(À"t -cs

OF P-AMINOTHIOLS ANTD THE REGULATION FIEPATIC OXALATE PRODUCTION

A Thesis submitted bY

PAUL WAYNE B¡,rcn B.Sc. (HoNS).

to the University of Adelaide

S outh Australia" Australia

for the degree of

DOCTOR OF PHILOSOPHY

Division of Clinical Biochemistry lnstitute of Medical and Veterinary Science

and

Deparnnent of PhYsiologY University of Adelaide

J¡xuenv 1995 flwo,J".l \'11r; This thesis is dedicated to the memory of my Grandfather

Tuorues C¡Ùrpsnrr Ancrmn

19thof October 1922 - 4thof October 1988 TABLE OT CON:INXTS

Sncrrox PAGE

Abstract

Declaration ur

Acknowledgements iv

Publications v

Presentations vi

Abbreviations vii

Cnaprnn Onn

INTRODUCTION

1.I IDIOPATHIC CALCIUM OXALATE IJROLITHIASIS I

T.2 TI{E PATHOGENESIS A}ID TREATMENT OF IDIOPATHIC 2 CALCIUM OXALATE IJROLITHIASIS

r.2.1 Tm RoLE OF URtr.IARY SiTPBNSATUNANO¡r D{ IDIOPATIilC 4 CercNn¿ OXATATE UROLMilASIS

l.2.l.l Urine Volume 5

I.2.I.2 Urinary Oxalate and Calcium 6

T.2,2 TI{E ROLE OF CRYSTAL INTßNORS IN IDIOPATHIC CETCN.NT¿ 9 UROLrl[ilASIS

L2.2.I Magnesium 10

1.2.2.2 Citrate ll

1.2.2.3 Glycosaminoglycans T2

1.2.2.4 Pyrophosphate 13

1.2.3 Trm Rors oF CRYSTAL PRoMoTERS IN IDIOPATHIC CATCNN¡ t4 URoLmlasIs

1.3 LITHOTRIPSY AS A TREATMENT FOR UROLITHIASIS l6 1.4 OXALATE: INTRODUCTION T7

1.5 EXOGENOUSLY DERTVED OXALATE A}ID IJRTNARY 19 OXALATE EXCRETION

I.6 ENDOGENOUS OXALATE PRODUCTION 20

1.6.1 TIrE CoNTRtsurroN oF (L)-AscoRBIc ACID METABOLISM To 2t ENDOGENOUS OXALATE PRODUCTION t.6.2 Trß Covrnnrmou oF Grvoxvrare METABOLISM To 23 ENDoGENOUS OXALATE PRODUCTION

1.6.2.1 Enzymes Catalysing the Oridation of Glyoxylate to Oxalate 24

I.6.2.1.1 Laçtz;tß Deþdrogenase 24

1.6.2.1.2 Gþollate Oxidase 26

1.6.2.1.3 Xanthine Oúdase 28

I.6.2.I.4 Comparnnentalisation of the Enzymes Catalysing the Oxidation of 28 Glycollate and Glyoxylate Resulting in Oxalate Production

1.7 PRECIJRSORS OF GLYOXY-I,ATE 29

I.8 PHYSIOLOGICAL PATFMAYS TI{AT DTVERT GLYOXYLATE 3l FROM OXALATE PRODUCTION

1.9 INHIBITION OF ENDOGENOUS OXALATE PRODUCTION BY 34 SELECTED COMPOTINDS

1.9.I PRELnÆ.IARY INVESTIGATiONS INTO THE TIIE DTVERSION OT 36 GtyoxvLRt¡ FRoM Oxer,err PRoDUcTIoN

t.l0 THIAZOLIDINE-2,4.DICARBOXYLIC ACID, TTIE (L)- 38 CYSTEINE-GLYOXY_I,ATE ADDUCT AI.ID (D)- ASPARTATE OXIDASE

I.I I SLMMARY AND AIMS OF THIS TFIESIS 40 crnprnn Two

Mernnrnl,s, METHODS AND ASSAYS

2.1 INTRODUCTION 44

2.2 MATERIALS 44 2.2.I REAGENTS 44

2.2.2 Souncr op ANn¿ers AND ïssu¡ Sernæres 45

2.2.3 ANITVÍALHOUSING 45

2.3 METHODS 45

2.3.I INWroSn-nns 45

2.3.r.r The Effect of Orally Administered OTC, (L)-Cysteine and (D)- 46 Penicillamine on Urinary Oxalate Excretion and the Risk of Calcium Omlate Urolithiasis in a Normooxaluric Rat Model

2.3.1.2 The Effect of OTC on Urinary Oxalate Excretion and Calcium 47 Oxalate Crystal Deposition in an Ethylene Glycol Induced Hyperoxaluri c Rat Model

2.3.1.3 The Effect of Q)-Cysteine and OTC on Urinary Oxalate Extetion 48 and the Risk of Calcium Oxalate Urolithiasis in a Glycollate Induced Hyperoxaluric Rat Model

2.3.1.4 The Effect of Chronic Sulphate bfusion on the Renal Clearance of 48 Oxalate

2.3.I.5 The Efect of Bolus Intraperitoneal Injections of the Cysteine- 50 Glyorylate Adduct and Sulphate on Unnary Oxalate Excretion

2.3.1.6 The Effect of a Bolus Intraperitoneal Injecfion of meso-Tartaric 5l Acid on the In vivo Metabolism of the Cysteine-Glyoxylate Adduct

2.3.I.7 Does an Intraperitoneal Injection of meso-Tartaric Acid Prevent 5l Metabolism of Endogenous Cysteine-Glyoxylate Adduct in Glycollate Hyperoxaluric Rats Given (L)-2-Oxothiazolidine-2- carborylic Acid (OTC)?

2.3.2 IuwrnoSruoms 52

2.3 .2.1 Hepatocyte Preparati on and Incab a fi on C ondi fions 52

2.3.2.2 Factors Affecting the Producfion of Oxalate and Carbon Dioride 53 from Glyorylate and Glycollate in Isolated Rat Hepatocytes

2.3.2.3 Investigations into The Effect of þ-Aminothiols on Oxalate and 54 Carbon Dioxide Producfion from Glycollate in Isolated Rat Hepatocytes

2.3.2.4 Formation oJ' the (L)-Cysteine-Glyoxylate Adduct from Glycollate 54 and (L)-Cysteine in Isolated Rat Hepatocytes 2.3.2.5 Investigations into The Effect of p-Aminothiols on Hepatic 55 Metabolism as Determined by Gluconeogenesis from (L)-Alanine and (L)-Lactdte in Isolated Rat Hepatocytes

2.3.2.6 A Kinetic Investigation of the En4tmes which Synthesise Oxalate 55 from Glycollate and Glyoxylate

2.3.2.7 The Effect of (D)-Penicillamine on Alanine Aminotransferase 56 Activity

2.3.2.8 Effect of (D)-Penicillamine Pretreatment on Oxalate and Carbon 57 Diotcide Production from Glycollate and Glyoxylate in Isolated Hepatocytes

2.3.2.9 Formation of the Cysteine-Glyoxylate Adductfrom Glycollate in the 57 Presence of Glycollate Oridase and Cysteine

2.4 ENZ\N\4E ISOLATION 58

2.4.1 IsoLAnoN oF OxALATE SyvnmsrsNc Acrrvny Fnorr¡ Rer Lrven 58

2,4,2 ISOT-ENO¡I OF MTTOCHONDRTAL Æ,.qNN.[E Aì,M.IOTRANSFERASE FROTr¡ 59 RAT LIVER

2.4.3 IsoLATroN oF (D)-ASIARTATE O>oasr 60

2,5 ASSAYS 60

2.5.T ANaTysss oF URINE AND PLASIUA 60

2.5.2 NIEASUREI!ßNT oF ¡t4c¡-CaruoN Dro)cDE AND ['4C]-OxALATE rN 61 I{EPATOC YTE StpgnNane¡rt' s

2.5.3 MEASUREIVGII"T oF [U-t4c]-Oxar¡,rs AND fH-METHoxy IN[nr nr 62 PTasvaAND URINE

2.5,4 IVIEASTIREIVßNI oF PYRIIVATE 63

2.5.5 DETERI!ff.IAltoN oF THE Kn.IEnc PRoPERTßS oF I{EPATc LacTeTe 63 DEHYDRoGENASE

2.5.6 MEASI'RET,ßNT oF GLYoXYLATE 64

2.5.7 DBTSPù/ü.IATION OF' TIIE KNSTIC PROPERTIES OF FIBPANC 64 Grycorlar¡ O>mase

2.5.8 MEASUREI!ßNT oF GLYCoLLATE 65

2.5,9 MEASUREMENT oF GLucoss 66

2.5.10 DETERmTATToN oF (D)-ASPARTATE O>

CHeprnn Tnnrn

TnHzol,mmn-2,4-DIcARBoxyLIc Acto, TrrE Cysrnnvn- Gr,yoxyr,nrn Anuucr

3.1 INTRODUCTION 68

3.2 PREPARATION OF TT{E CYSTEINE-GLYOXY_I,ATE ADDUCT 69

J.J ENZII,TATIC MEASI.]REMENT OF TT{E CYSTEINE. 69 GLYOXY-LATE ADDUCT

3.4 GAS CHROMATOGRAPHICII\4ASS SPECTROSCOPIC Æ.IALYSIS 7t OF IJRINE AND PLASMA FOR DETECTION OF THE CYSTEINE. GLYOXY_LATE ADDUCT

3.5 CHARACTERISATION OF TI{E CYSTEINE-GLYOXY_I,ATE 72 ADDUCT

3.6 RENAL CLEARANICE A}ID EVIDENCE FOR IN VTVO 76 METABOLISM OF THE CYSTEINE.GLYOXY-I,ATE ADDUCT

Crnprnn Foun

I¡¡vtvo Sruorns

4.T INTRODUCTION 79

4.2 RESULTS 80

4.2.I I}TTER-ÐGERIMENTAL VARIATIoN tr.I DETARY MINERAI INTAKE AND 82 URbIARY ELECTRoLYIE EXCRETON

4.2.2 THE EFFEcT or OTC oN URwaRy Oxarars Excnnnotr AND THE 82 RISK oF C.qrcruv Ox¡rer¡ UROLTilASIS

4.2.2.I Long Term OTC Administrafion in a Normooxaluric Rat Model 83

4.2.2.2 Short Term OTC Administrafion in a Normooxaluric Rat Model 84

4.2.2.3 Long Term OTC Administration in an Ethylene Glycol 85 Hype roxaluri c Rat Mode I 4.2.2.4 short Term orc Administration in a Grycollate Hyperoxaluric Rat 86 Model

4.2.3 TTæ ETTSCT OF (L).CYSTEINE ON URINARY OXELETS EXCRETION 89 ANDTT{E RISK OF CETCNN¡ OXATAIE UNOMIESS

4.2.3.1 short Term (L)-cysteine Administration in a Normooxaluric Rat 89 Model

4.2.3.2 short Term Q)-cysteine Administration in a Glycollate 90 Hyperomluric Rat Model

4.2.4 TTM ETTSCT OF (D)-PEMCILI.AMINE ON URINARY OXAI.A,ITE 91 EXCNSTIONAND TI{E RISK OF CET,CTUU OXAI.A'TE I]ROLITI{IASIS

4.2.4.1 short Term Administration in a Normooxaluric Rst Model 9l

4.2.5 TIæ ETTSCT OF SULPHATE ON URINARY OXALATE EXCNTNON 93

4.2.5.1 rhe Effect of chronic sutphate Infusion on the Renal clearance 93 and Tissue Distribution of Oxalate

4.2.5.2 The Efect of a Bolus Intraperitoneal Injection of sulphate on 94 Urinary Omlate Excretion

4.3 DISCUSSION 94

4.3.1 TID EFFECT oF oTC A}TD (L).CYSTEINE oN URINARY OXALATE 94 EXCRETIONAND TTTE RISK OF CETCNN¿ OXETETS UROLI¡ilASIS

4.3,2 TTü EFFECT OF SULPHATE AND ACDTIRIA ON URINARY OXAIATE 101 Excn¡uo¡r

4.3.3 THE EFFECT oF (D)-pENrcrLrAMrNE oN URTNARY OxAr-A:rE 103 ExcnrnoNAND TT{E RISK oF Calcnru oxarars UROLTHASIS

Cmprnn FryE

Irvwrno Sruurns

5.I INTRODUCTION 105

5.2 RESULTS 106

5.2.1 STUDIES \vlm ISOLATED Rar H¡parocylEs 106

5.2.t.r The Efect of (L)-Cysteine on Oxalate and Carbon Dioride 107 Production from Glycollate: Evidence þr (L)_Cysteine_ Glyoxylate Adduct Formation in Isolated Rat Hepatocytes 5.2.1.2 The Effect of þ-Aminothiols on Oxalate and Carbon Dioride 108 Productionfrom Glycollate in Isolated Rat Hepatocytes

5.2.t.3 The Effect of (L)-Cysteine and (D)-penicillamine on Carbon 108 Dioxide and oxalate Production from Gtycoilate in Isorated Rat Hepatocytes

5.2.1.4 The Effect of B-Aminothiols on Hepatic Metabolism as Determíned 109 by Gluconeogenesis from (L)-Alanine and (L)-I-actate in Isolated Rat Hepatocytes

comparison production 5.2.r.5 of oxalate and carbon Dioride from ll0 Glyoxylate and Glycollate in Isolated Rat Hepatocytes

5.2.r.6 Effect of (D)-Penicillamine pretreatment on omlate and carbon Lt2 Dioxide Productionfrom Glycollate and Gtyorylate in Isolated Rat Hepatocytes

5.2.2 STIJDES WTIT{ RECoNSTTTTED ENZYME SYSTEMS tt2

5.2.2.L Formation of the cysteine-Glyoxytate Adductfrom Glycollate in the r12 Presence of Glycollate Oridase and Cysteine

5.2.2.2 A Kinetic Invesfigafion of the Enzymes wh¡ch sytthesise oxalate 113 from Glycollate and Glyoxylate

5.2.2.2.1 Lactzte Dehydrogenase 113 5.2.2.2.2 Glycollate Oxidase lt4

5.2.2.3 The Effect of (D)-Penicillamine on Aranine:Glyoxylate and 115 Alanine : a-Keto glutarte Aminotransferas e Activitlt

5.3 DISCUSSION rl6

5.3.I TIü EFFEcT oT B-AwvoTHIoLS, F-AMINoTT{IoL ÀIETABoLITES AND ll6 ENZYNß CorRclons oN oxelqre AND CansoN DIo)OE PRODUCTION FROM GLYCOLLATE IN ISOI-A,TED RET FIEPATOCYTES

2 5.3 FoR\4ATIoN oF THE CYSTEI.TE.GLYoXYI-A,TE AIoucT FRoM 124 GIycoI n¡ IN TIìE PRESENCE oF GLYCoLI.A,TE o>oasn aup Cysr¡nm

5.3.3 TIü EFFEcT on B-AlvmvorHrols oN FIEIATTc METABoLTsM AS t2s DerERNm.rED By GLUcoNEocENESTs FRoM (L)-ALAN[.IE AND (L)_ LacTaTT IN ISoLATED T{AT FIEPAToCYTES

5.3.4 CowarusoN oF OXALATE AND CARBoN DIo)OE PRoDUcÏoN r28 FROM GTYOXYLETS A}ID GLYCOTT¡,T¡ IN ISOLATED RAT FIEPATocrlES 5.3.5 CnanecreRrsATroN oF TIIE ENZytuÆs wHrcH Syvnæsrse Oxerar¡ 13l FRoM Gl-ycotI.ers ext GryoxyLATE

Cnarrnn Sx

COxCr,uoING REMARKS

6.1 SYNOPSIS AND CONCLUSTONS OF THIS THESIS 135

6.1.1 TIm (L)-Cysre[.rE-Gryoxyt-ATE A¡oucr: SyNlIüsß, IsoLATroN, 135 CHARAcTERISATIoN AND METABoLISM

6.1.2 REGu.AnoN oF URnrARy Oxemrr By B-Arvü{orHroLS 137

6.1.3 N{ncl{¿¡tsrr,r oF THE REGLTLATIoN oF El.DocsNous Oxntnre 139 PRoDUCToN

6.2 CLINICAL IMPLICATIONS 142

6.2.1 MaNacBtævr or Carcnru OXALATE URoLr[ilASrs 142

6.2.2 Pn¡vBNmoN oF Oxarosn rN Sugr¡crs wrrl{ pRIrdARy 143 HYTEnoXAUIRIA

6.2.3 IATRocEMc HypERoxALuRrA Folrownvc AomusrnanoN oF (D)- r44 PEMCILLAMINE: IrwTTcenoNS FoR Srmrscrs wTrII W[,soTg,S DISEASE

6.3 FLTTURE STUDIES r46

6.4 CONCLUDING STATEMENT r47

Brnr,rocneprry I49 to 172 AssrRAcr

Of the numerous risk factors for calcium oxalate urolithiasis, oxalate is tÌre most important as small reductions in urinary oxalate excretion can markedly reduce the risk of calcium oxalate urolithiasis. The investþations in this thesis centre on the use of B- aminothiols (cysteine, cysteamine and penicillamine) to decrease endogenous oxalate production by forming adducts with glyoxylate, the immediate precursor of oxalate.

The investigations include, the preparation, characterisation and metabolism of the cysteine-glyorylate adduct (Chapter 3), the ability of B-aminothiols to decrease urinary oxalate excretion under normo- and hyperoxaluric settings (Chapter 4) and in vitro studies with hepatocytes to establish the mechanism by which B-aminothiols regulate hepatic oxalate production and hence urinary oxalate excretion (Chapter 5). The latter includes an examination of the pathways regulating endogenous oxalate production.

Different diastereomeric ratios, i.e. cis- andtrans-, ofthe cysteine-glyoxylate adduct were prepared and characterised. At pH 7 .4 the ratio of cls- to trans- was 5 1% to 49Yo whtle at pH 1.4 the preparation was found to be 100% trans-. Although tn vivo studies (i.p. injection of adduct) indicated that considerable adduct met¿bolism occurs, onty 2Yo of the metabolised dose appeared in the urine as oxalate.

The ability of orally administered (L)-cysteine, (D)-penicillamine a¡d (L)-2- oxothiazolidine4-carboxylic acid (OTC, an intracellular (L)-cysteine delivery drug) to decrease urinary oxalate excretion in rats under normo- and hyperoxaluric conditions (ethylene glycol or glycollate induced) was investigated.

Under normooxaluric conditions, OTC significantly decreased urinary oxalate, 30Yo (long term administration, p<0.05) and l0% (short term administration, p<0.05); but (L)- cysteine did not, 9% (short term administration, p=0.22). ln contrast, (D)-penicillamine significantly increase urinary oxalate excretion, 68% (short term administration, p<0.05); in association with decreased plasma alanine, 34% (p<0.01); and aspartate, 23o/o (p<0.001); aminotransferase activity. In vitro studies indicate that (D)-perucillamine exerts its effect through inhibition of glyoxylate:alanine aminotransferase via interference with pyridoxal phosphate, the result being increased availability of glyoxylate for oxidation to oxalate. Under mild hyperoxaluric conditions (glycollate induced, urinary oxalate 3x normal) short term administration of both OTC and (L)-cysteine was shown to be effective in reducing urinary oxalate, 39% (p<0.001) and 3l% (p<0.001) respectively. After extensive investigation (including the use of gas chromatography/mass spectroscopy) evidence for the presence of adduct in the urine of these rats could not be found. This was consistent with earlier studies which indicated that extensive metabolism of adduct would occur at the levels expected to be produced endogenously.

OTC was also investigated under more severe hyperoxaluric conditions (ethylene glycol induced, urinary oxalate 18x normal). Significant reductions in urinary oxalate were not observed and this was attributed to the 5 fold difference in the dose of ethylene glycol and OTC. Hence OTC could not supply sufficient cysteine for adduct formation under these conditions.

Isolated rat hepatocytes were used to further investigate the effect of p-aminothiols on oxalate production from glycollate, which is metabolised within the cell to glyoxylate and oxalate. Addition of extracellular glyoxylate was inappropriate in this setting due to the potential for extracellular formation of adduct. Adduct formation was shown to be proportional to the decrease in oxalate production, the first confirmation of the postulated mechanism by which p-aminothiols reduce oxalate production and excretion.

Further investigation of the roles of gþollate oxidase and lactate deþdrogenase using isolated hepatocytes and a reconstituted enzyme systems indicate that hepatic oxalate production is dependant on compartmentalisation between peroxisomal (glycollate oxidase) and cytosolic (lactate dehydrogenase) metabolism.

Evidence was prevented to suggest that at physiological substrate levels lactate deþdrogenase is the enzyme responsible for oxidation of exogenous glyoxylate to oxalate while glycollate oxidase is responsible for the two step oxidation of glycollate to oxalate. Hence, in vivo gþollate oxidase may be the enzyme responsible for the major portion of oxalate production.

These studies indicate that cysteine delivery drugs like OTC have the potential to aid rn management of calcium oxalate stone disease presumably through formation of the (L)- cysteine-gþxylate adduct, thereby reducing endogenous oxalate production and urinary oxalate excretion.

1l DBcrmeuoN

This thesis contains no material which has been accepted for the awa¡d of any other degree or diploma in any universþ or other tertiary institution and , to the best of my knowledge and beliet contains no material previously published or written by another persofi, except where reference has been made in the text.

I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying.

Date ..iJ..7/.?Í. Signatur AcrxowrpocEMENTS

Firstly I would like to thank my supervisors Dr Allan Rofe, Dr Renze Bais and Associate patie'nce and Professor Gany Scroop. Special thanks is given to Dr Allan Rofe for his guidance throughout the course of this work. I am greatful to Dr' Peter Coyle and Mr' Jeftey philcox for their constructive criticism and guidance. I would also like to thank the staff of the Division of Clinical Biochemistry at the Institute of Medical and am Veterinary Science for their support and technical assistance tbroughout this work. I zupport indebted to Dr Renze Bais, Dr Atlan Rofe and Dr Kim Bannister for the financial given to me in the form of an NH & MRC research scholarship; Grant N' 92064. I

would like to thank my parents, Coral and Graham Baker, for their love and support over the years. Finally, I would like to thank my wife, Michaela" for her continual love,

encouragement and commitnent.

lv PusucRuoNS

Baker, P.\ü., Coyle, P., Bais, R. and Rofe, A.M. (1993) lnfluence of season, age, and sex on renal stone formation in South Australia. Med .J. Aust.,l59 390'392'

Baker, P.W., Bais, R. and Rofe, A.M. (1994) Formation of the (L)-cysteine-glyoxylate adduct is the mechanism by which (L)-cysteine reduces oxalate production from gþollate. Biochem. J., 302; 753-757.

Baker, P.W., Bais, R. and Rofe, A.M. (1994) The efficacy of (L)-2-oxothiazolidine4-

carboxylate (OTC) and (L)-cysteine in reducing urinary oxalate excretion. J. UroI.,l52: 2139-2146.

Baker, P.W., Tilley, M.H., Rofe, A.M. and Bais, R. (1992) Reduction of urinary oxalate

excretion by the oral administration of the cysteine precursor (L)-2-oxothiazolidine-4- carboxylic acid. ln: Urol¡thiasis, Proceedings of the 7th. International Conference on Urolithiasis (Ryall, R. ed.) Plenum Press, New York, Pg.7l.

Baker, P.W., Coyle, P., Rofe, A.M. and Bais, R. (1992) lncreased risk of calcium

oxalate stones in young females. ln: Urolithiasis, Proceedings of the 7th. International

Conference on (Irolithiasis (Ryall, R. ed.). Plenum Press, New York, Pg. 435'

Baker, p.W., Bais, R. and Rofe, A.M. Calcium oxalate urolithiasis and endogenous

oxalate production. Crit. Rev. Ctin. Lab..lci. (lnvited Review; Submitted)

Baker, P.W., Bais, R. and Rofe, A.M. B-Aminothiols and the regulation of hepatic

oxalate production in rats. Toxicol' App. Pharmacol. (Submitted)

v PnBssx-rATroNs

Baker, P.W., Tilley, M.H., Rofe, A.M. and Bais. R.: Reduction of urimry oxalate excretion by the oral administration of the cysteine precursor (L)-2oxothiazolidine4- carboxylic acid. 7th. International Conference on Urolithiasis, Cairns, August 1992.

Baker. P.W., Coyle, P., Rofe, A.M. and Bais, R.: Increased risk of calcium oxalate stones in young females. 7th. International Conference on Urolithiasis, Cains, August

1992.

Baker. P.W., Tilley, M.H., Rofe, A.M. and Bais, R.: Reduction of urinary oxalate excretion by the oral administration of the cysteine precursor (L)-2-oxothiazolidine4- carboxylic acid. The Australian Society for Medical Researcb" Adelaide, May 1992.

Baker. P.W., Rofe, A.M. and Bais, R.: The effectiveness of (L)-cysteine and (L)'2- oxothiazolidine4-carboxylic acid in reducing urinary oxalate excretion. A comparative study. The Australian Society for Medical Research, Adelaide, June 1993.

Baker. P.W., Rofe, A.M. and Bais, R.: B-Aminothiols and the regulation of hepatic oxalate production in rats. The Australian Society for Medical Research, Adelaide, June t994.

Baker. P.W., Rofe, A.M. and Bais, R.: Formation ofthe (L)-cysteine-glyoxylate adduct rs the mechanism by which (L)-cysteine reduces oxalate production from glycollate. Australian Society for Biochemistry and Molecular Biology, Gold Coast, September t994.

vr ASSREVTATToNS

OTC (L) -2 -oxothi azolidine -2,4 -dicarboxylic acid

BSTFA N, O-å is-(trimethylsilyl) -trifl uoroacetamide TMCS trimethylchloro silane NMR nuclear magnetic resonance

GC/IVÍS gas chromatography/mass spectroscopy KBr potassium bromide i.p. intraperitoneal cts- isomer with adjacent like ligand or functional group trans- isomer with non-adjacent (opposite) like ligand or functional group meso- a prefix indicating that a substance is optically inactive due to intra-

molecular compensation o.p.m- oscillations/minute p.p.m. parts/million c.p.m. counts/minute r.p.m. revolutions/minute a.m.u. atomic mass units V-* maximum enryme velocþ under the stated conditions Ki inhibition constant; concentration of inhibitor at which half maximal

enzyme inhibition is achieved under the stated conditions K. Michaelis constant; concentration of substrate af which half mæ

the affinity of the substrate for the active site of the enzlme

Pk" acid disassociation constant; pH value at which above an acid will

disassociate to a proton and it conjugate base

P"r calculated probability of stone formatton

vll CnnprBn ONe

INrnooucrIoN

1.1 IDIOPATHIC CALCruM OXALATE UROLITHIASIS

Throughout time civilisation has been plagued by urolithiasis; to date the oldest recorded urinary stone, 4000 to 5000 years 8.C., was removed from the grave of a 16 year old boy in El Amrah,Egwt (Michell, 1989; van Aswegan and Plessis, 1992; Menon and Koul,

1992). In industrialised countries, urolithiasis is now one of the most common disorders of the urinary tract with annual hospital admission rates ranging ftom 0.1 to 20 per

10,000 (Watts, 1933; Smith, 1989; Robertson, 1993) and as a consequence, urolithiasis incurs a considerable social and economic cost. ln the United States in 1986 an estimated two billion dollars \ilas spent on stone fragmentation and removal alone (Curhan et al.,

1993). Although the mortality rate is low, urolithiasis is commonly associated with pain that is legendary in its severity (Asper, 1984). This pain is produced when the stone moves slowly down the urster resulting in ureteral spasm and stone passage; the degree of ureteral sp¿Nm is independent of stone size. Approximately 65Yo of stones are spontaneously passed (Robertson, 1984). On average the prevalence of urolithiasis in males is twice that in females, wtth 12% of males who reach the age of 60 having already presented with at least one stone (Robertson, 1993). However, females have a higher incidence of magnesium ammonium phosphate stones, commonly referred to as infection stones. In both males and females, urolithiasis occurs between the ages of 30 and 70 wittl

I the incidence of urolithiasis in males peaking between 45 to 55 years of age and rn females much earlier, around 30 to 40 years of age (Robertson, 1984; Baker et aL.,1993).

Globally, calcium stones (mixed calcium oxalate/calcium phosphate) are the most predominant stone type accounting for 6I+4Yo of total stones followed by uric acid,

23+4Yo; magnesium ammonium phosphate, I3+2Yo; xanthine, 2,Sdihydroxyadenine, sodium urate and the drug triamterene, ?tlYo; and cystine, I+l%o: (Watts, 1983; Smith er al., 1990; Coe and Favus, 1991; Robertson, 1993; Baker et al', 1993). Due to the predominance of calcium oxalate stones, many studies have investigated their aetiology and epidemiology to aid in the development of effective treatnents and medical prevention programs (Pyratr, 1979; Rofe et al.,1986; Smith et al.,1990; Coe and Favus, 1991; Su et al., 1991; Khan, 1991; Menon and Koul, L992). These studies concentrating on a wide range of demographics (geography, climate, culture, level of affluence, gender, age, race) have identified the major factors essential for a better understanding of the mechanisms of calcium oxalate urolithiasis.

1.2 THE PATHOGENESIS AND TREATMENT OF IDIOPATHIC

CALCruM OXALATE UROLITHIASIS

Stone formation is considered to be the result of abnormal crystalluria caused by subtle

changes in urine composition, as mild crystalluria is often observed in the urine of normal

non-stone forming individuals. These changes can be categonsed as follows; a) increased

urinary saturation with the relevant stone forming electrolytes (Welshman and

McGeowin, 1976;Wong et al., 1992), b) decreasedurinary excretion of substances that

inhibit crystal nucleation, growth and aggregation (citrate, pyrophosphate, magnesium

2 and glycosaminoglycans) (Welshman and McGeowin, t9?6; Michell, 1989; Grases et al',

1992) and c) increased urinary excretion of substa¡ces that promote crystal growth by

(Coe acting as heterogeneous nucleants (uric acid, mucoproteins and certain drugt) et al',

1975; Grover et al., 1990; Grases et al., 1992). One or any combination of these changes may be found in an individual presenting with urolithiasis. However, as the manifestation of these changes is dependant on a range of factors, such as genetic predisposition, dietary intake, climate, level of affluence, fluid intake, disease state, age, physiological abnormalrty and gender, it is often not possible to clearly identify which factors are responsible for the presentation of the condition. Although the susceptibility of an individual to calcium oxalate urolithiasis can be attributed, in some cases to recognised d,isorders, in almost 90% of cases it is idiopathic in nature. Of the disorders which cause calcium oxalate urolithiasis, hyperparathyroidism is the most predominant, accounting lor I\%oof all cases of calcium oxalate urolithiasis (Robertson, 1993).

Idiopathic calcium oxalate urolithiasis is a subcategory of calcium oxalate urolithiasls thar was first proposed by Pak et al. n 1974 (Smith, 1990). Idiopathic calcium oxalate urolithiasis is diagnosed when all other abnormalities, such as absorptive hypercalciuria,

renal hypercalciuria and prirnary hyperoxaluria, have been eliminated. This thesis is

primarily concerned with idiopathic calcium oxalate urolithiasis as at least 70%o of

subjects who form calcium oxalate stones have this disorder (Smith, 1990). ln general

the main urinary risk factors for calcium oxalate urolithiasis have been identified as

reduced urine volume, increased excretion of oxalate, increased excretion of uric acid,

reduced excretion of glycosaminoglycans, increased urine pH, increased excretion of

calcium and decreased excretion ofcitrate (Robertson. 1984; Pak et al',1985). A risk

factor model developed by Robertson demonstrates that even subtle changes in the above

fbctors can markedly affect the relative risk of calcium oxalate urolithiasis (Figure 1.1).

3 20 Volume O¡alâþ

15 þ o o cË GAGS f¡i üro Uric Acid ú pH q) Calcium (É rl) & 5

0 a -3 .J -l 0l 3 S.D. Units from Normal Mean Value

Figure 1.1 The 'risk curyes' for the pe formation of õut-"i"r" utotittriasis. The values aie r of süandard Ñì"tã" (. lt *iO above and below the m in the normal male popuiation (Adapted from Robertson, 1984). The mechanism by which these risk factors increase the susceptibilrty of an individual towa¡ds calcium oxalate urolithiasis, and the treatnents required to correct each abnormality and prevent furttrer stone formation are described in the following sections'

A pictorial representation of the factors which influence the risk of calcium oxalate urolithiasis is given in Figure 1.2.

T,2.I TID ROLE oF URINARY SupeRSaTuRATioN IN IDIOPATHIC CETCNTU OXALATE

URomlesIs

The levels of urinary saturatior¡ with respect to stone formation, have been categorised as

follows; a) the undersaturated zone where complete ionic dissolution exists, b) the

metast¿ble zone where heterogeneous nucleation, crystal growth and induced nucleation

occur and c) the ove¡saturated zone where homogeneous nucleation occurs (Figure 1.3)

(Finlayson, 1978; Robertson, 1984; Coe and Favus, 1992). These zones are bounded by

two thermodynamic entities, the solubility product and the formation product. The

solubility product is the upper boundary for the undersaturated zone whilst the formation

product is the upper boundary for the metastable zone. The solubility product, Ko, is the

most important of these boundaries and is described as the product of the concentration of

the ions of a dissolved electrolyte when in equilibrium with undissolved substance. When

the solubilþ product for a given compound is exceeded in a solution, some of it is

precipitated until the product of the concentration of the ions (the activitv product) falls to

the value of the solubilþ product. Normally the activity product of calcium and oxalate

in urine exceed the solubilþ product and thus crystals of calcium oxalate may be

observed in urine of normal non-stone forming individuals (Fleisch. 1978; Coe and

Favus, 1991). Urinary supersaturation with ions that promote calcium oxalate

4 Diet¿ry Factors which r¡fluence th€ Risk of Caloium Oxalatc Urolithiasis Oþlalate Fæ& (i.e. As@óate, Plrire ùd Pyrimidine NEIætid6), CiÞq Magnsim a¡d Phosphate

Factors Influcncing Oxalate Upøko from the Ga¡troinfcstinal Lu¡ncn Lwißl pH, Calcirr, MsgrEsiuL Epithelial Vito¡nin D Levels ud Absorption Inteti¡nl Flon I Faecal Elimination of Calcium O)

Eleobolytes which Undergo Substrates Available for the Endogenous Rapid Renal Excretion Production ofOxalate and Urate o,€late Caldun, lvlsgn€iwf Cdbohydr¿úô md Prote¡D, arid Püino ùd CiF¿te PhßphåtemdUEte Pyrimirlino Nudætide

Oxalate

Uric Acid

Urinary Risk Factors Changc in the Risk of Calcium for Calcium oxalatc Urolithiasis Associated with Oxalatc Urolithiasis an Increasc in the Urinary Risk Factor O)øl€úo I Calcim 1 Urin€vollre J UTiEPH I CitEte t M4leiu t U¡ic Acid I Srephæphate t

Figure 1.2 Pictorial representation of the factors which influence the risk of calcium oxalate urolithiasis. 1 Unstable Zone Homogeneous Nucleation

Ê Formation Product (K¡) o

ÉË 1 CË (A Zone èo Meastable 6ø o) (.)k Solubility Product (IÇo)

1 Undersaturated Zone Crystal Dissolution

Figure 1.3 The levels of urinary saturation with respect to stone formæion (Adapted from Robertson, 1984). urolithiasis can occur by, a) decreased urine volume due to inadequate fluid intake or excess insensible loss of fluid or b) increased excretion of lithogenic substances.

1.2.1.1 Urine Volume

Under varying levels of hydration the nephrons are efficient in regulating plasma osmolarity at the expense of urine osmolarity; plasma osmolarity ranges from 280 to

295mOsm/kg whilst urine osmolarity ranges from SOmOsm/kg with over hydration to l4OOmOsm/kg with under hydration (Coe and Favus, 1991). Thus, a strong relationship exists berween the level of hydration and the level of urinary saturation as shown by the increased incidence of calcium oxalate, calcium phosphate and particularly uric acid stones in subjects working and living in hot snvironments (Bateson, 1973; Robertson ¿f al.,1974; Baker et al.,1993; Borghi et al., 1993). Hence, the ñrst line of treatnent for calcium oxalate stone formers is to increase the daily urine ouput to at least 3Llday

(Finlayson, 1974; Pyrah, 1979; Pak et al., 1980; Watts, 1983; Ackermann, 1990;

Robertson, 1993). Fluid therapy has been shown to be an effective treatnent in approximately 60%o of individuals with calcium oxalate urolithiasis as it brings the activþ product of calcium oxalate below the solubility product, preventing crystal nucleation and stone formation (Smith, 1990). The use of fluid therapy as a first line treaûnent is further justified by the observation that a 2 fold increase in urine volume decreases the activity product of calcium oxalate formation by 30o/' whilst increasing the formation product by 30%o, decreasing the relative risk of stone formation by 50% overall

(Pak et al., 1980; Curhan et al., 1993).

5 I.2.I.2 Urinary Oxalate and Calcium

Although urine volume is a central factor in the formation of calcium oxalate urolithiasis,

subjects afflicted with this condition, unless living in arid climates, do not generally have reduced urine volumes (Johansson et al., 1980; Nicar et al., 1983; Pak et al., 1985;

Cupisti et a1.,I992;Borghi et a\.,1993; Wong et al., 1992), rather hypercalciuria and/or

mild hyperoxaluria appear to be the most common causes. In the past, much attention

had been focused on hypercalciuria as the most important risk factor for calcium oxalate

urolithiasis as mild hyperoxaluria has been shown to be present in only 15 to 50% of

subjects (Tiselius, 1980; Smith, 1990; Edwards et al., 1990), whilst hypercalciuria is

present in 30 to 60% (Lucke, 1979\. However, hyperoxaluria is now known to be the

more important of the two factors as subtle changes in urinary oxalate excretion markedly

affect the rate of calcium oxalate precipitation (Fþre 1.4) (Robertson, 1993)'

Hyperoxaluria may be dietary in origin resulting from, a) ingestion of foods rich rn

oxalate and oxalate precursors, such as chocolate, protein, brussel sprouts, parsley,

mushrooms, coffee and grapes (Hams and Richardson, 1980; Laker, 1983; Balcke et al-,

1989;Williams and WilsorU 1990) or b) ingestion of oxalate precursors such as ethylene

glycol, xylitol, dietary refined sugars and ascorbic acid (Gessner et aL.,1961; Thomas ef

at., 1976; Ha¡nett et a1.,1976; Gambardella and Richardson, 1977; Rofe e¡ al., 1977,

1979, 1980 and 1986a; Winek et al.,1978; James et al.,1982 Conyers, 1985; James et

al., 1985; Bais e¡ al., 1985; Williams and Wilson, 1990; Urivetzþ et al., 1992). In

practice however, the most common dietary causes of mild hyperoxaluria appear to be

increased gastrointestinal oxalate absorption due to either a high dietary oxalate/calcium

ratio or increased gastrointestinal absorption of calcium (Marshall et al., 1972;

Robertson and Peacock, 1980; Erickson et al., 1984; Coe and Favus, 1992, Robertson,

6 1.0 .l o v 0.9 13 0.) (ú Þ. o o) H U.b o. c) d ct X o 0.4

O (Ë (-) É 0.2 É X à 0.0

1.5

o

! e c.)q Formation (A 1.0 c.) Product 4CÉ oX J () (tl (J 0.5 ()

c) Stone Forming Range Stone Forming Range à0 l------+ o J # Normal Range Normal Range Solubility 0.0 Product

5r0 150 0.5 1.0 1.5 Calcium (mmol/L) Oxalate (mmol/L)

Figure L.4 The effect of independently increasing urina¡y calcium or oxalate concentration on (lo\¡/er panels) the relative supersaturation of urine with respect to calcium oxalate and (upper panels) the ma;

excretion (Marshall et al., 1972; Hodgkinson, 1978; Brinkley et al., I98l; Berg et al.,

1936, Nakada et a1.,1988). Hence, dietary management can play an important role in

alleviating the hyperoxaluria observed in some subjects with calcium oxalate urolithiasis

caused through increased intestinal absorption of calcium and oxalate (Williams and

Wilson, 1990; Smith,I99l; Brown and Wolfson,1993; Curhan et aL.,1993). At present

management of dietary induced hyperoxaluria involves concomitant reductions in the

dietary intake of oxalate, potential oxalate precursors and calcium (Hodgkinson, 1978;

Rao ef al., 1982; Erickson et al., 1984; Robertson, 1993). However, the success of

dietary management, depends on the degree of compliance.

Abnormalities in erythrocyte oxalate tr'2nsport have been shown to occu¡ in 80% of

subjects with calcium oxalate urolithiasis (Bægio et aL, 1984a; Baggio et al., 1986;

Gambaro and Baggio, 1992). Although these abnormalities may be the source of

hyperoxaluria in some subjects, in light of the observation that only 15 to 50% of subjects

with calcium oxalate urolithiasis exhibit hyperoxaluria, the significance of this

observation is unclear. The abnormality is thought to be due to high rates of

phosphorylation of the erythrocyte band 3 anion exchanger resulting in increased

transmembrane oxalate exchange (Baggto et al., 1984b; Baggio et a1.,1986;Bagg¡o et

al., 1990). The rate of band 3 transporter phosphorylation in the erythrocytes of these

subjects is increased due to decreased er¡Ìrocyte membrane glycosaminoglycan content

which has been shown to inhibit this phosphorylation (Baggio et al., 1990: Baggio et al.,

l99la; Gambaro and Baggio, 1992).

7 Although this abnormality was originally demonstrated in the erythrocyte band 3 anion transporter, evidence is mounting that the same abnormality may be present in other anion transporters, i.e. those devoted to oxalate transport in the proximal tubules. Studies with monoclonal antibodies raised against erythrocyte band 3 have demonstrated the presence of an anion transporter (which does not transport oxalate) in the basolateral membrane of c¿ intercalated cells in the collecting tubules that is encoded by the s¿me gene as band 3

(Borsatti, 1991). In addition, transport studies have demonstrated that proximal tubula¡ cells cont¿in at least two anion transporters, involved in transcellular oxalate transport, that share with band 3 protein all its functional properties (Borsatti, 1991). [n support of the hypothesis that a systemic oxalate transport defect, similar to the one observed in erythrocytes, is an underlying cause of calcium oxalate urolithiasis, oral administration of glycosaminoglycans to calcium oxalate stone formers with this transport defect has been shown to, a) reduce the increased rate of erythrocyte band 3 oxalate exchange (Bagglo et al., I99Ia) and b) reduce the hyperoxaluria associated with this transport abnormality

(Baggio et al.,l99Ib).

lncreased endogenous production of oxalate is another factor thought to be responsible for the hyperoxaluria observed in some subjects with calcium oxalate urolithiasis. If hyperoxaluria is accompanied by raised urinary excretion of glycollate the condition is

said to be due to a met¿bolic abnormalrty and not high intake or absorption of dietary

oxalate (Gills and Rose, 1986). An enzyme defect in man similar, although milder, to that observed in primary hyperoxaluria type I, is though to be the underlying abnormality

in some patients with mild hyperoxaluria, as administration of pyridoxine returns urinary

oxalate excretion to normal (Gills and Rose, 1986). Pyridoxine supplementation elevates

endogenous pyridoxal phosphate, a transamination cofactor, thus diverting glyoxylate

from oxalate production by increasing its transamination to glycine. However, not all

8 subjects respond well to pyridoxine administration, suggesting the presence of other underlying met¿bolic abnormalities which are yet to be identified (Gills and Rose, 1986;

Edwards et aL.,1990).

Whether the hyperoxaluria observed in subjects with calcium oxalate urolithiasis is due to diet, transport abnormalities or melabolic disturbances, the importance of management of urinary oxalate excretion through reductions in endogenous oxalate production cannot be overlooked. As endogenous oxalate production, under normal conditions, accounts for

85 to 95%o of urinary oxalate, the regulation of endogenous oxalate production is vital in controlling unnary oxalate excretion (Smith, l99l). This regulation can be achieved primarily through diversion of the immediate precursors of oxalate, namely glycollate and glyoxylate, from oxalate production (Bais er al.,l99la).

1.2.2 TI{E ROLE oF CRYSTAL INTMNORS IN IDIOPATHIC CETCNryT UROLITilASiS

Magnesium, citrate, pyrophosphate and glycosaminoglycans are all agents present ur urine which have been shown to aid in the prevention of calcium oxalate urolithiasis

(reviewed by Robertson, L993, Fleisch, 1978, Grases et a|.,1992, Coe and Favus, L992).

These compounds act primarily by inhibiting crystal nucleation, growth and aqgregation, thus reducing the tendency towards abnormal crystalluria. Urine from normal non-stone forming subjects is known to contain these inhibitors (Drach et al., 1985a), however, numerous studies have demonstrated reduced urinary levels of these inhibitors in subjects with calcium oxalate urolithiasis (Dent and Sutor, 1971; Welshman and McGeowin,

1976; Smith et al., 1979; Nicar et al., 1983;Pak et aL.,1985; Kok ef al., 1986; Conte et a|.,1989; Grases et al.,l99O; Cupisti et al., 1992; Khan ¿f a|.,1993).

9 Recent studies by Grases et al. htgltlighted,the fact that urine is a complex heterogeneous solution, the composition of which can vary from subject to subject in terms of its ability to inhibit crystal nucleation, growth and aggregation. Moreover, these studies demonstrated that a substance in the urine of one subject, which has a strong inhibitory effect against calcium oxalate urolithiasis, may have an insignifica¡t effect in the urine of another subject (Grases et al., 1988 and 1989). Hence, in order to determine the most effective inhibitor of calcium oxalate crystallisation for a particular subject, the urine of that individual must be used.

1.2.2.I Magnesium

Magnesium prevents calcium oxalate crystal nucleation by complexing oxalate in the urine forming soluble magnesium oxalate, thus reducing the calcium oxalate activity product and the tendency towards nucleation (Flammarsten, 1929; Meyer and Smith,

1975; Barilla et a1.,1978; Li et al., 1985; Kohri et al., 1988; Coe and Favus, 1992;

Khan et al., 1993). However, inhibition of calcium oxalate nucleation by magnesium only occurs at concentrations greater than lmmoVL. At concentrations of 0.I¡"umol/L to

0.lmmol,/L, magnesium has been shown to promote calcium oxalate crystal growth (Ryall et al., lgSl). Thus, magnesium can both inhibit and promote calcium oxalate crystallisation. As the average adult daily urinary excretion of magnesium is greater than

lmmol/L (Geigy Scientific Tables) it appears that magnesium may play a vital role in protection against calcium oxalate urolithiasis, although reports as to the extent of its

contribution are conflicting (Fleisch, 1978; Bataille et aL.,1985; Michell, 1989; Coe and

Favus, 1992; Robertson, 1993). Hypomagnesuria or an increased urinary

calcium/magnesium ratio have been shown in a number of subjects with calcium oxalate

10 u¡olittriasis, supporting the hypothesis that magnesium plays an important role in the prevention of this condition (Smith et a1.,1979; Johansson e/ al., 1,980;Batzille et al.,

1985; Drach et al., 19S5b). Moreover, magnesium therapy has been shown to be beneficial inthe prevention of calcium oxalate urolithiasis (Johansson et al.,1980; Sato et al., L992;I(han et al.,1993).

1.2.2.2 Citrate

Citrate has been reported as a major risk factor in the pathogenesis of calcium oxalate urolithiasis, with hypocitraturia being present in 30 to 50%o of subjects with the condition

(Smith et a|.,1979; Nicar ør aL.,1983; Robertson, 1984; Tiselius and Larsson, 1985; Pak et al.,1985; Cupisti et al., L992). Citrate prevents calcium oxalate crystal nucleation by complexing calcium in the urine, forming soluble calcium citrate, thereby reducing the calcium oxalate activity product and the tendency towards nucleation (Pak et al., 1982;

Nicar et al., 1983; Pak, 1990). ln addition, citrate inhibits calcium oxalate crystal growth through binding to active crystal faces preventing further calcium oxalate deposition (Ryall et al., 1981; Nicar et al., 1983; Abdulhadi et a1.,1993;Khan et al.,

1993; Histome et al., 1993; Barcelo et al., 1993). Thus, citrate provides a major contribution to the total inhibitory activity of urine with respect to calcium oxalate crystallisation (Bisaz et al., L978; Fleisch, 1978; Robertson, 1993). The fact that women have higher urinary citrate levels may in part explain why the incidence of calcium oxalate urolithiasis in woman is 65% less than that in men (Shorr et a1.,1942; Ryall er al.- 1987 Baker et a1.,1993). lndeed, citrate therapy has been shown to be effective in the prevention of calcium oxalate urolithiasis, presumably by increasing urinary citrate exc¡etion (Herrmann et al.,1992; Pak er al.,1992; Abdulhadi et a|.,1993;Barcelo et al.,

tl 1993). However, this increase in urinary citrate excretion is probably the result of an increase in renal citrate production due to alkalinisation and not the ingestion of citrate itself (Robertson, 1993).

L2.2.3 GlycosaminoglYcans

Gþosaminoglycans (speciñcally chondroitin, dermatan and heparin sulphates) are

known to inhibit calcium oxalate nucleation and crystal growth (Ryall et al., I98l

Baggio et aI., l99¡a; Grover et al., 1992), however their ability to inhibit calpium oxalate

crystal aggregation is many-fold greater at the concentration which they are present in

urine (Ryall et aI., 1981; Robertson, 1993). lndeed, gþosaminoglycans have been

reported to acco¡nt for the major portion of inhibitory activity against aggregation of

calcium oxalate crystals in urine (Fleisch, 1978; Bowyer et al',1979; Ryall et al',l98l)'

There are conflicting reports as to whether a urinary deficiency of glycosaminoglycans

exists in patients with calcium oxalate urolithiasis (Martelli et al., 1985; Hwang et al.,

1988; Robertson, 1993). This may in part relate to the observations that, a)

hyperuricosuria occurs in 15 to 30%oof subjects with calcium oxalate urolithiasis (Coe,

1978; Ettinger, 1989; Coe and Faws, 1991; Robertson, 1993) and b) uric acid can

attenuate the inhibitory activrty of urinary glycosaminoglycans towards calcium oxalate

crystal growth and aggregation (Coe, 1978; Brockis et a\.,1980; Conte et a\.,1989; Coe

and Favus, 1992; Grover et al.,1992).

A recent study by Shum and Gehel demonstrated that chrondroitin sulphate isolated from

the urine of stone formers enhanced calcium oxalate crystal nucleation from urine

ultrafiltrates when compared to chrond¡oitin sulphate isolated from controls (Shum and

t2 Goehl, 1993). This study also showed that heparin sulphate isolated from controls showed higher crystal growth inhibitory activity than that isolated from stone formers.

Thus, significant differences may exist in the ability of the various glycosaminoglycan fractions in urine to inhibit and promote stone formation.

L2.2.4 Pyrophosphate

Although, pyrophosphate has been shown to inhibit growth arrd aggregalion of calcium oxalate crystals, there are conflicting reports as to whether it plays a major role in the prevention of calcium oxalate urolithiasis (Fleisch and Bisaz, 1962; Russell et al., 1964

Robertson et al., L973 Fleisch, 1973; Smith et a1.,1979; Ryall et al.,I98l; Grases ef al., 1989; Coe and Favus, 1991; Hennequin er al., 1993\. To further complicate the issue, pyrophosphate has been shown to induce crystallisation of calcium oxalate dihydrate under physiological conditions (Grases et al., 1993). It was suggested by

Fleisch that pyrophosphate inhibited calcium oxalate and calcium phosphate crystal growth by binding to calcium ions on the active crystal face (1978). Indeed, using scanning microscopy pyrophosphate has recently been shown to bind directly to calcium on the surface of calcium oxalate monohydrate crystals, rather than to calcium in solution, providing a mechanism for its inhibitory effects (Shirane and Kagawa, 1993); pyrophosphate did not bind to calcium on the surface of calcium oxalate dihydrate crystals.

Reduced urinary pyrophosphate excretion can be observed in stone fbrmrng subjects

(Russell et al., 1964; Singh et al., 1985; Sharma et al., 1992). However, it has been shown that hypopyrophosphaturia may not be a contmon symptom of calcium oxalate

l3 urolithiasis, a conclusion supported by other investigators (Fleisch, 1978; Schwille et al., l98S). The observation that under certain conditions, i.e. posþarandial, spot or 24 hour urinary sample; hypopyrophosphaturia can be observed in young females and older male normocalciuric and hypercalciuric subjects suggest that consideration need be given to the particular type of urine specimen obtained when investigating urinary pyrophosphate status

Although recent studies suggest that hypopyrophosphaturia does not have a major effect on calcium oxalate crystallisation and stone formation, mild increases in urinary pyrophosphate excretion are thought to have a protective effect against calcium oxalate urolithiasis (Fleisch, 1978; Robertson, 1993). Urinary pyrophosphaæ excretion can be increased through oral administration of orthophosphate (Russell et al., 1964 and 1976;

Fleisch, 1978).

I.2.3 TIIE ROLE OF CRYSTAL PROTT¡OTSRS IN IDIOPATHIC CATCN-N¿ UROTr|I¡ASIS

Calcium phosphate, certain mucoproteins and uric acid are thought to be promotory factors in the pathogenesis of calcium oxalate urolithiasis, with moderately raised levels

being observed in stone formers (Fleisct¡ 1978; Robertson, 1993). These substances are

said to act primarily by providing a nidus for crystal growth. Hence, pure calcium

oxalate stones are rarely found, instead calcium oxalate stones contain various arnounts

of uric acid, calcium phosphate and protenaceous material. Calcium phosphate which

crystallises readily at pH 6, considered to be the physiological pH of urine; is known to

promote heterogenous nucleation and growth of calcium oxalate (Baumarur et al., 1989;

Grases et al., 1993). These observations support the hypothesis that calcium phosphate

t4 plays a major role in the heterogeneous nucleation of calcium oxalate under physiological conditions

Analysis of calculi from stone formers has shown the stone matrix to consist essentially

of protein-carbohydrate complexes of which a protein-polysaccharide known as 'matrix

substance A' may account for as much as 85% of the total matrix (Robertson, 1993).

However, the exact mechanism/s by which these mucoproteins become entangled in the

stone matrix is/are controversial (Michell, 1989). Of the promoters of calcium oxalate

crystallisation, uric acid and calcium phosphate have received the most attention.

Microcrystals of uric acid can induce calcium oxalate monohydrate crystal growth from a

metastable supersaturated solution of calcium oxalate, a process lnown as epita,ry

(Meyer et al., L976). This induced growth is thought to be one of the mechanisms

responsible for the increased incidence of calcium oxalate urolithiasis in subjects with

gout and hyperuricosuria. The prevalence of hyperuricosuria in subjects with calcium

oxalate urolithiasis is reported to be between 20 to 30%o (Coe, 1978). Binding of urinary

glycosaminoglycans by colloidal uric acid, with a subsequent decrease in the inhibitory

activity of urine towards stone formation, is thought to be another mechanism by which

uric acid promotes calcium oxalate urolithiasis (Grover et al.' 1990).

The inconclusive results obtained from treatment of calcium oxalate stone formers with

allopurinol, a competitive inhibitor of xanthine oxidase, suggest that uric acid does not

play a major role in the induction of calcium oxalate urolithiasis (Robertson, 1993).

Other investigators have concluded that uric acid itself is not an outstanding predisposing

factor for calcium oxalate urolithiasis (Conte et a1.,1989). Thus, uric acid may play a

15 role in promoting the formation of calcium oxalate stones in vivo but the exact mechanism and the degree to which it contributes is still unclear.

Gambaro et al. have recently identified an abnormality in urate transport, similar to that

seen in oxalate transport, in the erythrocytes of subjects with calcium oxalate urolithiasis

(Garrrbaro et al., 1993). From these findings it is suggested that this abnormality may be the underlying cause of the hyperuricosuria observed in subjects with calcium oxalate

urolithiasis. This hypothesis still needs further investigation.

1.3 LITHOTRIPSY AS A TREATMENT FOR UROLITHIASIS

The introduction of lithotripsy in the 1980's heralded a revolution in the treatnent of

urolithiasis such that surgical intervention is now required in less than 5Yo of cases

(Bernstein, 1989). Although lithotripsy is effective in greater than95Yo of cases (Segura,

1989; Kelley, 1990), the need for regular metabolic evaluation and subsequent dietary or

medicinal management of individuals with recurrent urolithiasis should not be overlooked

(Pak, 1989). This is clearly evident in subjects with calcium urolithiasis as the recurrence

rate after an initial calcium stone has been shown to be l4%o at I year, 35Yo at 5 years

and52o/' at l0 years without any form of management (Uribarri et a1.,1989). Further

justification for use of dietary or medicinal intervention, in preference to end stage

surgical intervention, can be seen in the observation that treatnent with lithotripsy, in the

short term, has been associated with intraparenchymal and perirenal haemorrhage,

hematuria, extreme pain, blockage of the ureter with stone fragments (steinstrasse) ar.rd

septicemia (Lingeman et al., 1989; Kelley, 1990). ln addition, loss of renal function,

l6 hypertension and increased rate ofnew stone formation are only now presenting as long term side effects of treatnent with lithotripsy (Lingeman et aI.,1989; Kelley, 1990).

L4 OXALATE: INTRODUCTION

Oxalate, the simplest dicarboxylic acid with pK"'s at 1.3 and 4.0 (Williams and

Wandzilah 1989; Williams and Wilson, 1990), is a met¿bolic end product in man which is excreted rapidly and almost exclusively in urine; 89 to 98% of an intravenous load of oxalate is excreted in the urine within 24 to 36 hours (Hodgkinson and Zarembski, 1968;

Tiselius, 1980). Oxalate, when present in excess, becomes pathologically significant due to the limited solubilþ of its calcium salt; the solubility of the free acid at 20oC is 878/L whilst the solubilþ of the calcium salt at pH 7 and l3oC is 6.7mglL (I{agler and

Herman, 1973; Williams and Smith, 1983; Williams and Wilson, 1990). Oxalate forms soluble salts with Li*, Na*, K*, Fe2* and Fe3*; all other salts are sparingly soluble in water

(Flodgkinson and Zarembski, 1968). lndeed ttre dangers of excess oxalate ingestion in man have been recognised since the beginning of the l9th century. In the early portion of the 20th century oxalate ra¡ked among the fi¡st three poisons in terms of the number of fatalities associated with its ingestion (Hodgkinson, 1977\.

Oxalate and its immediate precursors glyoxylate and glycollate are cotnmonly found ur vegetables and fruits due to their intermediary role in photosynthesis (tlarris and

Richardson, 1980; Brinkley et al., 1981). ln normal subjects, endogenous oxalate production from dietary oxalate precursors constitutes 85 to 95yo of urinary oxalate, with the residual 5 to lsyo being derived through absorption of dietary oxalate (exogenous) from the gastrointestinal tract. lngestion of dietary oxalate precursors (glucose,

t'7 tryptothan, tyrosine, phenylalanine, xylitol, ascorbate, glycollate, glyoxylate, glycine,

hydroxypynrvate) and non-dietary oxalate precufsors (ethylene glycol and

methoxyflurane), genetic disorders (primary hyperoxaluria) and vitamin deficiencies

(vitamins A, Br and Bo) can all increase endogenous oxalate production (Dean et al.,

1968; Liao and Richardson, 1972; Hagler and Herman, 1973; Thomas et al., 1976;

Hannett et al., 1976; Rofe et al., 1976, 1977, 1979, 1980, 1986a and 1986b;

Gambardella and Richardson, 1977;Harris and Richardson, 1980; James et al-,1982 and

1985; Conyers, 1985; Bais ef at., 1985; Williams and Wilson, 1990; Conyers et al.,

1990). Excess ingestion of oxalate or increased absorption of dietary oxalate from the

gastrointestinal tract can increase the exogenous component of urinary oxalate.

A distinct correlation exists between urina¡y oxalate excretion and the pathological

severity of the symptoms associated with elevated plasma/urinary oxalate levels. Normal

subjects excrete on average 0.19r0.02mmo1/day of oxalate, whilst recurrent calcium

oxalate stone formers excrete on average 0.35+0.06mmoVday. Subjects with primary

hyperoxaluria who, due to increased endogenous oxalate production excrete between 1.0

to 2.6mmol/day of oxalate, represent the extreme. The common symptom of primary

hyperoxaluria, systemic oxalosis, produces renal failure resulting in the death of over

80% of subjects with this disorder before the age of 20 (Gibbs and Watts , 1970l' Williams

and Smith, 1983; Wanders et aL,1987; Edwards et al., 1990).

l8 1.5 EXOGENOUSLY DERIVED OXALATE AND URINARY

OXALATE EXCRETION

Initially oxalate was thought to be absorbed from the gastrointestinal tract by passive diffi.rsion (Binder, 1974; Hodgkinson, 1977; Tiselius, 1980; Knickelbein and Dobbins,

1990; Williams and Wilson, 1990). However, recent studies using buffers containing calciunu a necessary ion for the maintenance of the integnty of the conductive pathways across gastrointestinal epithelium, have demonstrated that oxalate is transported by an energy dependant process in the rat and rabbit colon, requiring chloride; and in the rabbit ileum, requiring hydroxyl, chloride, formate or oxaloacetate; (Freel et al., I98};Hatch et al., L984; Knickelbein et al., 1985; Williams and Wandzilak, 1989; Knickelbein and

Dobbins, 1990). The major site of oxalate absorption is said to be the small intestine and the rate of oxalate absorption has been shown to increase with increasing oxalate concentration (Binder, 1974;Prcnen et al., 1984; Tiselius, 1980).

Although the average dietary intake of oxalate has been shown to range from 0.8 to

2.OmmoVday (Zarembski and Hodgkinson, 1962; Williams and Wandzilak, 1989) not all of this oxalate will be available for abso¡ption. The amount of oxalate absorbed from the diet is dependant on the bioavailability of oxalate in the intestinal lumen. Luminal oxalate bioavailability is paralleled by oxalate solubility as oxalate in its ionic form is readily available for transport. Hence oxalate bioavailability is determined by luminal pH and calcium content. Fortunately the intestinal lumen is alkaline and as a result the majority of ihgested oxalate is complexed by calcium, resulting in poor absorption and subsequent faecal excretion (Hagler and Herman, 1973; Hodgkinson, 1977; V/illiams and Wandzilak,

1989). However, on a low calcium diet, oxalate complexation by calcium in the intestinal lumen is decreased, enabling increased absorption of oxalate resulting in hyperoxaluria.

l9 Conversely, reductions in urinary oxalate excretion are observed on a high calcium diet

(Zarembski and Hodgkinson, 1969). Oral ingestion of l,25-dihydroxyvitamin D has been shown to increase absorption of calcium from the intestinal lumer¡ increasing oxalate absorption resulting in hyperoxaluria (Erickson et al., 1984). The relationship between calcium and oxalate absorption from the intestinal lumen is clearly demonstrated by the fact ttrat 50% of subjects presenting with calcium oxalate urolithiasis have hypercalciuria with a mild secondary hyperoxaluria (Baggio et a1.,1983). The hypercalciuria in these subjects is primarily due to a combination of disturbances in renal calcium handling and bone calcium deposition/reabsorption which ultimately result in increased intestinal absorption of calcium in order to maintain homeostasis (Zarembski and Hodgkinson,

1969; Hodgkinson, 1978; Robertson, 1984; Smith, 1990; Menon and Koul, 1992).

1.6 ENDOGENOUS OXALATE PRODUCTION

Ascorbate and gþxylate are considered to be the immediate precursors contributing to the endogenous production of oxalate. ln addition, glycollate is thought to be directly

oúdised to oxalate by glycollate deþdrogenase (Fry and Richardson, 1979), however

this latter finding has not been unequivocally confirmed nor has the physiological

significance of this enrqe (Yanagawa et al., 1990). 'vVhilst the pathways that result in

endogenous oxalate production from glyoxylate and its precursors are well characterised,

much of tlte current information regarding the endogenous conversion of ascorbate to

oxalate is contradictory or ill defined.

20 1.6.1 TnB CovrnnuÏoN oF (L)-Asconstc Acn METABoLISM To ENDoGENOUS

OXALATE PROOUCTTON

The relative contribution of endogenous ascorbate met¿bolism to urinary oxalate excretion remains a controversial subject, with values ranging from l0 to 50%

(Zarembski and Hodgkinson, 1969; Hagler and Herman, 1973; HodgktnsorL 1977,

Gambardella and Richardson, 1977; Tiselius, 1980; Schmidt et al., l98l; Chalmers er al., 1985 and 1986; Williams and Wandzilatq 1989). A literature survey reveals that the most common figure of ascorbate conversion to oxalate, approximately 4l+6Yo, has been perpetuated from a study in the early 1960's involving one apparentþ normal subject

(Atkins et a1.,1964). The individual studied was in fact LTkg lighter than the average rveight for his height and age (Geigy Scientific Tables). Another study in the late 1950's

investigating ascorbate conversion to oxalate in three subjects with clinical disorders, multiple lipomatosis, multiple sclerosis and recurrent breast cancer with metastases;

suggested a conversion of 4l+3yo (Flellrnan and Burns, 1958). A study in the late 1960's

investigating ascorbate conversion to oxalate in fwo males, with no obvious clinical

disorder, suggested a conversion of22+I%o (Baker et al.,1966). Thus, based on previous

datz, it can be concluded that insufficient information exists to make a confident

slatement as to the contribution of endogenous ascorbate metabolism to urinary oxalate

excretion. ln addition, the observations that, a) ascorbate is oxidised non-enzymatically

to oxalate posUnicturition, b) some apparently healthy subjects have an abnormality in

their metabolism of ascorbate to oxalate and c) many assays for urinary oxalate are

affected by ascorbate, further complicate the issue (Atkins et al.,1964: Conyers, 1985,

Chalmers et a|.,1985; Rivers, 1987; Butz et aL.,1994).

2l Although ascorbate conversion to oxalate is though to be non-enrymatic,little is known about its site, mechanism and function. It is lnown that oxalate is derived from carbons

I and 2 of ascorbate (Curtin and King, 1955; Hellman and Burns, 1958; Baker et al.,

1962;Banay and Dimant,1962; Kallner et al.,1979; Farinelli and Richardsoa 1983). A pathway for the conversion of ascorbate to oxalate and th¡eonate involving dehydroascorbate and 2,3-diketogulonate as intermediates has been proposed (Figure

1.5). Although dehydroascorbate and 2,3diketogulonate have been suggested as intermediates in the conversion of ascorbate to oxalate, some workers doubt whether they

toCO, exist as free intermediates based wholly on the observation that no respiratory is produced in the metabolism of [taC]-ascorbate (Baker et al., 1966). It is for this reason that glyoxylate is thought not to be an intermediate in tlie conversion of ascorbate to toCO, oxalate. Clearþ, whilst uncertainty still exists as to the percentage of respiratory

which is produced in the metabolism of [taC]-ascorbate, values range from 0 to 60%o

(Curtin and King, 1955; Hellrnan and Burns, 1958; Atkins et al., 1964); free

dehydroascorbate, 2,3-diketogulonate and glyoxylate cannot be ruled out ¿ls

intermediates in the met¿bolism of ascorbate

Controversy also exists as to whether ingestion of an excessive amount of ascorbate can

increase endogenous oxalate production and urinary oxalate excretion (Hodgkinson,

1977; Schmidt et al., 1981; Conyers, 1985; Rivers, 1987; Williams and Wilson, 1990;

Urivetzky et al., 1992). However, careful examination of the recent literature suggests

that excessive ascorbate intake does not markedly increase either parameter (Schmidt er

al., I98l; Conyers, 1985; Rrvers, 1987; Williams and Wilson, 1990). ln general,

ingestion of large doses of ascorbate (several grams per day) result in only small

increases in urinary oxalate excretion (Lamden and Chrystowski, 1954; Taloguchi et al.,

1966; Takenouchi et al., 1966; Tiselius and Almgard, 1977; Schmidt et al., l98I;

22 z- Fructose xylitol Y Fructose- l -PhosPhate ---l Elhylene Glycol ) Xylulose Glyceraldehyde <---/ \^ ) Xylulose- I -phosphate ,- GlYcercle € or Xylulose-5-phosphate ú --\ Hydroxypyntvate

Etranolamine cr-Keto¿cids Phenylalanine and Tyosine Hydroxypyruvafe / Glycollate ct-Keto1-hydroxyglutarate Hydroxyproline

Gþcollate Laêt¿tc Dehydmgenase Dehydrogenaso Indolepyruvate TryPtolhan

Pynrvate Serine =-- GlYcine Glyoxylate Formic Acid + CO, L¿ctate Dehydrcgewte Glycollote Oxidaso Xonthine Oxidase cr-Ketoglutarate

Oxalate a-Hydro:

Purines and Pyrimidines

(L)-Ascorbate ------> (L)-Dehydroascorbic Acid 2,3-Dketo-(L)-gulonic Acid

oxalogenic substrates' Figure 1.5 pathways involved in glyoxylate catabolism and oxalate formation from carbohydrates, amino acids and other Wandzilak et al., 1994; Butz et al., 1994; Conyers et aI., 1990), suggesting that thls pathway is operating maximally under normal conditions in most individuals. This conclusion is supported by the following observations, a) an inverse relationship between the absorption of ascorbate from the gastrointestinal tract and the dose administered and b) a proportional relationship between the amount of unmetabolised ascorbate appearing in the urine and the dose administered (Lamden and Chrystowski, 1954; Kallner et al.,

L97 9; Conyers, I 985 ; Rivers, 1987).

A recent study by Singh er a/. demonstrated that ascorbate by itself does not cause significant calcification or stone formation (1993). However, under hypercalciuric

(induced by calcium carbonate feeding) and hyperoxaluric (induced by sodium oxalate feeding) conditions, ascorbate was found to exacerbate calcification and stone formation

(Si"gh et aL.,1993).

1.6.2 THS CONRIBUTION OF GLYOXYLATE METABOLISM TO ENDOGENOUS OXATETE

PRoDUcTIoN

In vitro studies have established that some 50 to 70%o of endogenous oxalate production results from the oxidation of glyoxylate which is derived from dietary glyoxylate or through metabolism of its many dietary precursors (Dean et al., 1968; Liao and

Richardson, 1972; Fainelli and Richardson, 1983; Williams and Wandzilak, 1989).

Glyoxylate oxidation to oxalate is catalysed by lactate dehydrogenase [E.C. l.I.L27), gþollate oúdase tE.C. 1.1.3.11 and xanthine oxidase [8.C. 1.2.3.21, however glyoxylate can also be reduced to glycollate by lactate dehydrogenase (Gibbs and Watts, 1966 and

1973; Romano and Cerra,1969; Warren, 1970; Schuman and Massey, 1971; Tateishi ef

23 al., 1977; Fry and Richardson, 1979; Bais et al., 1989; Yanagawa et al., 1990).

Glyoxylate functions as a reductive substrate for gþollate oxidase because in aqueous solutions it is largely present as a hydrate (Asker and Davies, 1983). Hepatectomy has

roCOz been shown to significantly reduce production from glycollate and glyo>cylate, SIYo

and 47Y' respectively; implicating the liver as the major site for endosenous oxidation of

oxalate precursors (Farinelli and Richardson, 1983). In rat liver, the oxidation of gþxylate to oxalate has been associated with peroxisomes and rough endoplasmic reticulum (Gibbs et al., L977). The activity associated *ith thrJggoso*gt is attributed to both glycollate oxidase and lactate dehydrogenase whilst the associated with th" r.o.gh r.ti*lum is attributed to lactate dehy_drogenase alone (Gibbs er ".dopl**i" aL, 1977).

I.6.2.I Enzymes Catalysing the Oridation of Glyoxylate to Oxalate

1.6.2.1.1 Lactate Dehydrogenase

Lactate dehydrogenase is a ubiquitous enz.qe found in all tissues to varying degrees;

intracellularly it is primarily located i" th".gyllgpJgg_. The oxidation and reduction of

glyoxylate, to oxalate and glycollate respectively, is catalysed by lactate dehydrogenase

requiring NADH and NAD*, as cofactors, respectively (Rofe et al., 1976 and 1986b;

Rofe and Edwards, l97S). Although, oxalate is a competitive inhibitor of glyoxylate

oxidation and an uncompetitive inhibitor of glyoxylate reduction, there is no subst¡ate

inhibition of lactate dehydrogenase by glyoxylate (Warren, 1970; Asker and Davies,

1983). Lactate dehydrogenase has been identified as the major enzyme catalysing the

oxidation of glyoxylate to oxalate in er¡hroc¡es and leucocytes (Smith et al.,l97l).

24 Lactate dehydrogenase has also been shown to be responsible for oxalate production from glyoxylate in the soluble fraction (100,0009 supernatarit fraction) of liver and heart

(Smith et a1.,1972; Gibbs and Watts, 1973). Furthermore, in the soluble fraction the

NAD*-dependant glyoxylate oxidation (lactate dehydrogenase) predominates over the

NAD*-independant oxidation in the soluble fraction (glycollate oxidase and xanthine oxidase) (Smith et a1.,1972; Gibbs and Watts, 1973).

Recent quantitative analysis of the total activities of lactate dehydrogenase, gþollate oxidase and xanthine oxidase from rat livers indicate that lactate dehydrogenase activity

(Yanagawa far exceeds that of the other two en4/mes et al., 1990). Eoy¡ever the actir4ry of lactate was determined at 9.0 as the substrate and

NAD* as the cofactor and as such bears no real relation to the of lactate dehydrogenase to oxidise under conditions. Although in vitro studies at pH 7.4, with a NAD* to NADH ratio of 0.3, indicate that the abilrty of lactate dehydrogenase to oúdise glyoxylate is many fold less than its ability to reduce glyoxylate

(Warren, I970),the cytosolic NAD* to NADH ratio in hepatocles is 725 (Williamson er al.,1967), tlrus one might expect cytosolic lactate dehydrogenase to preferentially oúdise glyoxylate to oxalate. However, this large excess of NAD* may still not be sufficient to d¡ive the lactate dehydrogenaseþlyoxylate system in the oxidative direction completely given that the intracellular lactate to pymvate ratio is 10 under conditions where the

NAD* to NADH ratio is of the order of 1000 (Park et aI.'Discussion in Regulation of

Hepatic Metabolism', 1973).

Early studies by Rofe et al., highlighted the importance of the intracellular NAD* to

NADH ratio for oxalate production from glyoxylate, presumably by lactate dehydrogenase (Rofb et al., 1976 and 1986b, Rofe and Edwards, 1978). Under

25 conditions of an increased intracellular NAD* to NADH ratio, caused by hydroxypyruvate, oxalate production from glyoxylate was increased, whilst a reduced ratio, caused by sorbitol, decreased oxalate production. Although the possibility that these manipulations were influencing FAD*IFADH, rather than NAD*AIADH cannot be ruled out.

1.6.2.L.2 Glycollate Oxidase

Glycollate oúdase is located in liver and flavin

mononucleotide for catalytic activity (Gibbs et al., 1977:. Fry and Richardson, 1979;

Fârinelli and Richardson, 1983). The oúdation of oxidase is

inhibited whilst the oxidation of IS

inhibited by oxalate (Richardson and Tolbert, 1961). Some investigators suggest, on the

basis of the following observations, that glycollate oúdase plays a major role in the

oúdation of glyoxylate to oxalate; a) glycollate oxidase activity is 30 to 50% higher in

male rat livers than in female rat livers; in male rats glycollate oxidase activity is

regulated by testosterone with castration significantly reducing hepatic levels, b) _di{.y

:uu:rth 2% glycollate oxaluria and oxalate kidneys of male but not female rats, c) male rats excrete 54%o more urinary oxalate thgn

female rats, d) hepatectomy reduced oxalate production from glycollate by 80%,

suggesting that in part this oxidation is catalysed by the enz¡rme glycollate oxidase, which

is essentially restricted to the liver and e) both Dl-phenyllactate and n-heptanoate, can

completely inhibit the conversion of glycollate to oxalate in isolated perfused rat livers,

these compounds inhibit glycollate oxidase but not lactate dehydrogenase (Silbergeld and

Carter, 1959: Richardson, 1964; Richardson, 1965; Liao and Richardson, 1973;

26 Gambardella and Richardson, 1978; Farinelli and Richardson, 1983). The above findings confirm that oxidase plays an important role in the oxidation of but state nothing about the role of oxidase in

Only one unconfirmed observation supports the conclusion that glycollate oúdase plays a major role in the oxidation of glyoxylate to oxalate, that is, gþxylate is oxidised to oxalate in isolated perfrrsed rat livers, which contain both gþollate oxidase and lactate dehydrogenase, but not in isolated perñrsed rat which contân

{ehydrogenase (Liao and Richardson, 1972).

Although glycollate and glyoxylate compete for the active site of glycollate oxidase

investigations into the kinetic properties of this en4me indicate that late is a

competitor for oxidation by glycollate oxidase (Asker and Davies, 1983). In additioru

since glyoxylate is a highly reactive molecule and a substrate for a number of

transaminases, its concentration in the peroúsomes compared to that of glycollate is

presumably low. Glycollate oxidation/glyoxylate oxidation ratios of 2500, 0.25mmol/L

glycollate and 0.025mmol/L glyoxylate; and 250,0.25mmo1/L glycollate and glyoxylate;

have been calculated indicating that if glycollate oúdase in the peroúsomes is actively

oxidising glycollate, it is unlikely that there will be significant oxidation of glyoxylate

(Asker and Davies, 1983), a conclusion supported by Yanagawa et al. (1990).

Studies by Rofe et al., htghlighted the importance of the intracellular FAD* to FADH2

ratio for oxalate production from glycollate, presumably by glycollate oxidase (1976)

Under conditions of an increased intracellular l"AD* to FADHz ratio, caused by

phenazine methosulphate, oxalate production from glycollate was increased.

27 1.6.2.1.3 Xanthine Oxidase

Xanthine oxidase is thought to play only a minor role in the conversion of glyoxylate to

levels observed in patients with the inherited SSlatgjts, a) normal urinary oxalate are disorder xanthinuria, in which xanthine oxidase activrty is absent (Gibbs and Watts,

1966), b) administration of allopurinol to human subjects does not decrease urinary oxalate excretion (Gibbs and Watts, 1966) and c) the kinetic parameters of xanthine oxidase are unfavorable for oúdation of glyoxylate especially in the presence of xanthine, its primary substrate (Yanagawa et a1.,1990'¡.

1.6.2.1.4 Comparfnentalisation of the Enzymes Catalysing the Oúdation of Gþollate

and Glyoxylate Resulting in Oxalate Production

Although cunently lactate dehydrogenase is thought to be the primary enzyme responsible

for the oxidation of glyoxylate to oxalate, glycollate oxidase is essential for the oxidatron

of glycollate to ¿ts glycollate is the primary intermediate in the production of

oxalate from many oxalate precursors such ð, serine, ethanolamine, tyrosine,

phenylanaline, hydroxypymvate and glycolaldehyde (Smith et al., 1972; Fatnelli and

Richardson, 1983).

ln 1978, Rofe a¡rd Edwards observed that amino-oxyacetzte, a potent inhibitor of liver

aminotransferase activþ, a) stimulated oxalate synthesis from glycollate, b) inhibited

oxalate synthesis from glyoxylate and c) inhibited the decarboxylation of both

compounds. A few years earlier Liao and Richardson observed that at low substrate

concentrations, more oxalate is produced from glycollate than from glyoxylate (1972). It

28 was from these observations that a comparfnentalised model for hepatic oxalate biosynthesis was proposed by and Edwards, the ¿ts

or site for oxalate synthesis. They also speculated that glycollate may be oxidised to oxalate in the peroxisomes without releasing glyoxylate as a free intermediate. The observation by Liao and Richardson that hydroxypynrvate stimulated oxidation of [l- tocl-glycollate to [laC]oxalate but inhibited oxidation of ¡U-l4c1-glyoxylate to ¡t4C1- oxalate supports this latter hypothesis (Liao and Richardson, 1978). Additional support for this hypothesis came from the discovery of an enzyme, namely glycollate.

e (Fry and

Richardson, 1979). However the existence of glycollate dehydrogenase has not been confirmed by other investigators (Yanagawa et aI., 1990).

Since the origrnal suggestion by Rofe and Edwards, the comparhnentalised model for oxalate production from glycollate and glyoxylate has gained support (Asker aad Davies,

1983; Yanagawa et al., 1990). Currently, in the liver, both peroxisomal glycollate oxidase and cytosolic lactate deþdrogenase are thought to cooperate in the production of oxalate from glycollate and gþxylate, respectively (Figure 1.6).

1.7 PRECURSORS OF GLYOXYLATE

The liver has been est¿blished as the major site of oúdation of glyoxylate precursors such as, glycollate, carbohydrates (xylitol, glucose), amino acids (glycine, phenylalanine, tyrosine, serine, hydroxyproline), ethylene glycol, etha¡olamine, glycolaldehyde and purines, resulting in oxalate production (Figure 1.5) (Dean et al., 1968: Cook and

Henderson, 1969; Liao and Richardson, 1972 and 1978; Hagler and Herman, 1973;

29 G lyco llate D ehydrogenas e______>

Glycollate Glycollate Glycollate Peroxisome Oxidase

Glycollate Oxidase a Glyoxylate o2 HrO,

Catalase or+Hro co2+H2o + Formic Acid

Cytosol

Lactate Dehydrogenase Lactate Dehydrogenase Glycollate Glyorylate Oxalate

NAD* NADH NAD* NADH

Figure 1.6 Present model for compartmentalisation of the enzymes involved in the production of oxalate, narnely glycollate oxidase and lact¿te dehydrogenase, from glycollate and glyoxylate (Asker and Davis, 1985; Yanagawa, 1990). Gambardella and RichardsorU 1977; IHarirs and Richardson' 1980; Varalashmi and

Richardson, 1983; Farinetli and Richardson, 1983; Williams and Wilson, 1990; Conyers et al.,1990). However, the immediate of in man are glycine,

(Dean et al., 1968;

Hodgkinson and Zarembski, 1968; Liao and Richardson, 1972; Hagler and Herman'

1973; Hodgkrnson, 1977; Gambardella and Richardson, 1978; Williams and WilsorL

1990; Conyers et al.,1990).

Three principle pathways are available fo¡ the conversion of to a) direct oxidation catalysed by glycine oxidase (D-amino acid oxidase), b) reversible transamination catalysed by glutamate, ornithine or (L)-alanine:glyoxylate aminotransfer¿rses or c) indirect oxidation via serine, ethanolamine, glycolaldehyde and glycollate (Ratrer et al.,1944; Weinhouse and Friedmann, 1951; Neims and Hellermann,

1962; Thompson and Richardson, 1967). Reversible transamination of glycine to glyoxylate is not a major pathway as the equilibrium of the transamination reactions favours glycine formation (Cammarata and Cohen, 1950; Weinhouse and Friedma¡n,

1951; Metzler et al., lg57). This is consistent with the furdittg that deficiency of

pyridoxine, a cofactor in transamination reactions, leads to increased rather than

decreased oxalate synthesis (Williams and Smith, 1933). Approximately 40%" of urinary

oxalate is thought to be derived from the glycine pool via glyoxylate in normal subjects,

with the glycine-serine pathway accounting for up to 80% of urinary oxalate, however no

recent confirmation of these results eústs (Hellman and Burns, 1958; Crawhall et al.,

1959; Dean et al., 1968; Hagler and Herman, 1973). Ihus, although only a small

fraction of is converted to oxalate, 0.lo/" of the total metabolic

of a fraction of oxalate is to be derived from

30 Glycollate is formed from the oxidation of glycolaldehyde which is catalysed by both aldehyde deþdrogenase and aldehyde oxidase (Maxwell and Topper, 196l). Precursors of glycolaldehyde include endogenous substrates such as ethanolamine, hydroxypymvate, serine and glycine and exogenous chemicals such as ethylene glycol and methoxyflurane

(Conyers et al., 1990). Glycolaldehyde can also exist bound to thiamine pyrophosphate

¿ls ¿ut intermediate in the transketolase reaction in the pentose phosphate pathway

(Williams and Smith, 1983).

cr-Keto-y-hydroxyglutaric acid, which is formed in the catabolism of hydro>ryproline, can be converted to glyoxylate and pynrvate by a-keto-y-hydroxyglutarate aldolase, however this enzyme þas only low activity in human tissue. Furthermore, normal urinary oxalate

levels are observed in subjects with hydroxyprolinuria and abnormal protein turnover. In

addition, administration of large doses of hydroxyproline to hyperoxaluric subjects does

not increase urinarv oxalate excretion. acid is

an immediate precursor to glyoxylate, it accounts for only a small

oxalate (Williams and Smith, 1983

1.8 PHYSIOLOGICAL PATHWAYS THAT DIVERT GLYOXYLATE

FROM OXALATE PRODUCTION

Glyoxylate is a highly reactive compound which has been show to participate in many

reactions in mammalian systems (Table 1.1) (Hodgklnson, 1977; Williams and Smith,

1983). Of the many reaction that divert glyoxylate from oxalate production, the two most

important are decarboxylation and transamination. The oxidation of glyoxylate to oxalate

31 oxidation Gþxylate , oxalate

Glyoxylate + (L) - Alanine "u , Glycine + Pynrvate Bt Gþxylate + (L)-Glutamate * , Glycine + c,-Ketoglutarate Bu Glyoxylate + (L)-Ornithine , , Glycinq + Glutamic-y-semialdeþde 86 Glyoxylate + OtherAminoAcids > Glycine + Ketoacids

Gþoylate -tH , Glycollate Bt, Glyoxylate + cr, - Ketoglutarate cc - Hydroxy - p - ketoadipate

Glyoxylate + Pyruvate € q, - Keto -y - hydroxyglutarate M, Glyorylate + CoA Formyl-S -CoA + CO2

Gþxylate + HzOz --r-----+ Formate + CO2

Glyoxylate + p - Aminothiols ------+ Thiazolidines

Ti¡ble 1.1 The reactions of glyoxylate in mammalian systems (Williams and Smith, 1e83). is a relatively minor pathway, with increased oxidation occurring only when glyoxylate levels are abnormally high (Weinhouse, 1955).

A major pathway for the metabolism of is its transamination to Three pyridoxal phosphate dependant aminotransferases which are present in mammalian liver catalyse this conversion, namely, glutamate, ornithine and (L)-alanine:glyoxylate aminotransferases (Thompson and Richardson, 1967). @ aminotransferase, which in man is predorninantþ located in the peroxisomes, in the mouse, cat, rat and dog it is located in the mitochondria; appears to be the most important of these enzymes (Rowsell et al., 1972; Nogucht et.al., 1978; Noguchi and TakadE

1979;Williams and Smith, 1983). Other amino acids, such as glutamine, phenylalanine, arginine and methionine, may be amino äorror., however they do not function as well as

(L)-alanine (Bais er al.,I99la) the transamination catalysed by these enzymes is reversible, in each case the equilibrium lies in favour of glycine formation (Weinhouse and Friedma¡n, 1951; Williams and Smith, 1983). The hyperoxaluria associated with pyridoxine deficiency highlights the physiological importance of the transamination pathway in oxalate production (Hodgkinson, 1977; Varalashmi a¡d Richardson, 1983;

Sharma et al., 1990). The functional significance of the glyoxylate transamination pathway is further highlighted by the observation that one third of subjects with primary hyperoxaluria type I have a unique protein sorting defect in which hepatic alanine:glyoxylate aminotra¡sferase, which is normally located in the peroxisome, is mistargeted to the mitochondria (Danpure and Jennings, 1988; Williams and Wandzilak,

1989; Purdue et al., l99l). Purdue et al. have shown that alanine:glyoxylate aminotransferase is mistargeted to the mitochondria due to three point mutations in its coding gene; each of these mutations specifies an amino acid substitution (1991)

32 Synergistic decarboxylation of glyoxylate and a-ketoglutarate by the o-ketoglutarate dehydrogenase complex is considered to be another important pathway for glyoxylate metabolism (O'Fallon and Brosemer, 1977; Hodgkinson, 1977). cL'

Ketoglutarate:glyoxylate carboligase is a thiamine pyrophosphate dependant enzyme which is located in the cytosolic and mitochondrial fractions of human liver. Thiamine deficiency decreases hepatic a-ketoglutarate:glyoxylate carboligase activity subsequently increasing hepatic glycollate oxidase and glycollate dehydrogenase activity which results in increased endogenous oxalate production (Sharma et al., 1990). The hyperoxaluria observed with thiamine deficiency is due to increased oxidation of both glycollate and glyoxylate to oxalate, due to increased activrty of hepatic glycollate oxidase and glycollate dehydrogenase, rather than decreased glyoxylate decarboxylation. A reduction

in the activity of a-ketoglutarate:glyoxylate carboligase does not significantþ affect

glyoxylate decarboxylation, as measured by respiratory carbon dioúde. Hence, the

physiological significance of glyoxylate decarboxylation by a-ketoglutarate:glyoxylate

carboligase remains uncertain (Hodgkinson, 1977; Stewart et al., 1981; Williams and

Smith, 1983) is non-en-4rmatically and spontaneously

to in the presence of hydrogen As most glyoxylate

originates from the oxidation of glycollate in the peroxisomes, which simultaneously

produces hydrogen peroxide, glyoxylate decarboxylation by hydrogen peroxide may be

the more important pathway for glyoxylate decarboxylation.

In the of can be reduced to

dehydrogenase. However it is thought that for every glyoxylate reduced one is

oúdised to oxalate. The fact that both glycollate and oxalate are

produced from glyoxylate in the presence of NAD* or NADH indicates that lactate

33 deþdrogenase and glyoxylate constitute a true oxidation-reduction system in the presence of nicotinamide nucleotide coenzymes (Romano and Cerra 1969).

1.9 INHIBITION OF ENDOGENOUS OXALATE PRODUCTION BY

SELECTED COMPOI.]NDS

As urinary oxalate is a dominant risk factor in the formation of calcium oxalate stones with 35 to 95Yo being derived endogenously, regulation of endogenous oxalate production would be the most appropriate mechanism to produce a change in urinary oxalate excretion. Although both ascorbate and late a¡e said to be the immediate precursors contributing to the endogenous production of oxalate, glyoxylate is though to be the most Thus, much effort has been devoted to finding metltods, enrymatic and chemical, which can divert glyoxylate from oxalate production.

Five percent pynrvate, administered orally, has been shown to be beneficial in the prevention of calcium oxalate urolithiasis in rats that were fed a diet containing 3% glycollate (Chow et a1.,1978). Although pynrvate has been shown to enhance oxalate production from glyoxylate in vifro (Romano and Cerra, 1969), when fed orally it decreases urinary oxalate excretion. The exact mechanism of this decrease in urinary oxalate excretion is unclear, however one of the following is thought to be responsible, a) pymvate may be acting as a competitive inhibitor of glyoxylate oxidation or b) pynrvate may interfere with the absorption of glycollate (Chow et aL.,1978).

Rooney et al. a series of 4-substituted 3-hydroxy-lH-pyrrole-2,5-dione derivatives to act as inhibitors of glycollate oxidase (1983), anumber of which were also shownto inhibit

34 oxalate production from glycollate in isolated perfused rat livers. Chronic administration of the 4-(4'-bromo[,1'biphenyl]a-yl) derivative to ethylene glycol hyperoxaluric rats over 58 days retumed urinary oxalate levels to normal.

Tyrosine administration to vitamin Bo deficient rats and subjects with calcium oxalate urolithiasis has been shown to reduce urinary oxalate excretion, however the underlying mechanism has not been elucidated (Zinsser and Karp, 1973\.

Taurine administration to glycollate induced hyperoxaluric rats has also been shown to reduce urinary oxalate excretion (Talware et al., 1985) by reducing hepatic gþollate oxidase activity; glycollate administration is lnown to increase hepatic glycollate oxidase activrty (Sharma et al.,1984).

O'Keeffe ¿f a/. demonstrated in vitro that glyoxylate is spontaneously decarboxylated by sulflrydryl compounds, like CoA and cysteine, and FMN. It was concluded, however, that intracellular concentrations of the sulphydryl compounds could not be increased sufficiently to make use of this reaction in vivo (O'Keeffe et al., 1973). Although reduction of hydroxypymvate to (L)-glycerate by lactate deþdrogenase leads to increased oúdation of glyoxylate to oxalate, (L)-glycerate infrrsions failed to inhibit the oxidation of glyoxylate to oxalate in rats (O'Keeffe et a1.,1973). Reduction of pynrvate to lactate, by lactate dehydrogenase, increases oxidation of glyoxylate to oxalate.

However ingestion of sodium laúate does not reduce urinary oxalate excretion in normal subjects (O'Keeffe et aL.,1973).

Administration of L, D, DL atd meso-tzrtric acid to glycollate induced hyperoxaluric rats has been shownto significantly decrease the hepatic activþ oflactate dehydrogenase and

35 glycollate oxidase and the renal activity of lactate dehydrogenase with concomitant reductions in unnary oxalate and calcium excretion (Selvam and Varalakshmi, 1989;

Selvam and Varalakshmi, 1990). In vitro however, L-tartrate was shown to significantly inhibit gþollate oxidase without a^ffecting lactate dehydrogenase.

Although the above attempts at reducing endogenous oxalate production were successful, in most instances, it is not clear whether these reductions we¡e achieved at the expense of accumulation of glycollate and gþxylate, substances which cause their own metabolic disturbances. Ideally, inhibition of endogenous oxalate would hcqf he achieved the rather than enz5rmalc inhibition resulting in substrate accumulation.

I.9.1 PRELIVßIARY INVESTGATONS INTO TTIE TIIE DIVSRSToN oF GLYOXYI-ATE

FROM OXET¡.TN PRODUCTION

Glyoxylate transamination and decarboxylation are known to be important pathways diverting glyoxylate from oxalate production (Rofe et al., 1976 and 1986b; Rofe and

Edwards, 1978). These pathways have recently been investigated in rat liver homogenates to determine whether their induction in vitro could, reduce oxalate production from glyoxylate (Bais et al., 1991a). Decarboxylation was shown to be an inappropriate pathway for diverting glyoxylate from oxalate production as, a greater amount of decarboxylation occurs from cr-ketoglutarate than from glyoxylate and cr- ketoglutarate did not significantly affect oxalate production from glyoxylate. Hence a range of amino donors were investigated for their abilrty to stimulate transamination of glyoxylate to glycine. Of the compounds investigated cysteine was shown to be an

36 effective amino donor demonstrating 93+7yo activrty when compared to alanine. In isolated rat hepatocytes cysteine was able to reduce oxalate production from glyoxylate by 85+10% (Figure 1.7). Initially this reduction in oxalate production was thought to be due to increased transamination of glyoxylate to glycine, however paper chromatographic analysis indicated the presence of another reaction product (Figure 1.8). The abilþ of glyoxylate to form thiazolidines with structurally appropriate B-aminothiols has long been lnown, thus formation of the cysteine-glyoxylate adduct, thiazolidine-2,4dicarboxylic acid, was suggested as the mechanism by which gþxylate was diverted from oxalate production. Cysteine was administered by i.p. injection to normo- and hyperoxaluric rats to investigate its effects on endogenous oxalate production. In all cases cysteine

administration was shown to significantly reduce urinary oxalate excretion (Bais et al.,

1991a and 1991b). Again formation of the cysteine-glyoxylate adduct was suggested as the mechanism through which these reductions occurred.

In accordance with the findings that cysteine can reduce oxalate production from glyoxylate, presumably through formation of the cysteine-glyoxylate adduct, Sharma and

Schwille have demonstrated that reduced glutathione, cysteamine and cysteine can reduce

oxalate production from glyoxylate (1992); these reductions were also thougbt to be due

to formation of late adducts Thus- it is sussested that oxalate oroduction

from glyoxylate may be regulated by free thiol coryPgeeds-Lnll399!þlarly. Again in

support of this hypothesis these investigators have recently shown that sulfite inhibits

oxalate production from glycollate and glyoxylate in vitro and from dichloroacetate in

vtvo (Sharma and Schwille, 1993). Again, these reductions were again thought to be due

to formation of sulfitgg,lyqlyþlq 3!!ldu-ct.

37 140

c) Ë r2o X o oà roo tro €Ë Bo '.=ão (n ()o 60 Eo\ ov i40 Cg (! ð20

0 åå6ËåËåÉ-85àÈðåFE a;¡rtso,ò Amino Donor o

Figure 1.7 The effect of 5mmol/L amino acids on the formation of oxalate from 5mmol/L [1-'aC]-glyoxylate by isolated rat hepatocytes (Adapted from Bais et al., l99la).

4 Glyo:rrylate Glycine Cysteine-Glyorrylate Adduct? (LlCysteine 3

J

I

0 4 o 6 Alanine

4 ! o ) o É(! & 0 Blank 4

)

0 Distance from Origin (aôitrary units)

Figure 1.8 Preliminary evidence that formation of the (L)-cysteine-glyoxylate adduct is the mechanism by which (L)-cysteine reduced oxalate production from glyoxylate (Adapted from Bais et al.,l99la). 1.10 THIAZOLIDINE-2,4-DICARBOXYLIC ACID, THE (L)- CYSTEINE.GLYOXYLATE ADDUCT AND (D)-

ASPARTATE OXIDASE

The ability of cysteine to form stable thiazolidines with aldeþdes has been known since the earþ 1900's. Under physiological conditions, hemimercaptals are normally formed by the reaction between aldehydes and mercaptans. However in the special case of cysteine, thiazolidines are formed due to the presence of an amino group B to ttre thiol group

(Schubert, L936aand b). Hemimercaptals, however, are still believed to be intermediates in the formation of thiazolidines fro cysteine and aldehydes (Schubert, 1936a and b;

Raûrer and Cla¡ke, 1937; Schomolka, 1957). The mechanism by which (L)-cysteine and glyoxylate are assumed to react to form cis- and trans-fhiazolidine-2,4dicarboxylic acid

is illustrated in Figure 1.9.

As the intracellular concentration of cysteine lies between 10 and 100¡rM and glyoxylate

between 0 and 200pM, adduct formation is thought to occur physiologically resulting in

greater than micromolar concentrations of adduct (Burns et a1.,1984; Hamilton, 1985;

Venkatesan and Hamilton, 1986). Although thiaz'olidines are stable at physiological pH's

(pesek and Frost, Ig75\, @l-a.pa.trte oxidase efficiently oxidises cis-adduct to À2-

thiazoline-2,4-dicarboxylic acid (Burns et al., 1984; Hamilton, 1985; Venkatesan and

Hamilton, 19S6) (Figure l.l0). (D)-Aspartate oxidase is a peroúsomal enzyme found in

many animals, rabbit, pig, beef, rat, sheep, cat, octopus and human; and tissues, liver,

kidneys, thyroid, intestine, lung, brain and nervous tissue (Hamilton, 1985).

38 C\ o \o o o \" Y -c C -c+ HOH,/\ HO H Glyoxylate

Cysùeine o o\"_." o nH-s- cf\- cH-c I OH HO H trl a\ I 'ör¡t.. H+ o -î-l- cH, -f c HO OH H ,$L /--Y ( ,.i*'^ o '-l ' o c c S- CH, -cH-c HO I I OH H N.q

-rr

H H

HOOC

H H

frazs-Adduct or or cis-Adduct H

H H H ,2

HOOC

Figure 1.9 Formation of trans- and cis-thiazolidlne-2_,4dicarboxylic- acil^^by coidensation of cysteine and glyoxylate (Schubeft, L936 Rafirer and Clarke, 1937; Schomolk4 1957). H H

(DtAspartate Oxidase ,-J""" / \ ¿coo* "oo"-"\",'"'? H o2 4ot c¡s-Adduct +¡to

H H H *,,.] _, HOO\ Hoo oH Hoo\" OH ,/ l*f / \ H \< cooH H cooH

I HH H

o o o o I lt c c -.q-cft-To"-" c-c-N-cI(cooÐ-CI1 -sH HO HO I NTl H

S-Oxalylcysteine N-Oxalylcysteine

Hydrolysis

o o o \/'c-c H-S- CH2 -CH-C ,/\ I OH HO OH liH'

Oxalate Cysteine

Figure 1.10 Oxidation of cis-thiazolidine-2,4dicarboxylic acid by (D)-aspartate oxidase and subsequent decomposition of the product to N- and S-oxalylcysteine then oxalate and cysteine (Bums et aL.,1984; Venkatesan and Hamilton, 1986) By analogy with other Ñ-thiazoline-2-carboxylic acids, A2-thiazoline-2,4-dtcarboxylic acid, the product of oxidation of the cis-adduct by (D)-aspartate oxidase, is thought to be stable to hydrolysis at37oC and pH 7 or above, but at pH 5 or below it undergoes rapid non-enzymatic hydrolyses to give a mixh¡re of N- and S-oxalylcysteines (Figure 1.10)

(Venkatesan and Hamilton, 1986). At pH's above l, N-oxalylcysteine predominates.

Skorc4mski and Hamilton have demonstrated significant quantities of N-oxalylcysteine in rat kidney, liver, brain, heart, muscle and fat suggesting that hydrolyses of thiazoline-2,4- dicarboxylic acid does occurs under physiological conditions (1986). The half-time for the nonænzymatic formation of N-oxalylcysteine under physiological conditions has been estimated to be of the order of a few minutes to an hour. As with most intermediates, the observed tissue concentration is dependent on the equilibrium between its rate of formation and its rate of destruction. N-Oxalylcysteine can be hydrolysed non- enzymatically to oxalate and cysteine (Figure l.l0), however it has been implied that there exists an enzyme to catalyse this hydrolysis (Skorczynski and Hamilton, 1986).

lndeed, in France the cysteine-gþxylate adduct has been marketed as the arginine salt,

Tiadilono, for use as a hepatoprotective and detoxicant drug approved for treaünent of hepatic diseases such as hepatitis and cirrhosis (Saigot, 1979; Raymond, 1981; Alary et al., I989a; Lamboeuf et al., 1990). The adduct is said to provides protection against hepatotoxic agents by delivering cysteine intracellularly, enabling maintenance of reduced glutathione levels and formation of adducts between cysteine and toxic aldeþdes such as, acetaldehyde and acetaminophen (Alary et al., 1989a and b; Lamboeuf et al., 1990).

Thus, in vivo and in vito the cysteine-glyoxylate adduct can be met¿bolised to cysteine and potentially oxalate.

39 Orally administered adduct is rapidly absorbed from the gastrointestinal tract and excreted in the urine, exhibiting very low toúcity (LDso (rats and mice) :29/kg). ÄJary et al. demonslrated that oral administration of a therapeutic dose of adduct (Z.7mglkg) to both rats and man resulted in urinary excretion of only 10% of the administered dose unchanged over 24 hours (1989a). However, increasing this dose to 22 and 45mglkg resulted in the excretion of 40 and 55%o of the administered dose unchanged ovet 24 hours. Thus, although adduct may be metabolised in vivo to cysteine and presumably oxalate, the pathway is saturable within the physiological range.

1.11 SUMMARY AND AIMS OF THIS THESIS

The following is a synopsis of calcium oxalate urolithiasis, its risk factors, its current methods of treatnent and the concept of the use of p-aminothiols in the regulation of hepatic oxalate production.

. ln industrialised countries, urolithiasis is now one ofthe most common disorders of the

urinary tract with annual hospital admission rates ranging from 0.1 to 20 per 10,000,

as a coru¡equence urolithiasis incurs a considerable social and economic cost; in 1986

an estimated two billion dollars was spent on stone fragmentation and removal alone

in the United St¿tes.

. Analysis of the distribution of the different classifications of renal calculi indicates

that 60 to 80% of renal calculi contain calcium oxalate. The main urinary risk factors

for calcium oxalate urolithiasis have been identified as reduced urine volume,

increased excretion of oxalate, increased excretion of uric acid, reduced excretion of

40 and decreased glycosaminoglycans, increased urine pFI, increased excretion of calcium

excretion ofcitrate.

as the most important . ln the past, much attention had been focused on hypercalciuria hyperoxaluria is now risk factor for idiopathic calcium oxalate urolithiasis' However,

in urinary oxalate known to be the more important of the fwo factors as subtle changes

precipitation. excretion markedly affect the rate of calcium oxalate

formers involve A principal form of management for idiopathic calcium oxalate stone

foods rich in calciunU increasing fluid int¿ke and restricting the dietary intake of

patrents are said to glycollate and glyoxylate, With this form of management 60o/o of

be treated with a stop forming calcium oxalate stones. The remaining 40o/o can

potassium citrate, myriad of drug therapies, including thiazide diuretics, pyridoxine,

underlying abnormality' orttrophosphate, allopurinol and others, depending on the

to reduce However, wittt

risk factor in endosenous oxalate production. As urinary oxalate is the most dominant

oxalate resulting in the formation of calcium oxalate stones, with a decrease in urinary drug that an exponential decrease in the risk of stone formation, the need for a

urinary oxalate excretion specifically reduces endogenous oxalate production and thus

is evident.

tract with only 5 to 15% . Normally oxalate is poorly absorbed from the gastrointestinal 85 95Yo is produced of dietary oxalate in the urine; the remaining to

production would be the most endogenously. Thus, inhibition of endogenous oxalate

appropriate mechanism to reduce urinary oxalate excretion'

4l . The regulation of oxalate rs to be in the

reactions which convert and to oxalate. Inhibition of lactate

oxidation to oxalate, is $þAt"gg43le, the major en4vme responsible for glyoxylate not a practical mecha¡ism to reduce endogenous oxalate production due to the

ubiquitous nature of this erizyme. In additiorl the transamination and decarboxylation

pathways, which divert glyoxylate from oxalate production, cannot be stimulated

sufEciently under physiological conditions to significantly decrease endogenous

oxalate production. However, like and

cysteamine, have the ability to form stable five membered thiazolidines with molecules

like structured

B-aminothiols may have tlterapeutic rn oxalate

production and hence urinary oxalate excretion.

. Although cysteine administered by i.p. injection has been shown to reduce urinary

oxalate excretion in rats the intracellular delivery of free cysteine is fraught with many

problems, due to the toúcity associated with the thiol group of cysteine, the

spontâneous oxidation of plasma cysteine to cystine and the lower rate of cystine

uptake into cells. acid (OTC) has been shown to

be an effective intracellular (L)-cysteine delivery compound and therefore has the

potential to limit endogenous oxalate production.

The aims of this thesis are as follows,

. to synthesise and isolate thiazolidine-2, 4-dicarboxylic acid. the late

adduct.

42 a characterise the adduct a its

under conditions and whether it exhibits unusual characteristics

oto determine the extent of adduct metabolism in

. to investigate the abilþ of orally administered B-aminothiols, namely (L)-cysteine,

(D)-penicillamine and OTC, to, a) decrease urinary oxalate excretion and b) atrect

other urinary risk factors associated with calcium oxalate stone form¿tion. These

compounds will be investigated using normo- and hyperoxaluric rat models and they

will be administered orally as they would be in a clinical setting.

. to confirm whether formation of the adduct is the mechanism

for the reduction in and urinary oxalate

excretion observed with and OTC.

¡ to use B-aminothiol-nucleophile adduct formation as a tool to further invesligate the

mechanisms responsible for oxalate production from gþollate and glyoxylate in

isolated rat hepatocytes and reconstituted en4rme systems.

. to investigate the enzymes responsible for oxalate production from glycollate and

glyoxylate in vivo using isolated rat hepatocytes and reconstituted enzymes systems.

43 CnaprBn Two

MRrpru¡rs, METHoDS Al.lD AssnYs

2.1 INTRODUCTION

p-aminothiols The studies reported in subsequent chapters investigate the effects of on

various aspects of hepatic oxalate production and urinary oxalate excretion' þ-9ÊÞ9t"

of and their metabolites on oxalate

Regulation of hepatic oxalate production by w¿ls

using isolated rat hePatocYtes and specific enzyme which the

conversion of and glyoxylate to oxalate. This chapter describes the materials

and methods employed in these studies and the ¿rssays used to measure enz)¡me activiües

and to identiff metabolites.

2.2 MATERTALS

2.2.I REAGENIS

All chemicals and biochemicals described throughout this thesis were of analytical grade

or better. The source of radiochemicals, enzymes and chemicals are listed in Tables 2.1,

2.2 and 2.3 respectively.

44 Radiochemicals Source aC¡-Glycollate tI _l Amersham, Australia

¡z-t4Cl-Glyorylate Amersham, Australia oC]-O*alate [Uf Amersham, Australia Methoxy-['Hl-Inulin ICN Radiochemicals, USA.

Tab\e 2.I Radiochemical used in the studies and their sources

Enzymes Sou¡ce

(L)-Lactate Dehydrogenase [E.C. l.I.L.2'71 Boehringer |v{ann heim, Australia

Glucose-6-phosphate Dehydrogenase [E. C. 1. 1. 1.49] Boehringer Ma¡nheim, Australia Hexokinase 8^C. 2.7 .l.Ll Boehringer Mannheim, Australia Oxalate Oxidase lE.C. 1.2.3.41 Boehringer Mannheim, Australia Oxalate Decarboxylase [E.C. 4. l. 1.3] Signa USA. Catalase [E.C. 1.11.1.6] Sigma. USA. Glycollate Oúdase [E.C. 1.1.3.15] Sigma USA. Collagenase H [E.C. 3.4.24.3] Boehringer Mannheim, Australia Peroxidase [E.C. 1. 11.1.7] Sigma USA.

@)-Aspartate Oxidase [E.C. 1.4.3. 1] In House Preparation, (Seaion 2.4.3)

Table 2.2 Enzymes used in the studies and their sources Chemicals Sou¡ce

Glycollate @ree Acid) Sigma, USA.

Glyoxylate @ree Acid) Sigma, USA. Oxalate (Free Acid) Sigma, USA.

Methylbenzethonium Hydroxide in lN Ethanol Sigma, USA.

(L)-Alanine @ree Acid) Calbiochem-Behringer, Australia

(,)-Lactate (Lithium SalÐ Boehringer Mannheim, Aust¡alia

Heparin Sodium Delta West, Australia

Adenosine-5'-triphosphate (Disodium Salt) Boehringer Mannheim, Australia

Nicotinamide-adenine Dinucleotide (Free Acid) Boehringer Mannheim, Australia

Nicotinamide-adenine Dinucleotide (Disodium Salt) Boehringer Man¡heim, Australia

Flavine Adenine Dinucleotide (Disodium Salt) Sigma, USA.

2, 6-Dichlorophenolindiphenol BDII, England

(L)-2 -Oxothiazolidine-4-carboxylate (Hydrochloride) Sigma, USA.

Thiazolidine-2,4-dicarbo>rrylic Acid (Hydrochloride) In House Preparation (L)-Cysteine (Hydrochloride) BDH, England

@)-Penicillamine (Free Acid) Sigma, USA. Pyridoxat Phosphate Boehringer Mannheim, Australia

Thiamine Pyrophosphate Calbiochem-Behringer, Australia

Nembutal (Pentabarbitone Sodium) Boehringer Ingelheim, Australia

Toluene BDH, England

2,5-Diphenyloxazole Koch-Light Laboratories. England

Emulsifier Safe Aqueous Scintillant Packard, USA. þruvate Boehringer Mannheim, Australia

@)-Glucose BDH, England DL-p-Phenyllactate Sigma, USA.

Potassium Ca¡bonate Sigma. USA.

meso-Tartaric Acid BDH, England Perchloric Acid Merck, Darmstadt DL-Malic Acid Calbiochem-Behringer, Aust¡al ia

Table 2.3. Chemicals used in the studies and their sources 2.2.2 Souncn op ANruers AND TISSUE Salærrs

Male Porton rats were used in the in vivo and. in vifro studies. These rats were purchased from the South Aústralian Government Deparbnent of Agriculture (Gilles Plains, South

Ausrralia). B_:gg t tdrr.gy obtained from the South Australian Meat Corporation (Gepps

Cross, South Australia) was used to isolate (D)-aspartate oxidase. All other €nzJ¡mes isolated as described throughout this thesis came from the livers of male Porton rats

2.2.3 ANIMALHOUST.IG

Rats were routinely housed on sawdust in plastic cages. The room temperature was kep

at a constant 22+2oC with the lights on a 10 hour hdntll4 hour dark cycle. Unless

specified, rats were fed joint stock ration ad libirum from Milling lndustries (Adelaide).

For studies investigating the effect of B-aminothiols on urinary oxalate excretion, rats

were housed in NALGENE metabolic cages.

2.3 METHODS

2.3.1 INVtvOSrUOns

For studies requiring metabolic gaces, rats were allowed to adapt by being placed rn

cages 3 days prior to the commencement of the study. The metabolic cages were washed

every second day to prevent bacterial contarnination. All diets rvere prepared from

45 powdered joint stock ration purchased from Milling Industries (Adelaide), unless

specified. The composition of the joint stock ration and the vitamin-mineral mx

contained in the ration are shown in Tables 2.4 and 2.5. The same batch of ration was

used for the duration of each study, however different batches were used between the

studies. It was not possible to use the same batch of ration for each study as these studies

prepared .r:fr"trr-.1gyga thryЀar period. The diets for the different studies were between 0.5 to by mixing B-aminothiol with the powdered joint stock ration to levels of

2.0%o, depending on the study and the B-aminothiol being tested. All dosages were then

expressed in terms of mmoVkg/day as calculated from the daily food intake, the molecular

pH, food intake, water mass of the B-aminothiol and the rat weights. Urine volume and

intake and body weight were recorded dailv, unless specified. 3mL alirygt's of the daily

urine collections were acidified the addition of l25pl of concentrated hydrochloric

acid. At the completion of each study, plasma was obtained for subsequent biochemical

analyses by cardiac puncture.

2.3.1.1 The Effect of OTC, (L)-Cysteine and (D)-Penicillamine on Urinary Oxalate

Excretion and the Risk of Calcium Oxalate Urolithiasis in a Normooxaluric

Rat Model

The short term OTC study was conducted in three parts, (i) a 5 day baseline, (ii) a 5 day

period in which OTC was given orally and (iii) a 5 day recovery period. The long term

OTC study was conducted in two parts, (i) a 5 day baseline and (ii) a 22 day period in

which OTC was given orally. The short term (L)-cysteine and (D)-penicillamine studies

were conducted in two parts, (i) a 5 day baseline and (ii) a 5 day period in which the

compounds were given orally. During the baseline and recovery periods, all rats were fed

46 Ingredient Amount (glID0gof dieQ

Whole Wheat 45.95

Whole Oats 15.0

Soþean Meal t2.37

Fish Meal 7.5

Lucerne Me¿l 5.5

Peas 5.0

Meat Meal 2.5

Tallow 2.0

Vitamin-mineral Mix " 0.55

Sodium Chloride 0.5

Choline Chloride 0.08

Lysine 0.05

Sunflower Oil 2.5

Blood Meal 0.5

a Table 2.4 Composition ofjoint stock ration (O'loughlin and Morris, 1994). Refer to Table 2.5 for the compositión of the vitamin-mineral mix' Ingredient Amount (melkgof dieQ

frans-Retinol 75

Cholecalciferol 0.05

RRR-o,-Tocopherol 50

Menadione 8.3

Cyanocobalamin t20 Thiamine 58 Riboflavin 10

Plridoxine 12

Calcium Pantothenate 20 Niacin 18 Folic Acid 2 Biotin 110 Zinc 6L Iron 66

Copper 5

Manganese roz

Molybdenum I

Iodine 0.6

Cobalt 420

Butylated Hydroryanisole 100

Table 2.5 Composition of the vitamin-mineral mix (O'Loughlin and Morris, 1994). the the control diet. During the period in which the compounds were administered,

control diet control rats were fed the control diet whilst the treated rats were fed the containing either OTC, (L)-cysteine or (D)-penicillamine; short term OTC study (L)- 5.57+0.0Smmolldaylkg,long term OTC study 3.92+0'l2mmoUdaylkg, short term cysteine study 3.71+0.l8mmol/daylkg and short term (D)-penicillamine study

2.22+0.l5mmoUdaYlkg.

Oxalate 2.3.1.2 The Effect of OTC on (Jrinary Oxalate Excretion and Calcium

Crystal Deposition in an EtþIene Glycol Induced Hyperoxaluric Rat Model

The long term effects of OTC on urinary oxalate excretion were investigated in an

study, rats were gthylene glycol hyperoxaluric rat model. For the duration of the 26 day

daY to housed individually in NALGENE metabolic cages for 6 hours every second

in enable urine collections for the remainder of the time the rats were housed collectively

ur plastic cages on sawdust; control and OTC treated groups in separate cages' Whilst

the metabolic cages rats did not have access to food, however they were gu^ Y"t"'-g(

rats were fed the !yy! The control rats were fed the control diet whilst the treated given the form of control diet containing OTC; 2.39+0.l5mmol4

joint Ethylene biscuits (3x3x lcm) which were prepared from the powdered stock ration.

Six glycol was administered at 0.75Vo in their drinking 12.2+0

whilst hourl urine volumes for rat second

this intake, water intake and were recorded At the competion of

volumes of experiment the kidneys, liver and spleen were removed and homogenised in l0

paper lmol./L sulphuric acid. The homogenates were then filtered with Watrnan filter

4',7 (Qualitative, Number l, 12.5cm) and analysis for calcium, magnesium and oxalate as described in Section 2.5.I

2.3.1.3 The Effect of Q)-Cysteine and OTC on Urinary Oxalate Excretion and theRisk

of Calcium Omlate Urolithiasis in a Glycollate Induced Hyperoxaluric Rat

Model

These studies were conducted in two parts, (i) a3 day baseline and (iÐ a 5 day period ur which the compounds were administered orally. During the adaptation and baseline periods, all rats were fed the control diet containing 1.9?i0

During the period in which the compounds were administered, the control rats were fed

the control diet whilst the treated rats were fed the control diet containing OTC or (L)-

cysteine; OTC study 7.94+0.06mmovdzy^

7.91+0.l0mmoVday/kg. Hence, the dose of OTC and (L)-cysteine administered were

equivalent on the basis of mmolldaylkg. At the completion of the (L)-cysteine study, the

liver and kidneys were removed and analysed for (D)-aspartate oxidase activity as

described in Section 2.5.10.

2.3.1.4 The Chronic on the Renal Clearance

Rats were anaesthetised with isoflurane/oxygen before polythene cannulae were placed rt

the external jugular vein, the cornmon carotid artery and the bladder (cannulae dimensions

: internal, 0.40mm; external, 0.80mm). A baseline arterial blood sample was taken and

the bladder was siphoned. 2mL of a solution containing ¡U-tocloxalate (600pCi/L),

48 ¡'FIl-methoxy inulin (450 ¡LCil/L\, sodium sulphate (35mmol/L) and sodium chloride

(7gmmol/L), in deionised water, was then infused via the jugular vein to determine whether sulphate had any effect on renal oxalate clearance. The control rats were infused with 2mL of a solution containing ¡U-toC1-oxalate (600pCi/L), ff{ methoxy inulin

(a5gpCi/L) and sodium chloride (140mmol/L), in deionised water. Thus, in a270gtat with an approximate blood volume of 20mL the sulphate infusate would increase tle blood sulphate level by approximately 8- to lO-fold. The sulphate load administered, mimics on average the amount of sulphate that is excreted in the urine per hour from rats given OTC and (L)-cysteine in the previous studies (Section 2.3.1.I'2.3.I.3). All infirsions contained physiological levels of sodium (l40mmol/L) to avoid stimulation of antidiuretic hormone secretion from the posterior pituitary gland. At appropriate times after infi¡sion of the test solution, 500pL of blood was sampled from the carotid artery into a syringe containing 20pL of heparin sulphate. 500¡rL of physiological saline was then infused into the jugular to maintain a constant blood volume. The blood was spun for I minute in a microcentrifuge with the resultant plasma being kept for analysis of [U- toCl-oxalate and [3F|-methoxy inulin as described in Section 2.5.3. Atthe completion of the study the bladder was siphoned and its contents were also kep for analysis of [U- toC]-oxalate and [3F!-methoxy inulin as described in Section 2.5.3. The rat was then killed rapidly, before recovery from anaesthesia, with an intravenous injection of nembutal (600pL). The liver, kidney and spleen were then removed and homogenised in

l0 volumes of lmol/L sulphuric acid. The homogenates were then filtered with Whaünan

filter paper (Qualitative, Number l, l2.5cm) and analysed for ¡U-toCloxalate and [3FI]-

methoxy inulin as described in Section 2.5.3

49 2.3.1.5 The Effect of Bolus Intraperitoneal Injections of the Cysteine-Glyoxylate

Adduct and Sulphate on Urinary Oxalate Excretion

Adduct Study - 2mL of physiological saline, pfJ^7.4,was given by i p. injection to each of slx male Porton rats (approximately 2809). The rats were ttren placed in metabolic cages for 24 hours to collect baseline urines. Whilst in the metabolic cages the rats were fasted.

Rat weights were recorded before they were placed into the mstabolic cages and after they were removed. After the collection period" the rats were housed communally on sawdust for a 48 hour recovery period. After the recovery period, each rat was given a 2mL i.p. injection of physiological saline containing 20mg (94p¡nol) of 5lYo-cis-adduct, 7 effective adduct dose 72tlmglkg. Agaitt the rats were in metabolic for 24 hours to collect treated urines. All urines were analysed for sulphate, oxalate, the cysteine-glyoxylate adduct, creatinine, calcium, phosphate, magnesium and uric acid and urine pH was recorded.

Sulphate Sndy - This study was performed using 6 male Porton rats (approximately

3009). The format of this study was identical to the adduct study, with the exception that

the second i.p. injection cont¿ined (450¡unol) of sodium sulphate. Uric acid,

magnesium and phosphate were not measured in this study' @e

administered in this ls to the average increase rn

observed upon administration of OTC or over a 24hour

50 2.3.1.6 The Effect of a Bolus Intraperitoneal Iniection of meso-Tartaric Acid on the In

Vivo Metabolism of the Cysteine-Glyorylate Adduct

Twelve male Porton rats (approximately 2309) were randomly divided into trruo goups; control and treated. For two days prior to the cornmencement of the study, the control group were each given a 2mL i.p. injection of physiological saline, pH 7.4, whilst the treatnent group were each given a 2nL i.p. injection of physiological saline containing

50mg (297¡rmol) of meso-tartanc acid, pH7.4. On the third day, the control gfoup were each given a 2mL i.p. injection of physiological saline containing 20mg (94¡^mol) of

5l%-cis-adduct, pH 7.4, whilst the treated group were each given aZmL i.p. injection of

physiological saline containing 20mg (94¡rnrol) of 5I%o'cis-adduct and 50mg (297pmol)

of meso-tartanc acid, PH 7.4; gffective adduct dose 89t3mg/kg. The rats were then

placed into metabolic cages for collection of a 17 hour urine specimen. The rats were

weighed prior to being placed into the metabolic cages. All urines were analysed t-or

sulphate, oxalate and the cysteine-gþxylate adduct. The urine of the control rats was

analysed for the cysteine-glyoxylate adduct the whilst adduct

levels in the meso-tzrtrate treated urines were relative of

adduct in the control and meso-tzrtrate rat urines as shown by GC/}/TS

2.3.1.7 Does an Intraperitoneal Injection of meso-Tartaric Acid Prevent Metabolism

of Endogenous Cysteine-Glyorylate Adduct in Glycollate Hyperoxaluric Rats

Given (L) -2-Oxothiazolidine-2-carb orylic Acid (OTC) ?

Twelve male Porton rats (approximately 2409) were randomly divided into fwo groups;

control and treated. Twenty four hours before commencement of the study the control

51 the group were each given a 2mL i.p. injection of physiological saline, pH 7 .4, whilst

containing treated group were each given a 2nL i.p, injection of physiological saline

the control 50mg (297¡unol) of meso-tartznc acid, pH 7.4. On the day of the study,

40mg of group were each given a 2nL i.p. injection of physiological saline containing glycollate (526ltmol) and 280mg of OTC (1900pmol), pH 7 '4, whilst the treated

of glycollate were each given a 2nL i.P of saline 40mg

(526pmol), 280mg of OTC (1900pmol) and 5

7.4. The rats were then placed into met¿bolic cages for collection of a hou¡

The rats were weighed prior to being placed into the metabolic cages' All urines were

analysed for sulphate, oxalate and the cysteine-glyoxylate adduct' Urinary cysteine-

glyorylate adduct was determined by GC/lvtS analysis'

2.3.2 l¡'tVrcnoSrulms

2.3 .2.1 Hepatocyte P reparati on and Incub ation C onditions

to Unless specified hepatocytes were isolated from fed male rats (Porton strain, 300

livers by a00g) by a modification of the original method of collagenase perfusion of the

Berry and Friend (Rofe er al., 1976). R¿ts were a¡aesthetised by i.p. injection of

nembutal (600pL). The isolation media contained O.05yo w/v collagenase, l0mmoL/L

I{EPES buffer pH 7.4, l30mmol/L sodium chloride, 4.7mmdyt- potassium chloride,

I.2mmol,/L magnesium sulphate, l.2mmolll potassium dihydrogen orthophosphate and

Z4mmo[I- sodium hydrogen carbonate through which carbogen (95o/o oxygen and 5o/o

carbon dioxide) had been bubbled for l0 minutes. The cells were incubated in isolation

media containing l.25mmoyl. anhydrous calcium cNoride (equilibrated with carbogen).

52 Cell viability was 85 to 95Yo as assessed by Blue Cells were incubated at a concentration of l0zcells/ml. lncubations were performed in glass scintillation vials which were flushed with carbogen, capped and incubated for 30 minutes at 37"C n a reciprocating shaking water bath at 80 o.p.m.. All incubations were performed in duplicate in a final volume of l.tml; lml- of hepatocytes and 100pL of effector. The stock solutions of substrate and effectors were prepared daily with the pH being adjusted to 7.4 with either sodium hydroxide or hydrochloric acid. For determination of ['oC]-

oxalate and ["C]-carbon dioxide incubations were stopped by the addition of 1.5mL of

cold lmol,/L citric acid, pH 3.0.

2.3.2.2 Factors , ffecting the Production of Oxalate and Carbon Dioxide from

Glyoxylate and Glycollate in Isolated Rat Hepatocytes

Hepatocles were incubated with l, 3 and 5mmoL/L glycollate (O.lpCi of [1-"C1-

glycollate) and glyoxylate (0.1¡rCi of [2-"C]-glyoxylate) to investþate the efficacy of

conversion of each precursor to oxalate and carbon dioxide. The effect of oxygen tension

on oxalate production from gþollate and glyoxylate was investigated by incubating

hepatocytes with 2mmol/L glycollate (0.1 FCi of 1- and 5mmol/L

glyoxylate (0.1¡rCi of [2- late in vials that had and had not been flushed with

carbogen prior to incubation. The effect of the addition of glyoxylate and glycollate on

the oxidation of glycollate and gþxylate respectively was investigated by adding cold

(t'C) 5 and lmmol/L glyoxylate and glycollate to samples containing I and 5mmolrI.

glycollate (0.lpci of [1-r+c]-glycollate) and glyoxylate (0.lpci of ¡zJ4cl-glyoxylate)

respectively.

53 2.3.2.3 lwestigations into The Efect of ftAminothiols on Oxalate and Carbon

Dioxide Productionfrom Glycollate in Isolated Rat Hepatocytes

Hepatocytes were incubated with 2mmoL gþollate (O.lpCi of [1-''C]-glycollate) for

investigations into the effects of p-aminothiols on oxalate and carbon dioxide production

from glycollate. (L)-Cysteine, @)-cysteine, (D)-penicillamine, orc

were tested at 5mmol/L, the (L)-cysteine-glyo>rylate adduct was tested at lmmol/L and

pyridoxal phosphate and thiamine pyrophosphaæ were tested at 0.5mmol/L. The

differing ability of (L)-cysteine and (D)-penicillamine to affect ca¡bon dioxide production

was further investigated by incubating hepatocytes with 0.1, 0.J, 1.0, 1.5 and 2.0mmol/L

gþollate (O.lpCi of [-"C]-glycollate) in the presence or absence of 5mmol/L (L)-

cysteine or (D)-penicillamine.

2.3.2.4 Formation of the (L)-Cysteine-Glyoxylate Adduct from Glycollate and (L)-

Cysteine in Isolated Rat Hepatocytes

Hepatocytes were incubated with 2mmo[ glycollate (0.1pCi of [t]'C1-glycollate) to

determine the ability of (L)-cysteine to affect oxalate and carbon dioúde production or

2mmo[ glycollate to determine the extent of (L)-cysteine-glyoxylate adduct formation.

(L)-Cysteine was tested at 1.0, 2.5 aú,5.Ommol/L. For determination of the

cysteine-glyoxylate adduct, incubations were stopped by boiling for 2 minutes.

54 2.3.2.5 Invesfigations into The Effect of ftAminothiols on Hepatic Metabolism as

Determined by Gluconeogenesis from (L)-Atanine and ft)-Lactate in Isolated

Rat HePatocYtes

Hepatocytes from 16 hour fasted Porton ratsl were incubated with lgmmol/L (L)-lactic acid or (L)-alanine for investigations into the effect of p-aminothiols (D)- on gluconeogenesis from (L)-lactate or (L)-alanine- (L)-Cysteine, @)-cysteine, penicillamine and OTC were tested at 5mmoUl, oxalate and glyoxylate were tested at

2mmol/L, the (L)-cysteine-gþxylate adduct and aminooxy acetate were tested at

lmmol/L and pyndoxal phosphate was tested at 0.5mmol/L. For determination

glucose, incubations were stopped bY the addition of 1.0mL of cold 5% perchloric acid.

2.3.2.6 A Kinetic Investigation of the Enzymes which Synthesise Omlate from

Glycollate and GlYoxYlate

A degree of uncertainty still eústs as to the roles of lactate dehydrogenase and gþollate

oxidase in oxalate production from glycollate and glyoxylate in vivo. To further

which investigate the role of these enzymes in oxalate production, an enzymatic system

(Section contains the majority of oxalate synthesising activity was isolated from rat liver

2.4.1). The kinetic parameters of these enzymes were established with glycollate and

by glycollate, glyoxylate as substrates. Substrate and product inhibition of these enzymes

glyoxylate and oxalate was also determined'

The dialysate containing rat liver glycollate oxidase and lactate dehydrogenase, as

described in Section 2.4.1, was used to investigate the kinetic properties of these enzymes.

55 K-'s, V^",*'s and Kis were calculated from Lineweaver-Burk plots (Michal, 1983;

Biochemistry, 1990) (Table 2.6). Lactate dehydrogenase and glycollate oxidase activity were determined as described in Sections 2.5.5 and2.5'7.

2.3.2.7 The Effect of (D)-Penicillamine on Alanine Aminotransferase Activity

To further investigate the mechanism by which (D)-penicillamine increases unnary^

oxalate excretion, the effect of (D)-penicillamine on the activitv of

aminotransferase and aminotransferase was determined at 8.0

arld. 37"C. Incubation of the mitochondrial aminotransferase with (D)-penicillamine,

alanine and glyoxylate or u.-ketoglutarate were performed in glass scintillation vials for

120 minutes at 37"C in a reciprocating shaking water bath at 80 o.p.m.. All samples

were performed in triplicate and were blanked against samples that did not contain either

(L)-alanine (enzyme activity studies) or glyoxylate ((D)-penicillamine-glyoxylate adduct

formation studies). For the study investigating (D)-penicillamine-glyoxylate adduct

formation, the aminotransferase was boiled for 3 minutes to remove any enzyme activity.

The aminotransferase (7.Smgof protein) was incubated in a media containing l00mmol/L

potassium dihydrogen phosphate, pH 8.0. Pyridoxal phosphate was not added to any of

the samples. A l0 minute preincubation with (D)-penicillamine was allowed before the

reaction was started by the addition of (L)-alanine and glyoxylate or cc-ketoglutarate. The

final volume in each vial was 444p.L. The incubation was stopped by the addition of

400pL of lmoL/L perchloric acid and treated according to the procedure of Wanders et al.

(1987) before measuring pyruvate generated (Section 2.5.4) or glyoxylate remaining

(Section 2.5.6).

56 Inhibition Type Slope Intercept Deterrnination of K¡

No Inhibitor Km 1 N/A V-"* V-o

Competitive Km I 1 (Slope)ry;*) t Inhibition V-.* V-* Ki (D(KL) I

Nonrcompetitive ( 11 (Intercept)(Vfio ) | 1+-lrl Inhibition V-r* \ Kr) KiI I -l Uncompetitive Km I (IntercepÐry;* ) I Inhibition V-"* Ki I I

Table 2.6 Formulas used in the derivation of enzyme kinetic pa¡ameters _from i-å**".r-Burk plots (Michal, 1983; Biochemistry, 1990). V,|u* refers to the V,n* with no inhibitor present. 2.3.2.8 Efect of (D)-Penicillamine Pretreatment on Oxalate and Carbon Dioxide

Productionfrom Glycollate and Glyorylate in Isolated Hepatocytes

To determine the effects of (D)-penicillamine pretreatnent on oxalate and carbon dioxide production from gþollate and glyoxylate, hepatocytes were incubated with 1, 3 and

5mmoL/L glycollate (O.lpCi of ¡t-toc1-glycollate) and glyoxylate (O.lmCi of l2:4cl- glyoxylate). Hepatocytes that had been pretreated with were

a single zn,L i of physiological saline, pH 7 .4, containing l00mg of

to rats at 48 and 24 hours to isolation ofthe which received an i.p. injection of saline were used as controls.

2.3.2.9 Formation of the Cysteine-Glyoxylate Adductfrom Glycollate in the Presence

of Glycollate Oridase and Cysteine

Some investigators suggest that glyoxylate in a 'free' form is not an intermediate in the oxidation ofglycollate to oxalate by glycollate oxidase as at low substrate concentrations more oxalate is produced from glycollate than glyoxylate by the perfused rat liver (Liao and Richards on, 1972). If glyoxylate is a 'free' intermediate in the oxidation of glycollate by glycollate oúdase, the presence of (L)-cysteine in the system will allow formation of the (L)-cysteine-glyoxylate adduct. The following study was performed to determine whether, a) gþxylate is a free intermediate in the oxidation of glycollate by glycollate oxidase and b) (L)-cysteine-glyoxylate adduct can be formed in a reconstituted system containing glycollate, glycollate oxidase and (L)-cysteine. lncubation of the glycollate oxidase suspension (Section 2.4.1) with glycollate and (L)-cysteine, no added NAD* or

NADH, were performed in 1.5mL eppendorf centrifuge tubes for 20 minutes at 37oC tn a

5',7 (0'46mg protein) reciprocating shaking water bath at 80 o.p.m.. Glycollate oxidase of pH was incubated in a media containing 100mmol/L potassium dihydrogen phosphate,

did not 7.4. All assays were performed in triplicate and blanked against samples that contain glycollate. The final volume in each vial was 520¡t'L. !!9 rncubatrqn ry?lllgpped by boiling and the supernatant a¡¡sayed for adduct formed (Section 3'3) or glycollate remaining (Section 2.5. 8)

2.4 ENZYME ISOLATION

2.4.1 ISOTAION OF OXALATE SYN|IßSNtr.\IG ACTTVIY FROM RAT LIVER

times The livers from 3 male Porton rats were sliced with a sharp blade and washed three

in cold l0mmol./L sodium phosphate buffer pH 7.0 containing 0.25mol/L sucrose. The

liver slices were then homogenised at high speed for 30 seconds in a Sorval Omnimix

giving 200mL of homogenate. The supernatant resulting from centrifugation of the

and homogenate for 5 minutes at 10009 was further homogenised in the cold for I minute,

then subjected to ultrasonication for 5 minutes using an ultrasonic probe' The

100009 supernatant resulting from centrifugation of the homogenate for l0 minutes at

10 was adjusted to pH 4.S with ZmdNI'. acetic acid and recentrifuged at 150009 for

minutes. The resultant supernatant was readjusted to pH 7'5 lvith sodium hydroúde'

Solid ammonium sulphate was then added to the supematant to 40, 60 a¡d 100%

saturation and the solutions centrifuged at 150009 for 10 minutes to yield the respective

precrpitates. The ammonium sulphate precipitates were suspended in 20mL of 5mmol/L

sodium phosphate buffer p]HZ.0 and dialysed for 16 hours agarnst 2L of the same buffer

with the buffer changed at 8 hours. The 40 to 60Yo fractton was cont¿in the

58 of oxidase and lactate activtty and a small level of xanthine oxidase activity. The for and used without further purification. Without added NAD* or NADH, lactate dehydrogenase did not oxidise or reduce glyoxylate to oxalate or glycollate respectively, this w¿ls shown spectrophotometrically. However, without added NAD* or NADF! the preparation produced oxalate from glycollate, this was shown using fl-taC]-glycollate. Over 30 or 60 minute incubations, pH 7.4 and 37oC with 100mmol./L potassium phosphate buffer, the

(0'lpCi suspension produced [toC]-oxalate from 0.1, 0.5, 1.0 and 2.0mmol/L glycollate

13'0, 16.9 of [1-t4c]-glycollate) atafate of 2.I,5.0, 9.8 and 7.5nmoU30min/mg and4.6,

and I 8.4nmoV60min/mg, respectively This observation suggests that elycollate oxidase

ca¡r oxidise to even in the presence of excess glycollate.

2.4,2 ISOL¡,ION OF MNOCHONDRI,AL AtANtr{E AIVMTOTNENSFERASE FN'OU R¿'T

LIVER

The livers from 3 male Porton rats were used to isolate hepatic mitochondria (Brand and

Hess, 1983). After the last wash, the mitochondrial fraction was suspended in 5 volumes

of 5mmol/L potassium phosphate buffer, pH 7.4. The suspended mitochond¡ia were

homogerused at 4oC by 15 second bursts for 2 minutes using an Llltra Turra"r. The

homogenised mitochondria were then centrifuged at 105,0009 for 30 minutes and the

pellet discarded. The mitochondrial supernatant contained 75mg/ml of protein and was

used without further Purification.

59 2.4.3 IsoLATIoN oF (D)-ASPARTATE O>oasB

Bovine kidney cortex and medulla (500-10009) were used to isolate (D).-aspartate oxidase as described by Nasu et al.; enzlme purification b (1982). ln step 2, sodium potassium tartrate powder was replaced with (Dl)-malic acid. After step 3, the dialysate was centrifuged at 200009 for 15 minutes. The resultant supernatant was assayed for protein and used without further purification.

2.5 ASSAYS

2.5.T ANALYSES OF URINEAND PLASIVÍA

Total urinary cysteine and cystine (cyst(e)ine) were measured colourimetrically on a

Cobas Bio Analyser (Roche, Sydney) as described by Chrastil (1990). Urinary sulphate

rryas measured turbidimetrically on a Cobas Bio Analyser as described by Lundquist et. al

(1930). Urinary phosphate, creatinine and uric acid were measured colourimetrically on

a Cobas Bio Analyser using kits purchased from Roche (SydnÐ, Trace (Melbourne) and

Roche (Sydney), respectively. Urinary calcium and magnesium were measured by atomic

absorption spectroscopy on a Perkin Elmer 3030 spectrophotometer (Cali et al., 1973).

Plasma biochemistry was measured using automated techniques (Technicon DAX, Bayer

Diagnostics, Sydney).

Urinary oxalate was measured ona as described by

Coyle et a/. (1939). The procedure for treaünent of the urines and standards and the

composition of the working solutions were as follows: ln a l0ml- centrifuge tube 2mL of

60 The sample saturated calcium sulphate was mixed with a lmIJ aliquot of acidified urine' was pH',ed to 7.0, made up to 10mL with absolute ethanol and allowed to stand

for 5 minutes and the overnight. After standing the sample was centrifuged at 2000r.p.m.

Smmol/L sulphuric acid supernatent was discarded. The pellet was taken up in lOmI, of

O'5mmoVl were and assayed according to Coyle et al.. Standards of 0- 125, 0'25 and

prepared through serial dilutions of a l0mmol/L stock solution. The colour reagent

contained 45.5mmol/L citrate buffer, pH 4.0, I26pno11l- 3-methyl-2-benzo-thiazolinone

peroxidase. The hydrazone, l.l2mmol/L dimethyt aniline and 820U/L of horseradish

oxalate starting reagent contained 41.6mmol/L citrate buffer, pH 4.0, and 1666U/L of

oxidase.

IN I{EPATOCYTE 2.5.2 MEASyRE¡VßNI oF II4C]-CARB9N DIg¡gDE AND II4C]-OXALATE

Swpnuatevr's

measured using methods described by Rofe ¡toc1-Carbon dioxide and ¡'oC1-o*alate were

acid to et at. (t976). Immediately following the addition of 1.5mL of cold lmol./L citric

containing the cell suspension in the incubation vial, a small inner vial (1.0x4.5cm ) vial 0.2,1L of lmol,/L hyamine hydroxide in methanol was inserted a¡d the incubation

capped. After 2 hours at room temperature the inner vial was removed, the exterior

production of washed with distilled water, and the radioactivity, representing metabolic

3.5mL of scintillant (0.4yo 2'5- [roc]-carbon dioxide, measured after the addition of

were then diphenyloxazole in toluene). Two lml portions of the residual cell suspension

in placed into separate scintillation vials. 0.1mL of 0.05U/mL oxalate decarboxylase

(as 400mmol./L citric acid, pH 4.5, was added to one vial, an irurer hyamine vial above vial capped' To the for metabolic ¡toc1-carbon dioxide) inserted and the incubation

6l added, an inner second vial only 0.lml of 400mmol/L citric acid buffer, pH 4.5, was

and the incubation hyamine vial (as above for met¿bolic ¡tocl-carbon dioxide) inserted vial capped. The latter vial was necessary for the determination of residual ¡toc1-carbon dioxide. The vials were incubatedat37"C for 2 hours in a reciprocating shaking water bath at g0 o.p.m.. After this time, the inner vials were removed, the outsides rinsed and

3.5mL of scintillant (0.4%2,5diphenyloxazole in toluene) added before measurement of

radioactivity. lnternal standards of lu-toc]-oxalate were included in each assay (Rofe er

at., 1976). The intemal sta¡dard contained l00pl of l.lmmol/L oxalate [10pCi of U-

toc1, 380¡rL of hepatocyte incubation buffer, pH7.4, and 520pL of lmol/L citric acid'

the pH 3.0. The amount of oxalate produced was calculated from the difference between

vials with and without oxalate decarboxylase'

2.5.3 MEASUREIßNI oF [U-14c]-Oxar,nrs AND fHj-METHoXY INULN IN PLASMA

AND URINE

clearance provides a The ratio of [U-"C1 -oxalate clearance to inulin

by measure of rinary oxalate and inulin were determined

(Packard, adding 50pL of urine in lOmI- of Emulsifier Safe Aqueous Scintillant USA')

toc and inulin before measurement of and 'H respectively. Likewise, plasma oxalate

Aqueous were determined by adding l00pl of urine in l0ml. of Emulsifier Safe toc 3H Scintillant (Packard, USA.) before measurement of and respectively'

62 2.5.4 MEASUREI!ßNT OF PYRI'VATE

Alanine aminotransferase was determined alanine conversion to

The conditions under which the incubations were performed are described rn -pyn¡vate.

Section 2.3.2.7. Pyruvate (in the presence and absence of glyoxylate) was determined

spectrophotometrically on a Cobas Bio Analyser at37"C by a modification of the method

described by Wanders et al. (1987). The buffer contained 227mmol/L Tris and 227ùtl

NADH, pH 8.3, whilst the starting reagent contained 550U/mL lactate dehydrogenase.

The final assay volume was 250¡rL; 25pL of samp\e,220¡tL of buffer and 5pL of sarting

reagent. The pynrvate assay wÍ¡s an endpoint ¿rssay measuring NADH disappearance

þynrvate reduction, NADH oxidation) at 340nm.

2.5.5 DETER},M.IA1]ON OF THE Ktr.TETIC PROPERTES OF I{EPATIC LACTA.]E

DEHYDROGENASE ACTTVTTY

As lactate deþdrogenas e in vivo is thought to play a major role in the oúdation of

glyoxylate to oxalate its kinetic properties were determined using -glyoxylate as a

substrate rather than lactate providing a more physiological picture. The kinetic

properttes of lactate were on a Cobas

Bio Analyser at 37oC by a modification of the method described by Bergmeyer et al-

(1983). The buffer contained l30mmol/L potassium dihydrogen phosphate for analysis at

p1g-7.4 or l30mmol/L boric acid for analysis at pH 9.0. ln addition, the buffer contained

either 3.Olmmoyl NAD* or 0.28mmoUl- NADH for studying lactate deþdrogenase

dependant glyoxylate oxidation or glyoxylate reduction, respectively. The starting

reagent was the reconstituted enzyme system (Section 2.4.I) con-taining lactate

63 samples contained dehydrogenase, gþollate oxidase and some xanthine oxidase. The

260pL; 50pL of glyoxylate in the range of 0 to l0mmovl. The final assay volume was

lactate deþdrogenase assay sample, 200pL of buffer and 10pL of starting reagent. The

(glyoxylate oxidation, was a rate assay over 4 minutes measuring NADH appearance

340nm; all NAD* reduction) or disappearance (glyoxylate reduction, NADH oxidation) at

rates were shown to be linear over the sampling time'

2.5.6 MEASUREI.,ßNT OF GLYOXYLATE

was determined on a Cobas Bio AnalYset at 37oC

(1970). The based on the method described by Romano and Cerra (1969) and Warren

0'28mmoVl buffer contained 130mmol/L potassium dihydrogen phosphate, pH 7.0, and NADH. The starting reagent contained 550U/mL of rabbit muscle lactate 200¡rL of buffer dehydrogenase. The final assay volume was 260pL; 50pL of sample,

4 minutes and l0pl of starting reagent. The glyoxylate assay was a rate assay over at 340nm; all measuring NADH disappearance (glyoxylate reduction; NADH oxidation)

rates were shown to be linear over the sampling time'

2.5.7 DETERMINATON oF THE KßIETIC PROPERTIES OF I{EPATIC GLYCOTTATE

O>oRse

ona The kinetic properties of glycollate oúdase were determined

Schuman er Cobas Bio Analyser at 37"C by a modification of the method descnbed by pH7'4, al. (1971). The buffer contained l30mmol/L potassium dihydrogen phosphate,

64 reagent was the reconstituted and 3.5mmovL 2,6-dtctlorophenolindophenol. The starting

and some xanthine enz;vme system containing lact¿te deþdrogenase, glycollate oxidase

0 lommol/L' oxidase. The samples contained glyoxylate or glycollate in the range of to 10pL of The final assay volume was 260pL; 50pL of sample, 200pL of buffer and

measuring starting reagent. The glycollate oúdase assay wÍls a rate assay over 4 minutes

be liûear over 2,6-dichlorophenolindophenol reduction at 610nm; all rates were shown to the sampling time.

2.5.8 MEASUREMENT OF GLYCOLLATE

at37"Cby a Glycollate was determined ona Bio

leaf glycollate modification of the method described by Schuman et al. :usir,g spinach

to rapidly oxidase (1971). When (L)-Cysteine was present in the samples, it was shown

(Basford and reduce 2,6dichlorophenolindophenol causing interference with the assay of Huennekens, 1955). Thus, when (L)-cysteine was present, a small volume

before boiling formaldehyde (final concentration of l0mmol/L) was added to the samples

(Ratner for 2 minutes enabling complexation of the residual (L)-cysteine and clarke,

pH 8'0, and Ig37). The buffer contained 130mmol/L potassium dihydrogen phosphate, of 3.5mmol/L 2,6-dichlorophenolindophenol. The starting reagent contained lU/mL

of sample, spinach leaf glycollate oxidase. The final assay volume was 260p'L;50pL

rate ¿lssay over 200pL of buffer and 10pL of starting reagent. The glycollate ¿u¡say was a

rates were 4 miriutes measuring 2,6-dichlorophenolindophenol reduction at 6l0nm; all

shown to be linear over the sampling time.

65 2.5.9 MEASURE},ßNI OF GLUCOSE

on the Abbot Biochromatic Analyser (D)-Glucose was determined spectrophotometrically

of Bergmeyer et al' (1974)' The buffer 100 at 37"Cby a modification of the method sodium acetzte' 2.3mmol/L NAD*, contained 150mmol/L Tris, pH 7.5, l6mmol/L mesenteroides glucose-6-phosphate 3.Ommoru ATP and 2.5IJ/Í]L of leuconostoc

dehydrogenase'Thestartrngreagentcontainedl4OU/mLoryeasthexokinase.Thefinal

200pL of buffer and 10pL of starting reagent' assay volume was 260pL; 50¡-rL of sample,

The glucose assay w¿u¡ an endpoint assay'

Acmrrrv IN LIVER A}rD KIDNEY 2.5.10 DElERtvm{AION OF (D)-ASPARTATE o>mess

HOMOGENATES

potassium dihydrogen phosphate' pH 7 '4' Fresh tissue lvas homogenised in l l0mmol/L

original mass of tissue (ie. 1g of tissue per and made up to l0 volumes relative to the

aliquot of homogenate was added 100pL of 10mL of homogenate buffer). To each lml

were incubated for I hour at37"C 55mmol/L 5l%:49%cis-'.ftans-adduct. The samples

inareciprocatingshakingwaterbathatS0o.p.m..Afterlhour,3mLof5%perchloric

were then centrifuged for 5 minutes at acid was added to each aliquot. The samples

3000r.p.m.inaHeraeuscentrifuge.lml.aliquotwerethenneutralisedwith250pLof

adduct as described in Section 3'3' 4M potassium dihydrogen carbonate and analysedfor

66 2.6 STATISTICAL A}{ALYSES

using the ¡vo sample t-test on Minit¿b All data was examined for statistical significance Significance is reported at the software (Minitab lnc., Pennsylvania" USA). !%, as On some graphs no confidence level. All values are expressed !ç4qJ-t.E.M..

the dimensions of the symbols' All data s.E.M. bars can be seen as they lie within

as changes in the average group excretlon relating to urinary para¡neters are expressed excrstion over the 5 daY the treatnent Period relative to the

plasma biochemistry refer to baseline, unless otherwise All data

values obtained on the last daY of each

67 Crnpren TrnsB

TTUNZOTNNM,-2,4.DICARBOXYLIC ACD, THE CYS:r¡'N'N'

Grvoxvl,nrg Aooucr

3.1 INTRODUCTION

by which Formation of the cysteine-glyoxylate adduct is the postulated mechanism

(see Chapter 4 and 5)' cysteine reduces oxalate production from glycollate and gþxylate its purist form and To confirm this postulate it was necessary to, a) prepare adduct in the detection of investigate its physical properties, b) develope a sensitive assay for

production in vivo aIld in adduct in various systems, c) investigate adduct metabolism and

adduct metabolism' vitro andd) determine what compounds are produced fromin vlvo

This chapter describes, a) a simple method for preparation of different diastereomenc

adduct' c) the mixfures of the (L)-cysteine-glyoxylate adduct, b) the characterisation of

the unusual assays developed for the detection of adduct in various systems, c)

and d) aspects of in epimerisation observed with adduct under physiological conditions

vivo adduct metabolism.

68 3.2 PREPARATION OF THE CYSTEINE.GLYOXYLATE ADDUCT

mixing l00o/o-cis-Thiazolidtne-2,4-dicarboxylic acid (l007rcis-adduct) was prepared by

glass equal amounts of aqueous l.6moL/L (L)-cysteine and glyoxylate in a scintillation

vial (pH not adjusted prior to addition, final pH 1.a). 5l%:49% cis-:trans-thiazolidine-

2,4dicarboxylic acid (517rcis-adduct) was prepared by mixing equal amounts of

(pH adjusted to aqueous l.6mol/L (L)-cysteine and glyoxylate in a glass scintillation vial

7.4 p¡.or to addition). After mixing, the vials were evacuated with nitrogen, sealed and

80 o.p.m'; the reaction allowed to proceed for 3 hours at 80oC in a shaking water bath at

to after a few minutes the solution turned bright yellow and after I hour adduct started

precipitate. At the completion of the reaction, the precipitate was collected and washed

precipitate the tfnee times with ethanol before freezedrying; SIo/rcis-adduct will not until

pH of the solution is lowered to 0.8 with concentrated HCL and cooled in an ice bath'

respectively- The precipitates were found to be pure 100%-cis-adduct or Slo/vcis-adduct

3.3 ENZYMATIC MEASUREME,NT OF THE CYSTEINE'

GLYOXYLATE ADDUCT

Bio The cysteine-glyoxylate adduct wÍls meÍ¡sured spectrophotometrically on a Cobas

is based Analyser using bovine kidney (D)-aspartate oúdase (Section 2'4'3)' This assay

minute period' on the rate of oúdation of cis-adduct by (D)-aspartate oxidase over a 40

physiological concentrations of cls-adduct ars an excellent substrate for (D)-aspartate

as the oxidase; trans-adducfis not a substrate (Flamilton, 1985). If fenicyanide is used

69 in absorbance at 420nm terminal electron acceptor for (D)-aspartate oúdase the decrease

(Dixon and Kenworttry, 1967)' corresponds to half the decrease in substrate concentration

@)-Aspa¡t¿te oxidase c5H3o4sN2- + 2Fe(cN)l- + 3H+ c5H6o4sN- + 2Fe(CN?- '

fenocyanide causing interference As cysteine was shown to rapidly reduce ferricyanide to

with formaldeþde (Ramer with the a¡isay, cysteine in the samples required complexation l0mmol/L) was added to and clarke, lg37). Thus, formaldehyde (final concentration,

to be stable to boiling' The the samples before boiling for 2 minutes; adduct was shown

containing adduct did not addition of this concentration of formaldehyde to samples

in the assay system' A standard significantþ alter the activity of (D)-aspartate oxidase

blanking solution to final curve was prepared by adding 5lolo-cls-adduct to an appropriate

The standard curve for concentrations of 0.1,0.2,0.3,04. and O.Smmol/L tot¿l adduct.

detection limit was at the adduct assay w¿ts linear between 0.0 and l.Smmol/L andthe with formaldehyde before least 0.05mmol/L. The standards and bla¡ks were also treated

because cysteine and boiting for 2 minutes. 5l7o-cis-Adduct was used as the standard

at pH 7'4 will glyoxylate reacting in the hepatocytes or the reconstituted enzyme system

samples an equal produce this diastereomeric ratio grving both sta¡rdards and

reagent contained, 376mmovl diastereomeric ratio of cis- to trans-. The colour liver catalase and potassium dihydrogen phosphate, pH 7 '4, l '45mU/L bovine 57'7¡mol[L FAD and 2.39mmolll potassium fenicyanide. The start reagent contained

and l'4mL of the (D)- (D)-aspartate oxidase suspension; i.e. 700¡rL of 173pmol/L FAD

oxidase were mixed at least 15 aspartate oxidase suspension. FAD and (D)-aspartate

of the FAD with the minutes prior to use and incubated at 30oc to allow recombination

volume was 207¡rL: 80pL of apoenzyme (Dixon and Kenworthy, |967). The final assay

'70 parameters for this assay sample, 55 pL of colour reagent and 72¡tL of start reagent. The on the Cobas Bio Analyser are shown in Table 3' l

3.4 GASCHROMATOGRAPHIC/\4ASSSPECTRoSCOPIC

ANALYSISoFURINEANDPLASMAFoRDETECTIoN

OF THE CYSTEINE.GLYOXYLATE ADDUCT

(D)-aspartate oxidase Although the cysteine-glyoxylate adduct assay using bovine kidney

was sensitive down to approximately 0.05mmoL/L in hepatocle and en'4/me

hyperoxaluric rats or supernatants, it could not be used to measure adduct in the urine of

rats treated v¡tth meso-tartaric acid due to the presence of (D)-aspartzte oxidase

inhibitors; oxalate, glyoxylate and meso-tarlanc acid. Thus, an alternative assay w¿ls

required to measure adduct in urine and plasma'

5890 gas Pooled urine and plasma samples were analysed on a Hewlett Packard

impact mass chromatograph linked to a Hewlett Packard 5988 quadrupole electron

gas was fitted with a spectrometer. The gas chromatograph used nitrogen as a carrier and

methyl silicon column which have an internal diamster of 0'25mm The column

giving retention temperature was ramped from 50 to 250"C over a 10 minute interval,

adduct' Selected ion times between 6.3 and, 6.7 minutes for the different stereoisomers of

were used to monitoring scans, which are more specific and sensitive than linear scans'

mass units detect adduct; linear scans identifi every ion between 40 and 400 atomic

monitoring scans' during a scan and as a consequence are less sensitive than selected ion

prepared by To ensure volatilþ for GC/IMS analysis, trimethylsilyl derivatives were

7l Cobas Parameter Value

l. Units owN 2. Calculation Factor 1000

3. Standatd 1 Concentration 0

4. Standard 2 Concentration 0

5. Standard 3 Concentration 0

6. Limit 2

7. Temperature (oC) 30.0

8 Type of Analysis 6

9. Wavelenglh (nm) 420

10 . Sample Volume (PL) 80

11. Diluent Volume (pL) 0

12. Reagent Volume (PL) 55

13. Incubation Time (Sec) 10

14. Start Reagent Volume (PL) 72

15. Time of First Reading (Sec) 0.5

16. Time Interval (Sec) 300 ,7 17. Number of Readings

18. Blanking Mode I

19. Printout Mode J

cis- isomer of Table 3.1 The Cobas Bio Analyser settings used for measurement of the A. ili-"Vrtãi"e-giyò"ytate addúct using (D)-aspartate oxidase and ferricyanide. on sensitivity required) evaporating an aliquot of urine or plasma (5 to 50pL; depending

adding l00pl of BSTFA to dryness with nitrogen at 90"c for 30 minutes before 30 minutes at (containing 1% TMCS as catalyst), capping each tubê and reacting for

the mass g00c. when analysing samples for adduct, using selected ion monitoring, 188,204,306anð' spectrophotometerscannedformassfragments of 73,86, I44,161,

specific for adduct 321 atomic mass units as these mass fragments a¡e known to be be free from (Figure 3.1). However, in urine the 86 mass fragment was shown to

(Figure 3.2a and This interference and was used to identiff the presence of adduct b)'

assay also had a detection limit of at least 0'05mmol/L'

from aqueous solution The gas chromatographic profile of fresh 5Io/ecis-adduct isolated

in ch¡omatogram correspond at pH 7.4 is shown in Figure 3.3. The first two peaks this last two peaks to the 2 different cls- stereoisomers of adduct (Fþre 1.9) whilst the (Figure 1.9); this was correspond to the 2 different trans- stereoisomers of adduct

determined frory these confirmed through analysis of l00o/rcis-adduct. It cannot be cis- trans- patterns which cis- or trans- stereoisomer cofresponds to which of the or

peaks.

3.5 CHARACTERISATION OF THE CYSTEINE-GLYOXYLATE

ADDUCT

by, capillary The purity of the preparation and the structure of adduct were confirmed

tube melting point determinations, proton nuclear magnetic resonance spectroscopy impact (proton NMR), KBr disk infra-red spectroscopY, Eæ chromatography/electron

'72 204

(a) 306

80P/o 86 o) 321 t5

300 350

t44 188

350 0 50 100 150 200 250 300 Mass/Charge (a.m.u.)

lyl derivative of silyl derivative of to the Peak at 5000000 5000000 MasVCharge of 73, 86, l4,16l, (a) Fiþ amu ,m00000 4000000 188, 204, 306 and 32L o'5 EB 3000000 HO --ao9 3000000 o< 2á 2000000 ã,9 2000000 Q> {J6 Ho. 1000000 É 1000000 âã

0 0 68 6.2 6.3 6.4 6.5 6.6 6.7 Retention Time (minutes)

3000 3000 MasVCharge of 86 amu Adduct (b) Ëþ æoo É, EE 2000 ! 2á o<ÞFi oËí rooo ,E 1000 Ç f, F-a 'C)

0 0 6.8 6.2 6.3 6.4 6.5 6.6 6.7 Retention Time (minutes)

Fisure 3.2aand,b Gas chromatographic scans of control urine (-----) and urine spiked ;ì"ttr ó.tñ"lfL of S¡Yo-cis-a¡dlct (-) (Section 3.4), a) scan c-omposed ot mass s.can composed of mass fragment ä;g"ätr s 6,144, toi, ttr_, 204,39ò and3}l *4 b) ã6: lgtt samples of póoled urine (n=6) were used to obtain these scans.

40000 40000 Fl 30000 o' 30000 É o cis-Adduct F> ()lJ rr¿r¡s-Adduot 20000 20000

cl o 10000 _o 10000 F

0 0 6.7 6.8 6.2 6.3 6.4 6.5 6.6 Retention Time (minutes)

Figure 3.3 Gas chromatographic s,9an of fresh 51%-cis-adduct isolated from aqueous .åi"tìó" ; pH Z + iSããtióit 3.4)._ This sgan clearly shows that 4 different ái*iålãiróträrs of adduct,2 cis- and,2 trort-, eúst underphysiological conditions' point mass spectroscopy and pIç determinations. For capillary tube melting

determinations, a small amount of adduct was worked down to the bottom of a sealed

capillary tube. The capillary tube was then placed in the capillary tube melting point

apparatus and the temperature was ramped to 220"C. Proton NMR of adduct were

obtained on a Bruker ACP 300MI{2. spectrometer using De-dimethyl sulfoxide as the

solvent and l% tetramethylsilane as the reference. Adduct stereochemistry was

confirmed by proton NMR (McMillan and Stoodley, 1968). lnfra-red spectrum were

prepared obtained on a Phillips SP3 100 spectrophotometer. KBr-adduct disks were by

mlxing KBr with adduct in a ratio of 10 to l. The mixture was ground into a fine powder

and placed into a l3mm Specac die before compression by S000kg. l007o-cis-Adduct

was trimethylsilated using BSTFA to eru¡ure volatilþ for gas chromatographic/mass

spectroscopic analysis which was performed as describe in Section 3.4. The pK's were

acid determined by titration of l}}o/vcis-adduct with sodium hydroúde or hydrochloric

whilst monitoring the pH with a glass electrode.

Sharp melting points corresponding to those observed by Forneau et al. (1971),

confirmed the purity of both diastereomeric preparations; 186oC fot l}}o/vcis-adduct and

188.C for 5lo/rcis-adduct. Upon reaching the melting point the samples bubbled

vigorously indicating release of carbon dioxide from the carboxylic acid functional

groups

The splitting patterns and chemical shifts observed in the proton NMR spectra of adduct

agree with predictions based on first principle for the suggested structure (Organic

Spectroscopy, 1987) provid.ing confirmation that at pH 7.4 and 1.4, (L)-cysteine and

(Figure glyoxylate react to give 5 lo/vcis-adduct (Figure 3.4a) and l}}o/ecis-adduct

3.4b), respectively. The chemical shift of Ho is furthest down field of all the protons on

73 cr3- trans- (a) cooH

H6 ttans-

crs- trans- cls- trans-

trans -Thiazolirtine-2,4-dicarboxylic Acid o)

H

H

2 '""'æoH

cis-Thiazolidine-2,4-dicarboxylic Acid (c)

H I H

H

54 48 4.2 3.6 3.0 2.4 Chemical Shift (ppm)

Figure 3.4a, b and c 300MHz thiazolidine-2,4-dica¡boxylic acid in D as reference; a) fresh 51%-cls-adduct with which chemical shifts), b) fresh adduct (Figure 3.4a), approximately 5 p.p.m.. This is caused by deshielding due to the strong electron withdrawing abilþ of both the carboxylic acid group, attached to carbon

2, and the adjacent sulphur atom. F{¿ appears at approximately 4.0 p.p.m. due to deshielding caused by the electron withdrawing carboxylic acid group attached to carbon

4. Hbappears as a triplet due to coupling between both It and H¿ which are magnetically unequivalent protons due to the dissymmetry around ca¡bon 4. IL a¡d H¿ are the furthest upfield, appearing at approximately 2.5 to 3.5 p.p.m., hence they are the least deshielded protons. Ft and FI¿ both appear as doublet of doublets; Ft couples with H¿ and FI¡ whilst

H¿ couples with Ét and t{á. Tlte 2" amine proton and the two carboxylic acid protons do not appear in the proton NMR spectra.

The infra-red spectra of both I}}Yo-cis- and 5I%o-cis-adduct (Figure 3.5) confirmed the presence of the secondary amine functional group (broad peak at 3600 to 3200cmt) and the carbonyl moieties on the carboxylic acid functional groups (sharp peak at 1680cmt)

(Organic Spectroscopy, 1987). The ratio of calculated mass to observed mass of ttre molecular ion of the trimethylsilyl derivative of I\\o/rcis-adduct (Figure 3.1), assuming

silation at both carboxylic acid functional groups, was 1.00, confirming the overall / elemental composition of adduct. In addition, the fragmentation patterns and the major

ion fragments confirm the overall molecular structure. Two pK values for lÙÙo/ecis-

adduct were found to be 3.90 and 9.25, corresponding to one of the carboxylic acid

functional groups and the secondary amine functional group, respectively. It is

reasonable to assume that the pK" for the other carboxylic acid functional group is below

2 as adduct is a strongly acidic dicarboxylic acid (ChemicalDataTables, 1987). These

values are in fairly good agreement with Alary et al. who clarm the pK"'s to be

approximately 1.2,4.0 and 8.0 (19S9b). Thus, at pH 7'4 adduct exists with both

74 100

80

() o -d 60

Ø É ctt H È40 H o\

20 H

0 1000 500 4000 3000 2000 1500 Frequency (cm-l)

s-adduct and ( "" ) the carbonvl moieW on at 3600 to-3200cm-l is group. dicarboxylic acid functional groups deprotonated and the secondary amine functional group protonated, hence adduct is a highly polar molecule under physiological condition'

Although, atpH1.4 when (L)-cysteine and glyoxylate initially react to produce 5lo/c-.cis'

trans-aúduct adduct, slow thermodynamic epimerisation of cls-adduct to the more stable

may occur. This was initially confirmed by the slow epimerisation of l}lo/ecis-adduct

in D6-dimethyl sulfoúde to 72o/o cls-adduct, over a 12 day period at room temperature,

as shown by proton nuclear magnetic resonance (Figure 3.4c)' Under physiological

by conditions, i.e. pH 7.4 and. 37"C, the epimerisation of cls-adduct was conñrmed

100% checking the kinetic properties of (D)-aspartate oxidase with fresh preparations of

and,5lo/rcis-adduct at different concentration, 0 to 2mmol/L; and different times (Fþre

3:6a and b and Table 3.2),20 and 140 minutes after preparation'

From the information in Table 3.2 it can be seen that cls-adduct exerts an inhibitory

effect on (D)-aspartate oxidase as the activity of (D)-aspartate oxidase at 20 minutes

with sIolrcis-adduct was 42+3yo gfeater than the activity with l00o/rcls-adduct'

Substrate inhibition of (D)-aspartate oxidase by cis-adduct, lvhen present at

concentrations greater than lmmol./L has been observed by other investigators

(Hamilton, l9S5). These results also show that considerable epimerisation of cls-adduct

to trans-addtct occurs at pH 7.4 as the V.o for (D)-aspartate oxidase increases over

time indicating that the concentration of cis-adduct decreases over time' Taking the

cis- to ratios of V-o at 20 and 140 minutes with 5l%-cis-adduct as substrate the ratio of

The trans-adduct can be calculated to be approximately 400/":60% at 140 minutes'

thermodynamrc epimerisation of Slo/vcis-adduct to 40o/o cls-adduct in vivo was

pH confirmed through i.p. injection of 20mg of S|o/vcis-adduct in physiological saline,

a pooled non-acidified 17 7 .4. to 6 male porton rats with subsequent GC/MS analysis of

75 (D)-Aspartate Oúdase ActivitY @)-AsPartate Oxidase ActivitY (¡rmoUmin/mg) (pmoVmin/mg) gg90Ôoo oêooPPP SgSEBET ãËsãBea 9 o o

ê Þ

o q

Fl ¡l o' o' É È Ê. Ê È c)l -ab

ô o= li vli

i-

ol.) ì')o hour urine sample (Figure 3.7). This slow thermodynamic epimerisation has also been

(D)-penicillamine- observed with 5,5-dimethyl-thiazolidine-2,4-dicarboxylic acid, the glyoxylate adduct (Bringmann et al.,1992).

3.6 RENAL CLEARANCE AND EVIDENCE FOR IN WVO METABOLISM OF TTTE CYSTEINE-GLYOXYLATE

ADDUCT

A single i.p. injection olSlo/vcis-adduct into rats (Section 2.3.1.5') resulted in excretion of 80t15% of the administered dose unmetabolised over a 24 hou¡. period' Urinary oxalate excretion was shown to increase significantly during the collection period (Table

3.3). , The increase in urinary oxalate excretion is thought to be due to metabolism of adduct by (D)-aspartate oxidase which subsequent breakdown of the intermediate thiazoline to oxalate and cysteine in vivo as shown in Figure 1.10. The small rise in urinary sulphate excretion, although not significant, supports this hypothesis.

To determine whether this increase in urinary oxalate excretion was due to in vivo metabolism of adduct, meso-tzrtrate, a potent inhibitor of (D)-aspartate oxidase was given simultaneouslywith adduct by i.p injectionto rats (Section 2.3.1.6). The control rats, rats that were not given meso-tartrate; excreted 70Y, of the administered dose

unmetabolised over a 17 hour period whilst the meso4artrate treated rats excreted l09olo

of the administered dose unchanged over the same period (Table 3.4). The percentage of

unmetabolised adduct excreted by the control rats, in this study, is in good agreement

with other investigators (Figure 3.S) (Alary et al., 1989a). This observation confirms

76 7o

51 20 1.18+0.02 78+1

100 20 0.91+0.01* 55+1'*

51 140 1.53+0.03 98+2

100 140 1.51+0.04 95+2

Table 3.2 )-aspartate oxidase with l}Oy' and Indirect evidence for expressed as ãpi-ã.i.utø -result¡_*g tlie mean+S from 5lolo-cls-adduct; + p<0.001.

120000 120000

100000 Fl 100000 40olccis-Adduct 60o/efians-Adduct ô' o 80000 g) (J 80000 60000 o o 60000 ,10000 o 6l 40000 É _o F 20000 20000

0 0 62 6.3 6.4 65 6.6 Retention Time (minute s)

Fizure 3.7 Confirmation of the epimerisation of 5lYo cis-adductto 40%o cis-adduct in ,¡-ío lS..tio n 2.3 .l .6) . Gas chromâtographic scan of pooled urine (n=6) from rats given an i.p. injection of SlYo cis-adduct. €c) 80 åË ãE 70 "obgR .É'! 60 EE 50 9

ã e.¡ 0 ú6ËÞ 0 80 90 100 0 10 20 30 405060 70 Adduct Dose (mglkg)

of unmetabolised adduct excreted in urine over 24 hours (o) (Alary et al., 1989) or 17 hours following i.p. injection adduct to rats. Parameter Control Treatment

Volume (mI ) 34.9+9.6 37.6!7.r pH 6.69r0.08 6.70t0.10

Oxalate (pmoUday 10.1r0.3 11.7+0.5 **

Sulphate (pmoUday) 234|rl 266!23

Adduct (pmoVday) 0.0+1.6 76.8+14.8 *

Table 3.3 The effect of a bolus i.p. injection of 5lo/o-cis-adduct on selected urinary parameters (Section of l?È.lmglkg. The .results.are expressed as the me different from control- period; *' pcb.Ot and ** p<0.0 were the only urinary electrolytes measured.

Pa¡ameter Control meso-Tartanc Acid

Volume (mL) r7.0Ð..9 I 1.3+3. I

pH 6.85f0.06 6.7910.08

Oxalate (pmoVlTHr.) 5.1110.23 4.28fl.25 *

Sulphate (pmoVl7Hr.) 111r8 95+16

Adduct (pmoVlTI{r.) 64.9+5.2 102.918.1 't

Thble 3.4 The effect of a bolus i.p. injection of meso-tartznc acid on the metabolism of cis-adduct in vivo as dete 2.3.1.6). Effective adduct respectively. The results different from control gro urinary electrol¡es measured. Again urinary that adduct is actively metabolised in vivo by (D)-aspartate oxidase'

period after a oxalate excretion was shown to increase significantly during the collection

is produced during the bolus i.p. injection of adduct (Table 3.4), confirming that oxalate

2Y" metabolism of adduct, however the increase in urinary oxalate could account for orly

of the adduct that was metabolised.

From this lack of conversion of adduct to oxalate we can conclude that either, a) N- and

Soxalylcysteine are not products of adduct met¿bolism in vivo' b) N- and S-

products oxalylcysteine do not undergo hydrolysis to oxalate or c) the majority of the of

a¿- adduct metabolism are not excreted in the urine. It is reasonable to ¿tssume that

(D)-aspartate thtazolne-Z,4dicarboxylic aci{ the product of the oxidation of adduct by

have found oxidase is hydrolysed to N- and S-oxalylcysteine in vivo as Skorczynsk,¿, et al.

fat significant quantities of N-oxalylcysteine in rat kidney, liver, brain, heart, muscle and

(Skorczynski and Hamilton, 1986). However, using radioactive tracers Nary et al' have

5 9% of the shown that when adduct was administered at a dose of Z2mg4

dose that was metabolised, 30% was found in the organs (highest percentages in the liver

and kidneys), 4o/o was eliminated in the faeces, 5Yo of the dose underwent pulmonary

elimination with the remaining dose, l6Yo, beng excreted in the urine as adduct

metabolites (19S9a). Given these findings and the lnownledge that oxalate is rapidly

cleared from the body, via the kidneys, it can be concluded that significant metabolism of

the cysteine-glyoxylate adduct to oxalate does not occut in vivo.

Although a pathway for the non-enzymatic ring opening and hydrolysis of other

thiazolidine.l-carboxylic acids has been shown to exist (W-lodek et al-, 1993)' it appears

unlikely that adduct can participate in such a pathway despite the unusual epimerisation

of adduct which suggests that under physiological conditions nontnzymatic ring opening

77 hemimercaptalformation and and closing can occur. Moreover, studies on the kinetics of

glyoxylate indicate that ring closure between p-aminothiols (cysteine and cysteamine) and

that ring closure is ring closure is many fold faster than hemimercaptal formation and our owr essentially irreversible (Fiøpatrick and Massey, 1982)' In addition'

existence of free observations and those of Burns et aI. (1984) cannot confirm the glyoxylate or cysteine in a solution containing adduct. Thus, under physiological

exist to any great conditions, the open chain conformation of adduct does not appeaf to

extent, hence hydrolysis to glyoxylate and cysteine does not occur.

78 Cneprsn Foun

I¡rr wt¡o Sruoms

4.1 INTRODUCTION

the most As explained in Chapter I urinary oxalate has been shown to be one of

levels of important risk factors in the formation of calcium oxalate stones with increased

calcium urinary oxalate being demonstrated in 15 to 50Yo of patients with idiopathic

The importance oxalate urolithiasis (Tiselius, 1980; Smith, 1990; Edwards et al.,1990)'

by the of urinary oxalate as a risk factor for calcium oxalate urolithiasis is highlighted

the risk of calcium observation that small reductions in urinary oxalate excretion decrease

is derived from oxalate stone formation exponentially. As the majority of urinary oxalate

that endogenous oxalate production, 85 to 95Yo; the need for a medicinal treatnent

specifically reduces endogenous oxalate production and urinary oxalate excretion is

evident.

on urinary oxalate In tlus Chapter, the effects of OTC, (L)-cysteine and (D)-penicillamine

investigated using excretion, and other risk factors for calcium oxalate urolithiasis, are

both normo- and hyperoxaluric rat models. As adduct formation is though t9 il g.'

e¡<9r9Ji91,l{entification o! q{duct Tgç!4g!4lþy_y-hi3Lçy-$epe reduces urinary-o;

administration of OTC, (L)-cysteine and (D) biochemistry was

any toxicity determined to asses the of these compounds and 1o identifr

'79 associated \¡/ith their administration. The cysteine-glyoxylate adduct was also

this administered over a short term to determine whether significant metabolism of

(L)-cysteine, and urine compound occurs. As sulphate, a major met¿bolite of OTC and

and pH have been implicated in renal oxalate transport, the effects of hypersulphaturia

aciduria (indirectly) on urinary oxalate excretion were investigated.

4.2 RESULTS

The primary aim of Ìhe in v¡vo studie yas to detery19 th9 effect of the administered

urinary oxalate exc¡gliqq s¡þsequgntly-thq qlk qf formation- of compounds 9n -and an caìcium oxalate stones. Qualitatively it is simple to make assumptions as to whether

the increase or decrease in one of the major urinary risk factors will increase or decrease

risk of calcium oxalate urolithiasis. However, as there are a number of important urinary

risk factors which may all change at a given time, it is difficult to make quantitative

interpretations about the overall effect of these changes on the risk of calcium oxalate

urolithiasis without an adequate model. A simple model for determining the relative

probability of calcium oxalate stone formation based upon the major urinary risk factors,

urine volume and pH and the urinary excretion of oxalate and calcium, has been

in developed by Robertson (Robertson, 1984). This model combines the overall changes

(Figure l the relative risk factors of stone formation, 6volun'", opg, ooxalare ild 6cot.ion,,, ' l),

to generate the relative probability of stone formation, Prt'

o Vo¡¡me . o pH , o Oxalate .G C"l"iu- Prf = I + oyo1urn".oOH. ooxalate . GC.lcium

80 l 1 using the Changes in the relative risk, o, are calculated from the risk curves in Figure '

deviations from overall changes in the individual risk factors when expressed as standard

urine volume the mean. Given the difference in urinary calcium and oxalate excretion and

Parameters from between humans and rats (Geigy Scientific Tables, 1981; Urinary

Chapter 4),

Urinary Parameter Human (Male) Rat Male)

Volume (ml/day) 690-2690 9-15 pH 5.3-7.2 6.5-7.4

Oxalate (pmoVday) 200-500 7.70-10.50

Calcium (pmoUdaY) 2500-7500 12.0-34.1

used and the fact that this model was developed using human urine it cannot be

quantitatively to predict the effects of OTC, (L)-cysteine and (D)-penicillamine on

calcium oxalate stone formation. Nevertheless, in order to gauge what effect the observed

subjects, changes might have on the probability of calcium oxalate urolithiasis in human

have been made, a) the maqrytu-@g!9þUe be the following assumptions -qgr]9 -wtl! rats Pç similar to those which might be observed in humans, b) for normooxaluric control

has been set at has been set at 30% and c) for glycollate hyperoxaluric control rats P*

90%. The hyperoxalunc control P value was set at 90olo as the urina_ry excretion of

oo*"'u,. value oxalate was greater than 3 standard deviations from the mean, hence the

was 19

8l 4.2.I N.MR-E)GERIMENTAL VARTATION IN DIETARY Mtr'IERAL INIAKE AND URINARY

ELECTROLYIE EXCRETION

Under normooxaluric conditions administration of OTC or (L)-cysteine did not significantly alter food intake, water intake or the rate of weight gain in any of these studies. Thus, the intake of calcium, phosphate, oxalate, magnesium and other impoftant electrolytes does not vary significantly between the control and treated goups within any of the experiments. However, basal food intake and urinary calciunL phosphate, magnesium and uric acid varied by as much as 16, 240, 190,95 and 140% respectively

in between the studies (Figure 4.1,4.3,4.9 and 4.13). These variation can be attributed

part to a) biological variation in the rats chosen for each b) variation in the initial

weight of the rats in each experiment and c) different food intake between the

experiments. More importantly however, these variation can be attributed to seasonal

variation in the electrolyte composition of the joint stock ration as the studies were

performed over a ttree year period. Despite interexperiment variation in the basal levels

of certain electrolYtes under normooxaluric conditions, the basal level of urinary oxalate

between the varies by less than 40%. Within an experiment, i.e. same rats,

same food and same dietary intake, the variation in urinary oxalate excretion was less

than25Y".

4.2.2 TTD EFFEcT oF OTC oN URINARY OXALATE EXCRETION AND TI{E RISK OF

C.qrcnn¡ Ox.qLarE URomlasls

Due to problems associated with the intracellular delivery of cysteine (Karlsen et al.'

lggl; Anderson and Meister, 1989; Whiæ et al.- 1993) and the observation that under

82 cer[ain conditions, premature and newborn infants as a result of low cystathionase activity and cinhotic patients; cysteine is an essential amino acid required during

have parenteral nutrition (chung et a1.,1990; coloso et al., 1991), many compounds been investigated for their potential as intracellular cysteine deliver agents' .Egq-q9t'

has studies one particular compound (L)-2-oxothiazolirline-4-carboxylic acid or oTc

oxidised by shown particular promise. OTC is transported into most cells where it is

(Williamson and cytosolic (L)-5oxoprolinase to yield cysteine and carbon dioxide

hepatic Meister, 1982; Meister et al., 1986), hence OTC delivers cysteine directly into the

cytosol.

(L)-5-oxoprolinas" + co2 + ADP + oTC + ATp + Hrg ' G)-cysteine 4

and Administration of OTC has been shown to significantly increase glqtqttri-q!ç,l4urin¡

et al', sulphate, indicative of its metabolism to cysteine (Williamson et al., 1982;Moslen

Taguchi et 1988; Anderson and Meister, 1989; Mesina et a1.,1989; Chung et al', 1990;

al., 1990; Coloso et al.,I99I; Taylor et al',1992) Orall administered OTC is Wicklf

minutes with a absorbed from the gastrointestinal tract, peaking in the plasma at 50

As- d9llyqr¡ subsequent peak of plasma cysteine at 120 minutes (Porta et al', I99I} 9JÇ

with the free thiol cysterne directly to the cell the extracellular toxtcity that is observed

group of cysteine is avoided.

4.Z.Z.l Long Term OTC Adminisftation in a Normooxaluric Rat Model

period in This study was conducted in ¡vo parts, (i) a 5 day baseline and (ii) a 2l day

this which oTC was administered orally (section2.3.Ll). The effective dose of oTC in

83 administered over a 2I day period OTC study was 3.92t0.l2mmoykg/dîy. Il_"" produced no significant çþanges in urine pH and volume, food and water intake or the urinary excretion of uric acid" magnesium, calcium, and creatinine (Figure

4.1). ln addition, OTC administration did not significantly alter weight gain; Control

3.g0l:0.34g/day and OTC treaæd 3.8lt0.3ldbv. Hg1-g.-l1qq[c3l] dtqg!" i" unnary oxalate was observed, 30o/o; (Figure 4.I and 4.2). At the completion of the treatnent period there were no significant differences in any of the plasma parameters ür

the OTC treated group when compared to the controls (Table 4.1). Although OTC

significantly decreased urinary oxalate excretion over the entirety of the treatrnent period,

it should be noted that in the fi¡al 7 days there was little difference in oxalate excretion

between the control and the OTC treated groups (Figure 4.2). The reason for this lack of

difference was found to be a small but not decrease in oxalate excretion in ttre

controls, rather than a change in oxalate excretion in the OTC treated group' In addition,

over the entire treatnent period there was no significant difference in the ability of OTC

to reduce oxalate excretion. When compared to the controls the reduction in urinary

oxalate excretion was 3.14+1.05p,moVday or 34Yo compared to basal levels (Figure 4.1).

Overall, the significant reduction in urinary oxalate excretion and the trend towards

reduced urinary calcium excretion and pH (Figure 4.1) reduced th" Ps from 3_0þto 5%:

It should be noted that over the first of the treaünent OTC decreased

unnary oxalate excretion sigqifica¡,r-lly ; 4'24t1.3}¡rmoVday, P<0'0 I'

4.2.2.2 Short Term oTc Administration in a Normooxaluric Rat Model

This study was conducted in th¡ee parts, (i) a 5 day baseline, (ii) a 5 day period in which

OTC was administered orally and (iii) a 5 day recovery penod (Section 2.3.I.1)' The

84 Food Inøke (elday)

Water Intake (rnl./day)

Urine Volume (ml/day)

Urine ptV0.1

Urinary Creatinine/l0 (pmoUdaY)

Urinary Ox¿late (pmoVdaY)

Urinâry Calcium/lO (pmoUdaY)

Urinary Phosphate/ I 00 ( pmoVday)

Urinary Magnesium/ 100 (pmoUdaY)

Urinary Uric Acid (pmoUdaY)

-8-6-4-202468 Change During the Treatment Period (units/day)

Pa¡ameter Baseline Value Food Intake (sJMv) t9.2fr.41 lVater Intake (slbv) 32.4Ð..64 Urine Volume (mVdav) 12.9Ð..11 Urine pH 7.18r0.10 Urinarv Creatinine (PmoVdaY) 76.rt+.1 Urinarv Oxalate (u.moUdaY) 9.28Ð;19 Urina¡v Calcium (pmoVdaY) 47.2X9.3 Urinarv Phosphate ( pmoUdaY) 288X24 Urina¡v Mapnesium ( pmoVdaY) 244t32 Urinarv Uric Acid (*moVdaY) 9.39X2.01 I c)

(l)((t ds o Eg E_1 ()- + fãÞ ag ,:Ìx=o . oe*f'l

o 'ÉÉEË Þo 'Ête go -r Éo¡ rì.EH H -$) -4 3< ()€9c¡ >,o -6 cE A -8 28 0 2 4 6 810 t2 t4 16 t8 20 22 24 26 Day of Study

on urinary oxalate excretion in anormooxaluric Figure The effect of long term (2.1 dayÐ oTC adr inistration ("s"tt_ug dose of.3.92+0.12mmol kg !ry) 4.2 as the mea¡r+S.E'M (n=6) for both rat model (Section 2.3.1.1,). The shadçd area on the graph indic;te'; th;5 à"y b;*lio" prrioì. rhãieiultí'ut t"pt".ó.d control (o) and OTC treated (o) groups' (L)-Cysteine Short Term Study Long Term OTC Short Term OTC Short Term @)-Penicillamine (L)-Cysteine Control Plasma Parameters Control OTC Control OTC Control @)-Penicillamine

Sodium (mmoVl-) 13611 t36Ð 146r0 146r1 t46tl 14610 l4lrl l4lrl 3.73+0.13 3.9810.14 Potassium (mmol/L) 4.42rn.19 4.48fl.15 4 t7fl.t9 4.20t0.21 104.510.4 104.0r0.4 Chloride (mmoVL) 99 3r1.5 99.0+2.0 109.8il.6 108.3+0.7 104.610.9 106.0r0.8 23.8fl.2 Bicarbonate (mmoVl-) 25 2+1.2 24.8+1.9 25.0ú.6 25.0r0.5 27.6L{.7 29.4!{.7 25.3r0.9 '7.921n.65 '7.60fr.12 Glucose (mnol/L) 10.0r0.6 I t.310.7 9.07f0.75 8.05r0.49 3.93fl.12 Urea (mmol/L) 5.31!J.52 4.8010.23 5.03r0.41 5.33r0.46 6.08i0.58 4.76fl.36 3.80f0.26 0.03r0.00 Crearinine (mmol/L) 0.0410.00 0.04f).00 0.03t().00 0.03r0.00 0.0410.00 0.03r0.00 0.03r0.00 0.0410.01 Urate (mmol/L) 0 03r0.00 0.03r0.00 0.07f0.00 0.0710.01 0.03f().00 1.9610.08 Phosphate (mmol/L) 2.29fl.05 2.1srO.08 2.60fr.07 2.5510.08 2.4010.03 2.38il.10 r.95r0.05 2.51r0.02 Calcium (mmol/L) 230fl.02 224L{.02 2.49Ð.03 2.5610.03 2.60fl.05 2.61r0.01 2.55r0.04

Albumin (g/L) 30.3r0.6 30.3r0.4 31.510.4 31.3r0.6 3t.2t{.4 31.010.0 32.6fr.5 33.310.3 59.2fl.9 Protein (g/L) 55.3r0.5 54 8+0.5 55.510.8 55.5+0.8 55.8+0.9 54.4r0.5 58.811.0

ALP (U/L) 2t'7t31 201+8 190r15 165i10 3 10129 3 18153

LD (UiL) 716X96 '742X94 298181 304+70 t6'lLzs t33rl0

ASr (U/L) 105.2t8.1 108.5+l 1.3 76.2+5.4 '72.6X4.4 55.8+1.53 64.8r4.05 ALT (U/L) 44.2X2.2 4'7.812.6 44.2t4.0 49.615.1

Anion Gap t6.2X1.3 16.3+1.2 15.3+1.0 16.710.5 17.611.1 14.610.9

on selected.plasma Table The effect of long term (2ldays) oTC and short term (5days) administration 4.1 The results are expressed as the parameters in non-hyperoxaluric ræs (S-eciio"-i.i1.l) All resultì wãre - p<0.05, ** p<0'02, **+ p<0'01 and mean+S.E.M. (n=6) *itrr tÏ. r*;pli;; òrtú" .rro.t term'(L)-cysteine study (n ++** p<0.001. effective dose of OTC in this study was 5.5710.08mmo1/kg/day. When administered

or the urinary over a 5 day period OTC did not significantly alter food and water intake

(Figure In excretion of uric acid, magnesium and creatinine or urine volume 4'3)' addition, OTC administration did not significantty alter weight gain; Control

2.84il0.38gl,clay and OTC treated, 2.74l:).67gtby. However, OTC administration

and 195 increased urinary sulphate, cyst(e)ine, calcium and phosphate, I57,59, 62

respectively; and decreased urinary oxalate and urine pH, 11 and 7% respectiveþ;

oxalate (Figure 4.3 and 4.4). When compared to the controls the reduction in urinary

(Figure ln the 5 excretion was 0.97+0.35pr.moVday ot ll%ocompared to basal levels 4'3)'

altered by day after oTC administration (recovery period), urinary pÍu¿rmeters that were

those of OTC administration returned to levels that were not significantly different from

the controls. Analysis of blood collected at the completion of the recovery period showed

OTC that there were no significant differences in any of the plasma parameters of the

treated group when compared to controls (Table 4.1). Overall, the sipificant reduction

in urinary oxalate excretion and pH (Figure 4.3) reduced the P.rfrom 30%to 4%'

Rat 4.2.2.3 Long Term oTC Administrafion in an Ethylene Glycol Hyperoxaluric

Model

excretion by Ethylene glycol at a dose of 12.2+0.5mmollkglðay increased urinary oxalate

12 fold when compared to normooxaluric control rats. Oral administration of ethylene to glycol increases urinary oxalate excretion as in vivo ethylene glycol is met¿bolised

glycolaldehyde, glycollate, glyoxylate and oxalate (Figure 1.5). The effective dose of

OTC in this study was 2.39+0.15mmoVkg/day. OTC administration over a 2l day

period to ethylene glycol hyperoxaluric rats did not significantly alter food and water

85 Food Intake (e/MV)

Water lntake (ml/day)

Urine Volume (ml-/daY)

Urine PlV0' I

Urinary Creatinine (pmoVdaY)

Urinary Oxalate (pmoVdaY)

Urinary Calcium (¡rmoUdaY)

Urina¡y Phosphate/l 00 ( pmoUdaY)

Urinary Magnesium/l 0 (PmoVdaY)

Urinary Uric Acid (pmoVdaY)

Urinary Cyst(e)ine/100 (¡rmoVday)

Urinary Sulphate/l00 (pmoVdaY)

-8-6-4-202468 Change During the Treaûnent Period (unitsiday)

Parameter Baseline Value Food Intake (s/d^v) 17.ùr0.0 Water Intake @/dav) 25.6fl.4 Urine Volume (mVday) 12.8r0.4 Urine pH 6.43Ð.04 Urinary Creatinine (pmoVdaY) '74.'7XL.5 Urinarv Oxalate ( moVday) 9.18r0. 16 Urinarv Calcium (pmoUday) t4.3+t.6 Urinary Phosphate (pmoVday) 848t33 Urinarv Magnesium (pmoVday) 476+I',7 Urinary Uric Acid (pmoVday) 5.'73fl.49 Urina¡y Cyst(e)ine (pmoUday) 52.7XL9 Urinarv Sulphate (rrmoVday; 267X4

Fizure 4.3 The effect of short term (5 5.57+O.08mmol/kg/day) on selected urin (Section 2.3.L1). The table contains pooled) whilst the graph inclicates th both control ( ¡ ) a¡rd OTC treated mean+S.E.M (n=6). Significant difference a

(.) ô- dsCJ É6 É9 .9E Þ--()-

'r€c¡9 0 :xËo I r¡l ?c aoøÊã -l 'trFÉct ÞÈ .a -. go -2 É c.¡ ñEEèO 3å Ee -3 >ìo cl a -4

0 a 4 6 8 l0 t2 t4 l6 Day of Study

in a normooxaluric Figure 4.4 The effect of shr rt term (5 days) OTC administration (effective-dose of-5.57+0-08mmol/kg/day) on-urinary oxalate excretion as the mean+S.E'M rat model (Section 2.;.1-.1;. rn. .rtù"á;t*. ;;üt; güptr i"¿i"ut" ttr" 5 day baseline and recovery [erióás. The reéults are expressed (n=6) for both control (o) and OTC treated (o) groups. creatinine or intake, urine volume or the urinary excretion of magnesium, phosphate,

not calcium, however urine pH was reducd 60/'; (Figre 4.5). Although OTC did significantly reduce urinary oxalate excretion a trend towards reduced excretion was evident. The inabilrty of OTC to significantly reduce urinary oxalate excretion in this srudy was possibly due to the degree of hyperoxaluria produced and S{]glgrygry

that glyoxylate øthylene glycol may have been produced from OTC_ letabolism lom did metabolism, thereby limiting the likelihood of adduct formation. OTC administration - not significantly alter weight gain; control oJft}.79gruay and oTc treated that ¡-ZZxO5Bg¡day. Analysis of blood collected at the completion of the study indicated

other OTC administration decreased plasma protein,.- 9%a tlryrmooëql-q!9--!9y9ls' all

rats plasma parameters were unaltered (Table 4.2). Compared to normooxaluric control

(Table 4.2), plasma ufea and creatinìne levels were significantly elevated at the

completion of this experiment indicative of renal impairment. ft9j*tt:l4qttq"9qql

ad-i"itttutiot *uv i" pufrÞIgþ!9g19ttt-"-{-eg:e-g of- T_qryry!.t.¡n1.y.rr_yrolr-orc of renal impairment. OTC administration did not significantly alter renal deposition

after 2l calcium, magnesium or oxalate (Figure 4.6). Renal calcium oxalate deposition

that days of ethylene glycol has also been observed by Kha¡ who demonstrated

significant administration of ethylene glycol to rats (0.75% in drinking water) produces

(Khan, 1991)' crystalluria by day t2 and crystal deposition in renal papilla by day 24

4.2.2.4 Short Term OTC Administrafion in a Glycollate Hyperoxaluric Rat Model

day period rn This study was conducted in rwo parts, (i) a 3 day baseline and (ii) a 5

which OTC was administered orally (Section 2.3.1.3). OTC and glycollate were

administered in this study at a dose of 7.94+0.06 and 1.92+0.06mmollkglday

86 Group Food Intake/s G/l8Hr)

Group Water lntake/lO (mll2aHr)

Urine Volume (mL/6Hr)

Urine pH

Urinary Creatinine (*moV6Hr)

Urinary Oxalate (pmoU6Hr)

Urinary Calcium (¡unoV6Hr)

Urinary Phosphate/ 10 ( PmoV6Hr)

Urinary Mapesium/l0 (¡rmoU6Hr)

05 l0 15 20 25 30 35 40 Parameter (units)

05. Study Non-Hyperoxaluric Ethylene Glycol Glycollate Glycollate (L)-Cysteine Plasma Pa¡ameters Control Cont¡ol OTC Confol OTC Confol

Sodium (mmoVl) 144+T 137+5 143+1 142+I l4zxl 13911 l39rl

Potassium (mmoL/L) 3.74t0.11 3.8510.14 3.78+0.09 4.2010.08 4.07+0.16 3.9010.18 4.30+0.34

Chloride (mmol/L) 106+1 104X2 106+1 103+l 10I+1 l0l+1 100+l

.*' ' Bica¡bonate (mmol/L) 25.9+0.4 20.5+l.l 20.7+2.0 3ûi?*.0:? '.t2.f . ,,.'aa,elto:48.' 1 Glucose (mmol/L) 8.65+0.33 9.17+0.81 8.43+0.72

Urea (mmol/L) 4.91+0.28 15.5312.08 28.80!12.71 5 1010.56 4.65+0.45

Creatinine (mmol/L) 0.03+0.00 0.15+0.05 0.22+0.10 0.03r0.00 0.03+0.00 0.02+0.00 0.03+0.00

Urate (mmoUf-) 0.06+0.00 0.12+0.06 0.07+0.00 0.07+0.01 0.0610.00 0.0510.01 0.0610.01

Phosphate (mmol/L) 2.3110.06 1.75t0.10 2.65+0.64 2.87+0.09 2.66+0.10 2.11+0.09 2.24!0.10

Calcium (mmol/L) 2.54!0.02 2.38+0.04 2.5l+0. t I 2.57.0.02 2.58+0.02 2.50t0.02 2.52+0.04

Albumin (g/L) 31.8r0.3 30.5+0.7 30 8+0.9 30.3+0.7 30.2+0.5 33::3È0.6

Protein (g/L) 56.8+0.5 51.2+0.9 50.3+1.0 54.8+1.8 53.9+0.7

ALP (UrL) 224+15 138+19 t23!23 324!25 29IXzl 18414 176+18

LD (U/L) 219+27 376!t23 436190 151¿35 t55L24 252!37 33t+127

AST (U/L) 65.5t2.4 87.0!t2.3 98.7t15.2 78.7!8.7 75.2t4.8 88.016.2 94.8+10.4

ALT (U/L) 4t.4+1.5 30.8+l.l 29.5+r.5 38,2!2.8 39.7t0.7

Anion Gap 15.810.5 18.8+2.1 20.1x2.6 12.3r0.8 11.9+0.6 13.5+0.6 12.5+0.6

and (L)-cysteine Table 4. s) OTC admi I hyperoxaluri days) OTC ters in a glyc (Séôtion 2.3.1 All results were obtained on adminis (n:17). Significant the last ." th the excepti controls differen P<0.01."*préõéa 20

15 ø

v) k 9toc) ã prctl

5

0 Liver as Kidney as Oxalate/5 Calcium Magnesium a%o of ao/o of (pmoVg) (pmoVg) (llmoVg) Body Weight Body Weight

(n) and OTC treated (ø) grouPs. respectively. Administration of glycollate at this dose increased urinary oxalate excretion by approximately 3 fold when compared to normooxaluric control rats' OTC administration over a 5 day period to glycollate hyperoxaluric rats did not significantly alter food and water intake, urine volume or the urinary excretion of creatinine, calcium

or phosphate (Figure 4.7). In addition, OTC administration did not significantly alter

weight gain; control2.52x0.27glday and oTC treated 2.24t0.279/day Hoyelgr qTç

pClq¡Ulllalrau-Ul-cryæ9!-g"¡ry su!p,-hate, W6 qqd d99rea¡qLqruç-p-{ and ¡¡rinq¡y

oxalate, 11 and 43olo respectively; (Figure 4.7 and 4.8). Whq99!PgI93lo-+J bu¡"l

level, the reduction in urinary oxalate excretion was 12.34+l.5SpmoUday

or 43yo (Figure 4.7). Analysis of blood collected at the completion of the study indicated

that OTC administration increased plasma bicarbonate and glucose, 7 and 25Yo

respectively; all other parameters were unaltered (Table 4.2). Although the significant

aciduria observed in this study suggests that the rats may have experienced a mild

metabolic acidosis, to normooxaluric control rats is reduced,

3r% indicative of a metabolic alkalosis (Table 4'2). The

reduced anion to be a function of than,O]T]C, however OTC

Over did exaggerate the effect l_.gq!g !g the ;igm{c_ant rlgrqase in p-lasma bicar-bonate

compensation of the original metabolic acidosis through carbon dioxide respiration and

renal bicarbonate regeneration cannot adequately explain the metabolic alkalosis as

restoration of pH is never possible in a simple acid./base disturbance (Tietz Textbook of

Clinical Chemistry). Hence, this alkalosis may be due to a systemrc poisoning of

acid./base homeostasis caused by the increased glycollate, glyoxylate and oxalate load, all

of which are poisons in their own right. The increase in plasma glucose may be attributed

to the gluconeogenic nature of OTC derived (L)-cysteine. Again overall, the sigmficant

reduction in urinary oxalate excretion and pH (Figure 4.7) reduced the P., from 90% to

I2%". Tltis significant reduction in the theoretical P.¡ is supported by the observation that r+

87 Food Intake (e/MV)

Water Intake (ml-/daY)

Urine Volume (ml-/daY)

Urine pH

Urinary Creatinine (pmoUdaY)

Urinary Oxalate/S (pmoVdaY)

Urinary Catcium/I0 (PmoUdaY)

Urinâry Phosphate/ 1 o0 (PmoUdaY)

Urinary Sulphate/ 100 (PmoVdaY)

-4 -3 -2 -t 0 1 2 3 4 5 6 7 8 Change During the Treatment Period (units/daY)

Pa¡ameter Baseline Value Food Intake (C/day) t'7.61{.2 Water Intake (g/daY) 28jll.Z Urine Volume (mVdaY) 19.l+1.2 Urine pH 6.85]{.04 '75.9tt.3 Urina¡y Creatinine (PmoVdaY) Urinary Oxalate (pmoVdaY) 28.811.6 Urinary Calcium (pmoVd¿Y) 66.519.0 Urina¡y Phosphate (PmoVdaY) 645-43 Urinary (pmoVday) 389115 l0 {.)

¿!- ds(I)R! ÉoitÉ 5 Ë-1()- XX fll.H c¿9 0 E9 *fu Og

FE -5 'ÉÉtr¡ ñl ÞÈ .=¿ l0 eðÉc¡ (Jb.EH â,1 (,€9cl -15 >rO .É A -20 'l 9 0 2 3 45 6 Day of Study

ox4late excretion in a glycollate Figure administration,(effectivg-l 9grg.o!.94+0.o6rnmol/kg/day) on urinary hypero ;i *¡¡;iiò;¡;*t.¡.r.Ð- The shaded atea ott-the graph indicátes the 3 day baseline period' The ró.'ott" control (o) and OTC treated (r) groups' rn under a light microscope signiñcant calcium

crystalluria was obsewed in fresh control urines whilst no (or very little) calcium oxalate

used to search for adduct in pooled t!9-9rine of OfC t¡eated -rats. The GC/NIS assay w¿rs the most sensitive daily urine samples from this study. Using selected ion monitoring,

Although adduct was not technique, adduct could not be found in the urine samples'

the natural substrate of 5- found in the pooled urine samples, high levels of 5-oxoproline,

(Figure As OTC has been shown oxoprolinase, were found on tinear GC/lvts runs 4.9).

inhibitor of utilisation of 5- to be a better substrate for S-oxoprolinase and an excellent

not surprising to find large oxoproline (Williamson and Meister, 1981 and 1982.), it is importantly' this quantities of 5-oxoproline in the urine of OTC treated rats. More cysteine by 5- obsewation confirms that oTC is being actively met¿bolised to

of this study' significant oxoprolinase as suggested by Meist er et aI-. At the completion

group suggesting that 8 days levels of oxalate could not be found in the kidneys of either this level of is not sufficient for renal calcium oxalate deposition to occur at

rats, renal calcium hyperoxaluria. lndeed, Khan has shown that in male Sprague-Dawley at least 200y' of the oxalate deposition does not occur until urinary oxalate exceeds

the normal level)' normal level (Khan, 1991) (in this study urinary oxalate is 180% of

simultaneous t'p' Intraperitoneal injection of meso-tzlrtaric acid 24 hours prior to

described in Section 2'3 'L '7 administrati on of meso-tzttaric acid, OTC and glycollate, as ' conditions' ÌIowev-er, did no enable detection of adduct in the urine under hyperoxaluric

given that the maximum reduction in urinary oxalate excretion, þtn"t uddu"t-&ilqtqt'

doses of OTC= (Figure 4-8) occurs approximatelY 48 hours after two consecutive 24 hour

window for maximal adduct and that onlv one 24 hour urine collection was taken, the

formation mav have been

88 oTc 20000 20000 (a) È 5-Oxoproline 5000 o' 15000 Þ =o C) o 10000 o 10000 o d 5000 5 5000 ê F-a 0 0 9.0 10.0 4.0 50 6.0 7.0 8.0 Retention Time (minutes)

156

Retention Trme ' 4.77 minutes o)

H 73 I

(cH3)3sio

5-Oxoproline

147 45 230 258

200 250 300 0 50 100 150 Mass/Charge (a.m.u.)

174 (c) Retention Tune - 5.73 minutes

73

2-Oxolhiazolidine'4-carboxyli c Acid (OTC)

t47 276 248 45 59

200 250 300 0 50 100 I50 Mass/Charge (a.m.u.)

from g¿¡s unne mass m¿¡ss EXCRETION AND TTIE ITISK 4.2.3 TI{E EFFECT oF (L)-CYSTEINE oN UPù'IARY OXal-erS

or. CATCNTU OXALATE UROLTTilASIS

Rat Model 4.2.3.1 Short Term (L)-cysteine Administration in a Normooxaluric

(ii) a 5 day period ur This study was conducted in rwo parts, (i) a 5 day baseline and

The effective dose of (L)- which (L)-cysterne was administered orally (Section 2.3-l'l)' (L)-Cysteine cysteine administered in this study was 3.?1+0.l8mmd/rglday.

urine volume or the urinary administration over a 5 day period did not significantly alter (Figure 4.10). Although excretion of uric apid, calcium, phosphate and creatinine

this was not statistically administration of (L)-cysteine decreased urinary oxalate' 8%; the reduction in significant (Figure 4.10 and 4.11). When compared to the controls,

basal levels urinary oxalate excretion was 0.98+0.78pmoVday or \Yo compared to

(Figure 4.10). However, (L)-cysteine administration increased urinary sulphate,

urine pFt l0%; cyst(e)ine and magnesium, 210, 63 and 137o respectively; and decreased

to have (Figure 4.10). At the completion of the treatnent period, (L)-cysteine was shown

and increased plasma decreased plasma glucose and urate, 22 and l7o/o respectively;

and the aciduria potassium, l8%; (Table 4.1). The increase in plasma potassium

metabolic acidosis, observed after administration of (L)-cysteine are indicative of

normal ranges' The reduction in although plasma bicarbonate concentratlons were within

conditions can be plasma glucose observed with (L)-cysteine under normooxaluric

to form a stable adduct' explained by the fact that (L)-cysteine and glucose are l,mown

1990; Gomezet al'' glucocysteine, under physiological conditions (Bjelton and Fransson,

groups' 1994). This highlights the fact that, in vivo, (L)-cysteine can bind free aldehyde

in plasma urate' Although like glyoxylate. No explanation can be given for the reduction

reduction in urinary oxalate excretion was not reduced by (L)-cysteine, the significant

89 Food Intake (e/daY)

Waterlntake (ml/day)

Urine Volume (ml/daY)

Urine PH

Urinary Creatinine (pmoVdaY)

Urtutary Oxalate (pmoVdaY)

Urinary Calciunt/lO

Urinary Phosphate/ 10 (PmoVdaY)

Urinary Magnesium/ 1 0 (PmoVdaY)

Urinary Uric Acid (¡rmoVdaY)

Urinary Cyst(e)inei l0 (¡rmoUday)

Urinâry Sulphatei 100 (¡rmoVdaY)

-4-20 2 4 6810 Change During the Tre¿trnent Period (units/day)

Parameter Baseline Value Food Intake (eldav) 19.61l).1 Water Intake (e/dav) 27.4+1.4 Urine Volume (mVday) t4.2!t.3 Urine pH 6.7tfl.11 Urinary Creatinine (pmoVday) 89.4+r.7 Urinary Oxalate (¡rmoVday) 12.2fl.4 Urina¡y Calcium (pmoVday) 41.0a4.8 Urinary Phosphate (pmoVday) 563+1 I Urinary Magnesium ( pmoVday) 36l+11 Urinary Uric Acid (pmoVday) t2.sfl.7 Urinary Cyst(e)ine ( pmoVday) 4r.8!4.7 Urinary Sulphate (pmoVday) t74t'l

mean+S.E.M (n=5). Significant difference 4 Ë¡Ë dsc¡¿E J co '-ElôÉ I o\'= (J-

'rEo¿9 I E9 * r¡l og o ãE 'ÉlaÉct -t ç7P gg -z HO Ebo õË -3 åå o€9 c.¡ >ìo 4 al o -5

0 ) 3 4 56 7 8 9 l0 ll Day of Study

dose of 3.71+0.18mmg!/kg/day) on urinary oxalate excretion in a Figure 4.ll The effect of shorr term (5 44Vs) -(L)-c-ysteine administration.(effective no"rmooxaluric rat model (Section 2.3.1. t). Ttie'shaded area on the graph indicates the 5 day baseline period. The results are expressed as the mean+S.E.M (n:5) for both control (o) and (L)-cysteine treated (o) groups. urine pH and the trend towards reduced urinary oxalate excretion (Figure 4'10) reduced the P.t from3}%oto2Yo

4.2.3.2 Short Term (L)-Cysteine Administration in a Glycollate Hyperoxaluric Rat

Model

in This study was conducted in ¡ro pans, (i) a 3 day baseline and (iÐ a 5 day period which (L)-cysteine was administered orally (Section 2.3.1.3). (L)-Cysteine and glycollate were administered in this study at a dose of 7.91+0.10 and 1.92+0.06mmolkg/My respectively. Again administr¿tion of glycollate at this dose increased urinary oxalate

excretion by approximately 3 fold when compa¡ed to normooxaluric control rats. (L)-

Cysteine administration over a 5 day period to glycollate hyperoxaluric rats did not

significantly alter food and water intake, urine volume or the urinary excretion of

phosphate (Figure 4.12). In addition, administration of (L)-cysteine did not significantþ

alter weight gain; Control 1.88t0.15úMy and (L)-cysteine treated 2.39t0.3791day.

However, (L)-cysteine administration increased urinary calcium and sulphate, 139 and

Z1g%respectively; and decreased urinary creatinine and oxalate and urine pH, I l, 3l and

20o/o respectively; (Figure 4.12 and 4.13). When compared to the basal hyperoxaluric

levels, the reduction in urinary oxalate excretion was 7.82+1.39¡"r.moVday or 3|o/o (Figure

4.IZ). Analysis of blood collected at the completion of the study indicated that (L)-

cysteine administration decreased plasma urea and albumin, 2l a¡d 5olo respectively, and

increased plasma bicarbonate and glucose, 6 and 2l%o respectively; and all other

parameters were unaltered (Table 4.2). TIte increase in plasma bicarbonate and glucose

were also observed in the OTC hyperoxaluric study and are explarned in Section 4.2.2.4.

No satisfactory explanation can be given for the reductions in plasma urea and albumin.

90 Food Intrke (e/day)

Water Intake (ml/day)

Urine Volume (ml/day)

Urine pH

Urinary Creatnine/ I 0 (pmoVday) *r[ f,

Urinary Oxalate/5 (pmoUday) þ

Udnary Calcium/ 10 (pmoVday)

Urinary Phosphatei 10 (pmoUday)

Urinary Sulphatei 100 (¡rmoVday) *i.

-6-4-20 ,, 46810 Change During the Treatment Period (units/day)

Parameter Baseline Value Food Intake (e/dav) 18.0r0.0 Water Intake (sldav) 27.2+r.3 Urine Volume (mUdav) 18.8+1.5 Urine pH 7.13r0.09 Urinary Creatinine ( pmoUday) rt2.6Ð..8 Urinarv Oxalate ( rrmoVday) 28.4+1.4 Urinary Calcium (pmoVday) 66.1+7.5 Urinary Phosphate ( umoVday¡ 564+38 Urinarv Sulphate (rrmoVday) 202+8

Figure 4.12 The effect of short term (5 day) (L)-cysteine administrati_on (effective dose ofJ.gt+O.t0mmollkg/day) on selected urinary pararneters in a glycollate hyperoxaluric (effective dose of f.gZlO.O0mmol/kg/day) rat model (Section 2].1.?).: The table groups whilst_the graph òontains the average ba.seline values-(3 days, both -pooled) indicates the average change during the treaûnent period for both congo_! _( I ) and (L)+ysteine treated(a) groups. The results are expres-s"-d- F th. mean+S.E.M (n=6). Significant difference from controls: * p<0.05 and ** p<0.001' l0 s) csc¡ arl éo 5 .eE E-=o- ñÞ c¡9 0 -=x=() *fÐ Os

6Ø4õ -5 'trÉ(ü ÞÈc0 .ãõ-á go l0 Fi c¡ EèO ðB 3l Ee l5 >ìO o -20 9 0 2 3 45 6 7 8 Day of Study

rn a Figure 4.13 The effect of short term (5_days)_(!)_-cvstelne administration (effective dose of 7.91+0.10mmoVkg/day) on gri¡rr5.y oxal{e excretion 3 dav baseline Èií"åir"t r,vp"ro*aurió-fãm""titã ã"ró àri.é¿ig'¡?t"í-"leg /!ay) ntmodelÌSectþn 2.3.13). The shaded area õn the graph indicates the TËiesults ure as the mean+S.E.M (n=6) ficr both control (o) and (L)-cysteine treated (r) groups. ;;"i"d- "*þt"r."d Overall, the significant reduction in urinary oxalate excretion and pH and the significant increase in urinary calcium excretion (Figure 4.12) reduced the P* from 90%' to 3l%'

This signifi c4¡lt reduction in the theoretical P.¡ was again supported by the observation that under a light microscope significant calcium oxalate crystalluria could be observed in

fresh control urines whilst no (or very little) calcium oxalate crystalluria was observed in

the urine of (L)-cysteine treated rats. The selected ion monitoring GC/I\4S assay wÍls

used to search for adduct in both pooled plasma (obtained on the last day) and urine

samples from this study. Although no adduct could be found in either the urine, on any

day of the study, or the plasma, on the final day of the study, ï4-fiJpt-Ievels of hepatic

(D)-¡spqpte oxidase u:lt]4ty wgle when calculated on the basis of Td l_._nul þ,1qrd,. total organ activity, the levels of (D)-aspartate oxidase found in the livers and kidneys

were 2430+213 and 204+46pmoUday/organ respectively; no significant difference

between control and (L)-cysteine treated rats.

4.2.4 Trü EFFECT oF (D)-PENTCTLLAMINE oN URINARY OxerRrs ExcnsuoNAND Tm

Rlsr op Carcnn¡ OxALATE Unotmlasts

4.2.4.1 Short Term Adùinistration in a Normooxaluric Rat Model

This study was conducted in rwo parts, (i) a 5 day baseline and (ii) a 5 day period rn

which (D)-penicillamine was administered orally (Section 2.3.1.1). (D)-Pemcillamine

administration over a 5 day period did not sigryficantly alter tþ9 u=rr¡4ry excretlçr4 of

ptto.pþ49, calcium, magnesium or uric acid, it did however decrease food intake, water

intake, urine volume and urine pH,41,46,64 and 8% respectively; (Figure 4.14)' In

addition, (D)-penicillamine administration decreased urinary creatinine and sulphate, 16

91 Food Inøke (g/day) á3

Waterlntake (ml/daY) {. {.

Urine Volume (ml/daY) J+

Urine PIVO.1 *

Urinary Creatinine/ 10 (pmoVdaY) *

Urinary Oxalate (pmoVdaY) *

Uri¡ary Calcium/ I 0 (pmoUdaY)

Urinary Phosphate/ 1 0 ( pmoVdaY)

Urinary Mapesium/l0 (pmoUdaY)

Urinary Uric Acid (pmoVdaY)

Urinary Cyst(e)ine/10 (pmoVdaY)

Urinary Sulphate/l0 (pmoVdaY) .t+ -10-8-6-4-2024681012 Change During the Treatment Period (units)

Parameter Baseline Value Food Intake (e/day) 16.9+1.0 Water Intake (e/day) 27.5+1.8 Urine Volume (mVdav) 13.'7t1.4 Urine pH 7.4910.18 Urina::y Creatinine (pmoVday) t02.3!4.3 Urina¡y Oxalate (pmoVday) 8.35+1.34 Urinary Calcium (pmoVday) 2t3r4.0 Urinary Phosphate (pmoVday) 462+t5 Urinary Masnesium ( pmoVday) 284+1',7 Urinary Uric Acid (pmoVday) 13.810.5 Urinary Cyst(e)ine (pmoVd¿y) 41.5+4.2 Urinary Sr.¡lphate (pmoUday) 22',7X20

Figure 4.14 The effect of short term (5 day) (D)-penicillamine doie of 2.22+0.I2mmollkg/day) on selected urinary parameter model (Section 2.3. ltains the average basel groups'pooled) wh the average.change during_the trea,fnent results are þericia for both con amine.treated (1) Croups. .The èxpressed as the me cant difference from controls: * p<0.05 and ** p<0.01. (Figttre and 4'15)' alrd 44yo respectively; and increased urinary oxalate, 74%; 4.I4 urinary oxalate excretion was when compared to the controls, $9_ inclease 4 The group treated 6.15+1.95pmol/day or 74o/o compared to basal levels (Figurc 4.14)'

Control with (D)-penicillamine failed to gain weight over the duration of the experiment,

Analysis of blood 2.8+0.4glday and (D)-Penicillamine treated -}.É}jg/day, p<0.01.

(D)-penicillamine administration collected at the completion of the sh,rdy indicated that

activity, decreased plasma alkaline phosphatase activity, aspartate aminotra¡sferase

respectively; and alanine aminotransferase activity and Cuz*,50,30,51 and 507%

gap, respectively; (Table increased plasma lactate dehydrogenase activity and the anion

treated 4.1). The reduction in plasma cu2*, control 18.22+0.58 and (D)-Penicillamine

3.0Gr0.57pgll; observed with (D)-penicillamine was not surprising as @)-penicillamine

(Glass et al', 1990; has been shown to be effective in the management of Wilson's disease

metabolism in Menara et al., lgg2). Wilson's disease is an inborn defect of copper

a nontoxic complex which there is a deficiency of caeruloplasmir¡ which normally forms

confirms that (D)- with copper, resulting in elevated systemic copper. This observation

(D)-Penicillamine penicillamine in vivo is actively scavenging electrophillic agents'

(Table 4.1)' The increased the anion gap indicative of an organic acid metabolic acidosis

darn?ge raised plasma activity of lactate dehydrogenasg !9 [rdicalive of -hqpatoeellqþr

can be attributed to the fact whilst the decrease phosphatase activitY

1992)' that (D)-penicillamine has been shown to-i4túlit tlú-q enz,yme (Menara et al',

significant reduction Overall, the signi-ficant increase in- urinary-oxalate-excretion and the

in urinary pH (Figure 4' 14) increased thç P,, from 30o/o to 64%o'

92 l6 c) .¿

C;, art Êrs É9 .98 Lz Þeo-

'ã€'t=o.¡ I JXo 8 oe*fq

FEÉ€ü 'É ÞÈø¡ ca 4 go Éo¡ EÞO õB E,.å o Ee >,o GI A -4 7 8 9 l0 ll 0 2 3 4 56 Day of Study

on uri rary oxalate excretion tn a The effect of short term (5 dayg @)-penicillamine administration (effective dose of 2.22+(.l2mmol/kg/day) Figure 4.15 ''ÍÍrè period. The rãsulti-are expresied as the mean+s'E'M normooxaluric rat model (Section 2.3.1.1). ,Írä¿"¿ area on thJËüh i;äi"utã irr"'j ¿"vïurèn* (n:6) for both control (o) and (D)-penicillamine treated (o) groups' 4.2.5 TTD EFFECT OF SULPTIATE ON URINARY OXETETE EXCN¡NON

As theoretical evidence exists to suggest that sulphate may effect renal oxalate transport these studies were performed to verifu that sulphate, which is a major metabolite of

upon cysteine, was not responsible for the reduction in urinary oxalate excretion observed administration of (L),cysteine or OTC

4.2.51 The Effect of Chronic Sulphate Infusion on the Renal Clearance and Tissue

Di s tri buti on of Oxa late

volume, The rate of oxalate and inulin clearance from plasma (Figure 4.16a and b), urine

were the urinary oxalate/inulin ratio and the oxalate/inulin ratio in the kidneys and spleen

not significantly affected by chronic sulphate infirsion (Figure 4'I7)' However, chronic

rate of sulphate infusions did increased the oxal¿æ/in¡lin ratio i¡ the liver; l4%. -As the

or disappearance of oxalate and inulin from plasma was not logarithmic, exponential

polynomial in nature, the data was linearised according to Dickerson et al- (Chemical

Principles); to obtain a linear plot from the plasma clearance datzn was set atZj '

Plot Rate Law Integrated Form Half-Life Linear I dC znr -l v.s f tv, = cn-l -=_rc"dt #=#'@-t)kt (n-)1{!-l

93 8000 (a)

7000

6.) € 6000 ËJpl_ 5ooo 18i5|.) 4)GI\ ÑH ð ä ¿ooo 5{) 3ooo Iéo E ão.çÉ -S .9 2ooo O

1000

0 25 0 5 l0 l5 20 Thme (minutes)

6000 o)

5000 0)

ã ¿ooo eo^{ É1ñ .t\ 4) qd¡ ci 3000 !ìE CH É K zooo o)H O 1000

0 25 0 ) l0 15 20 Trme (minutes)

Fisure 4.16a and The effect of sulphate b 3H-inulin a)-raC-oxalate and b) frogr the results are expressed as the mean+S.E.M infused groups. toc-O*alate Inñrsed/lOó (c.p.m.)

tH-Inutin Infi:sed/lo6 (c.p.n)

t4C0xalate Rate of Clearance fiom ¡rmin-t) Plasma/10{ ((c,p.m./500p'L)-r

3H-hulinClearance Rate of from Plasma/10¡ ((c.p.m./500¡rl-¡tnmin-t ¡

t, Plæma 'oc-Oxalate (minutes)

3H-Inulin t, Plasma (minutes)

Urine Volume (mL)

t oC-oxalateÆI-Inulin Urinary

t4C-Oxalate/3H-lnulin Liver (%o c.p.m'/ g)

toc-Oxalatei'H-Inulin Kidney (% c.p.m./ g)

Spleen'ocÐxalate/tH-Inulin (Yo c.p.m./ g)

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Parameter (units)

Fisure 4.17 The effect of chronic sulphate inütin from the plasma compartnent,2'l thg 3H accumulation of raC oxalate and inuli 2.3.I.4). The results are expressed as the sulphate (c) infused groups. 4.2.5.2 The Effect of a Bolus Intraperitoneal Injection of Sulphate on (Jrinary Omlate

Excretion

A single bolus i.p. injection of sulphate, at a dose which would mimic the average

increase in urinary sulphate excretion upon OTC and (L)-cysteine administration'

significantly increased urinary sulphate excretion such that T7+llyo of the administered

dose was recovered over 24 hours (Fþre 4.18). However, the i.p. injection did not

significantly change urine pH and volume or the unnary excretion of oxalate, calcium or

creatinine suggesting that renal oxaþ-te and sulphate trïNPo{ are indgpqnderr! (Figure

4.18).

4.3 DISCUSSION

4.3.1 THE EFFEcT oF OTC AND (L)-CysrEINE oN URINARY OXALATE ExcRETioN

AND TTIE RISK OF C¿¡,CNN¡ OXALATE UROLITHIASIS

Although previous studies have shown that acute i.p. injections of cysteine can

significantly reduce hepatic oxalate production and urinary oxalate excretion (Bus et al.,

l99la and b), therapeutic administration of cysteine, whether i.p. or orally, for the

prevention of calcium oxalate urolithiasis may be inappropriate due to the adverse

pharmacological properties of cysteine. Cysteine administration to young rats has been

shown to cause significant brain atrophy and destruction of hypothalamic neurones and

retina (Harper et al.,1970; Olney and Hu, 1970; Olney et al.,l97I; Karlsen et al',l98l;

Cooper et al., l9S2). Moreover a recent study evaluating the toxicity of both (L)-

cysteine and OTC in neonatal rats demonstrated that intravenous doses of both

94 Urine Volume/l0 (nl/daY)

Urine pH

Urinary Creatinine/lO0 (PmoVdaY)

Urinary Oxalate/ 10 (pmoUdaY)

Urinary Calcium/lO (pmoVd¿Y)

Urinary Sulphate/l00 (pmoVdaY)

012 345 678 Parameter (unis)

Figure 4.18 The effect of a single bolus i.p. inje d urinary oãä*.t.n (section z.i t.s\. Tñe results Í¡re exp (n=6) for ütltätt"iþi *ipnuæ (n) intused groups. controls: r, p<0.01. ^A compounds at doses of L.52 and 1.80g/kg respectively resulted in mortality rates of 80o/o

and l0o/o, respectively, indicating that OTC is less toxic than (L)-cysteine (ltihite et al',

1993). Indeed, in the normooxaluric studies, administration of (L)'cysteine at a lower dose (3.7lmmol/xdcur1'l produced significant changes in certain plasma biochemical parameters whilst administration of OTC at a higher dose (5.57 and 3.92mmoU}rglday) did not, adding further credence to the belief that more toxic than

(Tables 4.2). These findings are similar to those observed by other investigators

(Anderson and Meister, 1989). In additioru occasionally, administration of (L)-cysteine

was seen to resulted in diarrhoea. Nevertheless, the effects of (L)-cysteine on urinary

oxalate excretion were still investigated as (L)-cysteine is the simplist B-aminothiol and

the compound from which the original observations were made. However, more attention

was given to the effects of OTC on urinary oxalate excretion and the risk of stone

formation.

Under normooxaluric conditions, administration of OTC, the int¡acellular

dq[i_vgr__c_omp,o1{rd and (L)-cysteine at equivalent doses (long term OTC and short term

(L)-cysteine studies, approximate dose of 3.8mmol4

manv fold more effective in (Sections

4.2.2.1and 4.2.3.1). Assuming that adduct formation is the mechanism by which (L)-

cysteine reduces urinary oxalate excretion, the observed difference in the efficacy of the

two compounds could be predicted given that OTC is more effective in elevating

rntracellular (L)-cysteine levels than (L)-cysteine itself (Anderson and Meister, 1989;

Chung et al., 1990; Taguchi et a1.,1990). ln part, this difference may be related to the

observation that, a) plasma cysteine is rapidly oxidised to cystine which has a four fold

slower rate of entry into hepatocytes than cysteine (Anonomous; Coloso et al., l99l;

Bjelton and Fransson, 1990; Ba¡rks and Stipanuk, 1994) and b) transport of OTC into the

95 hepatocyte is 2 fold faster than that of cystine (Coloso et a1.,1991; Banks and Stipanuk,

1994).

More importantly though, is the observation that when cystine is transported into cells it is rapidly reduced to cysteine leading to an immediate elevation of intracellular cysüeine

(Anonomous). However, as increased levels of free intracellular cysteine can be detrimental to cells, excess cysteine is rapidly oxidised to cysteine sulfinate, by cysteine dioxygenase. Cysteine sulfinate is then catabolised to either taurine and carbon dioxide or pymvate, sulphate and ammonium (Daniels and Stipanuk, 1982; Reed and Beatty,

1980). Maximum induction of cysteine dioxygenase occurs rapidly when the intracellular cysteine concentration reaches the induction level of 0.2 to O.3mmol of cysteineþ of tissue (Reed and Beatty, 1980). Indeed, when OTC was administered at a 50%o higher dose (Section 4.2.2.2) than (L)-cysteine (Section 4.2.3.I), under normooxaluric conditions, proportionally less sulphate was excreted in the urine indicating that cysteine is catabolised to sulphate more readily than OTC; 35% of both doses were excreted in the urine as sulphate. The quantþ of inorganic sulphate recovered in the urine in both experiments is in accordance with that found by Krijgsheld et. al. (1981) upon administration of SmmoVkg (L)-cysteine. Irrespective of the difference in urinary sulphate excretion, in the normooxaluric studies, these results show that both OTC and

(L)-cysteine are efficiently metabolised to sulphate, indicating that intracellular levels of

(L)-cysteine were elevated for time. Hence, formation of the (L)-cysteine- glyoxylate adduct would have been expected to increase relative to the increase in intracellular (L)-cysteine and glyoxylate concentrations.

The differential metabolism of OTC and cysteine to pymvate, sulphate and taurine has been observed by other investigators in isolated hepatocytes. These urvestigators

96 demonstrated that production of the major metabolites of cysteine, glutathione, sulphate

and taurine, did not increase as OTC increased but did as cysteine increased (Stipanuk ef at., 1992); under conditions of low cysteine availabilþ, glutathione, sulphate and taurine

production accounted for 90, 10 and l% of cysteine catabolism whilst under conditions of

high cysteine availabilþ, glutathione, sulphate and t¿urine production accounted for 19,

47 a¡d34%' of cysteine cat¿bolism. The latter findings have been confirmed by Banks

and Stipanuk (1994). Clearly the above observations suggest that OTC is more likely to

provide a steady but slow supply of (L)-cysteine into the cell, enabling glyoxylate to be

complexed as it is formed, whereas cystine provides a rapid supply of (L)-cysteine into

the cell, dramatically increasing the intracellular levels of (L)-cysteine resulting in its

rapid conversion to taurine and carbon dioxide or pynrvate, sulphate and ammonia and its

subsequent removal from the cell. This view is furttrer supported by the observation that

in the glycollate hyperoxaluric studies, administration of OTC and (L)-cysteine at

equivalent doses resulted in a 43%o greater increase in plasma glucose from OTC than

from (L)-cysteine (cysteine is a gluconeogenic amino acid) (Fþre 5.7).

Despite the difference in the abilþ of both OTC and (L)-cysteine to reduce urinary

oxalate excretion, under normooxaluric conditions, both compounds produced significant

reductions in the probability of stone formation based on the model by Robertson; overall

OTC and (L)-cysteine reduced P.¡ from 30 to 4o/o. lt$o¡en administration of (L)-

cysteine at a similar dose to OTC did not significantly reduce urinary oxalate excretion,

the reduction in urinary pH was consistently 2 fold greater than that observed with OTC

(Figure 4.1, 4.3,4.7, 4.10 and 4.12). Administration of both compounds results m

'i\ significant aciduria due to their acidic nature Reduced urinary pH is advantageous for

the prevention of calcium oxalate crystalluria and stone formation due to the increased

ylgÞ{rry qq!4cl_lt olalate u,nder ac!dic__c_onditr9,!_r, This observation highlights the

97 of importance of þo[pq1!L91eþ!"_¡¡rq1g1!g pH as major risk factors in the formation calcium oxalate urolithiasis. This 2 fold difference in the abilþ of the compounds to produce aciduria is due to the fact that (L)-cysteine was administered in the hydrochloride form, i.e. thiol and carboxylic acid groups protonated and amine group as a hydrochloride

salt, whereas OTC was administered as the free acid. Hence, upon dissociation in the plasma, OTC releases one proton whilst (L)-cysteine releases three.

The significant increases in urinary calcium (Section 4.2.2.2 and 4.2.3.2), magnesium

(Section 4.2.3.1) and (Section 4.2.2.2) following administration of OTC and

(L)-cysteine, in some of the is thought to be the result of aciduria, as aciduria has

been shown to elevate the urinary excretion of these ions (Ullrich and Murer, 1982;

Robertson, 1976). Reduced reabsorption of calcium and phosphate in the renal tubules is

though to be the mechanism by which aciduria (metabolic acidosis) results in

hypercalciuria and hyperphosphaturia (Robertson, 1976; Fluids and Electrolytes). In the

case ofphosphate it has been postulated that aciduria can alter renal phosphate transport

in three different ways, a) it could alter the dissociation of phosphate, hence atrecting

transport, if the transport system prefers one ionic species over the other, b) it may alter

ttre affinity or maximal transport rate of the transport system, and c) it may alter

intracellular milieu whereby the ratio of NAD* to NADH could be altered, secondarily

affecting phosphate reabsorption across the brush border membrane. Unlike calcium and

phosphate the effect of aciduria on urinary magnesium excretion is variable producing

either an increase or no change (Robertson, 1976). Although significant aciduria was

observed in all experiments hypercalciuria, magnesiuria and phosphaturia were not. This

fact may be related to the different calcium, magnesium and phosphate status of the

animals in the different studies: basal urinary calcium, magnesium and phosphate varied

by as much as 240,95 and 1907o respectively; (Figure 4.1, 4.3,4.9 and 4. t3).

98 The difference in efficacy between OTC and (L)-cysteine to reduce urinary oxalate under normooxaluric conditions was also observed under hyperoxaluric conditions. At identical doses (7.9mmol4

Althougb under normooxaluric conditions, more cysteine was catabolised to sulphate than

OTC, the opposite was true under hyperoxaluric conditions as 35 and 22Yo of the respective doses of OTC and (L)-cysteine were excreted in the urine as sulphate. This observation suggests that under hyperoxalauric conditions either, a) the transport of (L)- cysteine into cells is much slower than OTC or b) the partitioning of met¿bolism of (L)- cysteine and OTC to sulphate, taurine and glutathione differs. Agg4,--?l-q!¡!u g- Éat adduct formation was the mechanism by which cysteine reduqed Uq44ry oxalate excretion, it appears unhkely that (L)-cysteine did not enter the ce_ll based on the observed

reductiqÂ. However, given that partitioning of the metabolism of (L)-cysteine to sulphate, . taurine and glutathione can change under conditions of altered cysteine availability whilst

partitioning of OTC metabolism cannot, the latter appears most likely (Stipanuk et al.,

reez).

Cellular glutathione stâtus may be the underlying factor producing this difference rn

partitioning of (L)-cysteine metabolism under norrno- and hyperoxaluric conditions.

Glutathione functions as a storage reservoir for cysteine and a detoxrfring agent by

combining preferentially with electrophillic metabolites (Prescott, 1982; Moldeus and

Jemstrom, 1983; Reed and Beatty, 1980) As oxidation of glycollate results in significant

production of glyoxylate and hydrogen peroxide (both of which are electrophillic

metabolites), administration of glycollate would be expected to produce glutathione

deficiency. Given that the supply of cysteine is the rate limiting step in glutathione

99 synthesis (Anonomous), under hyperoxaluric conditions excess catabolism of cysteine to sulphate would be avoided by the increased need for glutathione resynthesis.

As noted in Sections 4.2.2.4 and 4.2.3'2, the (L)-cysteine-glyoxylate adduct could not be detected in the urine of gþollate rats which had been given either OTC or

(L)-cysteine. This observation suggests that either, a) adduct formation is not the mechanism by which (L)-cysteine reduces urinary oxalate excretion or b) if adduct is formed in vivo then it is completely metabolised. Assuming that the reduction in urinary oxalate excretion is due to adduct formation, then in the two hyperoxaluric studies the amount of adduct formed daily would be 10¡"mol (Figures 4.8 and 4.12'¡. Hence, given that, a) significant levels of (D)-aspartate oxidase activity were found in the livers,

2431¡:¡¡¡oltday[iver; and kidneys, 200pmoVday/kidneys; of rats in this study and b) adduct is thought to be the natural substrate for (D)-aspartate oxidase (Hamilton, 1985), the latter option appears most likely. Under normal conditions, peroxisomal glycollate oxidase (Fry and Richardson, 1979;Fainelli and Richardson, 1983; Gibbs et al.' 1977) is thought to produces the majority of glyoxylate, which is then oxidised to oxalate, hence the peroxisomes may be a major site for adduct formation given the permeability of the peroxisomes to (L)-cystetne. ¿ts oxidase is also located in the

(Hamilton, 1985), _4. pqtliþillty exists that adduct is both produced and met¿bolised in the

Alary et al. demonstrated that when adduct is administered orally to rats at a dose of 5 to

lOmgikg only 14 to20o/o of the dose is excreted unchanged in the urine over a 24 hour

period (1989a). If adduct is formed in the glycollate hyperoxaluric studies at a rate of

lgpmoVday, if given in a single bolus dose, this would equate to ímgkgldav. At this

dose, according to Alury, only I5%o of the dose would be expected to be excreted in the

100 adduct is urine unmetabohs_gd -ofg_24 hours. Ho¡vever in the þypero¡aluric studies, administered as a bolus dose, rather it would be expected to form gradually over the day with subsequent met¿bolism as shown in Section 3.6. Hence, even if adduct formation ts the mechanism by which (L)rysteine reduces urinary oxalate excretion, adduct would still not be excreted in the urine in significant enough levels to permit its detection, even under hyperoxaluric conditions.

4.3.2 TID EFFECT OF SULPHATE AND ACIDURI,A ON URINARY OXEI¿TS EXCRETON

Although adduct formation is the most likely cause for the reduction in urinary oxalate

excretion, the possibility that the changes in urinary sulphate and pH may affect renal

oxalate handling cannot be overlooked. Under normal conditions the renal clearance of

freeþ fiþre{_a1 and g)gþ!_e__999f1s__r_?_p_4_y-. _wil! 9¡alat9 b"Tq bgttr t$ _qfol¡ruli

actively secreted in the proximal tubules (Weinman et al., 1978; Kuo and A¡onson,

prgximal tubule 1988), þqw91er o¡f1te can_ gndergo bidiregtrol3l llnsport _- q.

(Weinman et al., 1978; Knight et al.,1979). If reduction in the glomerular filtration rate

is responsible for the changes in urinary oxalate excretion, this would be reflected by

changes in urinlV creatimne, but this was not the case. Thus, if the changes in renal

oxalate handling are responsible for the observed reduction in urinary oxalate excretion,

these changes are most likely to occur at the site of active oxalate excretion.

For active secretion to occur, oxalate must undergo transcellular transport requlnng a

the transporter on þqth the sides of the cell, for entry from

blood stream, and on the luminal side of the cell, for exit into the glomerular filtrate

Oxalate uptake into renal cortical cells is known to occur via the sulphateibicarbonate

101 exchanger with oxalate being exchanged for either bicarbonate or sulphate (Prichard and

Renfro, 1982; Kuo and Aronson, 1938). Thus, intracellular sulphate and bicarbonate can provide a driving force for oxalate uptake at the basolateral membrane' As the renal

and tubules are the most important sites for bicarbonate (Clinical Chemistry in Diagnosis

Treatment) and sulphate (Ullrich et al',1980; Brazy and Dennis, 1981) reabsorption, the

driving force for basolateral oxalate uptake is quite evident'

(Lucke Sulphate is absorbed at the luminal membrane by a sodium dependant transporter

et al., 1979; Ullrich et al., 1980; Braey and Dennis, 1981; Schneider et al'' 1984;

Turner, l9S4). Bicarbonate is absorbed passively at the tubular lumen in the form of

carbon dioúde. lntracellular carbonic anhydrase on the luminal membrane converts

carbon dioxide and water into bicarbonate and ff (Clinical Chemistry in Diagnosis and

Treatment). Once inside the cell, oxalate transport at the luminal membrane into the

glomerular filtrate is thought to occur by the chloride/formate/hydroxide anion exchanger

(Karniski et a1.,1987; Yamakawa and Kawamura, 1990). In series, these transporters

provide a pathway for transcellular oxalate transport resulting in its active secretion in the

proximal tubules. If these transporters are involved in active secretion, as suggested by

other investigators, then clearly changes in urinary sulphate excretion and urine pH may

alter the rate of active oxalate secretion in turn affecting urinary oxalate excretion.

lndeed, increased concentrations of sulphate in the tubular lumen (3 to 5 fold) have been

shown to decrease (Z to 6 fold) the T- for sulphate reabsorption by the luminal sodium

dependanttransporter (Frick et al., 1984).

Speculation in terms of the whole animal model is important in any discussion on possible

mechanism by which (L)-cysteine and OTC may reduce urinary oxalate excretion.

Hence, the effect of sulphate on plasma oxalate clearance and urinarv oxalate excretion

t02 was investigated. The first of these investigations clearly demonstrated that sulphate does not significantly affect the rate of clearance of oxalate from the plasma and its subsequent

the rate of appearance in the urine (Section 4.2.5.1). This finding was confirmed by

second investigation described which demonstrated that when given by i'p' injection,

(Section sulphate did not alter the rate ofurinary oxalate excretion over a 24 hour period

4.2.s.2). Th.vt?y that sulphate is not an important regulatory factor for unnary oxalate

excretion is supported by the lack of any significant association between urinary oxalate

and sulphate excretion, and the change in both urinary oxalate and sulphate excretion

upon administration of OTC or (L)-cysteine.

Similarly, the reduction in urine pH observed upon administration of OTC and (L)-

qnteine does not appear to be the cause of the reduction in urinary oxalate excretion as

OTC was more effective in reducing urinary oxalate excretion whilst (L)-cysteine was

more effective in reducing urine pH. Again, the lack of assoclatron

between urinary oxalate excretion and urine pH, and the change in both urinary oxalate

excretion and urine pH upon administration of OTC or (L)-cysteine supports this view

4.3.3 Tm Er¡SCr OF (D)-PEMCTLLAMTNE ON URINARY OXALATE EXCRETIONAND THE

Rrx or Carcnn¡ Oxarnre URoLrmasls

Some investigators have suggested that due to its greater nucleophilicrty (D)-

penicillamine may be more useful than either (L)- or (D)-cysteine in the prevention of

calcium oxalate urolithiasis, through formation of the (D)-penicillamine-glyoxylate

adduct (Bringmann et al.,l99l arñ 1992). However, short term oral administration of

(D)-penicillamine significantly increased urinary oxalate excretion. As noted in Chapter

103 I (Section 1.8), pyridoxal phosphate dependant transamination of glyoxylate is one of the major pathways for glyoxylate metabolism, as such it prevents the oxidation of glyoxylate to oxalate. However, (D)-penicillamine is known to bind both en4rme bound and free pyridoxal phosphate altering its structure, its ability to facilit¿te group transfer in tra¡samination reactions and its abilþ to conjugate with apoenzymes (Schonbeck er a/.,

1975; Waner and Nyska, 1991). Hence, administration of (D)-penicillamine would be

expected to bind both free and enzyme bound pyndoxal phosphate, decreasing the activity

of glyoxylate aminotransferases, mcreasrng late to oxalate.

is further supported by the reduced activrty of aspartate aminotransferase and alanine

aminotransferase (Table 4 in the plasma of (D)-penicillamine treated rats

In man and experimental animals, pyridoxal phosphate deficiency results in hyperoxaluria

(Williams et al., 1967). The metabolic disorder Primary Hyperoxaluria Type 1

highlights the importance of gþxylate transamination in regulating endogenous oxalate

production. This disorder is characterised by hyperoxaluria and systemic oxalosis due to

either, reduced glyoxylate aminotransferase activity or mistargeting of the

aminotransferase to the mitochondria (in humans alanine:glyoxylate aminotransferase is

peroxisomal) (Williams et a1.,1967 and 1983; Danpure and Jennings, 1988; Purdue er

al., l99L; Danpure, l99l). ln some cases pyridoxal phosphate is used successfully to

increase the activity of the aminotransferase thereby reducing endogenous oxalate

production (Gibbs and Watts, 1970; Watts, 1983; Yendt and Cohanim, 1985). Tþq.,

unlike OTC and (L)-cysteine, (D)-penicillamine is an unsuitable agent for reducing-the

endogenous production ofoxalate and hence urinary oxalate excretion.

104 Cuaprpn Fve

It't wrno Sruom,s

5.1 INTRODUCTION

be In vivo studies in the previous chapter demonstrated that urinary oxalate excretion can

(L)-cysteine reduced through oral administration of (L)+ysteine and the intracellular

no delivery compound, OTC, in both normo- and hyperoxaluric rat models. Although (L)- conclusive evidence was presented that adduct formation is the mechanism by which

be cysteine and OTC reduced urinary oxalate excretion, adduct formation is believed to

the most likely mechanism. As small d."tjË1r_*.qlr}l oxalate excretiorr can reduce

the risk of calcium oxalate urolithiasis in an nuuuler, the reductions rr u*ry

oxalate excretion observed with ôTC, and (L)qr-qe1¡.g to a lesser extent, are of cliniqal

significance

Although to date no definitive evidence has been presented, the liver is thought to be the

major site for formation of the cysteine-glyoxylate adduct. This chapter investigates

whether formation of the cysteine-glyoxylate adduct is the mechanism by which cysteine

reduces oxalate production from glycollate in hepatocytes and the abilþ of P-

reduce oxalate production aminothiols, B-aminothiol prodrugs and enzyme cofactors to

from glycollate in hepatocytes. As the tested compounds mav produce some degree of

(L)-lactate and cellular toxicity, the effect of these compounds on gluconeogenesis from

(L)-alanine was determined. Gluconeogenesis was used to determine the cellular toxicity

105 of the tested compounds as it is a fundamental indicator of cellular function requiring coordination between the cytosolic and mitochondrial comparÍnents. As considerable controversy still exists as to which enzymes are responsible from oxalate production from glycollate and glyoxylate, the pathways involved in oxalate production from glycollate and gþxylate a¡e investigated using hepatocytes and enryme preparations. The effect of

(D)-penicillamine on transamination and oxalate production from glycollate was also

(D)- determined to veriff that the increase in urinary oxalate excretion observed with penicillamine treatnent (Chapter 4) is due to (D)-penicillamine-pyridoxal phosphate binding with a subsequent reduction in glyoxylate transamination.

5.2 RESULTS

5.2.I STU¡MS WIn{ ISOI-A.TED RAT FIEPATOCYTES

Glycollate is preferentially used as the oxalate precursor when investigating the effects of

p-aminothiols on oxalate production, as it requires transport into the cells before it is

metabolised in the peroxisomes by glycollate oxidase to glyoxylate. If adduct formation

glyoxylate is is the mechanism by which B-aminothiols affect oxalate production, when

used as the oxalate precursor, adduct formation could occur either inside or outside the

cell whereas any reduction in oxalate production from glycollate observed with B-

aminothiols can essentially be attributed to intracellular adduct formation.

106 5.Z.l.l The Effect of (L)-Cysteine on Oxalate and Carbon Dioride Productionfrom

Glycollate: Evidence for (L)-Cysteine-Glyoxylate Adduct Formation in Isolated

Rat HePatocYtes

Hepatocytes incubated in the absence of produced oxalate and carbon dioxide from 2mmol/L glycollate a) a rats of 82?¡42 and 63+3nmoV30minl'l07cells, respectively. However, incubation in the presence of 1.0, 2.5 and 5.Ommol/L (L)-cysteine decreased oxalate production from 2mmo[ gþollate, 114+16, 20I+24 and

:-filZlnmoy30min/l07cells res_p_ectiveþ (Figure 5.1); whilst carbon dioxide production was not significantly affected. The reduction in oxalate production observed with 1'0, 2.5

and 5.Ommol/L (L)-cysteine was accompanied by an increase

adduct levels, 16l+7,265+28 and 364+1lnmoV30min/l07cells; respectively (Figure 5.1)'

A significant correlation between the cha¡ge in oxalate productign and thg-fg¡mation of

the (L)-cysteine-glyoxylate adduct was shown to exist (Figure 5 '2);

Adduct Formation (nmov30min/l07cells) = (-0.42\ + (1.58)(Reduction in oxalate) -

(0.00l3)(Reduction in Oxalate)2 (r = 0.99)

This observation supports the view that (L)+ysteine-glyoxylate adduct formation is the

most likely mechanism by which (L)-cysteine decreases oxalate production from eJv:Wit,t,g

t0'7 900

* gâ 7s0 96 It iË õç 600 * 'çå€E ox ãÈ 450 * ô= ^o9dÉ lf o.9 3oo -!r'(€5 9 * do * ðÀ t5o

0 0.0 1.0 2.0 3.0 4.0 5.0 (L)-Cysteine (mmol/L)

and 5.0mmol/L (L)rcysteine on oxalate (n) and isolated lmL at 3 difference from controls: * p<0.01 and ** p<0.001.

450

Ø 400 =oG¡ I 350

3oo cl5 E 250

200 oá d É 150 L o r00 (J E50

0 0 50 l0o I50 200 250 300 350 400 Reduction in Oxalate Production (nmoV30mir/107cells)

Figure 5.2 The relationship between formæion of the (I) utrã th" reduction in oxalæe production from glycollæe ob isolæed rat hepatocytes (Sectìon 2.3.2.4). The results are (n=a) The bróken line indicates the line of equivalence. Production 5.2.1.2 The Effect of þAminothiols on Oxalate and Carbon Dioxide from

Glycollate in Isolated Rat Hepatocytes

incubated with 2mmoVl. glycollate produced In the absence of effectors, lreplto-g¡tgl oxalate and carbon dioxide at a rate of 750+90 and 87+9nmoV30min/107cells, respectiv,ely Altn:ggþ¡rll1q4 oTq *¡ sulghat¡, t119!! ll')*y{eine-glyo¡vlate adduct and O.5mmol/L thiamine pyrgphosphate þa{ ¡ro etre4 q-9I¿l?lgproductio-n from

2mmoL oxalate production was decreased 5mmol/L 4l*2o/o;

(D)-cysteine , 27+5Yo; and (D)-penicillamine, 27+2Yo; and O.5mmol/L pyridoxal

phosphate, 9+Io/o; (Figure 5.3). Carbon dioxide production from 2mmol/L gþollate was

I}0o/vcis- decreased by 5mmol/L (D)-penicillamine, 2ù+5%; and increased by lmmol/L

(L)-cysteine-glyoxylate adduct, l0+2%o: (Fþre 5' 3)'

5.2.1.3 The Effect of (L)-Cysteine and (D)-Penicillamine on Carbon Dioxide and

Oxalate Producfion from Glycollate in Isolated Rat Hepatocytes

In the absence of (L)-cysteine or (D)-penicillamine, oxalate and carbon dioxide

production by hepatocytes from 0.1, 0.5, 1.0, L5 and 2.0mmo[ glycollate inc¡eased in a

linear manner (Figure 5.4),

COz (nmoV3 gmtu/ I gTcells) = (2. 64+ 1.75) + (49. 39+ 1.5 6)(Glycollate) (r0. 99)

oxalate (nmov30min/l07cells) = (-20.99+15.32) + (359.38+l3.69XGlycollate) (r=0.99)

108 Oxalate or Carbon Dioúde Production (% Control) Oxalate or Carbon Dioxide Production (nmol/3 Omin/ I 0Tcells) 8à338 ¡J ñaàuô'l æ o o r. =c88888 a) o *+** 5 o 3 U .) c) -.D ** o 5v o ãaãoÞ- H o t-ts(Dl: ç Èl P ci qe t¿PJig Þ o * ìã3 cr il o ***+ 5 Ë ;EåYOo',9n o (¡): trl =Þ o U) oid- ã Ø_9 - E l^H Þ --ic¡a a 3 o Þ¿DP- 5 +ê^Éc)É uì? (D X'è+ F Ê- ** 'Þv È è "3ó'fJ ô t ,ú',.J IF i. ü NJ:f u o /.¿ Þx =trJ^+vu Ø. ; J È! * Þ-' o € ããi ã ã o1" o o Xõ- rÉ x ã.p Ë o iØØ-Ø-- Ë ÈÉ(D(r o (DE¡^. Þ Hãó o Again 5mmol/L (L)-cysteine did not signiñcantly alter carbon dioúde production from

0.1, 0.5, 1.0, 1.5 and 2.Ommol/L gþollate, (Fþre 5.5), however 5mmol/L (D)- penicillamine significantþ reduced carbon dioxide production, linear reduction from 45+4 to 23+2Yo; (Figure 5.5). Oxalate production from 0.1, 0'5, l'0, l'5 and 2.0mmol/L glycollate was decreased by both 5mmol/L (L)-cysteine and (D)-penicillamine, linear reductions from 69+3 to26+LYoand 54+3 to 38+3% respectively; (Figure 5'6)'

5.2.1.4 The Effect of ftAminothiols on Hepatic Metabolism as Determined by

Gluconeogenesis from (L)-Alanine and (L)-Lactate in Isolated Rat

Hepatocytes

As the tested compounds may produce some degree of cellular toxicity, the effect of these

compounds on gluconeogenesis from (L)-lactate and (L)-alanine \ryas determined-

cellular of the tested compounds as it _GlUçqlg.9gqry_s:tqwas used to determine the toxicity

is a fundamental indicator of cellular function requiring coorriination between the

cytosolic and mitochondrial comparbnents. To achieve the optimum conditions for

gluconeogenic studies hepatocytes were isolated from &glgllgalelA4qq=çrls' &{Tg

depletes hepatic glycogen stores to rely on for their

g!99-ry, Hepatocytes incubated with the various effectors produced significant ryqpty "f quantities of (D)-glucose from l0mmoL/L (L)-lactate and (L)-alanine and 5mmoyl (D)-

and (L)

respectively; (Figure 5.7). Aminooxy acetic acid, a transamination inhibitor, was shown

not to be glucogenic and in fact decreased basel gluconeogenesis, 104*lolo compared to

(L)-alanine; (Figure 5.7). Gluconeogenesis from l0mmol/L (L)Jactate was decreased by

5mmol/L (D)-cysteine, 5g+4yo; (D)-penicillamine, l2+3%o; and OTC, I4+lo/o; Zmì|ld

109 L20

âo Ë r00 (Jo * s It ã80o lf * () + lÉ E60 J} Þi () E 840 a o €20frt Q

0.1 0.5 1.0 1.5 2.0 Glycollate (mmol/L)

Figure 5.5 The effect of Smmol/L dioxide production from Incubations were 107 cells/ml at 3 the absence of or (D)-penicillamine produced ca¡bon dioxide at a rate of 10Gr6nmoU3Omin/l Tcells Results ¿re expressed as mean*S.E.M (n=3). Significant difference from controls: * p<0.02 and ** p<0.01

lÉ 80 * *

o lc Ë60 lÉ * o lÉ O \ê lf tÉ

o

840 .tt E o P. (,)

-E'ct 20 X

0 0.1 0.5 I.0 1.5 2.0 Glycollate (mmol/L)

eine (l) or (D)-penicillamine (n) on oxalate hepatocytes (Séction 2.3.2.3). I¡cubations foi 30 minutes. Incubations in the absence ed oxalate from 2.0mmol/L glycollúe at a ults are expressed as mean*S.E.M (n=3). Significant difference from controls: * p<0.01' 1800 Ø .t+ l+ 1600 9Ê Ì-I E r,+oo

clo t200 Ê rooo l+ É 800 lf o t+ O l+ Þ 600 * €o pr ,+00 (.) * Ø I zoo Yo tft+ vô ** -200 (L)-Lactate (D)-Cysteine Adduct (DÞPC Oxalate PP (L)-Alanine (L)-CYsteine OTC Glyoxylate AOA

Figure hepato for 30 from controls: * P:0.13, ** P=0.06, **+ PP stand for (D)-penicillamine, amin respectively.

9f t20 ta o

loo .tf R l+ l* J JÈ 1ã Þts 80 tro 9() 9ro ¡o\ õU * õv tç * .- 0) t+ * o.H JÊ €ã 40 rt O-i * + Oiì l+ lf* * * * tÈ d)ì * It lÉ 20 .tê 3= .tÉ Bã Yo # *-rê

20 (L)-Cysteine Adduct (D)-PC Oxalate PP (D)-Cysteine oTc Glyoxylate AOA

(D)-gluco.s: frgm Figure 5.8 *iolq compounds .oq thg production-of l0îrmolil or (L)-alanìne ( tr ) by isòlated ræ hepatgcl't_e,s (Section 2.3.2.5). perfórmed with 107 cells/ml at 37oC for 30 minutes. lncubaiion r produced (D)-gl (L)-alanine at a Results are expre p<0.05, ** p<0.0 (D)-penicillamine 93+lYo; glyoxylate, 85+Io/o; and oxalate, 8l+2Yo; and lmM aminooxy acetic acid'

(Figure 5.8). Gluconeogenesis from l0mmoL/L (L)-alanine was decreased by 5mmol/L

(D)-penicillamin e, StlYo; and (L)-cysterne, T*Io/o;2mM glyoxylate, 57+3%o; and oxalate,

gg+3o/o; and lmM aminooxy acetic acid, 106+2Yo; and increased by lmmoVl (L)-

cysteine-glyoxylate adduct, I?tI%o; (Figure 5. 8).

5.2.L5 Comparison of Oxalate and Carbon Dioride Production from Glyoxylate and

Glycollate in Isolated Rat Hepatocytes

Incubation of with 1, 3 and SmmoU[- or resulted in a

lirrear increase in carbon dioúde production (Figure 5.9) and a nonJinear increase rn

oxalate 0) from both substrates. However, over the range tested

carbon dioxide production was significantly higher from glycollate than from gþxyllte

(Figure 5.9). Again at lower substrate levels (I ?4 IT",ol/L) oxalatg_produgtjg!!_ltlas

whilst at 5mmoL/L substrate the

rates ofoxalate (Figure 5.10)

Hepatocytes that had been flushed with oxygen prior to incubation produced oxalate at a

rate of 723+34 and I 179*l38nmoV30min/l07cells and carbon dioxide at a rate of 69+5

and 5l+4nmoV3gmfu/lg7cells when incubated with 2mmol,/L glycollate and 5mmol/L

glyoxylate respectively. Hepatocytes that were not flushed with carbogen prior to

incubation produced significantly less carbon dioúde from both glycollate and glyoxylate,

53+lyo and 30+l% respectively. Although, failing to flush the incubation vial with

carbogen prior to incubation did not significantly change oxalate production from

glyoxylate, oxalate production from glycollate was reduced, 5A5%. Flushing the

110 140

t20

o Ëa too €o)o() ÀÞ 80 €È'tE 'Ãg 60 a?OE €]a 40 (Jcl

20

0

0.0 1.0 2.0 3.0 40 5.0 Glycollate or Glyoxylate (mmol/L)

Figure 5.9 ncentration on the rate of carbon dioúde piõãuction (:) -.isolated ta!_hgatg"ryJ (Section i.il.Z>. I 107 cells/ml at 37oC for 30 minutes' Results are

1600

1400

1200 ¿Ø Ìi-t¡ 1000 €-o-à ÈË 8oo ()ô Ëe ãã 600 ðE 400

200

0

0.0 1.0 2.O 3.0 40 50 Glycollate or Glyoxylate (mmol/L)

Figure substrate concentration on the rate of oxalate prõàuct gxyl{e (:) in.isolaæd ra!_!t^e¡aqoc4e¡ (Section 2.2.2.2.¡ d with 107 cells/mL at 37oC for 30 minutes. Results (n=3). incubation vial with carbogen prior to incubation significantly increases the oxygen tension, favouring glycollate and glyoxylate oxidation by peroxisomal flavin linked oxygen dependant glycollate oúdase. However, failing to flush the incubation vial with carbogen prior to incubation results in significantly lower oxygen tension, favouring glyoxylate oúdation and reduction by cytosolic nicotinamide linked lact¿te dehydrogenase.

For investigations into the effect of unlabelled glycollate or glyoxylate on [raC]-oxalate

and carbon dioxide production from [1aC]-glycollate and glyoxylate, hepatocytes

incubated with, a) I and 5mmoUL ¡t-'4C1-glycollate produced oxalate at a rate of 448+32

and l4l5+42nmoV30min/107cells, respectively and carbon dioxide at a rate of 3?Ì2 anrd

l15+llnmoV3gmin/lQ7cells, respectively (Figure 5.11 and 5.12; Control Levels), and b)

1 and 5mmol/L [2-r4C]-glyoxylate produced oxalate at a. fate of 347+47 and

l5l0+5lnmoV30min/l07cells, respectively and carbon dioxide at a rate of 23+3 and

86+8nmoV30min/107cells, respectively (Figure 5.ll and 5.12; Control Levels). Excess

unlabelled oxalate in the samples was shown not to significantly affect the [raC]oxalate

¿rssay. Addition of 5 and lmmol/L unlabelled glyoxylate to samples containing I and

55+5%; 5mmol,/L ¡t4c1-gtycotlate decreased [tac]-oxalate production, l9+4% and

respectively (Figure 5.ll), but did not significantly decrease [raC]-carbon dioxide

production (Figure 5.12). Addition of 5 and lmmol./L unlabelled glycollate to samples

containing I and 5mmol/L ¡taC1-glyoxylate decreased [r4C]-oxalate production, 34+6yo

and,23+3Yo: respectively (Figure 5.11), and [taC]-carbon dioúde production, 2I+I%o and'

6+.1%o; respectively (Figure 5. 12)'

111 1800

1600

I400 .tf IF -vl t200 Ë8 oc:. 1000 ãr .tF .¡Ê ÞÈ JÉ oiE 800 !lo 'd=Ecl 600 X= * ^É 400 * 200

0

-200 Control --> lmM Gc' 5mM Gc* lmM Gx* 5mM Gx* Treâtment -> (+5mM Gx) (+lmM Gx) (+5mM Gc) (+lmM Gc)

glycollate on r glyorrylate, treafrnent (m). . Results a¡e expressed as mean+S.E.M._(n:3). Signficant difference from co_ntrols:_* p<0.05, t* p

150

r25 o -û !6 loo .,ë o-- lJr- c,¡ Þ XÞ 'Ãqoo ¿O ots )u (! (J ,tf JÊ 25

0 Control --¡ lmM Gc' 5mM Gc* lmM Gx' 5mM Gx* Trêat¡nent --) (+5mM Gx) (+lmM Gx) (+5mM Gc) (+lmM Gc)

Fieure 5.12 glYcollate on [l{C]-carbon or glyoxylate, iespéciwely, treaünent (E). Incubations Results a¡e expressed ¿¡s mean+S.E.M. (n=3). Signficant difference from controls: * p<0.05, ** p

Productionfrom Glycollate and Glyoxylate in Isolated Rat Hepatocytes

Although carbon dioxide production from 1, 3 and 5mmol/L glyoxylate was increased by pretreatnent with (D)-penicillamine, this trend did not reach significance (Figure 5.13).

Carbon dioxide production from 1, 3 and 5mmol/L glycollate was completely unaffected by (D)-penicillamine pretreatrnent (Figure 5.13). Alternatively (D)-penicillamine pretreatnent increased oxalate production from l, 3 and Smmol/L glycollate, 49+4,25+3 and Ig+2o/o respectively; and I and 3mmoL/L glyoxylate, 75+3 a¡d 74+2yo respectiveþ;

(Fþre 5.14). Oxalate production from Smmol/L glyoxylate was not significantþ affected by (D)-penicillamine pretreatnent (Figure 5' 14).

5.2.2 Srums wrlr{ I{EcoNSTITUTED ENZYÌ'ß Svsrsþfs

5.2.2.I Formation of the Cysteine-Glyoxylate Adduct from Glycollate in the Presence

of Glycollate Oxidase and Cysteine

This study was performed to determine, a) if glyoxylate is a free intermediate in the oxidation of glycollate by glycollate oxi.lase and b) whether cysteine-glyoxylate adduct formation could occur from glycollate and (L)-cysteine in the presence of glycollate oxidase. Significant quantities of (L)-cysteine-glyoxylate adduct could be detected in the r:""1{tqfqjyyrng_ly$rn_$ection 2.4.1) cgltainins_(!l-gsqgt, w_hilst no adduct could be detected in the system without (L (Figure 5.l5). The presence o!(L)-

in the reconstituted system did not affect the rate of glycollate grdatll yr-tfre ralge !ç-sJ-cd;, --

rt2 150

125 o -cs.9 ãõ loo ä-o I+Ê d)> 13.= 1< xti oo ãS -O €ÊõE 50 (.) t<

0 1.0 3.0 5.0 1.0 3.0 5.0 Glycollate (mmol/L) Glyoxylate (mmol/L)

Figure 5.13 The effect of (D)-penicillamine pretreaûnent o:r ca¡bon dioxide production from glycollate or glyoxylate in isolated rat hepatocytes (Section 2.3.2.8); conirol (l) and pietreatrnent (n). Incubations were performed with 107 cells/ml al37oC for 30 minutes. Results are expressed as mean*S.E.M. (n=a).

2000 *

lÊ 1500

-ø L €8()c:. 39 öè o.E 000 qô ;ã6 d= XX *

500 .tt

0 1.0 3.0 5.0 1.0 3.0 5.0 Glycollate (mmol/L) Glyoxylate (mmol/L)

Figure 5.14 The effect of (D)-penicillamine pretreatrnent on oxalate production from glycollate or glyoxylate in isolated rat hepatocytes (Section 2.3.2.8); control (l) and þrètreatment (a). lncubations were peformed with 107 cells/ml at 37oC for 30 minutes. Results are expressed as mean+S.E.M. (n=a). Significant difference from controls: + p<0.05. 0.4

C)

=().= 0.3 >ìÉ \JX oEL\ oi_ o2 'Elv(!- Ëö È€.9Ë Ë'l E 0 E=o

0.0 0.0 0.2 0.4 0.6 08 1.0 Glycollate (mmol/L)

late adduct in a reconstituted enzyme glycollæe and glycollate oúdase (o) and absence of steine (l) and the 20 minute. Results ples, pool ofthree rat livers).

0.30

0

õlo 0 20 o 1

0 5 Ec) o l¡i 0. l0 o rt 005

0.00 0.00 0.05 0. l0 0.15 0.20 0.25 0.30 0.35 0.40 Glycollate Oxidised (pmoV20min)

Figure 5.16 The relationship between adduct formation and glycollate oxidation obierved in the reconstituted enzyme system containing glycollate oxidase, glycollate and (L)-cysteine (Section2.3.2.9\. The results Í¡re expressed as mean*S.E.M: (triplicate samples, pool of three rat livers). The broken line indicæes the line of equivalence . Glycollate oúdation with cysteine (pmol) = (-0.01+0.02) + (0.96+0.07)(Glycollate

Oxidation without Cysteine) (r = 0.99)

Although a significant correlation was observed between adduct formation and glycollate oxidatior¡ at glycollate levels above l.5¡r.mol adduct formation could not adequately account for all of the glycollate oxidised (Figure 5.16);

Adduct Formation (pnol) = 1.67(Glycollate Oúdised) - 5.60(Glycollate Oxidised)z +

7.78(Gþollate Oxidised)' (r = 1.00)

s.2.2.2 A Kinetic Investigation of the Enzymes which Synthesise Oxalate from

Glycollate and GlYoxYlate

5.2.2.2.1 Lactate Dehydrogenase

At pH 7.4 and 37oC, wittÌ glyoxylate as a substrato, V-o (Reduction) and V,no

(Oúdation) are 18.7+0.4 and 0.5+0.0¡^tmoVmin/mg respectively indicating that 4 !9yie1

substrate levels reduction ls

oxidation (oxalate production) (Figure 5.17). However, K. (Reduction) and K*

(Oxidation) are 10.8+0 3 and 0.6+0.0mmol/L that lactate

has almost a 20 in the oúdative

direction (oxalate production) than in the reductive direction (glycollate production). At

pH 9.0 and 37oC, the opposite is true, V-* (Reduction) and V* (Oúdation) are 7'5+0.1

and 66.5+l.gnmoUmin/mg respectively indicating that at lower substrate levels glyoxylate

reduction is slower ttran oxidation (Figure 5.18). ln addition, K- (Reduction) and K.

Il3 1.0 q,rr o l) () (D E 0.) 0.8 Reduction (Du \Jd È à' :Éô .= bo oE 0.6 Eç <è SH o-¡ É :JO ee< do {> öiÉ oa ã g Þo= E9r' rtlEv .ä >' Oxidation (.) o â 0.2 x. (L, A'È (É () o (Ë ¡ 0.0 0 0.0 0.5 1.0 1.5 2.0 Glyoxylate (mmoVl-)

Fig s o (o) of g-lY-oxYlate bY rat at and 2'5'5)'-Results are .M rat livers)' Note the scale is ¡rmol/min/mg.

40 10 r{ o a) À: o (D Oxidation B8 U \-,d 30 (D >l È .¿ üt 'H'ô^ã ()E O <è =To=' c)E 20 !J(Dáø Ø! Ë' û)É Ëh boH 4 gc Reduction Y>< Ë .ä c) 10 o Ê2 X (.) ô. Ë Þ (!o o -l 0 0

0.0 0.5 1,0 1.5 2.0 Glyoxylate (mmol/L)

Fig s of the reduction (-n) andoú4ati9n-(o) of g-ly-gxylate by 37oC (Section 2-.1.2'6 and 2'5 Results rat at pH 9.0 and '5)' - are .M.^(duplicate s¿rmples, pool of three rat livers). Note the scale is nmol/min/mg. the atrnitv (oxidation) are 1.02+0.01 and 1.73+0.04mmovl respectively indicating that

(glycollate of lactate dehydrogenase towa¡ds glyoxylate in the reductive direction (oxalate production) is almost 2 fold greater than it is in the oxidative direction production).

ra¡ge l0 to Based on the enzyme kinetic data and a glyoxylate concentration in the of

l0Opmol/L, the ratio of glyoxylate oxidation to reduction under physiological conditions

At pH 7'4 and is 0.3 and 0.4 at pH 7 .4 and4.5 and 5.5 at pH 9.0 (Fþre 5.19 and 5.20).

ability of 37"C the presence of glycollate in the system did not significantþ alter the

However, lactate dehydrogenase to oúdise or reduce glyoxylate (Figure 5.21 and 5.22)' of oxalate was shown by Lineweaver-Burk plots to be a non-competitive inhibitor

glyoxylate reduction, K 2.26+0.04mmol/L (Figure 5.23); and a strong competitive

inhibitor of glyoxylate oúdation, K, 29.8+6.5pmol/L (Figure 5.24).

5.2.2.2.2 Glycollate Oxidase

(Glyoxylate) and At pH 7.4 and,37"C, with glyoxylate and glycollate as substrates, V-o

at V-o (Glycollate) are Z.5I+0.20 and 10.9+0.5nmovmin/mg respectively indicating that

lower substrate levels glycollate oxidation (glyoxylate production) is frster than

(Glyoxylate) and glyoxylate oxidation (oxalate production) (Figure 5.25). However, K-

K. (Glycollate) are 1310+140 and 195+l4pmol/L indicating that glycollate oxidase has

for glyoxylate almost a 7 fold greatef affinity for glycollate (glyoxylate production) than

(oxalate productiori). Again based on the enzyme kinetic data and a glycollate or

glyoxylate concentration in the range of l0 to 100pmol./L, the ratio of glycollate to

pH 7 glyoxylate oxidation under physiological conditions is approximately 23 to 26 at '4

tt4 0.5

t o'¿ ()

€c) & 0.3 èo (! '! o.2 o(.) (É >r X o ð o.t

0.0 0.001 0.01 0.1 I Glyoxylate (mmol/L)

Figure 5.19 The relati xyla!€_oxidation and reduction (dõrived from Figure 5 at^ plf 7.4 and 37oC-(Section ).Z.Z.A and 2.5.5)l The of glyoxylate that is though to exist intracellularly.

8

7 o ()

G) 6

o

5 oX C) c! 4 X o (, 3

, 0.001 0.01 0. I I Glyoxylate (mmol/L)

Figure 5.20 The relationship between thg ra_te of glyoxylat_e_ oxidaúon and reduction (dãrived from Figure 5.18) by lactzte dehydrogenasq at pH 9 0 and- 37oC-(Section 2.3.2.6 and 2.5.5). The shaded area indicates the level of glyoxylate that is though to exrst intracellularly. 7 o oX q) Ø 6 c! oo9oa PE õÐâF 5 c¡ .! ('E o ¡ó o 4

()

J 0.0 0.5 1.0 l'5 2'0 l/GlYorylate (L/mmol)

Ficure 5.21 The effect of 0.0 (r), 0.5 (o), 1'0 (a) and 1'5mmol/L-(o) a and 37oC ;*ijdt;;"f glyoxylate by lactàé dehvdrogenase -æ.pH.! {.i"tt are.ipresred as mean+S.E.M. (duplicate samples, liven).-ãl.S.Sl,

2.5 o o () ú 2.0 (.ì) Ø d 9oa oo 1.5 €tr à-a ãÐâF l¿'ã 1.0 crt tr () (! I

o 0.5

() 0.0 00 0.5 l-0 1.5 2.0 l/Glyoxylate (Limmol)

Figure 5.22 The effect of 0.0 (¡),9..5 (o), l'0 (a) and--1Jrymo!/I.-(o) i"ã""iið" glyoxylate by l"cùé dehydrogenas_e_.1 pFf 7 .4..and 37oC Z.S.S)."f {eéuhi are eipressed as the mean+S.E.M. (duplicate samp rat-J livers). 2.5 o ()

c.) ú 2.0 () 6 CË qoãc)^ .EE 1.5 ào -É0)Él o.È dE 1.0 () (! J o 0.5

o

0.0 -05 0.0 0.5 1.0 1.5 2.0 liGlyorylate (L/mmol)

Figure 5.23 The effect of 0'0 (n reduction of glyoxylate by lactate ' and 2.5.5\. Results are exPressed rat livers).

150 o €C! X t25

I() €Ø C)^ 100 èI)=

'i3 = .qôòoé 75 t-{ r d).=

(J 50 ¡(! o 25 o

0

0.0 0.5 1.0 t.5 20 l/Glyoxylate (L/mmol)

Figure 5.24 The effe and 1.0mm on the oúdation of elioxvlate by lactate H 7.4 and 2'3'2'6 and 2'5'5)' zu;iffi; ãípi"ir.¿ (duplicate fthree ræ livers). l0 2.0 rt) o 6J c) o o o 8 Þ 1.5 t¡ 6 Glycollate o ^X FÊ ËÞ¡ËÊ 'Ës 6 AØ ()H *o <'E 1.0 É> c¡Þ ='t) (!ÉØo ã< 4 EHXi/ 6,ä o Glyoxylate o C) 0.5 GI , o o() E >r ai) o ö 0.0 0 0.0 0.2 0.4 0'6 0.8 l'0 Glycollaæ or Glyoxylate (mmol/L)

of the oúdation of glycollate (o) and glyoxylate (¡) bI 37oC (Sectionl3.l.6 and2'5'7). Results are expressed amples, pool ofthree rat livers).

30

€2i X o >' õzo 0) ó

815>ì õ o Ét0o rt(! osX

0

0.001 o.ol o.I Glycollate or Glyoxylate (mmol/L)

between the rate o glycollate oxidase indicates the level (Figure 5.26). At pH 7.4 and 37'C glyoxylate was shown to be an uncompetitive inhibitor of glycollate oxidase, K, 2.34+0.02mmoL; as shown by a Lineweaver-Burk plot (Fþre 5.27).

5.2.2.3 The Efect of @)-Penicillamine on Alanine:Glyoxylate and Alanine:a-

Ke t o glutarte Aminoff ansfe ras e Activi ty

(D)-Penicillamine at 5 and l0mmol/L decreased the of

5.28\, however this inhitition was not linear with respect to the

p{!t, be due to concentration of glyoxYlate. This non-linear behaviour was -sþW{r,-rn to formation of 5,5dimethy the (D)-penicillamine- glyoxylate adduct as addition of (D)-penicillamine to a solution containing glyoxylate reduced the levels of free glyoxylate stoichiometrically (Figure 5.29). Thus, when corrected for the concentration of free glyoxylate by assuming a I to I (D)-penicillamine to glyoxylate binding ratio, 5 and l0mmol/L (D)-penicillamine inhibited the activity of

alanine:glyoxylate aminotransferase by 29+5% arrd 32+7o/o respectively. To avoid the

complication of substrate binding by (D)-penicillamine as observed wittì

alanine:glyoxylate aminotransferase, the effect of 5 and l0mmol/L (D)-perucillamine on

the activity of alanine:a-ketoglutarate was determined; (D)-penicillamine does not form

an adduct with a-ketoglutarate as it is a ketone not an aldehyde. (D)-Penicillamine at 5

and lgmmol/L inhibited the activity of alanine:a-ketoglutarate aminotransferase by

33+3o/o and, 52+lo/o, respectively (Figure 5 . 3 0).

115 200

0) R' 180 o (J 160 .ao>ì^ 6-e <ò 140 d)H 6) '!]a€É o 120 c.)

()o 100 >r o

80 3.0 0.0 0.5 1.0 1.5 2.O 2.5 l/Glycollate (L/mmol)

Fieure 5.27 The effect of 0.0 (a), 0.5 (o), 1.0 (a) and l.5mmoL/L (9) elyo)ryI4-on the (Sections 2.3 and ;fid;hil"f glycollate by glycòú'ate oùá'ase æ_ pH.1..a .9nd 37oC '2'6 t.5rt R"r.rlís'-. e*preisãd as the mean+S.E.M. (duplicate samples, pool of three rat livers).

36

c)230 ,0.) 9Ð E€ :.E 24 =i <

0

0 5 10 l5 20 Glyorylate (mmoVl-)

Fisure 5.28 The effect of 0 (o), 5 (l) and l0mmol/L (a) D-penicillamine on the activity ãiä*i"",glyoxylate aminolransferase at pH 8O and 37oC (Section 2.3.2.7 aultd2'4'2)' rat liven). n"r"tG -"'.ípréssed as the mean+S.E.M. (duplicate samples, pool ofthree 25

20 .l o

15 c) d >\ X o >ì 0

()C) fri 5

0 20 0 5 l0 l5 Total Glyoxylate (mmol/L)

on o adduct in the (¡) amine under '.37'o exPressed as

42

o>, (.) .= YÉ n ;< ä6

0 6 7 0 a 3 4 5 cú-Ketoglutarate (mmol/L)

Figure 5.30 The effect of 0.0 (o), 0.5 (¡) an! l.5mmol,/L (a) Dleqcilaminegl F" of ¡at ine:a-tãioglutaraìe'áminoìránsfelale-4 ql 8 0 and 37oC (Sectioy213.27 ;ä"ci*try i.4.ti.T"sults a¡é eipressed as the mean+S.E.M.-(duplicate samples, pool ofthree rat livers) 5.3 DISCUSSION

AND ENZYME 5.3.1 TIü EFFECT oF B-AMINOTHIOLS, B-AMINOTHIOL METABoLmS

CopectORs ON Ox¡rare AND Can¡OU DIO)OE PRODUCION FROM

GTYCOITATS IN ISOLATED RET I{¡PATOCYTES

form cyclic condensation The abilþ of B-aminothiols, like cysteine and penicillamine, to

products with compounds containing aldeþde functional groups, such as formaldeþde,

glyoxylate and acetaldehyde, has beenknown for many years (Schubett, 1936a and b,

Raûrer and Clarke, 1937; Schmolka and Spoerri, 1957). However recently attention has

turned to exploiting these condensation reactions in biological systems to protect against

higtly reactive and toxic molecules containing aldehyde functional groups (Crawhill er

al., 1963; Crawhill and Thompson, 1965; Nagasawa et al., 1978; Poole et al., 1990;

Bringmann et al.,I99I; Sharma and Schwille,1992).

In the liver, glycollate is thought to be oxidised to oxalate indirectly by cooperation

between peroxisomal glycollate oxidase, which oxidises glycollate to glyoxylate, and

cytosolic lactate dehydrogenase, which oxidises glyoxylate to oxalate (Rofe and Edwards'

l9Z8; Asker and Davis, 1983). However, some investigators believe that glycollate

oxidase may be responsible for oxalate production from glyoxylate which is produced by

the peroxisomal oxidation of glycollate. Altlfo:gþr--glygglþLe-&þLdr-9gqas9 is said to

catalyse the direct conversion of glycollate to oxalate, the

oxalate production invivo remains uncertain. Regardless, if 'free'glyoxylate is produced

in the oxidation of glycollate by glycollate oxidase, then increasing the cytosolic or

peroxisomal concentration of p-aminothiols, such as (D)-penicillamine and (L)- and (D)-

ll6 tn cysteine, would be expected to increase the rate of glyoxylate condensation resulting

increased B-aminothiols-glyoxylate adduct formation.

The sigruficant correlation between the increase in (L)-cysteine-glyoxylate adduct

formation and the reduction in oxalate production (Figure 5.2) supports the view that

adduct formation is the mechanism by which (L)-cy,q!e-ine reduces oxalate production

A.,onogg"g_[49_T_!gluto"t. The observation that adduct form¿tion accounted for

significantly more glyoxylate than the reduction in oxalate can be explained by the fact

that normally the majority of gþxylate is diverted from oxalate production by

transarnination or decarboxylation (Metzler et a1.,1957; O',Fallon and Brosemer, 1977,

Hodgkinson, 1977; Bais et al., 1991a), hence the additional adduct comes from

gþxylate that under normal conditions would undergo transamination or

decarboxylation.

By analogy with (L)-cysteine, the reduction in oxalate production from glycollate

observed with (D)-cysteine must also be accompanied by a rise in formation of the (D)-

cysteine-glyoxylate adducts as adduct formation is not affected by cysteine

stereochemistry. However, the observation that (L)-cysteine was 59%o more effective at

reducing oxalate production from glycollate than (D)-cysteine (Figure 5.3) is attributed to

the fact that (D)-cysteine is met¿bolised to sulphate and pymvate more rapidly than (L)-

cysteine (Krijgsheld et al.,1981). This finding is supported by our own studies in which

the glucogenic capacity of (D)-cysteine was shown to be approximately 2 fold greater

than that of (L)-cysteine (pyruvate being the gluconeogenic precursor) (Figure 5'7)'

Thus, although adduct formation between (D)-cysteine and glyoxylate may occur

intracellularly, smaller amounts of adduct would be expected to form.

rt7 Due to the greater nucleophilicþ of (D)-penicillamine, formation of the (D)- faster than penicillamine-glyoxylate adduct would be expected to be at least 5-fold formation of the (L)-cysteine-glyoxylate adduct under physiological conditions from (Schonbeck et al., 1975). Equilibrium constants for forming thiazolidine derivatives

107 for pyridoxal phosphate and aminothiols are high, ranging from 106 for cysteine to

be penicillamine (Friedman, lg77). The greater nucleophilicÛ of (D)-penicillamine can

adducts' seen when preparing both the (D)-penicillamine- and (L)-cysteine-glyoxylate

form and Although it takes I hour at 80"c for the (L)-cysteine-glyoxylate adduct to (D)- precipitate (Section 3.2), under the same conditions it takes just 20 minutes for the

penicillamine-glyoxylate adduct to form and precipitate. Hence, (D)-penicillamine-

(D)- glyoxylate adduct formation would also be expected to be the mechanism by which

penicillamine reduced oxalate production from glycollate in isolated hepatocytes'

Formation of the (D)-penicillamine-glyoxylate adduct under physiological conditions was

also confirmed indirectþ by investigating the effect of (D)-penicillamine on free

glyoxylate levels (Figure 5 . 29 ; Section 5 .2.2'3) .

Despite the fact that penicillamine is a better nucleophile than cysteine, the reduction tn

oxalate production from glycollate observed with (D)-penicillamine was 34%" less than

the fact that that observed with (L)-cysteine (Figure 5.3). This difference is attributed to

(D)-penicillamine is an avid scavenger of cysteine, pyridoxal phosphate, hydrogen

peroúde, copper and other compounds/enzymes containing free aldehyde or thiol

et functional groups (Jaffe, 1969; Schonbeck et al., 1975; Bergstrom et al-, 1980; Statte

al., 1984; Ioyce et al., 1989; McQuaid et al., 1992,Ltt and combs, 1992\. Hence,

the same stoichiometncly speaking, more (D)-penicillamine would be required to achieve

level of free thiol intracellularly when comparison to (L)-cysteine.

118 produce As carbon dioxide results from glyoxylate decarboxylation, glycollate can only

oxidase' ca¡bon dioxide if it is first oxidised to glyoxylate by peroxisomal glycollate

and non- Glyoxylate decarboxylation has been shown to be occur both enzymatically

Williams enzymatically (Richardson and Tolbert, 1961; O',Fallon and Brosemer, 1977;

is catalysed by o- and Smith, 1983; Bais et al.,l99la). The enzymatic decarboxylation

in ketoglutarate-glyoxylate carboligase whilst the nonrenzymatic decarboxylation occurs

(D)-penicillamine the peroxisomes and is known to be hydrogen peroxide dependant. As

has the is an avid scavenger of hydrogen peroxide (Staite et al., 1984 and 1985), it

potential to reduce glyoxylate decarboxylation causing increased glyoxylate (D)- transamination and oxidation to glycine a¡d oxalate respectively' Indee'd"

(Figure and 5'5)' penicillamine does reduce ca¡bon dioúde production from gþollate 5'3

Thus, (Dlg9ru-c,itl.4qlgry3y.fi9t þ1ve be94 as g@ctive as: (L){vsteine in reducing

oxalate production foom as it reduced glyoxylate decarboxylation therebY

the and oxidation of glyoxYlate to oxalate.

to Richardson et al. observed a phenomena similar to this with the addition of catalase

purified glycollate oxidase. Upon addition of catalase to the purified enz ørre, oxalate

production was increased from glyoxylate and production of carbon dioxide and formate

were decreased (Richardson and Tolbert, 1961)'

(D)-Penicillamine is used widely in the treatnent of Wilson's disease, heavy metal

poisoning, cystinuria and systemic sclerosis (Crawhill et al., 1963; Crawhill and

er Thompson, 1965; Lodemann, l98l; Staite er aI.,1984; Menara et al.,1992; McQuaid

al., 1992) However, (D)-penicillamtne administration has been associate4 with vitamln

PYridoxal Bo and (Jaffe, 1969; Joyce, 1990)

phosphate, the transamination cofactor, is derived from vitamin Bo' This deficiency ß

phosphate, in due to (D)-penicillamine binding both free and enzyme associated pyridoxal

119 apoenzymes and in the first instance altering its structure and its ability to conjugate with

(Schonbeck et al'' the second instance altering its ability to facilitate group transfer

thr,gl¡gh clqþüon 1975). Thus, reduce q4q;as4elion qþy449ï4ph9ryh4-e,rndi-ry911y,-i"gfçO-i"-g oxr-dali9¡-qfulvory1q1e-t9 ql{lle:

(D)-penicillamine This is supported by the observation that oral administration of

decrease the activity of increased urinary oxalate excretion (Figure 4.14 and 4'15) and

(Table ln addition' (D)- both aspartate and alanine aminotransferase in the plasma 4'2)' penicillamine in v¡tro was shown to reduce the activity of alanine:glyoxylate

adduct aminotra¡sferase primarily through formation of ttre (D)-penicillamine-glyoxylate

the (Frgure 5.28 and 5.29). However direct interaction between (D)-penicillamine and

a-ketoglutarate tránsaminase, rather than substrate binding, could be detected' When

(D)-penicillamine, was used as the amino acceptor, to avoid substrate binding by

significant inhibition of alanine:a-ketoglutarate aminotransferase was observed'

(Figure 5'30)' confirming the direct interaction between penicillamine and the en4çme

The most convincing evidence that (D)-penicillamine increases endogenous oxalate

pretreatnent production by decreasing glyoxylate transamination was the observation that

hepatocytes led to of rats with (D)-peniciltamine at 48 and 24 hours prior to isolation of

(Figures 5' 13 and 5' t4)' No increased oxalate production from gþollate and glyoxylate

the fact that glycine data on glyoxylate transamination to glycine is presented here due to

a small portion of the which is derived from glyoxylate transamination accounts for only

total glycrne pool. Hence measurement of small changes in glyoxylate transamination

under these experimental conditions is very difficult'

It can be concluded from the above dat¿ that (D)-penicillamine increases endogenous

oxalate by reducing both glyoxYlate

t20 pyridoxal peroxide scavenging (in the short term) and glyoxylate transamination through

more phosphate scavenging (in the long term). Thus, although (D)-penicillamine is

than (L)- effective in forming thiazolidines with glyoxylate and other carbonyl compounds or (D)-cysteine, it is this strong nucleophilicity which pfevents it from being a useful treatnent for the reduction ofendogenous oxalate production.

cofactor in the transamination of glyoxy-late to lYndoxat is an important

glycine by alanine:glYoxYlate aminotransferase (Metzler et aL, 1957; Gbbs and Watts,

1970; Tiselius and Almga¡4 1977; Yendt and cohanim, 1985; Gill and Rose, 1986;

Edwards et al., 1990). Pyqidoxal phosphate deficiency is known to result in excess

oxalate the importance of both pyndoxal pbqsphate and the

(Sharma et al', tr:ùrsaminati_on pattrw4y in di191ing ClVoxylaq-from oxalate prgductio-n

1990; Edwards, 1990). The reduction in oxalate production from glycollate observed

with pyndoxal phosphate (Fþre 5.3) is thought to be due to increased glyoxylate

transamination to glycine by alq¡-ut-lg,g.lyoxylate aminotransferase highlighting he

importance of homeost¿sis in oxalate prodlction.

OTC is known to be a¡r effective iniraeellular (L)-çyqteinç de[ivery compound being

metabolised in the cytosol bY to S-carboxycysterne which is sporrtaneously

(Anderson and Meister, 1987) However, over a 30 4.:g9gryþf94 !q-(-L)-:ylle-i¡p-

minute incubation OTC was unable to reduce oxalate

r'ggelylglþle (Fþre s.¡) torn rygggs:Te ${ -'llfficj-e41Q)-c¡!teT9-*3!I'!-q"99q the metabolism of OTC to enable formation of the (L)

adduct. The inability of OTC to produce significant quantities of (L)-cysteine over the 30

minute incubation period could be explained by the observations that the cellular uptake

rate for OTC into isolated rat hepatocytes is 43olo slower than that for cysteine,

t2r suggesting that in the short term uptake of OTC may be a rate limiting step in its utilisation in freshly isolated rat hepatocytes (Coloso et a1.,1991), this finding has been confirmed by other investigators (Banks and Stipanuk, L994\.

The observation that greater than physiological levels of sulphate did not affect oxalate production from glycollate (Figure 5.3) in the isolated hepatocytes was not surpnsrng, as no evidence could be found that sulphate is important in the regulation of oxalate production. This observation confilms the view that the increase ur heqa-tic sulnhatgt

upon administration of cystelge o¡ would not significantly affect oxalate 9-b¡erved _O_JC=, production in the liver,

Thiamine pyrophosphate is an important cofactor for glyoxylate decarboxylatì-or-r b¡r mitochond¡ial a-ketoglutarate:glyoxylate carboligase- Thiamtne deficiency has been shown to result in hyperoxaluria by an_ln_ctq¿lse rn the hgpalic activrty of glycollate oxidase and glycollate dehydrogenase and a concomitant decrease in the activity of a- ketoglutarate:glyoxylate carboxylase (Stervart et al., 1981; Sharma et al., 1990).

Although thiamine deficiency decreases hepatic carboligase activity. no resultant change in respiratory carbon dioxide production from glyoxylate is observed (Stewart et al',

1981). Thus, cr-ketoglutarate:glyoxylate carboligase is thought to play only a minor role

rn diverting glyoxylate from oxalale production. Moreover, the hyperoxaluria associated

with thiamine deficiency appears to be due to increased oxidation of glycollate to

glyoxylate and oxalate by glycollate oxidase and glycollate dehydrogenase respectively,

rather than decreased decarboxylation of glyoxylate by o-ketoglutarate:glyoxylate

carboligase. this is consistent with thiamlne pyrophosphate not sigmficantly affecting

carbon dioxide or oxalate production from glycollate in isolated hepatoc¡es (Figure 5.3).

t22 As noted earlier, carbon dioxide production from glycollate can only occur if glycollate

oxidation results in the release of free glyoxylate which is then decarboxylated'

Gtyoxylate decarboxylation can occur enzymatically in the mitochondria catalysed by ct-

ketoglutarate:glyoxylate carboligase (O'Fallon and Brosemer, 1977 Bais ¿¡ al., l99la)

or through non-enzymatic hydrogen peroxide dependant decarboxylation in the

peroxisomes (Richardson and Tolbert, 1961). As glyoxylate and hydrogen peroxide are

to both products of the oúdation of glycollate by glycollate oxidase, they can be assumed

non- be present near the active site of the enzyme in relatively high concentrations. Thus,

en-4rmatic decarboxylation of glyoxylate by hydrogen peroxide, is thought to be the major

pathway for carbon dioxide production from glycollate, rather than mitochondrial

decarboxylation by ct-ketoglutarate:glyoxylate carboligase. This hlpothesis is supported

by the observation that; a) the reduction in oxalate production from glycollate observed

with (L)-cysteine, due to (L)-çysteine-glyoxylate adduct formation, is not accompanied by

a reduction in qarþon, dioxide productioq, suggesting that the majority of carbon dioxide

production occurs before formation of oxalate or adduct (Figure 5.3 and 5.5)' b) (D)-

penicillamine, a known hydrogen peroxide scave_ng.-er, can- decrqpqe carbon dioxide

production from glycollate (Figure 5.3 and 5.5) and c) the (L)-cysteine-glyoxylate adduct

is metabolised in the peroxisome by (D)-aspartate oxrdase, producing hydrogen ryhich peroxide, increased carbon dioxide production (Figure 5 3). More importantly,

localisation of a-ketoglutarate:glyoxylate carboligase in the mitochondria (O'Fallon and

Brosemer, lg77) would reduce the likelihood for mitochondrial enzymatic

decarboxylation of peroxisomal glyoxylate which is produced from glycollate because the

glyoxylate would be oúdised or reduced to oxalate or glycollate by lactate dehydrogenase

in the cytosol before reaching the mitochondria. Hence, the majglty.of carbon dioxide

produced from glycollate is probably due to hydrogen peroxide dependant non-enzymatic

decarboxylation of glyoxylate in the peroxisomes.

t23 Carbon dioxide production from glyoxylate appears to be only a minor pathway for tlre catabolism of glyoxylate that is derived from glycollate as incubation of hepatocytes with glycollate resulted in the production of approximately 10 fold more oxalate than carbon dioxide (Frgure 5.4, 5.9 and 5.10).

5.3,2 FORMATON OF TIIE CYSTEI.IE.GLYOXYLATE ADDUCT FROM GI.YCOITETB W

TTIE PRESENCE OF GLYCOLLATE O>OASB A}ID CYSTEINE

part Although lactate deþdrogenase w¿rs present in the reconstituted system it played no

in the oúdation or reduction of glyoxylate as NAD* and NADH were not added to the

system. Formation of the (L)-cysteine-glyoxylate adduct from glycollate in the presence

of glycollate oxidase and (L)-cysteine (Fþre 5.15 and 5.16) again confirmed that

gþxylate is a free intermediate in the oxidation of glycollate by gþollate oxidase.

Thus, glyoxylate formed in the peroúsomes from glycollate could undergo condensation

with (L)-cysteine to form the adduct The observation that (L)-cysteine did not (L)- significantly alter the oúdation ele_!yeell49_þr etvggtt-{9 rflas" :}_ss_1{1that

cysteine does no!-affeg!-th9,¡_119-qt9*{etglyo1ylate frg1 thq substrate gleft qf glvcolþe

oxidase. Moreover, it can be concluded that free glyoxylqlg_{9e_¡ not regql_{9 the agtivtiy

of glycollate oúdase as adduct formation would have reduced the level of free

and the thereby reducing any inhibitory effect of glyoxylate on glycol-lale .oxi--d45e activity

rate of glycollate oúdatio,n. This view is further supported by the observation that

glyoxylate was only a weak uncompetitive inhibitor of glycollate oxidation (K'

2. 3 4+0. O2mmol/L) by glycollate oúdase (Section 5 .2.2'2.2).

t24 At high glycollate concentrations, (L)-cysteine-glyoxylate formation could not account fbr all of the glycollate that was oxidised to glyoxylate (Figure 5.16). This can be explained by the observation that the enzyme preparation, without added NAD*, can produce oxalate from glycollate (Section 2.4.I). Hence, the additional glyoxylate was most likely

oxidised to oxalate by glycollate oxidase in the reconstituted system.

DETERMINED BY 5.3.3 TrD EFFECT OF B-AMTNOTHTOLS ON Í{EPATIC METABOLISMAS

GLUcoNEocENESTs FRoM (L)-ArANßIE AND (L)-LAcTATE IN ISOLATED I{AT

HSPRTOCYTES

req]¡iring Gluconeogenesis is a fi,rndamental indicator of basic cellula¡ -function

coordination between the cytosolic and mitochondrial comparfnen!9. The effect of the

(L)-lactate (L)-alanine was thus selected B-aminothiols on gluconeogenesis from and

investigated to determine whether their administration to reduce endogenous oxalate

produ_ction would have a detrimental effect on cellula-r firnction. As (L)-lactate, (L)-

alanine and (L)- and (D)-cysteine are all known to participate in gluconeogenesis, the

production of (D)-glucose from these substrates (Figure 5.7) confirms the metabolic

integrity of the isolated hepatocyte. The significant reduction in basel glucose production

observed with aminooxy acetic acid (Figure 5.7), atransaminase inhibitor, also validates

the integrity of the system

(D)-Aspartate oxidase is a peroúsomal enzyme found in many animals, rabbit, pig, beef,

rat, mouse, sheep, cat, octopus and human; and tissues, liver, kidneys, thyroid, intestine,

lung, brain and nervous tissue; (Dixon and Kenworthy, 1967; Yusko and Neims, 1973;

Davies and Johnson, 1975; Jaroszewicz, 1975; Nasu ¿f al., 1982; Hamilton, 1985; Negri

t25 ef ai., et al., I98ï;Yamada et al., 1989; Zaar et al., 1989; Beard, 1990; Van Veldhoven l99l; D'Aniello et aI., I993a and b; Kera et al., 1993; Nagasaki, 1994). The

is peroxisomal metabolism of the (L)-cysteine-glyoxylate adduct by (D)-aspartate oxidase known to produce Â2-thiazoline-2,4-dicarboxylate which is thought to be degraded to

1979; Raymond, 1981; 1Jary et al., I989a and b; Lamboeuf et al.' :Y:19.ry(Saigot,

1990), however little is l,rnown about the pathways by which thiazolines are degraded to cysteine. Â2-Thiazoline-2,4dcarboxylate has the necessary carbon skeleton to produce

(D)-glucose. Without active enzJrme degradation, A2-ttriazoline-2,4-dtcarboxylate and

(Venkatesan thiazolines in general are stable at neutral to alkaline pH's for hours to days

(L)- and Hamilton, 1986). Hence, the small aÍiount of (D)-glucose produced from the

the cysteine-glyoxylate adduct (Figure 5.7) suggests that either, a) entry of adduct into c.lls is rate limiting or b) enzymatic breakdown of À2-thiazoline-2,4dicarboxylate to

cysteine, and oxalate, does not occur to any great extent in the liver.

Although metabolism of OTC results in the production of (L)-cysteine, which is a

glucogenic amino acid, insignificant amounts of (D)-glucose are produced under these

conditions (Figure 5.7). This observation is attributed to the slow rate of entry of OTC

into the hepatocyte (Coloso et al., 1991) and again supports the view that OTÇ

(L)- metabolism over the 30 minute incubation does not sufficient quantities of

cïtllng (D)-Penicillamine is said to undergo no metabolic degradation in mammals

(Perrett, 1977; Planas-Bohne, 1981), as hepatocytes do not posses the pathways that

(D)- enable the carbon skeleton to be separated from the thiol functional group of

penicillamine. Thus, (D)-penicillamrne did not produce significant quantities of (D)-

glucose. (D)-Cysteine produced 44o/o mote (D)-glucose when compared to (L)-cysteine

(Figure 5.7) rndicative of the faster catabolism of (D)-cysteine to pyruvate and sulphate.

This view is supported by Krijgsheld et al. who demonstrated that urinary sulphate

t26 excretion after oral administration of (D)+ysteine is 66Yohtgher than after administration of (L)-cysteine (Krijgsheld et al., 1981).

produced As (D)-penicillamine, OTC, (L)-cysteine and the (L)-cysteine-glyoxylate adduct

tbat these only minor changes in (D)-glucose production (Frgure 5.8), it can be concluded

However' the compounds do not produce major perturbations in cellular metabolism'

(D)+ysteine 5g+4y" decrease in (D)-glucose production from (L)-lactate caused by

(L)-lactate to (Figure 5.8) suggests that (D)-cysteine interferes with the conversion of

(L)-alanine; pynrvate as (D)-cysteine produced no change in (D)-glucose production from

pynrvate alanine is converted to pynrvate via transamination whilst lactate is converted to

through reduction. Thus, the rapid conversion of (D)-cysæine to pynrvate and sulphaæ

arid its interference in the production of (D)-glucose from (L)Jactate makes it an

glyoxylate' inappropriate compound for controlling oxalate metabolism from glycollate or

(D)-glucose Moreover, the observation that glyoxylate and oxalate significantly reduced

production from both (L)-lactate and (L)-alanine (Figure 5.8) confirms that they have a

regulatory influence on gluconeogenesis. It is not surprising that oxalate inhibits

pynrvate gluconeogenesis from both (L)-alamne and (L)-lactate as it is known to inhibit

(Yount and carboxylase the first enzyme in the gluconeogenic pathway from pynrvate

Harris, l9S0). Pymvate carboxylase converts pynrvate to oxaloacetate which is then

converted to phosphoenolpynrvate.

t27 PRODUCTON FROM 5.3.4 COTW,qRISON OF OXALATE ANO CANSON DIO)OE

Gryoxyr¡tg ¡¡{D GIycoLLATE nI ISOLATED RAT FIEPATOCYTES

are produced from The observations that signiñcantly more oxalate and carbon dioúde

ca¡bon dioxide glycollate than glyoxylate suggest that the systems for oxalate and

of glycollaæ) and production from a) endogenous glyoxylate þroduced through oxidation

The greater b) exogenous glyoxylate (added to the cell) are enzymatically different'

has been noted ability of the liver to produce oxalate from glycollate than from glyoxylate

This difference in oxalate before (Liao and Richardson, 1972; Rofe and Edwards, 1978). of production from glyoxylate and glycollate has been attributed to compafbnentalisation

grown popularity over the two systems (Rofe and Edwards, 1978), a theory that has in

oxalate by lactate the years. Currently exogenous glyoxylate is thought to be oúdised to

oúdised in the deþdrogenase in the cytosol whilst glycollate is thought first to be

peroúsomal peroxisome to glyoxylate which can then be oxidised to oxalate by either

and glycollate oxidase (Richardson and Tolbert, 1961; Liao and Richardson, I972;Fry

Richardson, 1979; Bais etal.,l989aandb; Yanagawa eta\.,1990)orcytosoliclactate

controversy dehydrogenase (Asker and Davis, 1983; Yanagùwa et al., 1990). However,

still exists as to whether peroxisomal glycollate oxidase or cytosolic lactate

to oxalate' deþdrogenase is responsible for the oxidation of endogenous glyoxylate

liver is slower than It may be that the rate of of glyoxylate into the

differencg in-g44-Pto9:fqttg fi'll-g: t*o g-ly99U-!49, I?qqq-t" ttt" tbserved m¿¡nner in which precursors , however this aPPears to be gr_v_eg_ 4-e* non¡Jgeq

(Figure 5.10) oxalate production from increases with substrate

is a far more labile This difference may also be related to the observation that

and amine gfoups species which can form hemimercaptals and schiff bases, with thiol

128 respectively, as well as participate in many enzymatic reactions including transamination

faster and decarboxylation (Table 1.1), whilst glycollate oúdation to oxalate may occur

and more directly within the confines of the peroxisome. Morgovel-,¡Ltr9-9ls-9¡yat¡94-¡þat

is increased conversion of glyoxylate to oxalate only occurs when the glyoxylate level

abnormally high suggests that the difference is purely enrymatic (Figure 5.10;

Weinhouse, 1955),

lndeed the observation tbat oxygen is essential for the oxidation of gþollate to oxalate

and carbon dioxide (Section 5.2.I.5) confirms that, a) an oúdase, ie. glycollaæ oxi{ase,

is responsible for the major portion of glycollate oxidation and that b) carbon dioxide

production from gþollate can only occur after gþollate has been oxidised to

glyoxylate. Moreover, the observation that the oxidation of exogenous glyoxylate to

oxalate is oxygen independent (Section 5.2.I.5) supports the view that lactate

deþdrogenase is the enz)¡me responsible.

The observation that unlabelled glyoxylate (1 or 5mmol/L) did not significantly affect

5.12) supports the view [raC1-carbon dioxide production from [lJaC]gþollate (Fþre

that carbon dioúde production from glycollate occurs rapidly and earþ during the

production of glyoxylate from glycollate. Despite the fact that glyoxylate is an

uncompetitive inhibitor of glycollate oxidase (K, 2.34+0.02mmo[; Section 5.2.2.2.2\,

exogenous glyoxylate did not alter the catal¡ic activity of peroxisomal glycollate oxidase.

Hence, it may be concluded that the majority of exogenous glyoxylate cannot enter the

peroúsome, if this was the case, glycollate oxidase would be inhibited given the observed

in vitro K¡ of glyoxylate and the level of unlabelled glyoxylate added.

r29 The observation that addition of 5mmoL/L unlabelled glyoxylate to lmmol/L Pl4Cl-

the 55% reduction glycollate decreased [raC]-oxalate production by 18% compared to observed when adding lmmol/L unlabelled glyoxylate to 5mmoL/L [2-taC]-glycollate

(Figure 5.11) suggest that at physiological concentrations, 9*ul1t9_p.odllgt_g_!g glycollate occurs rapidly and maY infact occur within the confines of the peroxisome, if

dilute the this was not the case the unlabelled glyorylate added to the hepatocYtes would

reduction m [ -taC] -glyoxylate diftrsing from the causmg a considerable

taC] I -oxalate production.

Although glycollate did not significantty a.ffect the ability of lactate deþdrogenase to

reduce or oxidise glyoxylate in vitro, oxalate was shown to be a non-competitive inhibitor

of glyoxylate reduction (K, 2.26+0.04mmo[; Section 5.2-2.2'L) and a strong

competitive inhibitor of glyoxylate oúdation (Kr 29.8+6.5¡rmol/L: Section 5.2.2.2.1).

Hence, the effects of unlabelled gþollate on [taC]-oxalate and ca¡bon dioxide production

by the unlabelled from [1aC]-glyoxylate can only be due to a) dilution of ¡taC1-glycollate

glycollate, b) dilution of [taC1-glyoxylate by endogenous unlabelled glyoxylate þroduced

through the peroxisomal oxidation of unlabelled glycollate) or c) competitive inhibition of

unlabelled oxalate the oxidation of [taC]-glyoxylate to [laC]oxalate by endogenous

(produced through the peroxisomal oxidation of unlabelled glycollate and glyoxylate)'

lndeed, substrate dilution by unlabelled glycollate and glyoxylate a¡rd product inhibition

by oxalate appear to be the mechanism by which unlabelled glycollate reduced oxalate

occurred was production from [2-raC]-glyoxylate, as the extent to which inhibition

(Figure inversely proportional to the ratio of unlabelled glycollate to [2-r4C]-glyoxylate

5.11 and 5.12).

130 5.3.5 CTTNNACTERISATiON OF TIIE EI\IZYT'ßS WHICH SYNrIIESISE OXET¿'TE FROM

GlvcoLlats a¡ro GIYoxYLATE

This study attempts to clariff the roles of glycollate oxidase and lactate dehydrogenase rn oxalate production from glycollate and glyoxylate.

The inabilþ of lactate deþdrogenase to oxidise glycollate to

(yanagawa et al., 1990\. Some investigators also believe that lactate dehydrogenase does

as analysis of the kinetics not play a major role in the conversion of _gþxylate to oxalate, of glyoxylate metabolism by lactate dehydrogenase indicates that the equilibrium lies in favour of glycollate production. Indeed my observations support this conclusion as under physiological conditions at 37"C, pH 7.4 and glyoxylate concentrations expected intracellularly (0.lmmol/L) glyoxylate reduction was 60 to 70o/o faster than glyoxylate oxidation (Figure 5.19) suggesting preferential reduction of glyoxylate by lactate dehydrogenase. However, examination of the Kn,'s for glyoxylate oúdation and reduction demonstrate that lact¿te dehydrogenase has a 2O-fold higher affinity for glyoxylate in the

oxidative direction under physiological conditions. Given that the NAD*/I.IADH ratio in the hepatic cytosol is 725 one would expect preferential oxidation of glyoxylate to

oxalate. However, the hepatic lactate (reduced) to pyruvate (oxidised) ratio is

consistently l0 (Park et al. 'Discussion in Regulation of Hepatic Metabolism', 1973)

suggesting that the NAD* to NADH ratio is not so important for determining the

equilibrium of the lactate dehydrogenase system a¡d that this system may work

preferentially in the reductive direction'

The strong competitive inhibition of glyoxylate oxidation by oxalate and the lack of

inhibition of glyoxylate reduction by glycollate (Section 5.2.2.2.1), support the view that

131 in vivo glyoxylate may be preferentially oxidised to glycollate by lactate dehydrogenase'

The effects of oxalate and glycollate on the oxrdation and reduction of glyoxylate by lactate dehydrogenase observed in this study are in agreement with other investigators

(Warren, 1970; Asker and Davies, 1983). As oxalate is known inhibit rmportant

intracellular oxalate levels need to remain below a cerüain level to maintaut necessary cellular processes. Hence, the strong competitive product inhibition of lactâte -+- dehydrogenase by oxalate suggests that lactate deþdrogenase may play some role in the oxidation of glyorylate to oxalate, with this product inhibition providing a regulatory mechanism to prevent cellular oxalate buildup.

While some investigators believe that lactate deþdrogenase is the major enzyme

responsible (Asker and Davis, 1983; Yanagawù et al., 1990)

others believe that glycollate oxidase is the responsible enzyme (Richardson and Tolbert,

1961; Liao and Richardson,1972; Fry and Richardson, 1979; Bais et al., 1989a and b).

Analysis of the enz.yme kinetic data described in this thesis suggest that glyoxylate is a

relatively poor competitor for the active site of glycollate oúdase as, a) the K.'s for

glyoxylate is l0 fold greater than that for glycollate and b) at physiological substrate

levels the rate of oxidation of glycollate by glycollate oxidase is approximately 23 to 26

fold faster than the rate of glyoxylate oxidation (Figure 5 .26). Thus, if glyoxylate should

happen to occupy the active site its oxidation to oxalate would take considerably longer

than the oxidation of glycollate to glyoxylate. Nevertheless, the observations that, a)

glyoxylate is a free intermediate in the oxidation of glycollate by glycollate oxidase

(Figure 5.15), b) glycollate is a better oxalate precursor than glyoxylate under

physiological conditions (Figure 5.26), c) exogenous unlabelled glyoxylate could not

significantly affect oxalate production from [t-r4C]-glycollate (Figure 5.ll) and d) the

glycollate oxidase suspension could produce oxalate from glycollate without added NAD*

132 (Section 2.4.1'), suggest that oúdation of glycollate by glycollate oúdase produces glyoxylate which can be further oxidised to oxalate by this enzyme.

An in vitro study by Yanagawa et al. tnto the formation of oxalate from glycollate by rat

and human liver, could not demonstrate oxalate production from gþollate in a

(Yanagawa reconstituted enzyme system containing hepatic glycollate oxidase and NAD*

et a1.,1990). However, when hepatic lactate dehydrogenase \4/¿ls added to the system,

is more significant oxalate production from gþollate could be observed. This anomaly

than likely due to the fact that l0mmol/L glycollate was used as the substrate and thus

any glyoxylate produced could not possibly compete for the active site under these

conditions. hese investþators noted that if glycollate oxidase is the enzyme responsible

for the production of oxalate, glyoxylate formed in the peroxisomal matrix would be

oxidised to oxalate in situ without being transported into the cytosol or other organelles.

In terms of the importance of peroúsomal metabolism to oxalate production they also

this noted that deficiency ofthe alanine:glyoxylate aminotransferase,

transamination ot gþine; leads enzyme is responsible for ,thg_4aj_olportlon of gly94yl49

to increased oxalate ln conclusion Yanagawa et al. stztedthat; if one

or assumes an in vivo condition in which the supply of glyoxylate and the met¿bolism

removal of glyoxylate in the peroxisomes are much slower than the activity of glycollate

oxidase, a high steady-state glyoxylate/glycollate ratio would be maintained in the

peroxisomes, allowing glYcollate oúdase to act on late.

Asker et al. have also investigated the kinetics of glycollate oxidase and lactate

1983). dehydrogenase to determine their abilþ to oúdise glyoxylate (Asker and Davies,

They concluded that when glycollate and glyoxylate concentrations are equal,

0.25mmol/L; the ratio of glycollate/glyoxylate oxidation is approximately 250' However,

133 substrate for a they also suggested that as glyoxylate is a highly reactive molecule and a number of transamination reactions, its concentration in the peroxisomes is presumably

ten low. Thus, for the situation when the concentration of glyoxylate, 0'025mmol/L; was fold lower than tlat of glycollate, 0.25mmol/L; the ratio of glycollate/glyoxylate

activities oxidation was estimated to be approximately 2500. However, in this study the of gþollate oxidase and lactate dehydrogenase \üere determined at pH 8'5 and in this

oxidase sense are not physiological comparable. In addition, ttre activity of glycollate

was shown to be pH dependant (Asker and Davies, 1983)'

Although reconstituted enzyme systems can provide useful insights into enzymatic

processes, it should be kept in mind that more often than not the enzymes a¡e studies

under conditions that do not mimic their normal environments, i-.. pFL surrounding

structures and substrate levels. Hence interpretation of the data from these studies should

or be made in conjunction with results obtained using whole animal models and cultured

freshly isolated cells.

t34 Cunpren Sx

CoNcl,uorNc RBtuems

This chapter gives an outline of the significant findings in this thesis, their clinical

p- implications and future directions which research into the regulatory effecß of aminothiols on urinary oxalate and management of calcium oxalate urolithiasis might be undertaken.

6.1 SYNOPSIS A}{D CONCLUSIONS OF THIS TIIESIS

6.1.1 Tm (L)-CYSTEINE-Gryoxyrers Apoucr: SYffiüsIS, ISolqTIo¡t,

CHANACTSRISATION AND METABOLISM

A simple method for synthesis and isolation of the (L)-cysteine-glyoxylate adduct was

developed. This method enabled preparation of different diasteriomeric mixtures of cls-

and trans-adduct, providing pure samples for the determination of the physical properties

of adduct and investigations into its behaviour under physiological conditions. To

confirm the structure and purity of adduct, preparations were characterised by melting

point and pK" determinations, KBr disk infra-red spectroscopy, gas chromatography/mass

spectroscopy and proton NMR spectroscopy. Results from these studies confirmed that

under physiological conditions, adduct has both carboxylic acid groups deprotonated and

the 2" amine group protonated, making it a highly polar molecule. Upon standing cis-

adduct was shown to epimerise to the more thermodynamically stable trans-adduct This

135 epimerisation was studied using enzSrmatic preparations of (D)-aspartate oxidase, proton

NMR spectroscopy and in r¡¡vo studies. The latter studies confirmed, as suggested by the

earlier in vitro studies, that in vivo 5l%-cis-adduct epimerises to the more

o/o-c thermodynamically stable 4 0 is -adduct'

To enable investigations into adduct formation and metabolism, ln vitro and in vivo, frvo

assays for the detection of under various conditions were developed. The first of

these assays was enzymatic using the flavin linked enryme (D)-aspartate oxidase to detect

adduct via coupling of adduct oxidation to ferricyanide. Although, this assay was ideal

for detecting adduct in reconstituted enzryme systems and hepatocyte supernatants it could

not detect adduct in concentrated urine. Hence, a second assay used gas

chromatography/mass spectroscopy in urine. The

GC/I\4S ¿rssay was as sensitive as the enzymatic assay. The enzymatic assay was used

for quantitative studies while the GC^4S assay was used for qualitative studies.

Studies involving i.p. injection of adduct with or without meso-tzrtaic acid, a (D)-

aspartate oxidase inhibitor, confirmed that significant adduct metabolism, most likely via

(D)-aspartate oxidase, occurs in vivo. Although some investigators have suggested that

adduct metabolism in vivo should result in the release of significant quantities of

glyoxylate or oxalate and cysteine, small increases in urinary sulphate and oxalate

excretion were observed. These finding confirm that although adduct is metabolised in

vivo to intermediate compounds, presumably Â2-thiazoline-2,44icarboxylate, N-

oxalylcysteine or S-oxalylcysteine, lþtg qqg=Le4{e- ar-e not catabo-lised-to -oè4lAtg and

sulphate in the Porton rat. It was concluded from these studies, that complete metabolism

136 of endosenouslv formgd adduct (i.e. adduct formed in vivo following administration of B- aminothiols to glycollate induced hyperoxaluric rats) could be expected to occur.

6.1.2 REGULATIoN oF URINARY Oxarere BY B-AMINoTHIoLS

drug, was found Of the B-aminothiols tested OTC, the delivery to be more effective at decreasing urinary oxalate excretion under both normo- and hyperoxaluric conditions (Table 6't). t{owever was found to be

in it was administered f&g[yg in, redugrng_ urine pH É¡q-QTÇ, due to the form which OTC reduced oxalate, (Table 6.1). , Althouglr þoth

compounds !EJ@"" in yrur¡ n! observed__up_ontr,eatment ryqfrrhe-se wæ 4n--equally sipnificant factor in lowering the risk of calcium oxalate urolithiasis Urine pH ßan

important risk factor as the solubility product for calcium oxalate lncreases with

decreasing unne thereby reducing the risk of formation of calcium oxalate

effective as in crystalisation. Althoug4 (L):-I{gt di_{_not appear to be q! -OTC in reducing urine pH r9$crne urinary 9Iu!19: th9= e-r9a1er gqicagY o{ (L)+y-steine for this in terms of the of calcium oxalate stone

formation (Table 6.1)

Based oî in vitro findings and theoretical speculation, some investigators have suggested

that both urinary sulphate and urine pH may be important factors in the regulation of

of OTÇ a,-t¿ (L)-cvsteine urinary oxalate. 4r._."þÞ9t. Ég qujg_ygqfy-!4eteþ:olitq

and administration of these compounds results in aciduria, studies were

performed to determine oxalate were due to

however failed to changes _in urinary-srr_lphate excretion and urine pH. These studies

137 Study Change in Change in Change in Urinary Oxalate Urine pH P"r

Long Term OTC -30% (p<0.05) 4% (N.S.) 30Voto 5%o Normooxaluric

Short Term OTC -11% (p<0.05) -7% (p<0.00r) 3ïo/oio 4Yo Normooxaluric

Short Term OTC 43% (p<0.001) -11% (p<0.001) 9Ùo/oto I2%o

Glycollate Hyperoxaluric

Shon Term (L)-CYsteine -8% (N.S.) -10% (p<0.001) 3Ùo/oto 2%o Normooxalu¡ic

Short Term (L)-Cysteine -31% (p<0.05) -20% (p<0.001) 90o/o to 3lo/o Glycollate Hyperoxaluric

Short Term @)-Penicillamine +74o/o (p<0.05) -8% (p<0.05) 3\o/oto 64Yo Normooxaluric

Table 6.1 The expected effect on the probability of stone formation (P"r) from the changes in urinary oxalate and urine pH observed upon OTC, (L)-cysteine and (D)- penicìlhmine administration. P5¡ wÍts determined using the risk model developed by Robertson and the assumptions described in Chapter 4. demonstrate any significant link between increased urinary sulphate excretion or aciduna

and the observed reduction in urinary oxalate excretion.

To determine whether (L)-cysteine-glyoxylæe adduct formation is the mechanism by

which (L)-cysteine reduces urinary oxalate excretion, OTC and (L)-cysteine were

administered during glycollate induced hyperoxaluria. Although the conditions

favourable for adduct formation, no (L late adduct could be ln

the urine or plasma of the rats in these studies Despite this, formation of the (L)-

cysteine-glyoxylate adduct, is still considered to be the mechanism responsible for the

reduction in urinary oxalate excretion. The fact that no adduct could be found in the

urine or plasma of these rats could be explained by the conclusion made in Chapter 3 that

I low levels of adduct are completely metabolised.

(D)-penicillamite urinary ln contrast to OTC and (L)-cysteine, increased oxalate _

excretion and decreased the plasma achvlty of both alanine and aspartate

aminotransferases. From this latter it

increased endogenous hepatic oxalate of

glyoxylate to glycine, thereby more to be (this

was confirmed in Chapter 5). Hence, (D)-penicillamine was shown to significantly

increase the risk of calcium oxalate urolithiasis (Table 6.1)'

138 6.1.3 MBcrn¡ustvt OF THE REGULAION oF ENDoGENoUs OXerarS PRoDUcIoN

in oxalate ln the first of these studies a significant correlation between the reduction

production from glycollate, in the presence of (L)-cysteine, and formation of the cysteine-

glyoxylate adduct was observed, provirting the first conclusive evidence that adduct

from formation is the mechanism by which (L)-cysteine reduces oxalate production

gþollate. This finding also gave support to the view that glyoxylate is a free

intermediate in the oxidation of glycollate to oxalate by gþollate oxidase. Given tlat

than adduct formation is not steriospecific and that penicillamine is a better nucleophile

reasonable assumed to be the cysteine, B-aminothiol-glyoxylate adduct formation w¿ls

production mechanism by which both (D)-cysteine and (D)-penicillamine reduced oxalate

from glycollate. Despite the fact that (L)-cysteine reduced oxalate production from

gþollate in these studies, OTC did not, although a trend towards reduced oxalate

production was evident. The inability of OTC to significantly reduce oxalate production

in these studies was attributed to the fact that sufficient quantities of (L)-cysteine could

not be released from OTC over the incubation period resulting in liüle adduct formation.

The observation that (D)-penicillamine reduced oxalate production from glycollate ut

(D)- hepatocytes (initial in vito study) was contradictory to the finding that oral

penicillamine administration increases urinary oxalate excretion. This inconsistency was

chronic believed to be due to the differing effects of acute (the initial in vifto study) and

(the in vivo study) (D)-penicillamine exposure. Under conditions of acute (D)-

penicillamine exposure, as achieved in the initial in vitro study, penicillamine-glyoxylate

adduct formation was ass¡med to be responsible for the reduction in oxalate production

(D)- from glycoll To clariff this inconsistency chronic exposure of hepatocytes to

penicillamine was achieved by giving i.p. injections of (D)-penicillamine to rats at 48 and

t39 24 hours prior to hepatocyte isolation. In comparison to control hepatocytes, the

more hepatocytes isolated from (D)-penicillamine pretreated rats produced significantly

chronic (D)- oxalate from glycollate or glyoxylate confirming the in vivo frndtng that

was penicillamine exposure results in increased rates of hepatic oxalate production. It

glyoxylate again postulated that this increase is due to inhibition by (D)-penicillamine of

glyoxylate transaminases resulting in increased glyoxylate availability and increased a) (D)- oxidation to oxalate. This postulate was supported by the observations that, penicillamine reduced the activity of hepatic alanine and a-ketoglutarate

alanine aminotransferases in vitro andb) (D)-penicillamine reduced the plasma activrty of

and aspartate aminotransferases in vivo.

a much úr agreement with the findings of other investigators, glycollate was found to be

the view better oxalate precursor than glyoxylate in isolated rat hepatocytes, supporting

and that two distinct enzymatic pathways exist for oxalate production from gþollate

glyoxylate. The oxygen dependence of oxalate production from glycollate implicated the

peroxisomal flavin-linked oxygendependant erÌzyme, glycollate oxidase, while the lack of

oxygen dependence for oxalate production from glyoxylate implicated the cytosolic

nicotinamideJinked enz1vme,lactate deþdrogenase. Furthermore, as glycollate oxidase is

is the major site solely located within the peroúsome, it was postulated that this organelle

for oxalate production in vivo, as glycollate is the major precursor of glyoxylate and

oxalate in vivo. This conclusion was supported by the findings from isotopic dilution

studies.

The knowledge that B-aminothiol-glyoxylate adduct formation prevents oxalate

production was used to further elucidate the mechanisms of carbon dioxide production

from glycollate in isolated rat hepatocytes. From these studies it was concluded that the

140 adduct majority of carbon dioxide production from glycollate occurs before oxalate or formation as no concomitant reduction in carbon dioúde was observed' Moreover'

implicated as hydrogen peroxide dependant peroúsomal glyoxylate decarboxylation was

glycollate, the pathway responsible for the majority of carbon dioxide production from given the affect of (D)-penicillamine, an avid hydrogen peroxide scavenger' on carbon dioxide production.

at Investigations into the kinetic properties of lactate deþdrogenase revealed that

physiological substrate levels, glyoxylate reduction was 2-fold faster than oxidation'

However lasfate dehydrogenase had a 20-fold greater atrnity for glyoxylate in the

oxidative direction. Nevertheless, it was concluded that the lactate dehydrogenase system

(reduced) to operates preferentially in the reductive direction as, a) the cytosolic lactate

pynrvate (oxidised) ratio is consistently 10 and b) in comparison to glycollate, glyoxylate

was a poor oxalate precursor. Another factor expected to contribute to the preferential

reduction of glyoxylate by lactate dehydrogenase was the strong competitive product

inhibition, K¡ 29.8pmoil/-,, ofglyoxylate oxidation by oxalate. Hence, it was concluded

that lactate dehydrogenase would produce more glycollate than oxalate from glyoxylate.

lnvestigations into the kinetic properties of glycollate oxidase revealed that at

physiological substrate levels glycollate oúdation was 25-fold faster than gþxylate

oxidation. ln addition, glycollate oxidase had a 7-fold greater affinity for glycollate than

for glyoxylate in the oxidation direction. Despite these findings, glycollate oxidase is still

believed to be capable of glyoxylate oxidation, and hence oxalate production, in the

presence of glycollate, a view supported by other investigators. Results presented in thls

thesis supporting this view are as follows: a) glycollate oxidase produced significant

quantities of oxalate from glycollate, b) glyoxylate was found to be an uncompetitive

L4l inhibitor of glycollate oxidation and c) glycollate is a better oxalate precursor than glyoxylate both in vivo andin viffo'

6.2 CLINICAL IMPLICATIONS

The primary aim of this thesis was to investigate the ability of B-aminothiols to decrease

major the risk of calcium oxalate urolithiasis, however during the course of this work two

unrelated findings of clinical significance were made'

6.2.T MANAGEMENT OF CALCIUM OXAI-ATE UROI.TTruASIS

Urolithiasis is one of the most common debilitating disorders of the urinary tract incurring

considerable social and economic cost. As 60 to 80% of renal calculi are composed of

calcium oxalate, prevention of this stone type would lead, not only to substantial

economic savings in terms of hospital admission time, treatnent and specialist attention

but would greatly improve the quality of life of individuals susceptible to this condition.

As urinary oxalate excretion and urine pH are two of the most important risk factor in the

formation of calcium oxalate calculi, the observed reductions in urinary oxalate and urine

pH resulting from administration of (L)-cysteine and OTC in this thesis are of obvious

clinical significance.

Although (L)-cysteine was shown to be effective at reducing urinary oxalate excretion,

and urine pH, its administration in a clinical setting would be inappropriate given the

t42 toúcity that has been shown to be asociated with its administration, both in this thests and by other investigators. Moreover, (L)-cysteine is an inefficient way to elevate intracellular (L)-cysteine levels given its pharmacological properties. OTC on the other hand was shown to have no effect on plasma biochemistry in any of the studies in this thesis. However, caution should be exercised when considering the clinical use of OTC in human subjects as recent studies have shown side effects at doses greater than approximately 0.4 to O.5mmol/kg (Porta et al.,I99I; Kalagian et al',1994\' These side effects include rashes, flusing, pruritus, lightheadedness, diminished ability to concentrate and drowsiness. Nevertheless in human subjects OTC at low doses, i'e. less than

0.2mmol&g, appears to be well tolerated.

6.2.2 PREVENTIoN oF OXALoSIS IN SIJBJECTS WTTH PNNUENY HYPEROXALURI,A

pnmary hyperoxaluria types I and II (PH I and II) are both rare lethal autosomal

recessively inherited inborn errors of metabolism which result in increased endogenous

lWitliams oxalate production (for a review see Watts, 1983 and and Smith, 1983). Most

cases of primary hyperoxaluria are type I and can be diagnosed on the basis of significant

hypergþollic aciduria and hyperoxaluria. Endogneous oxalate production is increased

in the case of PH I due to a deficiency of the hepatic peroxisomal enzyme

alanine:glyoxylate aminotransferase, while in PH II hepatic (D)-glycerate dehydrogenase

is the deficient en4rme. The lack of transamination of glyoxylate to glycine in PH I leads

to a build up of glyoxylate and glycollate and increased glyoxylate oúdation to oxalate.

lncreased rates of endogenous oxalate production can cause calcium oxalate deposits at

sites of high calcium concentration, like the growing ends of bone, the myocardium, the

143 tunica media of muscular arteries and arterioles and most importantly, the renal tissues'

As the condition progresses, renal deposition of calcium oxalate reduces the glomerular

filtration rate resulting in uremi4 oliguria and increased systemic oxalosis' ln general,

and die subjects with primary hyperoxaluria present with symptoms in the first decade

before they are 20 years ofage.

As the major clinical variant of primary hyperoxaluria is due to a deficieny of

alanine:glyoxylate aminotransferase, resulting in increased glyoxylate oxidation to

importance in limiting the oxalate, B-aminothiols like OTC might be of major clinical

greater than effects of the disease. Given that urinary glyoxylate excretion is 3 to 5 times

normal in pH I (Williams and Smith, 1983), a systemic excess of glyoxylate is presumed

to- exist, leading to increased rates of glyoxylate oxidation and the resultant

- adducts with glyoxylate, hyperoxaluria. Hence, B-aminothiols would be expected to form

reducing oxalate production, calcium oxalate deposition and systemic oxalosis.

6.2.3 IATROGENIC HYPEROXALURTA FOI,TOWNqC A¡M'USTNANON OF (D)'

PENICtr,LAI.,,ü.TE: IIIæITCRTIONS FOR SI'BJECTS WTTH WILSON,S DISEASE

Wilson's disease is a rare inborn error of copper met¿bolism in which there is a

deficiency of caeruloplasmin, the protein responsible for copper binding, resulting in

elevated systemic copper levels. TreaÍnent of Wilson's disease is centred on the use of

(D)-penicillamine, an active scavenger of copper and other electrophiles, to reduce the

systemic copper load and increase urinary copper excretion. (D)-penicillamine

administration however, is known to result in vitamin 86 deficieny, thus pyridoxine is

administered simultaneouslY.

r44 shown to ln this thesis chronic (D)-penicillamine exposure both ln vivo and in vitro was

From reduce aminot¡ansferase activity and increase endogenous oxalate production'

oxalate production these observations, it was postulated that (D)-penicillamine increased of by reducing glyoxylate aminotransferase activity, thereby increasing the availability

glyoxylate and its subsequent oxidation to oxalate'

In a clinical setting, concomitant administration of (D)-penicillamine (300mg 4 times

excretion daily) and pyridoxine (300mg/day) has been shown to increase urinary oxalate

(Floppe et al., 1993); in one individual urinary oxalate rose from O'25mmo|day to

0.87mmovday and was O.55mmoVday after 12 months of t¡eatnent. Although the

the authors claimed that vitamin Bo deficiency appears to be an unlikely cause of

plasma transaminase hyBeroxaluria as pyridoxine was administered, they did not measure (D)- levels or other indicators of vitamin Bo homeostasis. Moreover, 4 fold more

penicillamine was administered than pyndoxine, hence vitamin Bo deficiency is likely

given that (D)-penicillamine is such an avid binder of pyridoxine, pyridoxal phosphate

this and vitamin B6 (Schonbeck et a1.,1975; Waner and Nyska, 1991)' Nevertheless,

observation conñrms that (D)-penicillamine administration to human subjects can

significantly increase endogenous oxalate production'

Apart from confirimg our in vivo observations, the finding that (D)-penicillamine

clinical administration to subjects with Wilson's disease increased urinary oxalate is of

prominent finding in significance given that, a) hypercalciuria and calcium urolithiasis are

subjects with Wilson's disease (Floppe et al., lgg3), b) (D)-penicillamine administration

is associated wrth a range of side effects, including fever, rash, lymphoadenopathy,

neutropenia, thrombocytopenia, autoimmune complications and neurological effects

(Menara et al., 1992) and c) there are alternative atoxic therapies available for the

t45 management of Wilson's disease (Menara et al., 1992). Thus (D)-penicillamine treaûnent would be expected to significantly increase the risk of calcium oxalate

u¡olithiasis in subjects with Wilson's disease, further reducing their quality of life and

perhaps even diminishing much needed renal function.

6.3 FUTURE STUDIES

a Development of a method for the extraction and isolation of adduct from urine and

plasma. This procedure would enable reliable quantitative measurement of low levels

of adduct in urine or plasma by the enrymatic (D)-aspartate oxidase assay without

interfering compounds .

a Development of a HPLC assay for the quantitative analysis and identification of

adduct and its metabolites in urine and plasma. This assay would enable quantitative

investigations into adduct metabolism in vivo and in vitro. The main aim of these

investigations would be identification of the major metabolites of adduct and their i¡a

vivo faþ.

a Sufficient evidence was presented in this thesis to warrant clinical trials of OTC and

effectiveness in the other intracellular B-aminothiol deliver drugs to determine their

prevention of calcium oxalate urolithiasis and the life th¡eatening systemic oxalate

observed in subjects with primary hyperoxaluria.

146 hepatic a Studies quantitating the kinetics, activity and intracellular locaction of both

peroxisomal glycollate oxidase and cytosolic lactate dehydrogenase to determine

which of these enz]¡mes is responsible for oxalate production in vivo.

a Studies into the role of gþollate dehydrogenase in oxalate production.

probe the a Studies using specific enzyme inhibitors, both ¡n vivo and in vitro, to

pathways thought to be responsible for a) oxalate production from glyoxylate and

glycollate and b) the diversion of glyoxylate and glycollate from oxalate production.

a Studies involving the isolation of the individual cellular comparünents in which

oxalate production from gþollate and glyoxylate are thought to occur, i'e ' cytosolic,

mitochondrial and peroúsomal compartnents. These studies would provide insights

into how the enzymes in these comparfnents operated and are controlled without the

influence of external factors.

a Further use of the novel reaction between p-aminothiol and glyoxylate to elucidate

ottrer aspects of 2 carbon metabolism

6.4 CONCLUDING STATEMENI

The studies presented in this thesis support the undertaking of clinical trials into the use

of p-aminothiols in preventing calcium oxalate urolithiasis and systemic oxalosis in

subjects with primary hyperoxaluria. The studies also support the view, held by other

r47 investigators, that peroxisomal oxidation of glycollate and glyoxylate, ia gþollate oúdase, is the major pathway responsible for endogenous oxalate production'

is strong Although, conclusive evidence for adduct formation in vivo was not found, there

primary in vitro evidence in support of the hypothesis that adduct formation is the

production and urinary mechanism by which B-aminothiols reduce endogenous oxalate

oxalate excretion as, a) adduct formation was demonstrated in isolated rat hepatocytes

and b) signiñcant adduct metabolism was shown to occur invivo.

l¡ çe¡çluding, the work presented in this thesis provides clear evidence to suggest that B- In aminothiols could be used clinically to regulate endogenous oxalate production.

additior¡ this thesis advances our understanding of the mechanism and

comparfinentalisation of the enzymes responsible for endogenous oxalate production'

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