared standard and sample was pipetted into labeled microcentrifuge tubes in duplicate. Next, 20lL of tris(2-carboxyetþl)-phosphine hydrochloride (TCEP) (see Solution Preparation Method in the Appendices, pp. 13 1) was

added and all samples were vortexed. The samples were incubated at room temperatue

for 30 minutes. Following incubation, 125 uL of 0.6 M perchloric acid (see Solution

Preparation Method in the Appendices, pp. 13 1) was added to all tubes to denature

sample proteins and the samples were centrifuged for 10'minutes at 14,000 x g. One

hundred microlitres of standard and sample supernatant was pipetted into new eppendorf

tubes and combined and vortexed with 200uL of 2 M potassium borate buffer (pH 10.5,

see Solution Preparation Method in the Appendices, pp. 130; containing 5 mM

ethylenediaminetetracetic acid (EDTA)) and 100 uL of SBDF (see Solution Preparation

Method in the Appendices, pp. 131). The tubes were incubated in a 60oC water bath for

one hour and then cooled in the refrigerator for 20 minutes. Finally, the standards/samples

were transferred to vials and 10 uL was injected onto the TpHPLC column. An isocratic 51 elution system was used consisting of 0.1M sodium acetate, pH 5, with 2 per cent methanol. The flow rate was 1.0 ml/min and the plasma homocysteine and cysteine concentrations were determined by fluorescence detection (Shimadzu, Mantech) with an excitation of 3 85 nm and an emission of 5 15 nm with a reference to an external standard curve.

4.6 Hepatic Total Homocysteine and Cysteine

Liver total homocysteine and cysteine extracted using the method of Farris and

Reed (1987), derivatizedwith SBDF and separated using the TpHPLC method of Araki and Sako (1987), with modifications as suggested by Gilf,rx et al. (1997).Similar as in plasma, thiols were quantified by fluorescence detection. Liver total homocysteine and cysteine was extracted. First, a 0.1 mM D, L-homocysteine/L-cysteine solution was prepared as described previously. Next, one gram of liver was homogenized in 10 ml of a l0% perchtoric acid solution and centrifirged at 14,000 x g for 30 minutes. Supernatant was decanted into new homogenizing tubes and weighed. To this, 4.1mI plus five drops of 4 M KOH was added to the extract, vortexed and re-weighed. The buffered samples were recentrifuged at 14,000 x g for 20 minutes. Fifty microlitres of standard/sample was combined with 50 ul of a one per cent TCEP solution in microcentrifuge tubes. Beyond this step, the standards/samples were processed and analyzed as previously described for homocysteine and cysteine measurement in plasma'

4.7 Preparation of Tissue Homogenates 52

Liver and kidney homogenates used in the analysis of CBS, CGL and MS were prepared similarly. Special care was taken when weighing kidney samples to ensure a cross-section of tissue was used for each homogenate preparation. One part tissue was added to four parts of 0.05M potassium phosphate, pH 6.9 (see Solution Preparation

Method in the Appendices, pp. 132) and homogenized with a Janke and Kunkel Ultra-

Turrax T25 homogenizer at medium-high speed for approximately 15 to 30 seconds. The homogenizing probe was inspected and embedded tissue was returned to the homogenate.

The mixture \¡/as homogenized again for the same length of time. The suspension was centrifuged at 4"C at 14,000 x g for 30 minutes and the siupematant removed. The supernatant was centrifuged for an additional 10 minutes and stored on ice until assayed.

Homogenate dilutions were prepared using the homogenizing buffer as the diluent.

4.8 Measurement of Tissue Protein

Tissue protein concentration were determined using a Bicinchoninic Acid (BCA) protein Assay Reagent kit (Pierce, Product No. 23225). This is a colorimetric assay based on the reduction of copper by peptide bonds in the protein under alkaline conditions and the reaction between the cupric form of copper and BCA. In brief, dilutions of a bovine serum albumen (BSA) stock standard were performed to make a protein standard curve with a working range of 20 to 2000 ug/ml. Next, 100 uL of the standards and samples were pipetted into labeled test tubes in duplicate. Two milliliters of a Working Reagent, which contained BCA and cupric sulphate, was added to each tube and vortexed. Samples were incubated in a heating block at37"C for 30 minutes and then cooled to room 53 temperature. Absorbencies were read at 562 nrn using a spectrophotometer (Spectronic

1001 Plus, Milton Roy). The blanks contained 0 ug/ml òf gSe and sample protein concentrations were determined against blank-subtracted values of the standard curve.

4.9 Analyses of Tissue Enzyme Activify

4.9.1 Assessment of Protein Concentration and Incubation Length for Optimizing Enzyme Activity

Optimization of assay conditions, specifically for protein concentration and incubation length, for CBS, CGL and MS was performed prior tissue enryme analysis.

CBS and MS activity were measured using endpoint assays and assessed for optimal protein and incubation length. CGL activity were measured using a kinetic assay and assessed for optimal protein only. Enzyme assays were performed as described below using various protein concentrations and incubation lengths. Protein concentrations of 0,

25,50,75 and 100 per cent for each enzyme and incubation lengths of 0, 10,20, 30 and

40 minutes for CBS and 0, 20,40 and 60 minutes for MS reviewed. Results for each enzyme were plotted against protein concentration and incubation length for curve comparisons.

4.9.2 Cystathionine Beta-Synthase

Cystathionine beta-synthase activity was measured using an endpoint assay which involved post-column derivatization with the fluorescent compound o-phthaldialdehyde

(OPA) and separation by rpHPLC. This assay was based on a method by Miller et al. 54

(1994) with slight alterations. Samples were prepared in duplicate with a corresponding blank. Microcentrifuge tubes were prepared to contain the following:20 uL of reaction mixture (0.1 M L-serine, 5 mM EDTA,2.5 mM D,L-propargylglycine (PPG) in 1 M

Trizma buffer, pH 8.4; see Solution Preparation Method in the Appendices, pp. 133), 20 uL of 2.5 mM pyridoxal-5'-phosphate, 100 uL deionized water and 20 uL of L- homocysteine, prepared from L-homocysteine-thiolactone (see Solution Preparation

Method in the Appendices, pp. i34). Cystathionine levels were measured in concentrated liver homogenate and 30 per cent-diluted kidney homogenate using homogenizing buffer.

Next, 20 uL of a25 per cent sulphosalicylic acid (SSA), pH 2 solution was added to all blanks prior to the addition of the same amount of tissue homogenate. Blanks were vortexed and immediately stored on ice. Twenty microlitres of tissue homogenate was added to each duplicate, vortexed and incubated in a heating block at 37oC. After 30 minutes, 20 ul of SSA was added to each tube, the samples vortexed and placed on ice.

Samples were centrifuged at 14,000 x g for 10 minutes. The supernatant was combined in a l: I ratio with 0.4 M sodium borate, pH 9.5 (see Solution Preparation Method in the

Appendices, pp. 134). An extemal standard of 0.5 mM L(+)cystathionine was prepared similarly for analysis. Cystathionine concentrations were determined by TpHPLC

(Shimadzo, Mantech) using a post-column o-phthaldiald.ehyde (OPA) method (Jones and

Gilligan 198; see Solution Preparation Method in the Appendices, pp. 134). Briefly, 100 uL of standard and sample was combined with 100 uL of OPA just prior to the autoinjection of 20 uL of the mixture onto the column. Initial solvent conditions consisted of 90 per cent 0.1 M sodium acetate buffer, pH 7 .2 (containing 9.5 per cent methanol and 55

0.5 per cent tetrahydrofuran (see Solution Preparation Method in the Appendices, pp.

134) and 10 per cent methanol delivered at a rate of I ml/min using a 4.5 X 25 cm C-l8

column (Waters, Fisher Scientific, Neapan, ON). CBS activity was expressed in nanomoles of cystathionine produced per hour per mg protein. .

4.9.3 Cystathionine Gamma-Lyase

Cystathionine gamma-lyase activity was measured using a coupled, kinetic

spectrophotometric assay, based on the method of Stipanuk (1979) with slight alterations.

The CGL catalyzed reaction was coupled with the degradation of CGl-endproduct alpha-

ketobutyrate by lactate dehydrogenase (LDH) The simultaneous oxidation of NADHto

NAD* via LDH and change in absorbency of the sample was considered proportional to

the CGL activity rate. Samples were prepared in duplicate with a corresponding blank. In

a final volume of 1 mL, the reaction mixture contained 0.05 M potassium phosphate

buffer, pH 6.9, 4 mM L-cystathionine, 0.125 mM pyridoxal-5'-phosphate, 0.32 mM

NADH and 1.5 units of LDH (see Solution Preparation Method in the Appendices, pp.

133). Boiling was required to dissolve the cystathionine in the sample-reaction cocktail;

the solution was cooled on ice prior to the addition of the remaining ingredients. Blank

reaction mixfures were prepared similarly but without L-cystathionine. Two millilitres of

the respective cocktails was added to cuvettes for all duplicate samples. Based on the

sample protein concentrations, 100 ul of liver homogenate and 150 ul of kidney

homogenate was added to the cuvettes and stirred gently using a glass rod. Declining

sample absorbance was measured at 340 nm over 20 minutes at a constant3T"C using a 56

Beckman Du-8 spectrophotometer. CGL activity is expressed as nanomoles of cysteine formed per hour per mg of protein.

4.9.4 Methionine Synthase

Methionine synthase activity was measured using an endpoint assay in which [5-

'4C]methionine produced, separated by anion-exchange chromatography and counted in a gamma-counter. The method was based on that of Koblin et al. (1981) but with slight alterations. Radio-labeled [5-'4C]-methyltetrahydrofolate (Amersham) was reconstituted in the original container using 2 mL of deionized water. Once fully dissolved, 200 uL aliquots were pipetted into 10 microcentrifuge tubes, freeze-dried and stored at -80"C.

Aliquots (100 ul) of a reaction mixture (containing 20 uM cyanocobalamin, 58 mM D,L- dithiothreitol, 0.5 mM S-adenosyl-L-methionine, 15 mM D, L-homocysteine, 14 mM B- mercaptoethanol, i mM methyltetrahydrofolate with 0.25 uCi of [5-'oC]- methyltetrahydrofolate and 175 mM phosphate buffer pH 7.5; see Solution Preparation

Method in the Appendices, pp. 132) and the respective homogenate supernatant were combined in microcentrifuge tubes in duplicate. Samples were topped with nitrogen, capped and incubated under foil in a37"C heating block for 15 minutes. Each reaction was stopped with 400 ul ice cold deionized water and samples were placed immediately on ice. The entire reaction mixture was added to the top of a drained AGl-X8 (200-400 mesh) chromatography column (Bio-Rad, Cat no. T3l-6212) and the effluent was collected in a scintillation vial. The column was washed three times with 500 ul deionized water so that the total effluent collected from the column was 2.1 mL. Ten milliliters of 57 scintillation cocktail was added and the samples were counted over five minutes using a liquid scintillation counter (Wallace Winspectral I4l4). Fifty microlitres of radio-labeled cocldail was counted in duplicate as an external standard. Blanks were treated identically to the samples except that the cold water was added prior to the supernatant and blanks were applied directly to the column with no incubation. Columns were washed with 2 ml of water three times between samples and discarded after approximately five sample separations. Sample radioactivity was measured in disintegrations per minute (DPMs) and converted to nanomoles of methionine produced per hour pef mg protein.

4.10 Statistical Analyses

The experiment was analyzed as a randomized complete block with blocking done on individual litters and treatment set as day of age. Statistical analysis was done by

ANOVA using SAS Analyst (SAS Institute Inc., 1999). Differences between means were determined using the protected-LSD method. Differences with an ø level of P<0.05 were considered to be statistically significant.

Log transformations for hepatic MS activity (per mg protein and per g liver), plasma methionine, alanine, glycine, histidine and lysine were necessary since a plot of the residuals versus predicted values revealed heterogeneity in the variability. 58

Chapter 5 - Results

5.1 Growth

All piglets, both terminated pigs and surviving members of the litters, appeared in good health throughout the entire trial. Average litter body weights increased significantly between day I and I 8 (P < .05) and day 19 and 26 (P < .05) as did the weights of the pigs selected for termination at each time point (Figure 5.1).

5.2 Plasma Homocysteine and Cysteine

Plasma homocysteine levels (Figure 5.2) more than doubled during the first two days of life and increased nearly seven-fold by day l8 (P. < .05). Concentrations declined slightly one day post-weaning and then further decreased to levels not significantly different from levels observed at birth.

Plasma cysteine levels (Figure 5.3) rose slowly but steadily during the first two and a half weeks of life with levels approximately doubling in concentration from day 0 to day I 8 (P < .05). Cysteine levels rose sharply between day 18 and day 19 (P < .05) but declined to a pre-weaning level by day 26 (P < .05). Significant block effects also were present for cysteine (P < .05).

5.3 Plasma Folate and Vitamin Btt

Plasma folate concentrations (Table 5.1) more than doubled between day 0 and day 1 (P < .05) and then remained constant between days 9 and26. Folate concentrations rose slightly beyond day 9 but the change was not significant. 59

Figure 5.1: Body Weight Averages of Litters and Terminated Pigs From Birth to Post-Weaning

rd c- þc "o il: (t) l¿Ã

'õ4o) =-o.) br Ou 9e c0 A-r-A a-^9#' o Litter averagea A O Terminalpig average

I I 0 10 15. 20 25 30 Time (day)

Data reported as LS Means +/- Standard Error Treatment Etfect for Litter Body Weight P<.0001 Treatment Effect for Terminated Pig Body Weight P<.0001 a,b,c,d means not followed by the sãme superscript are significantly different by P<.05 All means are composed of at least 7 replicates 60

Figure 5.2: Plasma Homocysteine in Developing Pigs From Birth to Post-Weaning

) o40 'õc

U) 830 E o = oËzo (õ o_

Data reported as LS Means +/- Standard Error Treatment Effect P<.0001 a,b means not followed by the same superscript are signifìcantly different by P<.05 All means are composed of at least 7 replicates 6l

Figure 5.3: Plasma cysteine in Developing Pigs From Birth to post-weaning

280

260 Tc 240 I

220 J q) TO l- 200 'a) P U) 180 Tb T' O (! I 160 E Ø o lct 0- 140 Iö 120 Ta 100 I

80 I 10 15 20 25 30 Time (day)

Data reported as LS Means +/- Standard Error Treatment Effect P<.0001 a,b,c means not followed by the same superscript are significantly different by P <.05 All means are composed of at least 7 replicates Table 5.1: Plasnr¡ Folate and Vitanrin 8,, in Developing Pigs From Birth to Post-Weaning

Day

0 I 9 t8 t9 26 SEM P-Value r'nt P-Value blo'k rb Folate (nM) 42 .9', lt0 .5h 82 .201' u3 ,41', t29.J t32,6h t3.8 .0004 .76

Vitamin 8,, (pM) 275.8^ 616 .gb 182 .8' 165 .z', r70.9" il0 .5" 39.9 <.0001 .90 a,b means within a row not followed by the same superscript are signifìcantly diflerent at P<.05 All means are composed of at least 7 replicates

o\ NJ 63

Plasma 8,, levels (Table 5.1) more than doubled between day 0 and day 1 (P < .05).

Concentrations declined sharply by day 9 (P < .05) and remained stable until the end of the trial.

5.4 Plasma Methionine, Taurine, Serine and Other Amino Acids

Plasma methionine, taurine and serine concentrations are listed in Table 5.2.

Plasma methionine concentrations increased slightly throughout the trial but the change was not statistically significant. Plasma serine concentrations remained constant between day 0 and day 9 (P < .05). Concentrations gradually declined and reached a significantly lower level by day l9 (P < .05) which was maintained until the end of the trial. Plasma taurine levels rose significantly between day 0 and day 9 (P < .05) after which levels remained constant to day 26.

A summary of the remaining amino acids and their plasma concentrations is listed in the Appendices, pp. 137. Except for citrulline and proline, no significant differences in concentration were observed. Block effects were also observed to be significant for isoleucine, proline and valine (P < .05).

5.5 Hepatic Total Homocysteine and Cysteine

Hepatic homocysteine concentrations (Figure 5.4) were not statistically different between day I and 26, however day 0 levels were significantly lower than day 9 and day

19 (P < .05). Hepatic cysteine concentrations (Figure 5.5) were stable from birth until

day 19 with a signif,rcant increase (P < .05) to day 26. Table 5.2: Plasma Methionine, Seiine nnd Taurine (uM) in Developing Pigs Fronr Birth to Post-Weaning

Day

0 I 9 l8 r9 26 SEM P-Vrll¡et"" P-V¿rlt¡el'l"tk

Methionine ll 4 l6 t2 l9 20 4l .06 40

Serine 1r 5ahc I 3 6"b" 192" 166'h l07b' 93' 15.5 0.001 21

Taurine I lgb 177',t', 252" lg5'h 135ub 149"b 26.9 0.02 .66 a,b,c rneans within a row not followed by the same superscript are signifìcantly different at P<.05 All means are composed of at least 7 replicates

Or 5 65

Figure 5.4: Hepatic Homocysteine in Developing Pigs From Birth to Post-Weaning

240

6' zzo o) ïo È zoo E T"¡ I 5 I' 180 .gg I Ø q 160 c) I"o E o i 140 I r' oG' #no r

100 051015202530 Time (day)

Data reported as LS Means +/- Standard Error Treatment Effect P=.005 a,b means not followed by the same superscript are signifìcantly different by P<.05 All means are composed on at least 7 replicates 66

Figure 5.5: Hepatic Cysteine in Developing Pigs From Birth to Post-Weaning

65

60 L o) I o) È55 I E) gso 'a) Ø O T" l" o45 Ò ö o o- q) JI I1, 40 I I t

35 051015202530 Time (day)

Data reported as LS Means +/- Standard Error Treatment Effect P=.0003 a,b means not followed by the same superscript are significantly different by P<.05 All means are composed on at least 7 replicates 67

5.6 Optimization of Enzyme Activity for Enryme Dilution and Incubation Length

Optimal protein concentrations and incubation lengths were established for CBS,

CGL and MS such that their measuïement of enzyme activity occurred along the linear

portion of the curve. These values are listed in the Appendices, pp. 134.

5.7 Cystathionine Beta-SYnthase

Hepatic CBS activity, as measured on a per mg protein basis (Figure 5.6)

increased signif,rcantly from day 0 to day 9 (P < .05). Levels increased slightly beyond day

per gram 9 but changes were not significant. Changes in hepatic CBS, measured on a

pp. liver basis were identical to that measured on a per mg protein basis (see Appendices,

13g). Total hepatic CBS activity (see Appendices, pp. 138) increased nearly eight-fold

(P < between day 0 and day 9 (P < .05) and almost two-fold between day 9 and day 26

.0s).

With the exception of day 0 where kidney CBS was greater than liver CBS, renal

activity measured on a per mg protein basis (Table 5.3) was approximately one-half to

one-third that of hepatic activity. Activity levels fluctuated slightly but remained

statistically unchanged between day 0 and day 26. Changes in renal CBS activity

measured on per gram of kidney basis (see Appendices, pp. 139) were identical to that

measured on a per mg protein basis. Total renal activity (see Appendices, pp' 139)

increased slightly between day 0 and day 26 but the change was not significant. Block

< effects for renal CBS, regardless of how activity was expressed, were significant (P

.05). 68

Figure 5.6: Hepatic CBS Activity in Developing Pigs From Birth to Post-Weaning

140

120 To" 'of- I o I Tu" ä 100 To" o) E r EBo r o E 560 I" .à €40 U) o20õ] f

10 15 20 25 30 Time (day)

Data reported as LS Means +Ê Standard Error Treatment Effect P<.0001 a,b,c means not followed by the same superscript are significantly different by P<.05 All means are composed on at least 7 replicates Table 5.3: Renal CBS, CGL and MS Activities (nmol product/hr/nrg protein) in Develo¡ling Pigs From Birth to Post-Weaning

Day

0 I 9 t8 t9 26 SEM P-Value t't P-Vâlt¡ebl"'k

') CBS 35 .3 32 3 8.3 32.5 39.5 37 .J 6 .0 .93 .00 r ¡b CGL 57.5" 44 .0b 42 J 49.7^t', 49 3^b 46.70b 3 .0 .02 .02

MS 6 .3 5 .0 4.7 5 .4 6.0 5.6 0 ,4 0.08 .0003 a,b means within a row not followed by the same superscript are significantly different at P<.05 All means are composed of at least 7 replicates

o\ \o 70

5.8 Cystathionine Gamma-Lyase

Hepatic CGL activity as measured on a per mg protein basis @igure 5.7) increased more than five-fold between day 0 and day 1 (P < .05). Enzyme activity declined stightly by day 9 and fluctuated at that level with no significant changes to day

26, except at day 18 (P < .05). Changes in hepatic CGL measured on a per gram of liver basis (see Appendices, pp. 140) were identical to that measured on a per mg protein basis.

Total liver CGL activity (see Appendices, pp. 140) increased significantly from day 0 to

day 9 (P < .05) and then remained unchanged to day 26'

With the exception of a small but significant decline on day I (P < .05), renal

CGL measured on a per mg protein basis maintained an activity level only slightly less

than day 26hepatic CGL th¡oughout the trial (Table 5.3). Changes in renal CGL

measured on a per gram of kidney basis (see Appendices, pp.141) were identical to that

measured on a per mg protein basis. Total renal CGL activity (see Appendices, pp. 141)

maintained a steady plateau from day 0 to day 9, rose significantly to day 18 (P < .05) and

then plateaued at the higher level until the end of the trial. Block effects for renal CGL,

regardless of how activity was expressed, were significant (P < .05).

5.9 Methionine Synthase

Hepatic MS activity as measured on a per mg protein basis (Figure 5.8) declined

two-fold by day 1 (P < .05). The decreasing trend in enzyme activity continued

throughout the entire trial. Changes in hepatic MS measured on a per gram of liver basis

(see Appendices, pp. Az)were identical to that measured on a per mg protein basis. 7l

Figure 5.7: Hepatic CGL Activity in Developing Pigs From Birth to Post-Weaning

120 T¡

'oe 100 I o o- To" T¡" o,, 80 ö Tu" ÈE I cltI a I cP60 C I = È40o J o Tu o20 I

10 15 20 25 30 Time (day)

Data reported as LS Means +/- Standard Error Treatment Effect P<.0001 a,b,c means not followed by the same superscript are significantly different by P<.05 All means are composed on at least 7 replicates 72

Figure 5.8: Hepatic MS Activity in Developing Pigs From Birth to Post-Weaning

Ê o 92s o- o) E \-c ¿v o E -915 .=

c) 10

CJ)

5

10 15 Time (day)

Data reported as LS Means +/- Standard Error Data log transformed for normality Treatment Effect P<.0001(based on log transformed data) a,b,c,d means not followed by the same superscript are significantly different by P<.05 All means are composed on at least 7 replicates 73

Total liver MS activity (see Appendices, pp. A2) declined between day 0 and I (P < .05) but returned to birth levels by day 18. Enzyme activity further decreased significantly one day post-weaning (P < .05) and continued to decline until the end of the trial

Renal MS activity, measured on a per mg protein basis (Table 5.3) and per gram kidney basis (see Appendices, pp. 143), was 20 to 25 per cent that of hepatic MS at birth.

Renal MS fluctuated slightly but remained statistically unchanged from day 0 to day 26.

Block effects were also present for renal MS measured on these basis (P < .05). Total kidney MS activity (see Appendices, pp. 1a3) increased slowly but significantly between day 0 and day 26 (P < .05).

5.10 Significance of Block Effects

As summarized in the Appendices, pp. 144, significant differences were found within assigned blocks (ie litters) for CBS, CGL and MS activity in the kidney, average litter body weight and the amino acids arginine, isoleucine, proline and valine. 14

Chapter 6 - Discussion

The purpose of this experiment was to create a static picture of the developmental patterns of the transsulphuration enzymes in the pig as arneans of assessing the enzymes' capacity to support porcine tissue cysteine requirements. Changes in CBS and CGL activity with age in rodents and human infants have been previously reported (Finkelstein

1967, Sturman et al. 1970, Kashiwamata 1971, Gaull et al. I97Z,Yolpe and Laster 1912,

Viña et al. 1995) and provide general developmental patterns suggestive of those that may be observed in pigs. However, species specific information on the developmental profiles of CBS and CGL in pigs is essential for several reasons. First, not only is it important to determine a piglet's capacity for endogenous cysteine synthesis, but also to establish the timeline as to when cysteine may be considered a nutritiqnally dispensable amino acid.

Zlotkinand Anderson (1982) have demonstrated that the kidney and adrenals of human infants contain steady, moderate levels of transsulphuration activity at birth in comparison to the initially low and increasing levels in the liver. They suggested cysteine may in fact be a dispensable amino acid right from birth and that renal and adrenal endogenous cysteine production is sufhcient to meet the neonates' cysteine requirements prior to complete hepatic enzyme activity. However whether this is the case in piglets remains to be determined. As well, increasing commercial popularity for early weaning in pigs makes understanding the developmental profile of CBS and CGL even more necessary to avoid potentially weaning the piglet and providing a cysteine-limited diet before its endogenous cysteine mechanisms are fully functional. Second, by understanding the developmental pattems of CBS and CGL, the observed changes in the 75 concentrations of homocysteine and cysteine may be more fully explained. Finally, a clear picture of the changes in CBS and CGL activity in piglets with age will further demonstrate similarities between pigs and humans and potentially verifu the use of the pig as a suitable model for studying sulphur amino acid nutrition in human infants. Based on results from the present trial, developmental delays al.ong the transsulphuration pathway do not appear to limit the piglets' ability to meet its cysteine requirements by weaning at day 18. Furthermore, this study offered potential explanations for the changes in homocysteine and cysteine concentrations in developing pigs and supports the use of the piglet as a model for human infant nutrition as it relates to sulphur amino acid metabolism.

6.1 Terminal Piglet and Litter Growth Patterns

The pattem of piglet growth observed in the present trial for both the terminal pigs and the surviving litter was similar to that of other trials (Leibbrandt et al. 1975,

Pettigrew et al. 1995) and suggests all piglets received sufficient amounts of milk and solid feed between day 0 and 18 and 19 and 26.The slight, though non-significant decline in body weight in piglets between day 18 and 19 has alsd been previously observed

(Leibbrandt et al. 1975). This pattern is suggested to be caused by the stress associated with the abrupt separation of the piglet from the sow and the temporary cessation of feed and water intake. The new environment and establishment of hierarchy that also takes place at weaning when litters of pigs are mixed together may also contribute to this observation (Henry 2001). The body weights of pigs randomly selected for termination 76 were within the standard deviation of the weights of the remaining litter and considered a good representative sample of the litter population at each time point.

6.2 Delayed Development of Enzyme Activity Along the Transsulphuration Pathway Characterization of CBS and CGL activity has been reported in othe¡ species however this is the first known experiment to provide a static picture of changes in transsulphuration activity in developing pigs. Analyses of the hepatic transsulphuration enzymes on day 0, 1 , 9, I 8, 19 and 26 support the acceptance of the alternative hypothesis: that developmental changes in CBS and CGL activity occtu in pigs between birth and day 26.The general pattern of development of CBS, summarizing the trends observed by other researchers (Finkelstein 1967, Kashiwamata 197I, Gaull et al. 1972,

Volpe and Laster 1972,Yanfuerts et al. 1995) is shown in Figure 6.1 and is similar to the gradual increase in hepatic CBS activity observed in pigs. Hepatic CBS activity in the neonatal pig was approximately one-quarter that measured post-weaning and similar to findings by Sturman et al. (1970) in rats. Premature human infant CBS activity (Gaull et al.1972) also was comparable to day 0 levels in newborn pigs. Enzyme activity levels in post-\.veaned pigs were between 115 and 120 nmollhrimg protein and within the range of adult CBS activity in rats (126 nmol/tr/mg protein; VanAerts et al. 1995), Rhesus monkeys (100-150 nmollhr/mg protein; Sturman etaI.l976b) and human adults (98 nmol/ln/mg protein; Gaull et al. 1972). The development pattern of hepatic CGL in pigs is also similar to the enryme profile observed in other spscies (Gaull et al. 1972,

Heinonen 1973, Sturman et al. 1976b, Zlotkin and Anderson 1982); an overall pattern of 77

Figure 6.1: General Pattern of Development for Hepatic CBS Activity

200 -cË iñ G 150 .EE o O 1oo Bo c/) o 10 o\ 50

lmmaturity Birth Maturity Stage of Life 78

Figure 6.2: General Pattern of Development for Hepatic CGL Activity

300

250 Ë iñ (Ît 200 ,= o 150 o 'õ o o- U) 100 rÞ o s 50

0 lmmaturity Birth Maturity Stage of Life 79 change is summarizedinFigure 6.2.The rapid spike and immediate decline to a stable activity level in pigs is comparable to that observed post-natally in rats (Heinonen L913).

Hepatic CGL activity in the piglet on Day 0 (19 nmollhrlmg protein) is within the range of activity measured in the Rhesus monkey during gestatìon and at parrurition (<20-40 nmolÆn/mg protein; Sturman etal.I976b). However, post-weaning hepatic CGL activity

(between 70 and 80 nmol/hr/mg protein) for pigs appears to be much lower than for rats

(1000-1200 nmol/hr/mg protein; Heinonen 1973) or Rhesus monkeys (125-200 nmol/hr/mg protein; Sturman et al. 1976b). However CGL activity at day 26 in the pig, was compffable to that observed at birth in full-term human infants (approximately

70nmol/tnlmg protein; Gaull et al. 1972, Heinonen and Räihä 7974,Zlotkin and

Anderson 1982).

Renal CBS and CGL activity in developing pigs remained relatively constant from birth to day 26, suggesting that adult-level enzyme activity may have been reached in utero. Renal CBS in pigs is approximately one-third the level of fully-developed hepatic

CBS activity, a ratio comparable to that reported by Finkelstein et al. (1971) in rats. Renal

CBS measured post-weaning (approximately 38 nmollhr/mg protein) appears

significantly lower than in other species with adult CBS activity in rats plateauing between 100 and 120 nmol/tr/mg protein (Finkelstein 1967). Renal CGL fluctuated more than CBS in pigs but its pattern was similar to that of developing monkeys whose hepatic

specific activity did not change significantly over time despite an apparent transient post- natal decline (Sturman et al. 1916b). Pig CGL values were somewhat lower than previously reported activity in rats (Sturman et al. I976b, Heinonen 1977) however data 80 collected by Mudd et al. (1965) and Finkelstein (1967) in full-grown rats was similar to that in pigs on day 26.

6.3 Maintenance of Hepatic Cysteine Concentrations

Hepatic cysteine concentrations did not significantly change from birth to weaning despite an overall increase in CBS and CGL activity with age (Hepatic cysteine level did significantly increase at day 26 and but this will be addressed in section 6.4). Stable hepatic cysteine levels in the present trial were unexpected given that transsulphuration enzymes in the liver of developing pigs were not at stable adult activity levels at birth.

However, this data suggests that the tissue requirements for cysteine in the developing pig are satisfied in the neonate, despite underdeveloped CBS and CGL activity. Malmezat et al. (2000a) reported similar findings in septic rats. Hepatic cystathionine concentrations were 80 per cent higher and CGL activity 17 per cent lower as compared to healthy, pair- fed controls. However, hepatic total cysteine concentrations remained unchanged between the infected and control groups (334 and 335nmol/g liver, respectively). Results by

Triguero et al. (1997) were comparable; PPGtreated rats had near-undetectable hepatic

CGL activity, elevated cystathionine, and constant cysteine concentrati ons. In vitro studies produced similar results as demonstrated by treating rat hepatocytes with increasing concentrations of PPG, a known inhibitor of CGL activity (Rao et al. 1990).

Production of [35S]cysteine from f3sS]methionine was not signif,rcantly affected by the inhibition of cellular CGL, despite enz-1me activity being reduced to 37 per cent of control levels. Viña et al. (1992) observed a similar effect in isolated hepatocytes of rats 81 suffering from surgical stress however, in contrast to prwious studies, hepatic cysteine concentrations increased 200 per cent in stressed versus normocontrol animals. Given inhibited transsulphuration, the stable and elevated hepatic cysteine levels reported suggest mechanisms other than endogenous cysteine synthesis must be involved in ensuring that the liver's requirements for cysteine are adequately met.

As discussed by Triguero et al. (1997), maintenance of intracellular cysteine concentrations of the liver may be influenced by several mechanisms including the transsulphuration pathway, cysteine uptake from plasma, proteolysis and the rate of cysteine utilization for taurine, sulphate and GSH synthesis. Constant hepatic cysteine levels, given limited transsulphuration, suggest the other mechanisms may be compensatory to ensure that the liver requirements are met during conditions that limit endogenous cysteine synthesis, such as sepsis (Malmezat et al. 2000a), surgical stress

(Viña et al. 1992), PPG treatment (Triguero et al. 1997) and incomplete enzyme development.

6.3.1 Cysteine Uptake From Plasma

Viña et al. (1992) demonstrated a significant reduction in circulating levels of

cystine in surgically-stressed rats as compared to control rats that did not undergo surgery

(18 versus 53 uM, respectively); given inhibited hepatic CGL activity, increased hepatic

uptake of cysteine would assist in meeting the liver's cysteine requirements. Increasing

plasma cysteine concentrations observed in developing pigs, from 97 to 250 uM, in the

present trial are in agreement with these results. Underdeveloped transsulphuration 82

erTzyme activity in the neonate, limited hepatic cysteine production and increased hepatic uptake from circulation could result in low plasma cysteine levels early in life. As transsulphuration activity increases, less hepatic uptake may be necessary and thus plasma cysteine concentrations may not be depleted to the same extent. Conversely, although not proposed by Triguero etal. (1997), decreased hepatic cysteine output early in life may also have had an influence on plasma cysteine concentrations and the maintenance of hepatic cysteine levels.

6.3.2 Proteolysis

Based on results by Triguero et al. (1997), protein degradation appears to assist in maintaining hepatic cysteine concentrations within a normal range when endogenous cysteine synthesis is limited. Although typical indicators of protein breakdown, such as plasma and urinary urea levels, were not assessed in developing pigs, plasma concentrations of citrulline, ornithine and arginine, intermediary amino acids of the wea cycle, were measured. Plasma citrulline fluctuated significantly between birth and day 26 in developing pigs with post-weaning levels (40-55 uM) only slightly less than those reported by Flynn et al. (2000) on day 21 (60 uM). However, Flynn et al. (2000) reported plasma citrulline levels in neonatal pigs to be significantly higher than those in the present trial and to decline with age. There were no significant changes in plasma arginine or ornithine concentrations in the present trial although a transient decrease in ornithine levels was observed, with near-significant differences in concentration between day 0 (185 uM) and day 26 (98 uM). Flynn et al. (2000) also reported significantly higher 83 plasma arginine levels in suckling pigs between day I and2I in comparison to present values as well as a pattern of decline, not observed here. Difference in lactation diets and analyical methods may account for some of the quantita;ive differences in plasma amino acids. The declining concentrations of ornithine observed in this trial are similar to the pattems observed by Flynn et al. (2000). Following from this, as the transsulphuration enzymes develop and their capacity to synthesize cysteine increases, less protein breakdown to maintain hepatic cysteine levels may be required and thus less amino acids funneled towards the urea cycle. Results by Triguero et al. (1997) are in agreement.

Hepatic levels of ornithine, citrulline and arginine increased when rats were treated with

PPG, inhibiting complete transsulphuration and endogenous cysteine synthesis. Unlike the majority of the other hepatic amino acids which increased in PPG-treated rats, alanine concentrations declined. Propargylglycine can inhibit alanine aminotransferase (Burnett et al. 1980), preventing the conversion of pymvate to alanine and its transport, via the blood, to the kidney, for amino group disposal. Although plasma alanine concentrations were not measured by Triguero et al. (1997), PPG treatment might have resulted in increased circulating alanine levels. In contrast, CBS and CGL activity development, an increased capacity to synthesize endogenous cysteine and the potential simultaneous decrease in protein degradation agrees with the observed downward trend in plasma alanine concentrations in aging pigs in the present trial. Triguero et al. (1997) further supported protein breakdown during inhibited endogenous cysteine synthesis by demonstrating a significant increase in urinary urea excretion (7mM) and plasma urea concentrations (16mM) in PPG-treated versus control rats (5mM and 7mM respectively). 84

6.3.3 Cysteine Utilization For Taurine Synthesis

The catabolism and conversion of cysteine to taurine will influence hepatic cysteine concentrations. The liver is the primary site of taurine synthesis, removing cysteine from the plasma and releasing taurine and sulphates via cysteine catabolism

(Garcia and Stipanuk 1992). In developing pigs, plasma taurine concentrations increase significantly between birth and day 9, from I 18 to 252 uM respectively. Taurine concentrations reported by Flynn et al. (2000) in pigs did not change between birth (130 uM) and day 21(127 uM) and were comparable to levels measured on day 0 in the present trial. Cho et al. (1984) observed increased plasma taurine by feeding rats cysteine concentrations above requirement levels. Taurine is considered the most abundant amino acid in so\¡r's' milk with concentrations increasing significantly between day 3 and 8 of lactation (Wu and Knabe 1994). This may partly contribute to the significant increase in plasma taurine measured post-natally in the present trial. Increased protein degradation, as a means of ensuring hepatic cysteine requirements are met given underdeveloped transsulphuration enryme activity, may also contribute to increased circulating taurine levels. Stipanuk et al. (1992) reported that cysteine availability to rat hepatocytes is a major determinant in the regulation of cysteine metabolism and that GSH formation was favored during low cysteine availability while taurine was favored during high availability. However, since taurine is not a component of protein, proteolysis would likely have an indirect effect on taurine via the release of cysteine. Breuillé et al. (1996) demonstrated that in septic rats, whole body content of cysteine is significantly increased as compiled to healthy pairfed controls. lncreased whole body protein catabolism, that 85 occurs during infection and trauma (Wolfe et al. 1989, Mansoor et al.1996, as referenced by Mercier et aI. 2002) and appears to support the maintenance of hepatic cysteine levels, may in fact be more than needed to maintain cysteine requirements and result in increased cysteine catabolism to taurine. Malmezat et al. (1998) reported that cysteine catabolism to sulphate was lower in septic rats while cysteine utilization for taurine in the liver was 81 per cent greater than in healthy pairfed controls. Furthermore Mercier et al. (2002) also demonstrated increased plasma taurine in dextran sulphate sodium-induced chronic inflammation in rats as compared with controls (230 and 209 uM). A similar effect may occur given underdeveloped transsulphuration enzqe activity and limited ability for endogenous cysteine production. The transient, non-significant decline in plasma taurine beginning on day 9 corresponds with the f,rrst observed day that both CBS and CGL are at

presumed a steady activity level. As transsulphuration enzyme activity stabilized at a adult level, compensatory mechanisms that may have ensured sufficient hepatic cysteine levels may be reduced, thereby decreasing total cysteine availability. Reduced cysteine availability may limit cysteine catabolism for taurine synthesis and divert cysteine towards protein and GSH production that take priority when cysteine is limited and/or sulphur amino acid intake is reduced (Stipanuk et al. 1992, Fukagawa et al' 1996).

Shoveller et al. (2003) reported decreasing plasma taurine levels with increasing methionine and excess cysteine intake in enterally and parenterally-fed pigs' As methionine availability increased up to its requirement level, incorporation of cysteine into protein increased, thereby decreasing the amount of cysteine shunted towards taurine

synthesis until methionine requirement levels were reached (Shoveller et al. 2003). 86

Although protein intake would presumably increase in the post-weaning diet, increasing protein requirements by the developing pig may be met by dietary and endogenous cysteine combined, thus maintaining taurine levels at a constant level to day 26. Hepatic taurine, released into circulation, may also be taken up by select tissues, such as the lungs

(Malmezat et al. 1998) and lymphocytes (Gaull 1986) for antioxidation purposes, or by the kidney as a means of removing excess methionine and cysteine from circulation

(Garcia and Stipanuk 1992). Daniels and Stipanuk (1982) demonstrated excess taurine was taken up by the kidney for excretion in rats fed excess dietary cysteine. However, renal taurine concentrations and urinary taurine levels were not measured in the present trial.

6.3.4 Speculation on the Involvement of Sulphate and GSH on Cysteine Levels

Although not measured in the present trial, changes in sulphate production and

GSH synthesis in response to alterations in cysteine availability would also be likely to influence hepatic cysteine flux. In septic animals, previously demonstrated as having inhibited transsulphuration activity yet constant hepatic cysteine concentrations

(Malmezat 2000a), Malmezat et al. (1998) observed significantly lower cysteine breakdown to sulphate in septic rats, limiting cysteine catabolism in support of meeting cysteine requirements. As well, these authors reported that part of the observed 38 per cent increase in plasma cysteine flux in septic rats resulted from an increase in the rate of

GSH degradation to cysteine in pairfed (33%) versus control (21%) animals, again another potential compensatory action against iimited endogenous cysteine synthesis 87

(Malmezat et al. 1998). Whether these homeostatic changes in support and maintenance of hepatic cysteine concentrations are similar in neonatal pigs with underdeveloped transsulphuration activity and endogenous cysteine synthesis, is unknown. Of greater concern however, may be whether the level of hepatic cysteine sustained between birth and day l9 in the developing pig is sufficient for adequate GSH synthesis' Substrate availability is proposed as the primary rate determinant of hepatic GSH with dietary conditions having a prominent effect on GSH production (Tateshi etal.1974)' Dietary cysteine availability to the piglet is limited (Wu and Knabe 1994) but the observed, constant level of hepatic cysteine implies that the developing pigs' hepatic requirements

proportion of are being satisfied. At cysteine intake levels near requirement, a large

of these available cysteine is used for GSH and protein synthesis, suggesting production

products to take priority over cysteine catabolism to taurine or sulphate (Stipanuk 2004)'

in part As mentioned taurine levels increased in the neonatal pig and although this may be

cysteine due to a high dietary taurine intake, increased cysteine availability facilitates

catabolism to taurine rather than conservation of GSH. (Stipanuk 2004)' Glutamate-

regulated by cysteine ligase catalyzesthe rate-limiting step in GSH synthesis and is highly

in feedback inhibition by GSH (Stipanuk 2004); increasing taurine concentrations

enzyme, thus developing pigs may suggest that GSH levels are adequate to suppress this

allowing increased cysteine catabolism to taurine. Further study is required however to

verify homeostatic changes in the movement of cysteine along these pathways in

developing pigs. 88

6.4 Increased Hepatic Cysteine Concentrations Post-Weaning

The only significant change observed in hepatic òysteine concentrations throughout the trial was post-weaning between day 19 and26. Weaning is considered a highly traumatic event to the developing neonate and, as a proposed result of cortisol released during this period, is recognizedto induced specific activity in some enzymes such as ornithine decarboxylase (Wu et al. 2000a), phosphate-dependent glutaminase and pynoline-5-carboxylate synthase (Wu et al. 2000b). However, since both CBS and CGL activity appeff to stabilize prior to weaning, increased transsulphuration flux to cysteine via a stress-induced increase in enzyme activity is unlikely. Continued hepatic uptake and/or proteolysis combined with endogenous cysteine synthesis is another possible explanation. However, despite extensive work demonstrating the effect of sepsis

(Malmezat et al. 2000a) and surgery ffiña ef al. 1992) on endogenous cysteine production, the long-term effects, such as to whether CGI activity, hepatic/plasma cysteine levels and/or plasma/urine urea concentrations retum to normal levels following the inhibition of transsulphuration is unknown. Release of cysteine from GSH post- weaning could influence hepatic cysteine at day 26 as it is known to contribute to plasma

cysteine flux (Malmezat et al. 2000a). Cho et al. (1984) demonstrated hepatic GSH to

serve as a reservoir of cysteine during cysteine depletion in rats. In agreement, Tateshi et

al. (1977) proposed GSH to be a better form of storage than free cysteine since GSH is

less auto-oxidizable than cysteine, its oxidized and reduced forms are more soluble than

cysteine/cystine and, nearly all tissues contain a mechanism for reducing oxidized GSH.

A post-weaning decline in cysteine catabolism is another possibility and would coincide 89 with the observed decline in plasma taurine concentrations in the developing pigs.

However, other factors may be involved in decreasing plisma taurine and results from the present trial cannot provide direct evidence for this. The proposed coordinate regulation via SAM between the remethylation and transsulphuration pathways may also enable the

developmental changes in MS activity to influence cysteine concentrations. Hepatic MS

activity is at the lowest point when hepatic cysteine levels rise. Reduced MS activity may

limit the rate of serine conversion to glycine and thus make more serine available for CBS

and cysteine production. More than likely, one or more of these possibilities and the

elevated CBS and CGL activity levels involved in endogenous cysteine synthesis would

have an additive effect on cysteine concentrations in the liver.

6.5 Development of MS in Neonatal Pigs

Similar to CBS and CGL, this is the first known characterization of the

developmental pattern of MS in neonatal pigs. Declining activity with age by hepatic MS

in pigs was similar to patterns observed in other species including rats, humans and

monkeys (Finkelstein etal.197I, Gaull etal.l973b, Sturman etaI.I976a, Sternowsþ et

al. 1976, Kalnitsky et al. 1982, VanAerts et al. 1995). A general sunmary of the patterns

of MS activity reported by these authors is shown in Figure 6.3. Hepatic MS activity in

pigs measured on day 0 was significantly greater than the highest hepatic activity levels

measured at birth in other species. In the neonatal pig, hepatic MS activity was 31

nmol/hr/mg protein as compared to the highest activity, measured in the fetal liver of

human infants (5 nmol/Ìu/mg protein, Gaull et al. I973b) and neonatal monkeys (7 90

Figure 6.3: General Pattern of Development Observed for Hepatic MS Activity

E 200 Ë i! (õ

150 .EE o o loo o-B U) r{- o -o o\ 50

Birth Stage of Life. 9l nmol/hr/mg protein, Sturman et al. 7976a), both being substantially lower. The hepatic

MS activity in pigs decreased significantly throughout the trial with levels reached on day

26 (4 nmollhrlmgprotein) being comparable to adult-level MS activity in rats (4 nmol/lu/mg protein, Finkelstein et al. l97l; 1 nmol/hrlmg protein, Gaull et al. 1973b) and monkeys (2 nmolÆr/mg protein, Sturman et al. 1976a). Renal MS activity of developing pigs was not significantly different from birth to post-weaning, ranging from 5 to 6

nmol/hrlmg protein. Sturman eT al. (1976a) also reported renal activity in neonatal and

adult Rhesus monkeys to be unchanged and similar to renal MS activity in the present

trial. Hepatic and renal MS activity in pigs on day 26 were comparable, a pattern similar

to that observed in humans (Gaull et al. 1973b). On the contrary, Finkelstein et al. (1971)

reported renal MS activity in adult rats to be approximately four-fold greater than hepatic

MS. Similarly, Sturman et al. (1976a) demonstrated renal MS as two-fold greater than

hepatic MS in Rhesus monkeys. No substantial increases in renal MS activity were

observed beyond birth for any species, suggesting renal MS in pigs is at an adult level and

the variation in reported liver-to-kidney MS activity ratios is due to species differences.

6.6 Potential Factors Influencing Homocysteine Flux Along the Remethylation Pathway

The methionine-metabolite homocysteine resides at a unique junction in

methionine metabolism and has several metabolic fates. Homocysteine produced via

SAM synthetase and SAH hydrolase can be either degraded by SAH hydrolase, BHMT,

CBS or MS with all four pathways simultaneously influencing homocysteine flux. The 92 development profiles of SAH hydrolase and BHMT have been previously reported with

SAH hydrolase declining up until birth in rats (VanAerts et al. 1995) and BHMT reaching adult levels immediately pre- or post-parlurition in humans and monkeys (Gaull et al.

1973b, Sturman et al. 1976a). Developmental pattems of these enzymes have not been previously demonstrated in pigs. Although these enzymes would be likely to affect homocysteine concentrations, characferization of their changes with age and their effects

on homocysteine were beyond the scope of the present trial. However, characterization of

CBS and MS developmental profiles in growing pigs in the present trial was established

not only because they can offer some explanation to the changes observed in

homocysteine, but also because of their combined potential to influence cysteine

synthesis (Finkelstein et al. 1971, Finkelstein and Martin 1984). While CBS is directly

involved in the transsulphuration pathway leading to cysteine, MS is proposed to be

involved in a coordinate regulation between the remethylation and transsulphuration

pathways via SAM (Selhub and Miller 1992). According to Finkelstein and Martin (1984)

remethylation and transsulphuration enzymes can be considered as competing for

available homocysteine with a reduction of activity along one pathway enabling more

effective use of homocysteine along the other. As such, changes in the activity of these

enzymes associated with aging ultimately may influence when the transsulphuration

pathway is fully capable of endogenous cysteine synthesis.

6.6.1 Enzyme Characteristics and Factors Potentially Influencing Homocysteine Concentrations 93

Hepatic homocysteine levels of developing pigs in the present trial fluctuated between approximately 130 to 200 nmol/g liver with no clear trends. These fluctuations are unexplainable based on observations in the present trial however a range of homocysteine levels may be normal. In humans, normal plasma homocysteine concentrations range between 5 and i5uM (House et al. 1999). Statistically, hepatic homocysteine concentrations in the developing pigs were stable between birth and day 26.

Hepatic homocysteine levels in neonatal pigs have not been previously determined.

Although measurement of homocysteine flux and all factors involved in maintaining stable homocysteine levels were beyond the scope of the-present trial, observed changes in MS and CBS combined with known enzyme characteristics offer potential insight into homocysteine metabolism in developing pigs. Several authors have demonstrated that MS responds to ensure sufficient methionine concentrations are available to the tissue

(Finkelstein et al. 1971, Finkelstein and Martin 1984, Finkelstein and Martin 1986).

Finkelstein and Martin (1986) reported an increase in hepatic MS, from 4 to 8.8 nmolimin/g liver, in rats fed decreasing levels of methionine. As well Finkelstein et al.

(1971) demonstrated MS activity was significantly lower in rats fed a high protein diet as compared with those fed a low protein diet followed by fasting. The Km value for MS has been reported to be approximately 0.06 mM while the Km value for CBS is reported in the range I to 25 mM (Finkelstein et al. 1990). These suggest that MS would likely and effectively compete with CBS over the limited free homocysteine available for methionine resynthesis. In the present trial hepatic MS activity is comparable to hepatic

CBS in pigs on day 0, while CBS activity is significantly greater than MS on day 1 with 94 the gap between the activity levels of the two enzymes widening with age. The relatively stable hepatic homocysteine, given extreme changes in MS and CBS, allows for the possibility that a decrease in the activity of MS with a low Km for homocysteine may be counterbalanced by the increasing activity of CBS with a higher Km for homocysteine.

Although the development of BHMT was not established in pigs, the Km value of BHMT is proposed to be approximately 0.002 mM (Finkelstein et aI.1972), suggesting the

ervyme would be operating at maximal capacity at given homocysteine concentrations.

Consideration of the full effects of enzymes involved in homocysteine degradation and their contribution to stable hepatic homocysteine levels cannot be completed however without characterizrng the activity of BHMT.

In line with evidence suggesting MS as being responsive to methionine

conservation and less affected by homocysteine concentrations, potentially limited

methionine availability to the neonatal pig would suppo{ high MS activity at birth.

Proteolysis, theorized as a primary contributor to stable hepatic cysteine concentrations in

the neonate (Triguero et al. 1997), would likely release some methionine to the liver;

however, the concentration of methionine in mammalian liver and muscle is low (170

umol/g protein) in comparison with other amino acids (Matthews 1999). According to

Wu and Knabe (1994), sow's colostrum and milk contain only trace amounts of free

methionine and the concentration of protein-bound methionine in the milk decreases

throughout the lactation period (Wu and Knabe 1994).In addition, metabolism of

ingested methionine by the mucosa of the small intestine has been demonstrated. Stoll et

al. (1998) reported that approximately 50 per cent of dietary methionine was lost in the 95 first pass by the portal-drain viscera in the intestine of milk protein-fed piglets while

Mitchell and Benevenga (1978) observed methionine transamination in small intestinal homogenates. Shoveller et al. (2003) also demonstrated ittut in the presence of excess cysteine, the parenteral requirement for methionine is approximately 70 per cent of the enteral requirement, suggesting the gut utilizes approximately 30 per cent of the sulphur amino acids provided. Taken together, high hepatic MS activity at birth may be in part an adaptive response to ensure hepatic methionine requirements are met early in life.

Measurement of hepatic methionine flux in developing pigs would better define the contributions of MS to de novo methionine synthesis and hepatic methionine concentrations.

The role of SAM in coordinating the remethylation and transsulphuration pathways (Selhub and Miller 1992) is yet another means by which homocysteine concentrations may be influenced. As a well-recognized allosteric inhibitor of MTHFR

(Kutzback and Stokstad 1971, Jencks and Matthews 1987) and an activator of CBS

(Finkelstein et al. 1975), SAM has been described as a "switch," acting to direct homocysteine either towards or a\ilay from methionine, depending on cellular requirements. Although the development profile of SAM was not the focus of the present trial, SAM's involvement in both remethylation (via MS) and transsulphuration (via

CBS) and their influence on homocysteine levels are valid considerations. In the present trial, SAM could influence homocysteine via several possible means; declining de novo methionine synthesis in aging pigs may lead to reduced SAM concentrations and CBS stimulation, thereby prolonging CBS development and limiting homocysteine removal is 96 one such possible scenario. Altematively, adequate methionine availability from proteolysis and the diet may cause SAM to accumulate, leading to suppressed MS and stimulated CBS similar to the pattem observed in the present trial, thereby shunting homocysteine along the transsulphuration pathway to cysteine.

6.6.2 Folate and Vitamin 8,, Availability For Complete MS Activity

Plasma folate and vitamin B,, were measured in developing pigs to test that these vitamins, both essential to the complete operation of MS,were sufficiently available to support full MS activity and not responsible for the decline in hepatic MS activity with age. Neither a folate nor a vitamin 8,, deficiency are likely to explain the rapid decline in

MS activity in the present trial. Plasma folate concentrations in the developing pig were lowest at birth (a3 nM) but rose to and were maintained at a stable plateau from the f,rrst post-natal day to post-weaning with levels ranging from 80 to 130 nM. A similar pattem of low circulating folate concentrations prior to the first intake of colostrum by neonatal pigs was supported by Natsuhori et al. (1996) and Barkow et al. (2001). ln contrast, Matte and Girard (1989) demonstrated that plasma folate concentrations in suckling pigs continued to increase well-beyond colostrum intake, rising steadily until day 16 of life.

Barkow et al. (2001) reported a similar though slightly higher range of plateau folate concentrations (140-175 nM) between day 2 and 28 of life as compared with the present trial. Although no stable folate level appeared to be reached in developing pigs according to Matte and Girard (1989), plasma folate concentrations between days 9 and 16 rose from 158 to 193 nM, respectively, and were comparable to plateau levels previously observed (Barkow et al. 2001). In contrast, O'Connor et al. (1989) reported plasma folate 97 valuesof 7-andZl-day oldpigletstobeapproximately3T andT2per centlessthanthat measured here. Differences in diet formulation and analytical methods may account for some of these differences. Although no significant changes in circulating folate concentrations in suckling pigs were reported by Barkow et al. (2001) beyond day 2, a gradual decline with age is evident and in agreement with the trend observed by

Natsuhori et al. (1996). These patterns correspond with and are likely in response to the decrease in milk folate concentrations that occur as lactation progresses in sows (Matte and Girard 1989, O'Connor et al. 1989, Natsuhori etal.1996, Barkow et al. 2001).

Although not statistically significant, the slight decrease in plasma folate of the present trial between day I and 9 may be a function of the rapid decline in milk folates that occurs during the first seven days of lactation (O'Connor et al. 1989). No further decreases in circulating folate were observed in this trial. Introduction of vitamin- supplemented creep feed and post-weaning diets to piglets was not performed in previous studies and provision of these diets to pre- and post-weaned pigs in the present study may have prevented this effect.

Plasma cobalamin in suckling pigs was constant throughout the entire trial period and with the exception of a significant two-fold increase in concentration between birth

(276 pld) and day 1 (617 pM), plasma levels ranged from 110 to 183 pM until day 26.In comparison, Simard et al. (2002) reported plasma cobalamin, measured in pigs on day 0 from sows fed graded levels of vitamin B,r, were nearly two-fold greater than levels measured in the present trial. Regardless of the sows' vitamin 8,, intake, plasma cobalamin concentrations of all piglets doubled on day 1, a pattem that is in consistent 98 with our observations. Quantitative differences may be due to methods of measuring plasma cobalamin concentrations. Circulating cobalamin levels measured in developing pigs in the present trial were greater than (on day 0 and 1) and comparable to (on day 9 to

26) those measured in early-weaned pigs between 17 and 32 days of age, considered to be consuming optimal levels of vitamin 8,, (House and Fletcher 2003). When graded levels of cobalamin were fed to early-weaned pigs, House and Fletcher (2003) reported that diefary supplementation greater than 35 uglkg did not influence plasma levels and circulating cobalamin was maintained between approximately 160 and 180 pM. These authors defined optimal cobalamin status as the level of dietary cobalamin needed to maximize plasma cobalamin and minimize plasma homocysteine (House and Fletcher

2003). This was achieved by supplementing the diet with 35 ug/kg cobalamin, a level comparable to that used in our creep feed and post-weaning diets.

The MS reaction is the only metabolic step known in which both vitamin 8,, and folate are required for complete enzyme activity (Sauer and Wilmanns 1,977). A vitamin

8,, def,rciency is known to inhibit the MS reaction and has been demonstrated by various authors (Sauer and Wilmawts t977, Doi et al. 1989, Yamada et al. 2000, Halstead et al.

2002). Cobalamin deficiency has also been proposed as the primary metabolic fault of inhibited MS activity and responsible for the functional folate deficiency that occurs as a result of trapping an increased proportion of folate as 5-MTHF and preventing its conversion to THF, the preferred storage form of folate in tissues (Sauer and Wilmarurs

1977, Shane and Stokstad 1985). Sauer and Wilmanns (1977) demonstrated MS in bone manow cells of humans to be significantly lower in cells subject to a cobalamin 99 deficiency as compared with control cells. Similarly, Doi et al. (1989) reported hepatic

MS activity in vitamin B,r-deficient rats to decrease to approximately five per cent of the activity of B,r-supplemented controls; more recent results by Yamada et al. (2000), were in agreement with these findings. Dietary folate deficiency and its effect on MS activity has received less attention than cobalamin however Shane and Stokstad (1985) suggested dietary folate deficiency, as compared to folate deficiency induced by inadequate levels of cobalamin, is likely just as important a physiological influence on MS activity. Although

Halstead et al. (2002) demonstrated that hepatic MS activity in micropigs fed a folate- deficient diet was not significantly different from animals fed a folate-sufficient diet,

Townsend et al. (2004) reported remethylation via MS was reduced by one-third in folate- restricted human intestinal cell cultures. In agreement, Finkelstein et al. (1984) further

demonstrated that MS activity was not measurable in rat hepatocytes incubated in a

medium without 5-MTHF.

6.6.3 Folate Availability for Serine Production

Folate is well-recognized as a primary cofactor required in the interconversion

reaction catalyzedby SHMT between glycine and serine. 5,10-MTHF availability has

been demonstrated as influential in serine production with Narkewicz et aI. (2002)

demonstrating that in fetal ovine hepatocytes, provision of excess 5,1O-MTHF resulted in

a direct reduction in serine utilization and a net increase in serine production as compared

with cells incubated with minimal 5-MTHF. In the present trial the pattem of change and

quantitative levels of plasma folate in developing pigs was comparable to that previously 100 observed (Matte and Girard 1989, Barkow et al. 2001), suggesting levels to be typical of

suckling pigs. Plasma serine concentrations did not significantly change between birth and day 19 however peak levels of t92 uM were reached on day 9, a level slightly lower than that observed from birth to day 21 of suckling piglets by Flynn et al. (2000). A

significant decline in serine concentration was observed however from day 9 to day 26

(93 uM). Folate concentrations did not change significantly during this time period and if anything, increased slightly, suggesting folate deficiency may not be the primary factor in this response. Furthermore, Cuskelly et al. (2001) reported serine synthesis to be increased during folate deficiency. These authors proposed that under conditions of a reduced rate of methyl group generation, 5,1O-MTHF is directed towards serine synthesis, potentially as a means of conserving one-carbon units for subsequent use in cellular metabolism (Cuskelly et al. 2001). Finkelstein and Martin (1986) demonstrated a

significant decrease in hepatic serine concentrations and.a significant increase in CBS

activity while the removal of 5-MTHF from an incubation medium of rat hepatocytes resulted in a34 per cent decrease in CBS activity. The decrease in circulating serine in the present trial is concurrent with hepatic CBS increasing to a presumed stable adult

activity level. Although not measured in the present study, proteolysis proposed as

influential to cysteine concentrations, may have increased methionine availability and movement of homocysteine along the transsulphuration pathway. Whether developmental

declines in MS activity would influence serine synthesis is unknown and further research

as to this effect is necessary. 101

6.7 Potential Explanations for the Significant Block Effects Observed

Significant differences between litters were observed in the present trial for some of the parameters measured, in particular renal CBS, CGL and MS activity measured per mg protein, g kidney and total kidney, average litter body weight and the amino acids arginine, isoleucine, proline and valine. In a randomized complete block design, the block is a set of experimental material that is presumed to be very uniform with each block containing all applied treatments. By this design it is hoped that slight differences within blocks will have an equal influence on all teatments in that block and thus prevent significant treatment differences as a result of exogenous effects. As block effects were observed in the present trial, it suggests that the chosen blocks may not have been as uniform as initially presumed. For example, older sows with a higher parity may have an effect on the developmental rates of renal enryme activities in the piglets with some

litters having overall greater enzyme activity at the same age as compared with other

litters. If dietary intake plays role in the development of CBS, CGL or MS, average litter

weights, or amino acid concentrations, differences in the number of piglets in the litter

competing for milk could have a similar effect. Since no clear pattem of significance in

relation to parity and litter size on day 1 is evident, observed block effects are more likely

to be a combination of genetic variation and minor envirbnmental differences between

individual sows and their subsequent litters.

6.8 Factors Influencing Plasma Homocysteine and Cysteine Patterns in Developing Pigs r02

In comparison with plasma homocysteine concentrations measured in early- weaned pigs by House and Fletcher (2003), circulating homocysteine levels in developing pigs at day 26 in the present trial were similar to that measured by these authors in older

animals, fed adequate amounts of B-vitamin cofactors. The pattem of change in plasma homocysteine concentrations in developing pigs has not been previously reported. Plasma

homocysteine concentrations are low at birth (7 uM) and increase to a maximum

concentration of 46 uM before declining to almost half that level by day 26 (19 uM).

House and Fletcher (2003) reported plasma homocysteine to range between 20 and 25 uM

when pigs were fed 35 ug/kg cobalamin or greater, a plasma level comparable to that

measured in the present trial where pigs were fed a post-weaning diet containing 45 ug/kg

cobalamin. Stangl et al. (2000) also reported circulating homocysteine concentrations to

be approxim ately 20 uM in pigs fed a diet containing adequate cobalamin and folate.

These studies, combined with data from the present trial, support the gradual

normalization of plasma homocysteine concentrations around 20 uM in developing pigs

at an adult level. However, the observed pattern of hyperhomocysteinemia in pigs, prior

to plasma levels reaching a presumed adult level, cannot be fully explained and is likely

due to a combination of factors.

Hyperhomocysteinemia, according to Refsum et al. (1998a), is a steady-state

condition characterized by increased homocysteine flux into tfre plasma and/or decreased

elimination of plasma homocysteine. Increased delivery of homocysteine into the plasma

is related to increased export from tissues as a result of an imbalance between

intracellular homocysteine synthesis and degradation. The tissues responsible for 103 homocysteine production and export to the plasma are not well known; however, homocysteine export has been demonstrated in human umbilical vein endothelial cells, murine lymphoma cells and human fibroblasts (Christensen et al. 1992, Christensen et al.

1994, Van der Molen et al. 7996, as referenced by Stead'et al. 2000). Homocysteine

export from human and rat hepatocytes have been measured in comparison with other

cells (lymphoma cells, fibroblasts and hepatoma cells; Christensen et al. 1991 as

referenced by Stead et al. 2000) and suggest the liver as a primary source of circulating

homocysteine (Christensen et al. 1991, Stead et al. 2000). Increased plasma homocysteine

may also be a result of an increase in the rate of transmethylation, a decrease in the rate of

transsulphuration to cystathionine, a decrease in the rate of remetþlation to methionine, a

decrease in the uptake and metabolism/excretion of homocysteine by the kidney

(Stipanuk 2004)o¡ a combination of these events.

Increasing plasma homocysteine concentrations in pigs in the present trial may be

the result of an imbalance in hepatic homocysteine metabolism temporarily caused by the

different rates of development and directional changes of MS and CBS. The lowest

plasma homocysteine concentration measured in developing pigs occurs at birth and

coincides with the highest level of hepatic MS activity. The rapid decrease in MS may be

reflected in the simultaneous increase in plasma homocysteine in pigs to day 18.

Although at low levels, homocysteine degradation via M.S remethylation is preferred over

CBS transsulphuration based on their respective Km values for homocysteine (Finkelstein

1990), the decrease in MS activity would likely limit homocysteine conversion to

methionine, regardless of its affinity for homocysteine. Increasing CBS activity, in 104 addition having nearly double the enzyme activity on day 1 as observed for MS at birth, would enable more homocysteine to be transsulphurated to cystathionine. However increasing CBS activity to a presumed adult level by day 9 did not curb increasing plasma homocysteine concentrations and other facto¡s are likely involved in the observed hyperhomocysteinemia.

Of particular interest is the transient decline in plasma homocysteine concentrations that occurs between day 18 and26. Although hepatic CBS activity has reached a steady activity plateau by this time and a lag in homocysteine degradation by

CBS is possible, B-vitamin nutrition in developing pigs should also be considered. At day

19, the piglets are receiving a vitamin-supplemented weaning diet, presumed to contain requirement levels of the B-vitamins, specifically those involved in homocysteine degradation. If the decline in plasma homocysteine is in response to an improved folate, cobalamin and/or vitamin Bu status, then pre-weaning availability of these vitamins to the piglet should be reconsidered in light of rising homocysteine concentrations. According to

Selhub et al. (1993),67 per cent of human cases of hyperhomocysteinemia are partially due to inadequate B-vitamin status and evidence for an inverse correlation between vitamin status and hyperhomocysteinemia is stronger and more consistent for folate than for vitamin 8,, or Bu (Stipanuk 1999). Changes in the plasma homocysteine concentrations in developing pigs suckling from a folate-supplemented sow have not been reported, however Barkow et al. (2001) demonstrated that colostral folate in sows' milk can be more than tripled with carryover increases in the circulating levels of folate in suckling pigs. Simard et al. (2002) reported that plasma homocysteine concentrations of 105 piglets suckling from B,r-supplemented sows from day 0 onward decreased as the level of

dietary 8,, increased. The pattern of increasing homocysteine levels with age was similar to that in the present trial. However, plasma homocysteine concentrations were lower

overall (3 uM on day 0 to29 uM on day 2I). Although plasma vitamin Bu was not

measured in the present trial, it will be looked at given that pyridoxine deficiencies can

cause increased plasma homocysteine in pigs (Smolin et al. 1983); the post-weaning diet

assumed to contain adequate vitamin Bu may have also influenced the post-weaning

decline in circulating homocysteine levels. Although these findings suggest the high

levels of homocysteine in piglets observed in the present trial may be potentially lowered

through improved pre-weaning B-vitamin nutrition, the pattern of hyperhomocysteinemia

in the neonate may be unrelated to vitamin status and more a function of other changes

relating to enzyme activitY'

Increased homocysteine export and uptake are other potential factors influencing

plasma homocysteine concentrations in developing pigs. Stead et al. (2000) demonstrated

that homocysteine export from the liver depended on the concentration of methionine in

the incubation medium. Hepatic methionine concentrations were not measured in

developing pigs however proteolysis and hepatic uptake of methionine, as suggested for

supporting hepatic cysteine concentrations (Triguero et al. 1997) are possibilities. A

gradual increase in plasma methionine may be a reflection of this effect. Bengtsson

(lg7l) reported a significant increase in serum methionine levels three days post-

parturition in piglets however plasma concentrations in the present trial remained

unchanged between day 0 and 26. Quantitative values obtained in this experiment were 106 appreciably lower than those measured previously in suckling pigs (Flynn et al. 2000) and may be a result of differences in nutrient intake or anal¡ical methods. Stead et al (2000) also demonstrated that incubation of hepatocytes with methionine and serine reduced homocysteine export to between 25 and 40 per cent, presumably by influencing homocysteine catabolism via the transsulphuration pathway. The kidney is recognized as

a major site for the removal and subsequent metabolism of plasma homocysteine (House

et al. 1997) while the liver is considered to be limited in this capacity (Hultberg 2003).

Renal MS and CBS activity were unchanged between birth and day 26, potentially due to

enzyme development having been reached in utero. Characterization of these enzymes in

the kidney of other species is limited however Sturman et al. (lg76a) reported renal MS

activity in Rhesus monkeys to be relatively stable throughout development and similar in

MS activity levels in the present trial. House etal. (1997) and Guttorrnsen et al. (1996)

proposed that, based on low rates of methionine synthesis measured using in vitro (tat

cells) and in vivo (humans) methods, the MS reaction is not as important to the removal

of homocysteine from the plasma in relation to transsulphuration. High renal CBS activity

as compared to MS measured in the present trial suggests this may be similar in

developing pigs but requires further confirmation. Urinary excretion of homocysteine is

related to the plasma homocysteine level and may contribute to the rapid decline in

plasma homocysteine concentrations observed post-weæring in pigs. However

homocysteine clearance via this mechanism is reported to represent less than 0.5 per cent

of total cellular homocysteine production and considered a minor means of homocysteine

elimination (Storch et al. 1988, Mudd and Poole 1975, as referenced by Refsum et al. t07

1998a, House et al. 1998).

Plasma cysteine paralleled the changes in plasma homocysteine for nearly the entire trial period. Cysteine levels were lowest at birth (97 uM) and rose significantly to a peak concentration on day 19 (210 uM), one day following the peak in plasma homocysteine concentrations, after which cysteine concentrations declined to a significantly lower level by day 26 (186 uM). Although flyoo et al. (2000) reported steady plasma cysteine levels in suckling pigs, concentrations at day 21 (158 uM) were comparable to those in the present study. As with plasma homocysteine, a combination of factors are likely to be involved in the observed pattern of change in the developing pig.

Proteolysis, proposed as a means of ensuring hepatic cysteine concentrations during sepsis- and PPG-induced CGL inhibition (Triguero et al. 1997, Malmezat et al. 2000a), may act similarly in neonatal pigs with underdeveloped transsulphuration enzyme activity. Muscle and protein breakdown may release cysteine into circulation and increase plasma cysteine in the neonate. This scenario would also increase cysteine availability for potential increased hepatic cysteine uptake, another proposed means of maintaining hepatic cysteine requirements when CBS and CGL are not completely developed

(Triguero et al. 1997). As endogenous cysteine synthesis mechanisms become more active, hepatic cysteine uptake may be reduced and increasing amounts of circulating cysteine, as observed in the present study, might be expected. The highest concentration of plasma cysteine was measured approximately 24 hours after weaning and replacement of the sows' low cysteine milk with higher protein post-weaning feed, may have also contributed to this observation. 108

The post-weaning decline in plasma cysteine concentrations, despite increased protein availability and an apparent capacity for endogenous cysteine synthesis, is also likely a function of various factors. Garcia and Stipanuk. (1992) demonstrated that in addition to the liver which removes approximately 12 uM cysteine from plasma, the splanchnic organs, including the small and large intestine, stomach and pancreas, also remove cysteine (5 uM) from circulation. Studies on isolated enterocytes suggest these cells have a limited capacity for transsulphuration (Lash et al. 1986 as referenced by

Garcia and Stipanuk 1992) and thus may require an exogenous source of cysteine.

Although Coloso and Stipanuk (19S9) suggest enterocytes' ability to degrade cysteine is limited, intestinal cells require GSH (Lash et al. 1986, Martensson et al. 1990, Reeds et

al. 1997) and are demonstrated to release significant amounts of GSH into the circulation

(Garcia and Stipanuk 1992). Adult-level endogenous cysteine synthesis combined with adequate protein intake post-weaning would presumably increase cysteine availability for protein, taurine and sulphate synthesis also. Using [35S]cysteine, Tateshi et al. (1981) demonstrated that in rat hepatocytes, cysteine formed from methionine was preferentially incorporated into hepatic proteins while cysteine provided as such was used more readily for GSH synthesis. Neither protein nor GSH production were measured in the present trial although both are suggested to be favored when cysteine concentrations are limited

(Stipanuk et al. 1992). Plasma taurine levels did not change post-weaning despite taurine

and sulphate production being the preferred mode of cysteine catabolism during high

cysteine availability (Stipanuk et al. 1992, Fukagawa et al. 1 996, as referenced by

Stipanuk 2004). As such, marginal sulphur amino acid intake post-weaning by pigs might 109 explain the decrease in plasma cysteine as cysteine is converted to GSH for storage

(Tateshi et al. 1977, Stipanuk et al. 1992) and used for protein synthesis. Measurement of both these parameters throughout piglet development would further help explain declining plasma cysteine concentrations.

6.9 Similarities Between Piglets and Human Infants With Regards to Sulphur Amino Acid Metabolism

Metabolic similarities between pigs and humans (Miller and Ullrey 1987),

specifically with regards to amino acid metabolism in piglets and humans infants have been previously demonstrated (Wykes et al. 1994, Darragh and Moughan 1995, Ball et al.

1996). Results of this study are in agreement and further support the use of the piglet as a

model for studying sulphur amino acid metabolism in human infants. Hepatic CBS, CGL

and MS activities in the human infants (Gaull et aL 1972, Zlotkin and Anderson 1982,

Kalnitsky et al. 1982) followed the same developmental pattern as those observed in

developing pigs. In absolute terms, hepatic CBS in premature human infants (around 24

nmol/lu/mg protein; Gaull et al. 1982) were comparable to that of piglets at birth (21

nmol/hr/mg protein) as were levels in mature humans (98 nmol/hrlmg protein; Gaull et al.

1982) versus pigs at 26 days of age (1i5-120 nmol/hr/mg protein). Hepatic CGL activity

in the developing pig at birth was slightly greater than that of premature human infants

(20 vs 0 nmol/lu/mg protein, respectively; Gaull et al. t982) although less than levels

measured in full-term infants at birth (approximately 66 nmollhrlmgprotein). Stable CGL

activity in both pigs and humans appears to be achieved within the first two days of life, 110 both reaching adult values within a similar range (70-130 nmoVhr/mg protein).

Similarities also exist for hepatic MS between pigs and humans although absolute levels at birth are significantly different. Hepatic MS in pigs on day 0 was 31 nmol/hrlmg protein and approximately six-fold greater than levels measured in human infants (Gaull et aI. I973b). While both species follow a similar decline in enzyme activity with age, enzyme activity of pigs at day 26 (4 nrnol/hrlmg protein) reaches a level comparable to that of adult humans (1 nmol/hr/mg protein; Gaull et aL l973b). Comparison of absolute rates of activity are less important than the relative changes in activity and with the exception of the hepatic MS between birth and day 26 pigs versus birth and adult levels in humans, enzyme activity development between humans and pigs is similar.

Despite similar developmental patterns of CBS, CGL and MS in pigs and humans,

changes in plasma homocysteine concentrations should also be considered. Plasma

homocysteine levels in fuIl-term human infants (6 uM, Hongsprabhas et al. 1999)

measured three days after birth were similar to levels measured on day 0 in piglets (7 uM)

in the present trial. Hongsprabhas et al. (1999) also reported an increase in plasma

homocysteine concentrations in human infants during the first few days of life, similar to

that observed in piglets. However, the increase in homocysteine in pigs was significantly

greater than that observed in humans, reaching plasma values of 46 uM, a concentration

that would be considered hyperhomocysteinemic by human standards. Normal plasma

homocysteine levels in human adults range between 5 and 15 uM, (House et al' 1999)

while in pigs, plasma homocysteine appears to remain around 20 uM (Stangl et al. 2000,

House and Fletcher 2003). Researchers using developing pigs as models of human infants 111 to study sulphur amino acid metabolism should to be aware of such differences in order to accurately predict the effects of nutritional treatments on homocysteine concentrations.

6.10 Developing Pigs and Their Potential to Respond to Disease Challenges

Enzyme activities and metabolite concentrations measured in the present study provide information on methionine metabolism in healtþ developing pigs. Based on the results of the present trial, physiological compensatory mechanisms, such as increased proteolysis , mày ensure that cysteine requirements of the pig are satisfied early in life when endogenous cysteine synthesis mechanisms are underdeveloped. If developing pigs were subject to a disease challenge, it is likely that the present measurements would be

altered although the direction of change may only be speculated. Malmezat et al. (2000a)

demonstrated that sepsis caused a significant decrease in hepatic CGL activity in rats

despite hepatic cysteine levels being maintained at healthy, control levels. Similarly, hepatic CGL in neonatal pigs subject to infection might remain suppressed at a lower

'Whether activity level for an extended period of time. infection could limit the complete

development of these enzymes and result in a lower adult-level transsulphuration enzyme

activity and endogenous cysteine synthesis is unknown. Increased protein and amino acid

catabolism that occurs during trauma and sepsis are used to increase synthesis of proteins,

including acute-phase proteins and proteins of the immune system (Breuillé et al. 1994b,

Rooyackers et al. 1994). Such proteolysis might assist in supporting septic piglets'

cysteine requirements as was suggested to occur in healthy, developing animals. Where as

Malmezat et al. (2000a) reported presumably adult CGL activity in septic rats to be tt2 depressed by 17 per cent, hepatic CGL in piglets on day 0 is already 73 per cent less than the levels on day 16. Infection would likely reduce this level even further and potentially inhibit endogenous cysteine synthesis altogether. However the large spike in hepatic CGL activity on day 1 and stabilizationat plateau levels in developing pigs, despite likely some inhibition by infection, could potentially support low endogenous cysteine synthesis; whether whole-body catabolism would be reduced, as is þroposed to occur during the development of the transsulphuration pathway, or remains elevated as during infection

(Breuillé et al. 1996), is unknown.

Hepatic GSH and taurine synthesis (Breuillé et al. lgg4a,Malmezat et al. 1998,

Malmezat et al. 2000b) and whole-body protein production (Breuillé et al. l994a,b) are increased in septic versus healtþ pairfed rats, and maintenance of hepatic cysteine concentrations suggests proteolysis is capable of satisSing the cysteine requirements in disease-challenged, adult animals. However, whether this would apply in disease- challenged neonatal pigs, which have little muscle for protein storage at birth and experience negative nitrogen balance during sepsis (N{rozek et al. 2000, Premer et al.

2002), has not been determined. Reduced feed intake during infection is common

(Breuillé et al. I994b, Malmezat et al. 2000a) and decreased protein intake combined with minimal protein stores may further try the neonate's ability to satisff hepatic cysteine requirements given underdeveloped and sepsis-suppressed endogenous cysteine synthesis. It is also unknown whether cysteine production from proteolysis during limited transsulphuration is shunted preferably towards protein, GSH or taurine synthesis during infection andlor if compensatory cysteine mechanisms have a maximum capacity to 113 maintain cysteine concentrations. A decrease in hepatic cysteine levels in septic piglets

with underdevloped transsulphuration enryme activity early in life might suggest that

compensatory mechanisms have reached their limit for maintaining hepatic cysteine

levels. Although this could be the case in post-weaned pigs, they may respond similar to

septic rats, which likely had adulrlevel but suppressed endogenous cysteine synthesis

mechanisms and compensatory mechanisms capable of cysteine maintenance (Malmezat

et al. 2000a).

6.11 Summary

The present study is the first to demonstrate the developmental pattems of the

transsulphuration enzymes CBS and CGL, as well as the remethylation enzyme MS in

healthy piglets from birth to post-weaning. While hepatic CBS and CGL activities

gradually increase with age to a relatively stable activity plateau post-weaning, hepatic

MS decreases rapidly during the first few days of life, reàching a low stable activity level

by day 26. Steady levels of CBS and CGL post-weaning and MS activity at day 26

comparable to the hepatic activity in other fully developed species suggest that enzyme

development along these pathways has been completed. These pattern are similar to those

previously observed in rats (Finkelstei n 1967 , Finkelstein et al. 1971, Kashiwam ata 197l,

Volpe and Laster I972,Heinonen l973,YanAerts et al. 1995) and monkeys (Sturman et

al.,l976a,b). More importantly, the developmental profiles of CBS, CGL and MS in pigs

and the adult-level specific activity levels reached post-weaning are similar to those

observed in humans (Gaull et al. l97l,Gaull et al. 1973b, Heinonen and Räihä Ig74, Il4

Kalnitsky et al. lgg2,Zlotkin and Anderson 1982). Characterization of the developmental

the patterns of vitamin cofactors and methionine and cysteine metabolites associated with transsulphuration and remethylation pathways offer further support to the changes

was observed in CBS, CGL and MS in growing piglets. Of interest, hepatic cysteine

(CBS, or unaffected by the age-related changes of enzymes involved directly CGL) indirectly (MS) in endogenous cysteine synthesis, suggesting that neonatal pigs have

early in life. compensatory mechanisms for supporting their cysteine requirements

for plasma Whether these mechanisms are partly responsible for the patterns observed

and whether cysteine and homocysteine that parallel each other throughout development,

during a disease the capacity of the piglet to maintain cysteine homeostasis is adequate

challenge, remains to be determined. 115

Chapter 7 - Future Research

While the present trial provides a static picture of the developmental patterns of

CBS, CGL and MS in developing pigs from birth until weaning, it is limited in its ability to describe the movement of metabolites along each pathway throughout development.

Measurement of metabolic flux is key to fully understanding how metabolic products are distributed between competing pathways and flux measurements in developing pigs would describe how changes in enzyme activity influence this distribution. For example, homocysteine flux measurements would allow increased understanding of how, during

enzyme development, the simultaneous decrease in MS activity and increase in CBS

activify affects homocysteine levels. Metabolic flux of methionine and cysteine would

offer similar information and provide a more complete explanation of the changes

observed in methionine, cysteine, and taurine concentrations. Characterization of the

developmental patterns of all enzymes in the neonatal pig relating directly or indirectly to

methionine degradation would be of interest however the profiles of the transmethylation

enzymes and BHMT would be particularly useful to bettèr understand changes in both

hepatic and circulating homocysteine concentrations. Investigation as to the mechanisms

involved in maintaining hepatic cysteine concentrations despite underdeveloped

endogenous cysteine mechanisms would also be of interest. The relationship between B-

vitamins, homocysteine and the activity of the remethylation and transsulphuration

enzymes has been well-studied (Selhub 1999, Refsum 2001, House et al. 2003, Braunaud

et al. 2003) presumably using stable enzyme activity levels. However the effect of a B-

vitamin deficiency on the development patterns of CBS, CGL and MS activity is 116 unknown and whether such deficiencies would influence the developing pigs' capacity for endogenous cysteine synthesis, either following treatment or in an ongoing deficient state, remains to be determined. Furthermore, given rising plasma homocysteine concentrations, the potential for inadequate B-vitamin nutrition pre-weaning remains a possibility. Given a greater susceptibility of neonates to infection as compared with older

on the animals, sufficient GSH availability is essential early in life. The effect of sepsis development profiles of cBS, cGL and MS, and the capacity of the body to maintain

GSH levels given underdeveloped endogenous cysteine synthesis requires investigation.

than Since protein synthesis has been demonstrated to have a higher priority for cysteine

GSH synthesis (Stipanuk et al. 1992,Lee et aL.2004 as referenced by Stipanuk 2004),

developing assurance of cysteine availability for GSH production during sepsis in rapidly

pigs would be especially important. Not only do results from the present trial offer some

development, explanation as to the changes observed in homocysteine metabolites during until but the development profiles established for CBS, CGL and MS in pig from birth

post-weaning offer a solid basis for extension of this research. t17

Chapter 8 - References

American Academy of Pediatrics, Committee on Nutrition. 1998. Nutritional Needs of Preterm Infants. In: Pediatric Nutrition Handbook. (Kleinman, R. E. ed). American Academy of Pediatrics. pp. 55-87.

Anderson, J. O., Wamick, R. E. and Dalai, R. K. i975. Replacing dietary methionine and cystine in chick diets with sulphate or other sulphur compounds. Poultry Sci. 54: tt22-r128.

Araki, A. and Sako, Y. 1987. Determination of free and total homocysteine in human plasma by high performance liquid chromatography with fluorescence detection. J. Chromatogr. 422: 43-52.

Ball, R. O., House, J. D., Wykes, L. J. and Pencharz,P.B. 1996. A piglet model for neonatal amino acid metabolism during total parenteral nutrition. In: Advances in Swine in Biomedical Research (Tumbleson, M. E., School, L. 8., eds). Plenum Press' New York, New York. pp 713-73L

Ballance, D. M. and House, J. D. 2001. Elevated plasma homocysteine concentrations during iron-deficiency anemia. Canadian Federation Biological Society Meeting. Ottawa, Ontario.

Barkow,8., Matte, J. J., Böhme, H. and Flachowsþ, G. 2001. lnfluence of folic acid supplements on the carry-over of folates from the sow to the piglet. Br. J' Nutr. 85: 179-

1 84.

Bender, D. A. 1999. Non-nutritional uses of vitamin Bu. Br. J. Nutr. Sl:7-20.

Benedikt, J., Roth-Maier, D. A. and Kirchgessner, M. 1996. Influence of dietary vitamin Bu supply during gravidity and lactation on total vitamin Bu concentration (Pyridoxine, pyridãxal and Pyridoxamine) in blood and mitk. Int. J. Vit. Nutr. Res. 66: 146-150.

Bengtsson, S. G. 1971. Serum free amino acids in piglets during the early postnatal period. J. Anim. Sci.32: 879-882.

Blom, W. and Huijmans, J. 1985. Differential diagnosis of (inherited) amino acid metabolism or transport disorders' Science Tools. 32:10-22.

Bolander-Gouaille, C. 2002. Focus on Homocysteine and the Vitamins Involved in Metabolism-2nd edition. Springer Verlag Publishing. France.

Braidman,I. P. and Rose, D.P. lgTL Effects of sex hormones on three glucocorticoid 118 inducible enzymes concerned with amino acid metabolism in rat liver. Endocrinology. 89: 1250-1255.

Breuillé, D., Arnal, M., Rabourdin, F., Rosé, F' and Obled, C. 1996. Increased cysteine requirements induced by sepsis' Clin. Nutr. 15 21. Abstract'

Breuillé, D., Malmezat,T.,Rosé, F., Pouyet, C' and Obled, C. L994a. Assessment of tissue glutathione status during experimental sepsis. Clin. Nutr' 13: 5. Abstract.

Breuillé, D., Rosé, F., Amal, M., Melin, C. and Obled, C. 1994b. Sepsis modifies the contribution of different organs to whole-body protein synthesis in rats' Clin' Sci. 86: 663-669.

Brunaud, L., Alberto, J.M., Ayav,4., Gerard, P., Namour, F., Antunes, L', Braun, M., Bronowicki, J. P., Bresler, L. and Gueant, J. L. 2003. Vitamin 8,, is a strong determinant of low methionine synthase activity and DNA hypomethylation in gastrectomized rats. Digestion. 68: 133-i40'

pig Burnett, G., Marcotte, P. and Walsh, C. 1980. Mechanism-based inactivation of heart L-alanine transaminase by L-propargylglycine. J. Biol. Chem. 255:3487-3491.

of and Byington, M. H., Howe, J. M. and Clark, H.E. 1972. Effect of different levels p.opðrtionr of methionine, cystine, choline, and inorganic sulphur on growth and body composition of young rats' J. Nutr. 102: 219'227 '

chiang, P. K, Gordon, R. K., Tal, J.,Zeng,G. C., DoCtOr, B. P., Pardhasaradhi, K. and McCaL, p. p. 1996. S-Adenosylmethionine and methylation. FASEB. 10: 471-80.

Cho, E. S., Johnson, N. and Snider, B. C. F. 1984. Tissue glutathione as a cyst(e)ine reservoir during cystine depletion in growing rats. J' Nutr. I 14: 1853-1862'

Christensen, 8., Refsum, H., Garras, A. and Ueland, P. M. 1992. Homocysteine various remethylation during nitrous oxide exposure of cells cultured in media containing concenirations of folates. J. Pharmacol. Exp. Ther.26t: 1096-1105.

Christensen, 8., Rosenblatt, D. S., Chu, R. C. and Ueland, P' M. 1994. Effect of methionine and nitrous oxide on homocysteine export and remethylation in fibroblasts from cystathionine synthase-deficient, cblG, and cblE patients. Pediatr' Res. 35: 3-9'

Christensen, 8., Refsuffi, H., Vintermyr, O. and Ueland, P' M. 1991. Homocysteine export from cells cultured in the presence of physiological or superfluous levels of måthionine: methionine loading of non-transformed, transformed, proliferating, and quiescent cells in culture. J' Cell Physiol. 146:52-62' r19

Chung, T. K. and Baker, D.H. 1992. Methionine requirement of pigs between 5 and 20 kilograms of body weight. J. Anim. Sci. 70: i857-1863.

Clark, E. S., Crissinger, K. D. and Granger, D. N. 1990.. Oxidant-induced increases in mucosal permeability in developing pigs. Pediatr. Res. 28: 28-30.

Coburn, S. P. 1994. A critical review of minimal Bu requirements for growth in various species with a proposed method of calculation. Vitam. Horm. 48:259-300.

Cole, D. E. C., Oulton, M., Stirk, L. J. and Magor, B. 1992. Increased inorganic sulphate concentrations in amniotic fluid. J. Perinat. Med. 20: 443-447.

Coloso, R. M. and Stipanuk, M. H. 1989. Metabolism of cyst(e)ine in rat enterocytes. J. Nutr. 119: 1914-1924.

Cuskelly, G. J., Stacpoole, P. W., Williamson, J., Baumgartner, T. G. and Gregory III, J' F. 2001. Deficiencies of folate and vitamin Bu exert distinct effects on homocysteine, serine, and methionine kinetics. Am. J. Physiol. Endocrinol. Metab.281: El182-E1190.

Daniels, K. M. and Stipanuk, M. H. 1982. The effect of dietary cysteine level on cysteine metabolism in rats. J. Nutr. ll2:2130-2141.

Darragh, A. J. and Moughan, P. J. 1995. The three-week-old piglet as a model animal for studying protein digestion in human infants. J. Pediat. Gastroent. Nutr. 21 :387-393.

Dicker-Brown, 4., Fonseca, V. 4., Fink, L. M. and Kern, P. A' 2001. The effect of glucose and insulin on the activity of methylene tetrahydrofolate reductase and cystathionine beta-synthase: studies in hepatocytes. Atherosclerosis. 158:297-30L

Dixon, M. M., Huang, S., Matthews, R. G. and Ludwig, M. 1996. The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylation of B,r. 4: 1263-1275. Abstract.

Doi, T., Kawata, T., Tadano, N., Iijima, T. and Maekawa, A. 1989. Effect of vitamin B' deficiency on S-adenosylmethionine metabolism in rats..J. Nutr. Sci. Vitaminol. 35: 1-9.

Doolittle, R. F. 1989. Redundancies in protein sequences. In: Prediction of Protein Structure and the Principles of Protein Conformation (Fasman, G. D. ed). Plenum Press. New York.pp:599-623.

Dröge, W. and Breitkreutz, R. 2000. Glutathione and immune function. Proc. Nutr. Soc. 59: 595-600. t20

Farris, M. V/. and Reed, D. J. 1987. High-performance liquid chromatography of thiols and disulphides: dinitrophenol derivatives. Meth. Enzymol. i43: 101-109.

Finkelstein, J. D. 1967. Methionine metabolism in mammals: Effects of age, diet and hormones on three enzymes of the pathway in rat tissues. Arch. Biochem. Biophys. i22: 583-590.

Finkelstein, J. D. 1990. Methionine metabolism in mammals. J. Nutr. Biochem. l:228- 237.

Finkelstein, J.D., Harris, B. J. and Kyle, W. E. 1972. Methionine metabolism in mammals: Kinetic study of betaine-homocysteine methyltransferase. Arch. Biochem. Biophys. 153 320-324.

Finkelstein, J. D. and Kyle. W. E. 1968. Ethanol effects on methionine metabolism in rat liver. Proc. Soc. Exp. Biol. Med. 129:497-501'

Finkelstein, J. D., Kyle, W. E. and Harris, B. J' 1971. Methionine metabolism in mammals. Regulation of homocysteine methyltransferases in rat tissue. Arch. Biochem' Biophys. 146:84-92.

Finkelstein, J. D., Kyle, W. 8., Marin, J. J. and Pick, A-M. 1975. Activation of cystathionine synthase by adenosylmethionine and adenosylethionine. Biochem. Biophys. Res. Comm.66: 8l-87.

Finkelstein, J. D. and Martin, J. J. 1984. Methionine metabolism in mammals: Distribution of homocysteine between competing pathways. J. Biol. Chem. 259 9508- 9513.

Finkelstein, J. D. and Martin, J. J. 1986. Methionine metabolism in mammals-adaptation to methionine excess. J. Biol. Chem. 261: 1582-1587.

Finkelstein, J.D., Martin, J. J. and Harris, B. J. 1988. Methionine metabolism in mammals; the methionine-sparing effect of cystine. J. Biol. Chem. 263 11750-11754.

Finkelstein, J. D. and Mudd, S. H. 1967. Transsulphuration in mammals; the methionine-sparing effect of cystine. J. Biol. Chem. 242:873-880.

Flynn, N. E., Knabe, D. 4., Mallick, B. K. and Wu, G. 2000. Postnatal changes of plasma amino acids in suckling pigs. J. Anim. Sci. 78: 2369-2375'

Frederick, G. L. and Brisson, G. J. 1961. Some observations on the relationship between vitamin 8,, and reproduction in swine. Can. J. Anim. Sci. 41 :212-219. T2T

Friedman, P. 4., Shia, M. A. and Wallace, J.K. 1977. A saturable high affinity binding site for transcobalamin ll-vitamin 8,, complexes in human placental membrane preparations. J. Clin. Invest. 59: 51-58.

Fukagawa, N. K., Ajami, A. M. and Young, V. R. 1996. Plasma methionine and cysteine kinetics in response to an intravenous glutathione infusion in adult humans. Am. J. Physiol. 27 0: 8209 -21 4.

Gallant, K. R. and Cherian, M. G. 1989. Metabolic changes in glutathione and metallothionein in newborn rat liver. J. Pharmacol. Exp. Ther.249: 63I-637.

Garcia, R. A. G. and Stipanuk, M. H. 1992. The splanchnic organs, liver and kidney have unique roles in the metabolism of sulphur amino acids and their metabolites in rats. J. Nutr. 122: 1693-1701.

Gaull, G. E. 1986. Taurine as a conditionally essential nutrient in man. J. Am. Coll. Nutr.5: I2I-125.

Gaull, G. E., Räihä, N. C. R, Saarikoski, S. and Sturman, J. A. l9l3a. Transfer of cyst(e)ine and methionine across the human placenta. Pediatr. Res. 7: 908-913.

Gaull, G. 8., Sturman, J. A. and Räihä, N. C. R. 1972. Development of mammalian sulphur metabolism: absence of cystathionase in human fetal tissues. Pediatr. Res. 6: 538- 547.

Gaull, G. E., von Berg, W., Räihä, N. C. R. and Sturman, J. A. 1973b. Development of methyltransferase activities of human fetal tissues. Pediatr. Res. 7: 527-533.

Gilfix, B. M., Blank, D. W. and Rosenblatt, D. S. 1997. Novel reductant for determination of total plasma homocysteine. Clin. Chem. 43: 687-688.

Girgis, S., Suh, J. R., Jolivet, J. and Stover, P. J. 1997. 5-Formyltetrahydrofolate regulate homocysteine remethylation in human neuroblastoma. J. Biol. Chem.272: 4729- 4734.

Glazenburg,8.J., Jekel-Halsema, L M., Baranczyk-Kuzmã, A., Krijgsheld, K. R' and Mulder, G. J. 1984. D-cysteine as a selective precursor for inorganic sulphate in the rat in vivo. Effect of D-cysteine on the sulphation of harmol. Biochem. Pharmacol 33: 625- 628.

Green, R. and Jacobsen, D. W. 1995. Clinical implications of hyperhomocysteinemia. In: Folate in Health and Disease. (Bailey, L. 8., ed). M. Dekker. New York, New York. 122

pp.75-124.

Grimble, R. F. 1992. Dietary manipulation of the inflammatory response. Proc. Nutr. Soc. 51 :285-294.

Grundy, S. M. 1999. Nutrition and Diet in the Management of Hyperlipidemia and Atherosclerosis. In: Modem Nutrition in Health and Disease, 9th edition. (Shils, M. E., Olson, J. 4., Shike, M., Ross, A. C., eds). V/illiams and Wilkins. Baltimore, Maryland. pp: 1199-1227.

Guan, X., Bequette, B. J., Calder, G., Ku, P. K., Ames, K. N. and Trottier, N. L. 2002. Amino acid availability affects amino acid flux and protein metabolism in the porcine mammary gland. J. Nutr. 132:1224-1234.

Guay, F., Matte, J. J., Girard, C. L., Palin, M-F., Giguère, A. and Laforest, J-P. 2002. Effects of folic acid and vitamin 8,, supplements on folate and homocysteine metabolism in pigs during early pregnancy. Br. J. Nutr. 88:253-263.

Guttormsen,4. 8., Schneede, J., Ueland, P. M. and Refsum, H. 1996. Kinetics of total plasma homocysteine in subjects with hyperhomocysteinemia due to folate or cobalamin deficiency. Am. J. Clin. Nutr. 63 194-202.

Halstead, C. H., Villanueva, J. 4., Devlin, A. M., Niemela, O', Parkkila, S', Garrow, T. 4., Wallock, L. M., Shigenaga, M. K., Melnyk, S. and James, S. J. 2002. Folate deficiency disturbs hepatic methionine metabolism and promotes liver injury in the ethanol-fed micropig. Proc. Natl. Acad. Sci. USA. 99:10072-10077.

Hayes, K. C., Carey, R. E. and Schmidt, S. Y. 1975a. Retinal degeneration associated with taurine deficiency in the cat. Science. 188: 949-95I.

Hayes, K. C., Rabin, A. R. and Bernson, E.L. I975b. An ultrastructural study of nutritionally induced and reversed retinal degeneration in cats. Am. J. Pathol. 78: 505- 524.

Heinonen, K. 1977. Effects of hypo- and hyperthyroidism on the activity of cystathionase in mammalian parenchymatous organs during early development. Experimentia. 33 : 1427 -1428.

Heinonen, K. 1973. Studies on cystathionase activity in rat liver and brain during development; effects of hormones and amino acids in vivo. Biochem. J. 136: 1011-1015.

Heinonen, K. and Räihä, N. C. R. 1974. Indt¡ction of cystathionase in human foetal 123 liver. Biochem. J. 144 607-609.

Henry, W. 2001. Managing weaning distress. Proceedings of the Manitoba Swine Seminar. 15: 101-125.

Hoffman, D. R., Marion, D. W., Cornatzer, W. E. and Duerre, J' A. 1980. S- adenosylmethionine and S-adenosylhomocysteine metabolism in isolated rat liver. Effects of L-methionine, L-homocysteine and adenosine. J. Biol. Chem. 255:10822-L0827 '

Hongsprabhas, P., Saboohi, F., Aranda, J. V., Claudette,'L. Bardin, Kovacs, L. 8., Papageorgiou, A. N. and Hoffer, L. J. 1999. Plasma homocysteine concentrations of preterm infants. Biol. Neonate. 7 6: 65-7 l.

House, J. D., Brosnan, M. E. and Brosnan, J. T. 1998. Renal uptake and excretion of homocysteine in rats with acute hyperhomocysteinemia. Kidney Int. 54: 160l-7.

House, J. D. and Fletcher, C. M. T. 2003. Response of early weaned piglets to graded levels of dietary cobalamin. Can. J. Anim. Sci. 83: 247-255.

House, J. D., Brosnan, M. E. and Brosnan, J . T. 1997 . Characterization of homocysteine metabolism in the rat kidney. Biochem' J.328:287-292.

Huang, R. F., Hsu, Y. C., Lin, H. L. and Y*g, F. L. 2001. Folate depletion and elevated plasma homocysteine promote oxidative stress in rat livers. J. Nutr. 131 : 33-38.

Hultberg, B. 2003. Modulation of extracellular homocysteine concentration in human cell lines. Clin. Chim. Acta. 330: 151-159.

Institute of Medicine. 2004. Dietary Reference Intake (DRI) for Water, Potassium, Sodium, Chloride and Sulphate. Available: [Accessed 04105104].

Iwata, S., Hori, T., Sato, N., Ueda-Taniguchi, Y., Yamabe, T., Nakamura, H., Masutani, H. and Yodoi, J. 1994. Thiol-mediated redox regulation of lymphocyte proliferation. Possible involvement of adult T-cell leukemia-derived factor and glutathione in transferrin receptor expression. J. Immun oI. | 52: 5 633 -5642.

Jacobs, R. L, House, J. D., Brosnan, M. E. and Brosnan, J. T. 1998. Effects Of streptozotocin-induced diabetes and of insulin treatment on homocysteine metabolism in the rat. Diabetes. 47:1967-1970.

Jacobs, R. L., Stead, L. M., Brosnan, M. E. and Brosnan, J. T' 2001. 124

Hyperglucagonemia in rats results in decreased plasma homocysteine and increased flux through the transsulphuration pathway in liver. J. Biol. Chem. 276: 43740-43747.

Jeffery, D. R. 1999. Nutrition and Diseases of the Nervous System. In: Modern Nutrition in Health and Disease, 9th edition. (Shils, M. E., Olson, J. 4., Shike, M., Ross, A. C., eds). Williams and Wilkins. Baltimore, Maryland.pp: 1543-1554.

Jencks, D. A. and Matthews, R. G. 1987. Allosteric inhibition of methylenetetrahydrofolate reductase by adenosylmethionine. J. Biol. Chem. 262:2485- 2493.

Jones, B. N. and Gilligan, J. P. 1983. o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physio lo gical fl uids. J. Chromat o gr. 266 : 47 I - 482.

Jones, D. B. and Stahly, T. S. 1999. Impact of amino acid nutrition during lactation on body nutrient mobilization and milk nutrient output in primiparous sows. J. Anim. Sci. 77: l5l3-22.

Kalnitsky, 4., Rosenblatt, D. and Zlotkin, S. 1982. Differences in liver folate enzyme patterns in premature and full-term infants. Pediatr. Res. 16: 628-631.

Karlsen, R. L., Gropova, D., Malthe-Sorenssen, D. and Fonnum, F. 1981' Morphological changes in rat brain induced by L-cysteine injection in newborn animals. Brain Res. 208:

1 67-1 80.

Kashiwamata, S. 1971. Brain cystathionine synthase: vitamin-Bu requirement for its enzymic reaction and changes in enzymic activity during early development of rats. Brain Res.30: 185-192.

K.ry, V., Bukovska, G. and Kraus, J.P. 1994. Transsulphuration depends on heme in addition to pyridoxal 5'-phosphate. J. Biol. Chem.269:25283'25288.

'W., Kim, Y. G., Kim, S. K., Kwon, J. Park, O. J', Kim, S' G., Kim, Y' C. and Lee, M. G' 2003. Effects of cysteine on amino acid concentrations and transsulphuration enzyme activities in rat liver with protein-calorie malnutrition. Life Sci.72:1171-1181.

Koblin, D. D., Watson, J. E., Deady, J. E., Stokstad, E. L. R' and Eger II, E. I. 1981. Inactivation of methionine synthase by nitrous oxide in mice. Anesthesiology. 54: 3 18- 324.

Kutzbach, C. and Stokstad, E. L. R. 1971. Mammalian metþlenetetrahydrofolate reductase: pafial purification, properties and inhibition by S-adenosylmethionine. 125

Biochim. Biophys. Acta. 250: 459-477.

Lakshmanan, F. L., Perera, W. D., Scrimshaw, N. S. and Young, V.R. 1976. Plasma and urinary amino acids and selected sulphur metabolites in young men fed a diet devoid of methionine and cysteine. Am. J. Clin. Nutr. 29:1367-I37I.

Lambert, B. D., Titgemeyer, E. C., Stokka, G. L., DeBey, B. M. and Löest, C. A. 2002. Methionine supply to growing steers affects hepatic activities of methionine synthase and betaine-homocysteine methyltransferase, but not cystathionine synthase. J. Nutr. 132: 2004-2009.

Lash, L. H., Hagen, T. M. and Jones, D. P. 1986. Exogenous glutathione protects intestinal epithelial cells from oxidative injury. Proc. Natl. Acad. Sci. USA. 83 4641- 4645.

Lavoie, J. C. and Chessex, P. 1998. Development of glutathione synthesis and Y- glutamyltranspeptidase activities in tissues from newborns. Free Rad. Biol. Med.24:994- 1001.

Leaf, G. and Neuberger, A. 1947.The effect of diet on the glutathione content of the liver. Biochem. J. 4l:280-287.

Lee, J. I., Londono, M.P., Hirschberger, L. L. and Stipanuk, M. H. 2004. Association of changes in cysteine dioxygenase and y-glutamylcysteine synthetase activities with cysteine and glutathione concentrations in rat liver. J. Nutr. Biochem. 15 ll2-T22.

Leibbrandt, V. D., Ewan, R. C., Speer, V. C. and Zimmerman, D. R' 1975' Effect of weaning and age at weaning on baby pig performance. J. Anim. Sci. 40: 1077-1080.

Leung-Toung, R., Li, W., Tam, T. F. and Karimian, K. 2002. Thiol-dependent enzymes and their inhibitors: a review. Cun. Med. Chem. 9:979-1002-

Lieber, C. S. and DeCarli, L. M. 1966. Study of agents for the prevention of the fatty liver produced by prolonged alcohol intake. Gastroenterology. 50 316-322.

Lindemann, M. D. and Korneg ay,E.T. 1989. Folic acid supplementation to diets of gestating-lactating swine over multiple parities. J. Anim. Sci. 67: 459-464.

Lindenbaum, J., Healton, E. B., Savage, D.G., Brust, J. C., Garrett, T. J., Podell,8.R., Parcell, P. D., Stabler, S. P. and Allen, R. H. 1988. Neuropsychiatric disorders caused by cobalamin deficiency in the absence of anemia or macrocytosis. N. Engl. J. Med. 218: t720-1728. r26

Loriette, C., Pierre, Y. and Chatagner, F. 1980. Effect of dietary cholic acid and cholesterol on liver and kidney cystathionase and cysteine sulphinate decarboxylase activities and taurine concentrations in the rat. Experientia. 36:285-286.

Lovett, T. D., Coffey, M. T., Miles, R. D. and Combs, G' E. 1986' Methionine, choline and suiphate interrelationships in the diet of weanling swine. J. Anim. Sci. 63: 467-471.

Lu, S. C. 1998. Regulation of hepatic glutathione synthesis. Sem. Liver Dis. 18: 331- 343.

Lu, S. C. 2000. S-Adenosylmethionine. Int. J. Biochem' Cell Biol. 32:291-395'

Lu, S-H. and Huang, P-C. 1997. Effects of casein and sow protein isolate on vitamin Bu nutritional status in rats. J. Biomed. Sci' 4: 120-124'

Mahan, D. C. and vallet, J.L. lgg7. vitamin and mineral transfer during fetal development and the early postnatal period in pigs. J. Anim. Sci. 75: 2731-2738.

Malloy, M. H., Rassin, D. K. and McGanity, W. J. i983' Maternal-fetal cyst(e)ine transfer. Biol. Neonate.44 l-9.

Malmezat, T., Breuillé, D., Poufi, C., Patreau Mirand, P. and Obled, C' 1998' Metabolism of cysteine is modified during the acute phase of sepsis in rats. J' Nutr. 128: 97 -105.

Malmezat, T., Breuillé, D., Pouyrt, C., Patreau Mirand, P. and Obled, C. 2000a' Methionine transsulphuration is increased during sepsis in rats. Am. J' Physiol' Endocrinol. Metab. 27 9 : El39l -1397 .

Malmezat,T., Breuillé, D., Capitain, P., Patreau Mirand, P. and Obled, C. 2000b' 130: Glutathione tumover is increased during the acute phase of sepsis in rats' J. Nutr. 1239-1246.

E., Mansoor, o., Beaufrère, B., Boirie, Y., Rallière, c., Taillandier, D., Aurousseau, components Schoeffler, p., Arnal, M. and Attaix, D. i996. Increased mRNA levels for pathways of the lysosomal, Cazt-activated and ATP-ubiquitin-dependent proteolytic in skeletai muscle from head trauma patients. Proc. Natl. Acad. Sci. USA. 93l.2714'2718.

Martensson, J. Jain, A. and Meister, A. 1990. Glutathione is required for intestinal function. Proc. Natl. Acad. Sci. USA. 87: 1715-1719'

Martinez,M., Cuskelly, G. J., Williamson, J., Toth, J. P. and Gregory III, J. F. 2000' and Vitamin Bu d"fi"i.ttcyin rats reduces hepatic serine hydroxymetþltransferase r27 cystathionine beta-synthase activities and rates of in vivo protein tumover, homocysteine remethylation and transsulphuration. J. Nutr. 130: 1115-1123'

Matte, J. J., Farmer, C., Girard, C. L. and Laforest, J. P. 1996. Dietary folic acid, uterine function and early embryonic development in sows. Can. J. Anim. Sci. 76: 427-433.

Matte, J. J. and Girard, C. L. 1989. Effects of intramuscular injections of folic acid during lactation on folates in serum and milk and performance of sows and piglets. J. Anim. Sci.67: 426-431.

Matte, J. J. and Girard, C.L. 1999. An estimation of the requirement for folic acid in gestating sows: the metabolic utilization of folates as a criterion of measurement. J. Anim. Sci.77: 159-165.

Matte, J. J., Girard, C. L. and Brisson, G. J. 1984. Folic acid and reproductive performance of sows. J. Anim. Sci. 59: t020-I025.

Matte, J. J., Girard, C,L. and Brisson, G. J. 1992. The role of folic acid in the nutrition of gestating and lactating primiparous sows. Livestock Prod. Sci. 32: I31-148.

Matte, J. J., Girard, C. L. and Sève, B. 2001. Effect of long-term parenteral administration of vitamin Bu on Bu status and some aspects of the glucose and protein metabolism of early-weaned pigs. Br. J. Nutr. 85: 11-21'

Matte, J. J., Girard , C.L. and Tremblay, G. F. 1993. Effect of long-term addition of folic acid on folate status, growth performance, puberly attainment, and reproductive capacity of gilts. J. Anim. sci. 71: 151-157.

Matthews, D. E. 1999. Protein and Amino Acids. In: Modern Nutrition in Health and Disease, 9th edition. (Shils, M. E., Olson, J. 4., Shike, M'., Ross, A. C., eds). Williams and Wilkins. Baltimore, Maryland. pp: 11-48.

McCully, K. S. 1975. Homocysteine, atherosclerosis and thrombosis: Implications for oral contraceptive users. Am. J. Clin' Nutr. 28:542-549

Meister, A. and Anderson, M. E. 1983. Glutathione. Annu. Rev. Biochem.52:7Il-760.

Mercier, S., Breuillé, D., Mosoni, L., Obled, C. and Mirànd, P.P ' 2002. Chronic inflammation alters protein metabolism in several organs of adult ¡ats. J. Nutr. 132: I92l- 1928.

Miller, J. W., Nadeau, M. R., Smith, J., Smith, D. and Selhub, J. 1994- Folate- deficiency-induced homocysteinaemia in rats: disruption of S-adenosylmethionine's co- 128 ofdinate regulation of homocysteine metabolism. Biochem' J - 298 415-419.

Miller, E. R. and Ullrey, D. E. 1987. The pig as a model for human nutrition. Ann. Rev' Nutr.7: 261-382.

Miller, L. T., watson, w. H., Kirlin, v/. G., ziegler,T. R. and Jones, D.P. 2002. Oxidation of the glutathione/glutathione disulphide redox state is induced by cysteine deflrciency in human colon carcinoma HT29 cells' J. Nutr. 132: 2302-2306.

Mitchell, A. D. and Benevenga, N. J. 1978. The role of transamination in methionine oxidation in the rat. J. Nutr' 108: 67-78.

Molloy,4.M., Orsi,8., Kennedy, D.G., Kennedy, S., Weir, D' G' and Scott, J' M' 1992. The relationship between the activity of methionine synthase and the ratio of S- adenosylmethionine to S-adenosylhomocysteine in the brain and other tissues of the pig. Biochem. Pharmacol. 44: 1349-1355.

Mosharov, E., Cranford, M. R. and Banerjee. R. 2000. The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulphuration pathway and its regulation by redox changes. Biochemistry. 39: 13005- i3011.

Mrozek, J. D., Georgieff, M. K., Blazer,8.R., Mammel, M. C' and Schwarzenberg, S' J' 2000. Effect of sepsis syndrome on neonatal protein and energy metabolism. J' Perinatol. 20:96-100.

Mudd, S. H., Finkelstein, J.D., Irreverre, F. and Laster, L. 1965. Transsulphuration in mammals; Microassays and tissue distributions of three enzymes of the pathway' J. Biol' Chem. 240:4382-4392.

Mudd, S. H. and Poole, J. R. 1975. Labile methyl balances for normal humans on various dietary regimens. Metabolism . 24: 721-735.

Nakagawa,H. lgTL studies on the enzymological basis of amino acid nutrition. physiólogical roles of serine dehydratase and cystathionine synthase in rat liver' Seikagaku. 43: I-22.

Narkewicz, M. R., Jones, G., Thompson, H., Kolhouse, F. and Fennessey, P.V. 2002. Folate cofactors regulate serine metabolism in fetal ovine hepatocytes' Pediatr. Res. 52: 589-594.

National Research Council. 1998. Nutrient Requirements of Swine, 10ù ed. National Academy Press. V/ashington, D' C. r29

Natsuhori, M., Shimoda, M. and Kokue, E. 1996. Altemation of plasma folates in gestating sows and newbom piglets' Am. J. Physiol. 270:R99-104'

Nelson, D. L. and Cox, M. M. 2000. Lehninger Principles of Biochemistry, 3'd ed. Worth Publishers. New York, New York.

Nielson, T. T., Trottier, N. L, Stein, H.H., Bellaver, C. and Easter, R. A. 2002. The effect of litter size and day of lactation on amino acid uptake by the porcine maÍìmary glands. J. Anim. Sci. 80: 2402'2411.

O'Connor, D. L., Picciano, M. F., Roos, M. A. and Easter, R. A. 1989. Iron and folate utilization in reproducing swine and their progeny. J. Nutr. 119: i984-1991'

Ohmori, H. and Yamamoto,I. 1982. A mechanism of the augmentation of antibody response in vitro by 2-mercaptoethanol: facilitation of cystine uptake in murine lymphocytes. Int. J. Immunopharmacol' 4: 47 5-419

J. and Friesen, K. owen, K. Q., Nelssen, J. L., Goodband, R. D., Tokach, M. D., Kats, L. G. 1995. Added dietary methionine in started diets containing spray-dried blood products. J. Anim. Sci. 73: 2647-2654'

Pearson, P. L., Klemcke, H. G., Christenson, R. K. and Vallet, J' L. 1998. Porcine endometrial haptocorrin: cloning of partial cDNA and expression from days 10 to 40 of gestation. Biol. Reprod. 58 (Suppll): 193.

Pettigrew, J. E., Walker, R. D. and White, M. E. 1995. The Early Weaned Pig: The Role of Age at Weaning, Health Status, and Diet. In: Animal Science Research and Development: Moving Toward a New Century. [van, M., ed). Centre for Food and AnimalResearch, Agriculture and Agri-Food Canada. Ottawa, ON.pp. 329-337 '

pohlandt, F. 1974. Cystine: a semi-essential amino acid in the newbom infant. Acta Pædiatr. Scand. 63: 801-804'

prathapasinghe, G. 4., Brosnan, M. E. and Brosnan, J. T. 2004. Intestinal metabolism of sulphui-containing amino acids in the rat. FASEB J. 19: Abstract#6125.

Premer, D. M., Goertz,R., Gerogieft M.K., Mammel, M' C' and Schwarzenberg, S' J' 2002. Muscle proteolysis and weight loss in neonatal rat model of sepsis syndrome. Inflammati on. 26: 97 -101.

Radcliffe, B. C. and Egatt, A. R. 1978. The effect of diet and of methionine loading on activity of enzymes in the transsulphuration pathway in sheep. Aust. J. Biol. Sci. 31: 105- 14. i30

Rao, A. M., Drake, M. R. and Stipanuk, M. H. 1990. The role of the transsulphuration pathway and of y-cystathionase activity in the formation of cysteine and sulphate from methionine in rat hepatocytes. J. Nutr. I20:837-845.

Rao, P. V., Garrow, T. 4., John, F., Garland, D., Millian, N. S. andZigler Jr., J. S. 1998. Betaine-homocysteine methyltransferase is a developmentally regulated enzyme crystallin in Rhesus monkey lens. J. Biol. Chem.273:30669-30674.

Ratnam, S., Maclean, K.N., Jacobs, R. L., Brosnan, M. 8., Kraus, J. P. and Brosnan, J. T. 2002. Hormonal regulation of cystathionine beta-synthase expression in liver. J. Biol. Chem. 277 : 42912-42918.

Redmond, H. P., Stapleton, P. P., Neary, P. and Bouchier-Hayes, D. 1998. Immunonutrition: The role of taurine. Nutrition. 14:599-604.

Reeds, P. J., Burrin, D. G., Jahoor, F., Wykes, L., Henry, J. and Frazer,E.M. 1997. Enteral glutamate is the preferential source for mucosal glutathione synthesis in fed piglets. Am. J. Physiol. 270:8413-418.

Refsum, H. 2001. Folate, vitamin B,, and homocysteine in relation to birth defects and pregnancy outcome. Br. J. Nutr. 85(Suppl 2): Sl09-113.

Refsum, H., Guttormsen, A. 8., Fiskerstrand, T. and Ueland, P. M. i998a. Hyperhomocysteinemia in terms of steady-state kinetics. Eur. J. Pediatr. 157(Suppl2): s45-49.

Refsum, H., Ueland, P. M., Nygard, O. and Vollset, S. E. 1998b. Homocysteine and cardiovascular disease. Annu. Rev. Medicine. 49 : 3 I -62.

Register, U. D., LaSorsa,4.M., Katsuyama, D. M. and Smith, H. M. 1959. Soluble sulphhydral changes in dietary and environmental stress. Am. J. Physiol. 197: 1353-1356.

Rodvien, R., Gillum, A. and Weintraub, L. R. I974. Decreased glutathione peroxidase activity secondary to severe iron deficiency: a possible mechanism responsible for the shortened life span of the iron-deficient red cell. Blood. 43:281-289.

Rooyackers, O. E., Saris, W. H. M., Soeters, P. B. and'Wagenmakers, A. J. M. 1994. Prolonged changes in protein and amino acid metabolism after zymosan treatment in rats. Clin. Sci. 87 619-626.

Rose, D. P. and Braidman,I. P. 1971. Excretion of tryptophan metabolites as affected by pregnancy, contraceptive steroids, and steroid hormones. Am. J. Clin. Nutr. 24: 673-683. 131

Roth, F. X. and Kirchgessner, M. 1989. Influence of the methionine:cystine relationship in the feed on the performance of growing pigs. J. Anim. Physiol. Anim. Nutr. 61 :265- 274.

Sasse, C. E. and Baker, D.H. 1974. Sulphur utilization-by the chick with emphasis on the effect of inorganic sulphate on the cystine-methionine interrelationship. J. Nutr. 104: 244-25r.

Sauer, H. and Wilmanns, W. 1977. Cobalamin dependent methionine synthesis and methyl-folate-trap in human vitamin 8,, deficiency. Br. J. Haematol. 36: 189-198. Abstract.

Saunderson,C.L.and MacKinlay, J. 1990. Changes in body-weight, composition and hepatic e4me activities in response to dietary methionine, betaine and choline levels in growing chicks. Br. J. Nutr. 63: 339-349.

Schoknecht, P. 4., Pond, 'W. G., Mersmann, H. J. and Maurer, R. R. 1993. Protein restriction during pregnancy affects postnatal growth in swine progeny. J. Nutr. 123: r8l8-1825.

Selhub, J. 1999. Homocysteine metabolism. Annu. Rev. Nutr. 19: 2I7-246.

Selhub, J., Jacques, P. F., Wilson, P. V/., Rush, D. and Rosenberg,I. H. 1993. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA. 270:2693-2698.

Selhub, J. and Miller, J. W. 1992. The pathogenesis of homocysteinemia: Intemrption of the coordinate regulation of S-adenosylmethionine of the remethylation and transsulphuration of homocysteine. J. Nutr. 55: 131-138.

Sen, C. K. 1997. Nutritional biochemistry of cellular glutathione. Nutr. Biochem. 8: 600-672.

Shane, B. and Stokstad, E. L. R. 1985. Vitamin B,r-folate interrelationships. Annu. Rev. Nutr. 5: 115-141

Shannon, B. and Demos, P. 1917 . Influence of estrogen treatment on hepatic cystathionine synthase and cystathionase of female rats fed varying levels of vitamin Bu. J. Nutr. 107 : 1255-1262.

Shoveller, A. K., Brunton, J. 4., House, J. D., Pencharz, P. B. and Ball, R. O' 2003. Dietary cysteine reduces the methionine requirement by an equal proportion in both parenterally and enterally fed piglets. J. Nutr. 133:4215-4224. r32

Sigman, J. 4., Sharky, M.L., Walsh, S. T., Pabofl, A.,Glucksman, M.J.and V/olfson, A. J. 2003. Involvement of surface cysteines in activity and multimer formation of thimet oligopeptidase. Protein Eng. 16: 623-268.

Simard, F., Guay, F., Laforest, J. P., Giguère, 4., Girard, C. L. and Matte, J. J. 2002' Dietary Vitamin 8,, supplements in gestating gilts and 8,, transfer to piglets during lactation. Joint Meeting of the American Society of Dairy Science, the American Society of Animal Science and the Canadian Society of Animal Science. Quebec City, QB. pp. 163. Abstract.

Smith, J. T. 1973. An optimal level of inorganic sulphate for the diet of a rat. J. Nutr. 103: 1008-101 1.

Smolin, L. 4., Crenshaw, T. D., Kurtycz, D. and Benevenga, N. J. 1983. Homocyst(e)ine accumulation in pigs fed diets deficient in vitamin Bu: Relationship to atherosclerosis. J. Nutr. I 13:2122-2133.

Stabler, S. P., Brass, E. P., Marcell, P. D. and Allen, R. H. 1991. Inhibition of cobalamin-dependent enzymes by cobalamin analogues in rats. J. Clin. Invest. 87:1422- 1430.

Stangl, G. L and Kirchgeßner, M. 1998. Effect of different degrees of moderate iron deficiency on the activities of tricarboxylic acid cycle enzymes, and the cytochrome oxidase, and the iron, copper, and zinc concentrations in rat tissue. Z. Emåihrungswiss. 37:260-268.

Stangl, G. I., Roth-Maier, D. A. and Kirchgeßner, M. 2000. Vitamin B', deficiency and hyperhomocysteinemia are partly ameliorated by cobalt and nickel supplementation in pigs. J. Nutr. 130: 3038-3044

Stead, L. M., Brosnan, M. E. and Brosnan, J. T. 2000. Characterization of homocysteine metabolism in the rat liver. Biochem. J. 350: 685-692'

Sternowsþ, H.J., Räihä, N. C. R. and Gaull, G. 1976. Methionine adenosyltransferase and transmethylation in fetal and neonatal lung of the human, monkey and rabbit. Pediatr. Res. 10: 545-550.

Stipanuk, M. H. 1979. Effect of excess dietary methionine on the catabolism of cysteine in rats. J. Nutr. 109:2126-2139.

Stipanuk, M. H. 1999. Homocysteine, Cysteine, and Taurine. In: Modern Nutrition in Health and Disease, 9th edition. (Shils, M. 8., Olson, J. A., Shike, M', Ross, A. C., eds). 'Williams and Wilkins. Baltimore, Maryland.pp: 543-558' 133

Stipanuk, M. H. 2004. Sulphur amino acid metabolism: Pathways for production and removal of homocysteine and cysteine. Annu. Rev. Nutr. 24: 539-577.

Stipanuk, M. H., Coloso, R. M., Garcia, R. A. G. and Banks, M.F. 1992. Cysteine concentration regulates cysteine metabolism to glutathione, sulphate and taurine in rat hepatocytes. J. Nutr. 122: 420-427 .

Stoll, 8., Henry, J., Reeds, P. J., Yu, H., Jahoor, F. and Burrin, D. G. 1998. Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets. J. Nutr. 128:606-614.

Storch, K. J.,'Wagner, D. 4., Burke, J. F. and Young, V. R. 1988. Quantitative study ln vivo of methionine cycle in humans using [methyl-2Hr] and [1-'3C]methionine. Am. J. Physiol. 258: 8322-331.

Sturman, J. 4., Cohen, P. A. and Gaull, G. E. 1969. Effects of deficiency on vitamin Bu in transsulphuration. Biochem' Med' 3: 244-251.

Sturman, J. A. and Chesney, R. W. 1995. Taurine in pediatric nutrition. Pediatr. Nutr. 42:879-897.

Sturman, J. 4., Gargano, A. D., Messing, J.M. and Imaki, H' 1986. Feline matemal taurine deficiency: effect on mother and offspring. J. Nutr. 116:655-667.

Sturman, J. 4., Gaull, G. E. and Niemann, W. H. 1976a. Activities of some enzymes involved in homocysteine methylation in brain, liver and kidney of the developing Rhesus monkey. J. Neurochem. 27 : 425-431.

Sturman, J. 4., Gaull, G. E. and Niemann, W. H. 1976b' Cystathionine synthesis and degradation in brain, liver and kidney of the developing monkey. J. Neurochem.26:457- 463.

Sturman, J. 4., Gaull, G. E. and Räihä, N. C. R. 1970. Absence of cystathionine in human fetal liver: Is cystine essential? Science. 169:74-76.

Sturman, J. 4., Gaull, G. E. and Räihä, N. C. R. 1975. DNA synthesis from the B- carbon of serine by fetal and mature human liver. Biol. Neonate. 27: 17 -22.

Sturman, J. A. and Messing, J. M. 1991. Dietary taurine content and feline reproduction and outcome. J. Nutr. I2l: 1195-1203.

3sS-methionine Sturman, J. 4., Niemann, W. H. and Gaull, G.E. 1973. Metabolism of and 35S-cystine in the pregnant Rhesus monkey. Biol. Neonate.22: 16-37. 134

Takeuchi, F.,Izuta, S., Tsubouchi, R. and Shibata,Y. 1991. Glutathione levels and related enzyme activities in vitamin 8-6 deficient rats fed a high methionine and low cystine diet. J. Nutr. 121: 1366-1373.

Tamura, T., Hong, K. H., Mizuno, Y., Johnston, K. E. and Keen, C.L. 1999. Folate and homocysteine metabolism in copper-deficient rats. Biochim. Biophys. Acta.1427: 351- 356.

Taoka, S., West, M. and Banerjee, R. 1999. Characterization of the heme and pyridoxal phosphate cofactors of human cystathionine beta-synthase reveals nonequivalent active sites. Biochemistry. 38: 2738-27 44.

Tateishi, N., Higashi, R., Shinya, S., Narsue, A. and Sakamoto, Y. I974' Studies on the regulation of glutathione level in rat liver. J. Biochem . 7 5: 93-103 '

Tateishi, N., Higashi, R., Narsue,4., Nakashima, K., Shiozaki, H. and Sakamoto, Y. T977 . Ptat liver glutathione: possible role as a reservoir of cysteine. J. Nutr. 107: 5 1-60.

Tateishi, N., Higashi, R., Narsue, 4., Hikata, K. and Sakamoto, Y. 1981. Relative contributions of sulphur atoms of dietary cysteine and methionine to rat liver glutathione and proteins. J. Biochem. 90: 1603-1610.

Thacker, P. A. 1999. Nutritional requirements of early-weaned pigs. Asian-Aus. J. Anim. Sci. l2: 976-987.

Thaler, R. C., Nelssen, J. L., Goodband, R. D. and Allee, G. L. 1989. Effect of dietary folic acid supplementation on sow performance through two parities. J. Anim. Sci. 67: 3360-3369.

Townsend, J. H., Davis, S. R., Mackey, A. D. and Gregory III, J. F. 2004. Folate deprivation reduces homocysteine remethylation in a human intestinal epithelial cell culture: role of serine in one-carbon metabolism. Am. J. Physiol. Gastrointest. Liver Physiol. 286: G588-595

Trachtman, H., Barbow, R., Sturman, J. A. and Finberg, L. 1988' Taurine and osmoregulation: Taurine is a cerebral osmoprotective molecule in chronic hyperatremic dehydration. Pediatr. Res. 23: 35-39.

Trembley, G. F., Matte, J. J., Dufour, J. J. and Brisson, G. J. 1989. Survivial rate and development of fetuses during the first 30 days of gestation after folic acid addition to a swine diet. J. Anim. Sci. 67: 724-732.

Triguero, 4., Barber, T., Garcia, C., Puertes, L R., Sastre, J. and Viña, J. R. 1997. Liver 13s

intracellular L-cysteine concentration is maintained after.inhibition of the transsulphuration pathway by propargylglycine. Br. J. Nutr. 78: 823-831.

Trottier, N. L., Shipley, C. F. and Easter, R. A. 1997. Plasma amino acid uptake by the mammary gland of the lactating sow. J. Anim. Sci. 75: 1266-1278.

Trugo, N. M. F., Ford, J. E. and Sansom, B. F. 1985b. Vitamin 8,, absorption in the neonatal piglet. 1. Studies in vivo on the influence of vitamin B,r-binding protein from sows' milk on the absorption of vitamin 8,, and related compounds. Br. J. Nutr. 54: 245- 255.

Trugo, N. M. F., Ford, J. E. and Salter, D. N. 1985a. Vitamin 8,, absorption in the neonatal piglet. 3. Influence of vitamin B,r-binding protein from sows' milk on uptake of vitamin 8,, by microvillus membrane vesicles prepared from small intestine of the piglet. Br. J. Nutr. 54: 269-283.

Tyson, J. E., Lasky, R. Flood, D., Mize, C. Picone, T. and Paule, C. L. 1989. Randomized trial of taurine supplementation for infants less than or equal to 1300-gram birth weight: Effect on auditory brainstem, evoked responses. Pediatrics. 83:406-4T5..

Vallet, J. L., Christenson, R. K. and Klemcke, H. G. 1998. Purification and characteÅzation of intrauterine folate-binding proteins from swine. Biol. Reprod. 59:

1 761-81 .

Vallet, J. L., Christenson, R. K. and Klemcke, H. G. 1999. A radioimmunoassay for porcine intrauterine folate binding protein. J. Anim. Sci. 77: 1236-1240.

VanAerts, L. A. G. J.M., Poirot, C. M., Herberts, C.4., Blom, H. J., De Abreu, R. 4., Trijbels, J. M. F., Eskes, T. K. A. 8., Peereboom-Stegeman, J. H. J. C. and Noordhoek, J. 1995. Development of methionine synthase, cystathionine-B-synthase and S- adenosylhomocysteine hydrolase during gestation in rats. J. Repro. Fert. 103: 227-232.

Van der Molen, E. F., Van den Heuvel, L. P. W. J., Te Poele Pothaff, M. T. W. 8., Monnens, L. A. H., Eskes, T. K. A. B. and Blom, H. J. 1996. The effect of folic acid on the homocysteine metabolism in human umbilical vein endothelial cells (HUVECs). Eur. J. Clin. Invest. 26:304-309.

Verjee, 2.H., Siddons, R. C., Bharaj, B. S. and Deana, R. 1975. A biochemical study of vitamin 8,, deficiency in the baboon. Int. J. Vitam. Nutr. Res. 45 273-283.

Viña, J., Gimenez, 4., Puertes, I. R., Gasco, E. and Viña, J. R. 1992. Impairment of cysteine synthesis from methionine in rats exposed to surgical stress. Br. J. Nutr. 68: 42I- 429. 136

Viña, J., Vento, M., García-Sala, F., Puertes, I' R., Gascó, E', Sastre, J., Asensi, M. and Pallardó, F. V. 1995. L-cysteine and glutathione metabolism are impaired in premature infants due to cystathionase deficiency. Am. J. Clin. Nuti'. 61:1067-1069.

Volpe, J. J. and Laster, L. 1972. Transsulphuration in fetal and postnatal mammalian liver and brain. Biol. Neonate.20:385-403'

Whiting, T. 2001. Isolated Weaning Technology: Incorporation Into the Canadian Code of Practice- Pigs. Manitoba Agriculture, Food and Rural Initiatives Website. Available at: http: fAccessed 031291041.

Wolfe, R. R., Jahoor, F. and Harti, W. H. 1989. Protein and amino acid metabolism after injury. Diabetes Metab. Rev. 5: 149-164.

Wright, C. E., Tallan, H. H. and Lin, Y. Y. 1986. Taurine: biological update. Annu. Rev. Biochem. 55:427-453.

Wu, G. 1998. Intestinal mucosal amino acid catabolism. J. Nutr. 128: 1249-1252.

Wu, G., Flynn, N. E., Knabe, D. A. and Jaeger, L. A. 2000a. A cortisol surge mediates the enhanced polyamine synthesis in porcine enterocytes during weaning. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 27 9 : R5 5 4-5 59.

Wu, G. and Knabe, D. A. 1994. Frce and protein-bound amino acids in sow's colostrum and milk. J. Nutr. 124:415-424'

'Wu, G., Meininger, C. J., Kelly, K., Watford, M. and Morris, S. M. 2000b. A cortisol surge mediates the enhanced expression of pig intestinal pynoline-5-carboxylate synthase during weaning. J. Nutr. t30 1914-1919.

Wykes, L. J., House, J. D., Ball, R. O. and Pencharz, P. B. 1994. Amino acid prfile and aromatic amino acid concentration in total parenteral nutrition: effect on growth, protein metabolism and aromatic amino acid metabolism in the neonatal piglet. Clin. Sci. 87:75- 84.

Xu,Z.,Byers, D.M., Palmer, F. 8., Spence, M. V/. and Cook, H. W. 1991. Serine utilization as a precursor of phosphatidylserine and alkenyl-þlasmenyl)-, alþI-, and acylethanolamine phosphoglycerides in cultured glioma cells. J. Biol Chem .266:2143- 2150.

Yamada, K., Kawata, T., Wada, M.,Isshiki, T., Onoda, J., Kawanishi, T., Kunou,4., Tadokoro, T., Tobimatsi, T., Maekawa, A. and Toraya, T. 2000. Extremely low activity 137 of methionine synthase in vitamin B,r-deficient rats may be related to effects on coenzymes stabilization rather than to changes in coenzyme induction. J. Nutr. 130: 1894- r 900.

Yamamoto, N., Tanaka, T. and Noguchi, T. 1995. Effect of cysteine on expression of cystathionine beta-synthase in the rat liver. J. Nutr. Sci. Vitaminol. 4l: I97-205.

Yamamoto, N., Tanaka, T. and Noguchi, T. 1996. The effect of a high-protein diet of cystathionine beta-synthase and its transcript levels in rat liver. J. Nutr. Sci. Vitaminol. 42:589-593.

Yamauchi, A. and Bloom, E. T. 1993. Requirement of thiol compounds as reducing agents of Il-2-mediated induction of LAK activity and proliferation of human NK cells. J. Immunol. 151 : 5535-5544.

Yamuachi, 4., Tsuyuki, S., Inamoto, T. and Yamaoka, Y. 1999' Liver immunity and glutathione. Antiox. Redox Signal. I : 245 -253.

Yip, R. and Dallman, P. R. 1996. Iron. In: Present Kno.wledge in Nutrition, 7'h edition. (Ziegler,E. E., Filer, L. J. ed). ILSI Press. Washington, DC. pp:277-292.

Young, M. lgTL Placental transport of free amino acids. In: Metabolic Processes in the Foetus and Newborn Infant. (Jonxis, J. H. P., Visser, H. K. A. and Traveistra, J. 4., eds). H. E. Stefert Knoese, N. V. Leiden. The Netherlands. pp.97.

Zlotkin, S. H. and Anderson, G. H. 1982. The development of cystathionase activity during the first year of life. Pediatr. Res. 16: 65-68'

Zlotkin, S. H., Bryan, M. H. and Anderson, G. H. 1981. Cysteine supplementation to cysteine-free intravenous feeding regimens in newborn infants. Am. J. Clin. Nutr. 34: 914-923. 138

Chapter 9 - Appendices

Solution Preparation Methods

2M Potassium Borate Buffer. pH 9.5 (Solution A) l. Dissolve ll2.22gpotassium hydroxide (KOH) in approximately 900mL of deionized water. Bring to a f,rnal volume of 1L using deionized water.

2. Add l23.66gboric acid and 300mL of the KOH solution to approximately 900mL of deionized water.

3. Dissolve the boric acid with constant stirring and low-medium heat'

4. Cool the solution only until it can be handled and bring the boric acid to a final volume of I L. Note that since 300mL of KOH was added to the boric acid, the final combined boric acid/KOH volume should be 1.3L.

5. Add KOH until pH 9.5 is reached.

6. Filter.

GDTA)) (Solution B)

1. Using Solution A, continue adding KOH until a pH of 10.5 is reached.

2. Calculate the amount of EDTA that must be added to make a 5mM EDTA solution (5mM EDTA : l.46glL).

3. Filter.

1. Make a l:20 dilution of Solution A using deionized water (50mL of Solution A + 950mL deionized water).

2. Calculate the amount of EDTA that must be added to make 2mM EDTA solution (0.s8sg/L) 139

3. Filtering is optional.

Tris (2-Carbox)¡eth)¡l)- Phosphine H)¿drochloride (TCEP) (Reducing A gent)

1. Make a 10 per cent TCEP solution by weighing 100mg of TCEP directly into a microcentrifuge tube and adding lml, of deionized water. A one per cent TCEP solution may be made using 10mL of deionized water.

2. Vortex.

0.6M Perchloric acid

1. Add 25.75mL of perchloric acid to approximately 400mL of deionized water and bring to a final volume of 500mL using deionized water.

7-Fluro-2-Oxa- 1 .3 -Diazole-4-Sulfonic Acid (SBDF)

1. Measure 1mg of SBDF into a glass test tube and add lmL of Solution A.

2. Vortex. Note that warming the solution may be necessary to dissolve the SBDF.

0.lM Sodium Acetate Buffer (pH 5. containing 2 per cent methanol) - Buffer A

1. Dissolve 13.6089 sodium acetate trihydrate in approximately 900mL deionized water. Bring solution to a final volume of 1L using deionized water to make a 0.1M sodium acetale solution.

2. Add 5.75mL glacial acetic acid to approximately 900mL deionized water. Bring to a final volume of 1L using deionized water to make a 0.1M glacial acetic acid solution.

3. Slowly add the 0.1M glacial acetic acid solution to the entire amount 0.1M sodium acetate solution until a pH of 5 is reached'

4. Measure the amount of the prepared pHed solution in a graduated cylinder. Calculate and add the appropriate amount of methanol to achieve a2 per cent methanol solution.

5. Filter. 140

0.05M Potassium Phosphate Buffer. pH 6.9

1. Dissolve 1.361g potassium phosphate monobasic in approximately 150mL of deionized water. Bring to a final volume of 200mL using deionized water to make a 0.05M potassium phosphate monobasic solution'

2. Dissolve l.7 2gpotassium phosphate dibasic in approximately 150mL of deionized water. Bring to a final volume of 200mL using deionized water to make a 0.05M potassium phosphate dibasic solution.

3. Add the 0.05M monobasic solution to the entire 0.05M dibasic solution until a pH of 6.9 is reached.

4. Store the solution at 4"C for up to one week.

Methionine Svnthase Reaction Mixture

1. Dissolve 13.2gpotassium phosphate monobasic in approximately 400mL of deionized \À/ater. Bring to a f,rnal volume of 500mL using deionized water to make a 0'05M potassium phosphate monobasic solution.

2. Dissolve i6.99 potassium phosphate dibasic in approximately 400mL of deionized water. Bring to afinal volume of 500mL using deionized water to make a 0.05M potassium phosphate dibasic solution.

pH 3. Add the 0.05M dibasic solution to the entire 0.05M monobasic solution until a of 7.3 is reached. This solution can be stored at 4"c for up to one week.

4. In approximately 50mL of 0.05 potassium phosphate buffer, pH7.3, dissolve 3mg çyunoóóbulamin, 233mgD, L-homocysteine, and 112.9uL B-mercaptoethanol' Bring the råaction mixture to final volume of 100mL using the 0.05 potassium phosphate buffer, pH7.3.

5. Weigh 2mgof S-adenosylmethionine directly into a small beaker. Using a pipette, add 4mL of the reaction mixture prepared in Step #4.

6. Weigh 89.4mg dithiothreitol into a weigh-boat and wash it into the same small beaker using 4mL of the reaction mixture prepared in Step #4. Cover and set aside'

water' Bring to a 7. Add 1 .981g sodium ascorbate to approximately 70ml of deionized final volume of 100mL using deionized water to make a 0.1M sodium ascorbate buffer. r4t

8. Weight 4.98mg metþltetrahydrofolate (calcium salt) into a microcentrifuge tube and add lml- of 0.1M sodium ascorbate. Vortex solution.

9. Add the entire lml- contents of the eppendorf to the 8mL reaction mixture solution set aside in Step #6. Bring the complete reaction mixture to a final volume of 1OmL using the solution prepared in Step #4.

Cystathionine Gamma-Lyase Reaction Mixture

1. Add 22.3mgof L(+)cystathionine (?) to approximately 15mL of 0.05M potassium phosphate buffer, pH 6.9 (see above for recipe), the buffer used for tissue homogenization

2. Weigh Z2mgof (PLP)into a microcentrifuge tube and add lml- of homogenizing buffer. Remove 38.5uL of this solution and add it to the mixture prepared in Step #1.

3. Weigh Z}mgof nicotinamide adenine dinucleotide (NADH) into a microcentrifuge tube and add 1mL of homogenizingbuffer. Remove 298uL of this solution and add it to the mixture prepared in SteP #1.

4. Calculate the correct amount of lactate dehydrogenase (LDH) to obtain 1.5units/ml and add that amount to the mixture prepared in Step #1'

5. Bring the completed reaction mixture to a final volume of 25mL using 0.05M potassium phosphate buffer, pH 6.9 and store on ice.

6. Reconstitute[5-r4C]-methyltetrahydrofolate in one microcentrifuge tube of using lmL of chilled reaction mixture. Vortex.

7. Combine the 1mL [5-r4C]-methyltetrahydrofolate solution with 3mL reaction mixture in a capped glass vial.

8. Vortex and store on ice.

Cystathionine B eta-Synthase Reaction Mixture

1. Dissolve IS.ITgTrizmabase in approximately 100mL deionized water. Bring to a final volume of 150mL using deionized water to make a 1M Trizma buffer, pH 8.4.

2. Dissolve 1.05g L-serine, 146mg EDTA and 28mg D, L-propargylglycine in approximately 60mL of 1M Triz.mabuffer. Bring to a final volume of 100mL using the same buffer. t42

L-Homocysteine

1. Weight l5.4mg L-homocysteine-thiolactone into a microcentrifuge tube.

2. Weigh 1.4039 potassium hydroxide (KOH) into a beaker and add iOmI- of deionized water to make aZ.SMKOH solution. Dissolve with constant stining.

3. Add 400uL of KOH to the eppendorf and vortex. Let mixture incubate at room temperature for five minutes.

4. Add 3.87mL of hydrochloric acid (HCl) to 6.13mL deionized water to make a45M HCI solution.

5. Combine 2.57mL of 4.5M HCI and 4.43m1of lM Trizmabuffer, pH 8.4 (see above). Add 600uL of mixture to the microcentrifuge tube to neutralize the solution.

0.4M Sodium Borate. pH 9.5

L Add 3.2g sodium hydroxide Q.iaOH) to approximately 150mL deionized water. Bring to a final volume of 200mL with deionized water to make a 0.4M NaOH solution.

Z. Add 4.95gboric acid to approximately 150mL deionized water and . Dissolve the boric acid with constant stining and low-medium heat. Bring to final volume of 200mL with deionized water to make a 0.4M boric acid solution'

3. Add the 0.4M NaOH solution to the entire 0.4M boric acid solution until a final concentration of 9.5 is reached.

4. Store solution at room temperature for up to one week.

o-Phthaldialdehyde Reagent

t. Weigh 5Omg o-phthaldialdehyde (OPA) into a microcentrifuge tube. Add I .25mL methanol to the tube.

2. Vortex t43

0.lM Sodium Acerate Buffer. pH 7.2 (containing 9.5% methanol and 0.5% tetrahydrofuran)

1 . Add 13.6089 sodium acetate trihydrate to approximately 900mL deionized water. Bring to a final volume of 1L using deionized water to make a 0.1M sodium acetate buffer,

2. Add 5.75mL glacial acetic acid to approximately 900mL deionized water. Bring to a final volume of 1L using deionized water to make a 0.lM glacial acetic acid buffer.

3. Slowly add the 0.1M acetic acid to the entire 0.1M sodium acetate until a pH of 7.2 is reached. Note that only a small amount of acid is required'

4. Measure 900mL of 0.1M sodium acetate, pH7.2 into a graduated cylinder and add 95mL methanol and 5mL tetrahydrofruan'

5. Filter. t44

Optimized Enzyme Dilutions and Incubation Lengths For Measuring CBS' CGL and MS

Enrymes Protein Concentration (mg/assay) Incubation Length (min)

CBS (liver) 9.1-1 8.1 20-40

CBS (kidney) 3.4-6.t 20-40

CGL (liver) 9.1-18.1 20

CGL (kidney) 6.7-r3.4 20

MS (liver) 9.1-1 8. l 10-20

MS (kidney) 6.7-13.4 20-60 r45

Plasma Amino Acids in Developing Pigs From Birth to Post-Weaning

Day

18 19 26 SEM P-Value P- uM 0 I 9 tmt Valueblotk

Alanine* 160 6l 109 119 t) 72- 22.7 .01 .43

Arginine 65 118 t36 126 93 80 18.8 .08 .07

Asparagine 172 ll0 195 146 ll3 t82 27.3 .15 .21

Aspartate 36 37 48 39 3l 36 5.7 .43 .96

Citrulline 29ù 20^b 17b zo^b 53u 44ub 7.9 .01 .66

Glutamate 191 202 224 211 t29 258 28.2 .06 .79

Glutamine 26r 384 457 332 209 374 65.9 .13 .87

Glycine* 675 562ub 558ub 4g7ub 341b 649 69.6 .004 .30

Histidine* t61 282 177 t49 160 r78 44.9 .54 .31

Isoleucine 69 70 121 7t 114 78 14.5 .04 .01

Leucine 97 2t5 179 160 122 t7s 31 1l .10

Lysine* 73 127 115 t24 55 139 27.9 .03 .71

Ornithine r85 177 108 142 101 98 22.2 .02 .76

Phenylalanin 83 110 73 69 84 85 I 1.9 .24 .t6 e

Proline 577^ 482^ 588" 609"b 734b gggb 69.6 .003 .02

Threonine** nl t07 226 t92 t79 186 27.0 .021 .91

Tryptophan 45 77 25 40 35 34 14.0 .t6 .52

Tyrosine 572 493 419 476 430 437 40.4 l1 .35

Valine 91 58 54 55 60 44 10.8 .08 .013 * Reported P-values reflect log transformed data ** Residuals do not follow a normal distribution a,b means within a row not followed by the same superscript are significantly different at P <.05 All means are composed of at least 7 replicates Hepatic CBS Activity in Developing Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue Basis

Day

Homocysteine produced 0 I I l8 t9 26 SE P-Valuc rn'l P-Valucl'lotk M

' nmol/lrlg liver I 15.9" 346.9b 451.6b" 470.5b" 586.5c 575.5b" 54.2 < .0001 .67

umol/tr/total liver 6.4u l5.5ub 47.5b" g l.3'd 74.1"d 92 .7d 8.9 <.0001 .82 a,b,c means within a row not followed by the same superscript are significantly different at P <.05 All means are composed of at least 7 replicates

À o\ Renal CBS Activity in Developing Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue Basis

Day r'' Homocysteine produced 0 t 9 l8 19 26 SE P-Value P-Valueblo'k M

nmol/hr/g kidney 176.6 r56.0 170.2 t70.4 r97.7 181.6 29.3 .94 .001

6.4b 1.0 .002 .05 umol/hr/total ki{ney 2.2 2.2ub , 3 .gob 6.1ub 6.6: , a,b méans within a row not followed by the same superscript are significantly different at P <.05 All means are composed of at least 7 replicates

\ì5 Hepatic CGL Activity in Developing Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue Basis

Day t*t Cysteine produced 0 I 9 18 t9 26 SE P-Value P-Valueblo"k M

nmolÆr/g liver 95.7^ 526.7b' 392.5b" 316.3' 379.8b" 35g.8b' 38.3 <.0001 .55

umol/hr/total liver 4.0 22.7^b 40.0b" 52.9" 47.5" 58.4' 4.9 <.0001 .43 a,b,c means within a row not followed by the same superscript are significantly different at P <.05 All means are composed of at least 7 replicates

è oo Renal CGL Activity in Developing Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue Basis

Day t'' Cysteine produced 0 1 9 18 19 26 SE P-Value P-Valueblo"k M

nmol/hr/g kidney 296.8" 220.\b 221.2b 249.4b 246.4ub 233.6^b 14.8 .02 .02 g.l b g.2b umollhr/total kidney 3.4 3.0u 4.7^ g.gb 0.5 <.0001 .02 @llowedbytheSamesuperscriptaresignificantlydifferentatP<.05 All means are composed of at least 7 replicates

\oÞ Hepatic MS Activity in DevelopÍng Pigs From Birth to Post-Weaning Reported on a Per Gram of Tissue and Total Tissue Basis

Day t'' Methionine produced 0 t 9 18 19 26 SE P-Value P-Valueblo"k M

nmol/trlg liver* r53.7^ 7g3b 54.7" 50.7" 43.2" 22.2d 6.64 <.0001 .21

umol/hr/total liver 6.3"b 3 .5" 5.6b" 8.3" 5.4b' 3.6" 0.5 <.0001 .55 *Reported P-values reflect log transformed data a,b,c means within a row not followed by the same superscript are significantly different at P <.05 All means are composed of at least 7 replicates

(â O pigs Per Gram of Tissue and Total Tissue Basis Renal MS Activity in Devetoping From Birth to Post-Weaning Reported on a

Day P-Value r'' P-Valueblo'k Methionine produced 0 I I 18 19 26 SE M

.0003 nmol/hr/g kidney 31.3 24.9 23.7 26.7 30.1 28.2 2.0 0.08 gg3.8b I < .0001 .03 nmol/hr/total kidneY 380.8' 328.3 526.2^ 9323b 1037.5b 78. esuperscriptaresignificantlydifferentatP<.05 All means are composed of at least 7 replicates

L¡r Summary of Block Effects and Visual Comparison with Litter Size at Day I and Sow Parify

Sow/Litter

I 2 3 4 5 6 7 8 SEM P-Value

b Kidney CBS (nmolÆr/mg protein) 17.g',b 25.0 53 .9" 56.3"b' 31.3"b 41.g"þ 45.1b" 15.5', 7 ,0* .0008

Kidney CBS (nmol/hrþ kidney) 99.3'b 127.5^b 269.5" 242.9^b" 156. l"b 205.8',b 233.gb" 77.6', 29.8* .0008

Kidney CBS (umol/hr/total kidney) 2.2 3.5 6.7 6.8 4.7 5.4 5.2 2.4 t.2 .05

Kidney CGL (nmol/hr/mg protein) 50.7.b 56.4', 48.1"b 4g.g'b 40.2b 50.2'b 51.2'b 39.3b 3.4 .02

Kidney CGL (nmol/hr/g kidney) 252.7"b 281.8" 239.0b 24g.l"b 201.2b 252.1"b 256.1^b 196.6b t7 .t .02

Kidney CGL (umolÆr/total kidney) 5.69"b 8.26" 5.96"b 5.84"b 5.03b 6.33"b 5.69'b 5.75"b 0.6 .02

Kidney MS (nmol/trr/mg protein) 5.3', 5.5b 5.2b 4 .6b 5.3b 4.7b g.0b 5.4b 0 .5 .0003

Kidney MS (nmol/hr/g kidney) 26.3" 27.6b 26.|b 23.0b 26 .4b 23.6b 40.0b 26.9b 2.3 .0003

Kidney MS (nmot/tr/total kidney) 6t7.2 871.0 61t.7 557.1 630. l 555.6 894.6 847.8 90 .l .03

Average Litter Weights 4.1. 4.7"b 4.3b. 4 .2b" 3.8' 3.8" J .9. 4.8" 0.r <.0001

Arginine t7t.l 76.6 78 .0 1 04.1 94.2 llr I I10.5 77.6 2t .7 .07

b Isoleucine 40.2' 79.7ú 95.I'b 753 120.3"b 643^b gl.2'b 50 .2b l6 .J .0r

Proline 960.8" 559.8b 547.0b 599.0'b 560.2b 650.1'b 703.7^b 605.5"b 80.4 .02

Valine 98.5" 743"6 65.3"b 40.gob 73.2b 33.6b 54.7^b 4l.g"b t2.4 ,0r

Litter size at day I l0 t2 l3 il t0 9 il r3

Parity 4 2 J 6 5

à,b,c means within a row not followed by the same superscript are significantly different at P < .05 All means are composed of at least 5 treatment values except kidney CBS (per mg protein and g kidney) which had four, resulting in a higher SEM average (¡ tJ