Screening for tMTHFR, Factor V Leiden and Hyperhomocyst(e)inemia: Emerging Propostic Factors in Myocardial Infarction ?

Jessica Jolly

A thesis submitted in conformity with the requhents for the degree of Master of Science

Graduate Department of Laboratoxy Medicine and Pathobiology University of Toronto

O Copyright by Jessica Jouy 1999

National Library 1+1 of Canada Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliogmphic Services services bibliographiques

395 Wellington Street 395, nie Wellington ûîtawaON K1AON4 OnawaON K1A ON4 Canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

.. u

Screening for tMTHFR, Factor V Leiden and Hyperhomocyst(e)inernia: Emerging Prognostic Factors in Myocardial Infarction ?

A thesis submiued in confonnity with the requirements for the degree of Master of Science

Graduate Depaiunent of Labontory Medicine and Pathobiology University of Toronto

1999

Jessica J o b

Abstract

Hyperhomocyst(e)inernia{HH(e)} is an independent risk factor for coronary anery disease (CAD),

but its prognostic value in patients with eaablished CAû, specifically myocardial infaraion (MI)

has not been defmed. We prospecrively studied the prognostic value of HH(e) among MI patients

enrolled in a pilot study at Sunnybrook Health Science Centre. The consensus Canadian reference

range of H(e) > 1 5.0 pmoVL was adopted in stratifying our patients as hyperhomoqst(e)inernic or

non-hyperhomocyst(e)inemic. An iz h p d composite outcome of death, recunent ischernia or

congestive hem faiiure was recorded in 37/53 patients. There were 22/53 subjects eligible for

thrombolytlc therapy, of whidi 13/22 were successfdy reperfused, 9/22 were not. There was no

association found benveen reperfusion and d m d h é methylenetetrahydrofolate reductase

@ - 0.69) or Factor V Leiden status. Hyperhomocyst(e)inemia appears to be an independent

prognostic indicator in MI. A decreased effectiveness of thrombolytic agents was observed in

hyperhomocyst(e)inemic patients. However, when we compared the proportion of non-

hyperhomocyst(e)inemics and hyperhomocyst(e)inemics that were successfully reperfused, the

difference was not statisticaüy significant (p-0.38). Nevertheless, our study showed that MI

patients having nonnal H(e) levels had m o Urnes an improved chance of successful reperfusion

compared to subjecrs with an elevated H(e) level. Clearly, sample sùe may have been our limiting

factor. We repoit that HH(e) may be predictive of adverse outcomes in MI.

Acknowledgments

As I reflea upon my experiences as a graduate student in the lu t eighteen months, 1 am rerninded

of many who made my days at Sunnybrook producrive a d interesting. Progress was catalyzed by a

multitude of individuals from the Division of Chical Pathology, Division of Cardiology and

Department of Pharmacy.

Dr. Maraano Reis- who believed in me from day one and provided me with a very exciting

project. He has a unique way of blendtig the roles of a friend, father and mentor into one. His

concem for others and ability to command respect resonates in bis charmer.

Dr. Ian Dubé- who provided me with precious feedbadc in both my cornmittee meetings.

Scott Walker- who accommodated me with lab shelter dbeit in the K - w i . basement. Perhaps he

was sheltering others from me ? Scott patientiy reviewed my manuscript and provided invaluable

assistance with the aatistid analyses of data in this thesis. He endured my innumerable questions,

which occasionah/ continued even at rnorning coffee gatherings.

Shirley Law and Danny Lau- Pharmacy QC lab Queen and King. They provided me wah

unconditional support and tolerated my idiosyncrasies, of which, they now know I have many !

Adriana Potichnyj- who intrduced me to the wondehl world of molecdar biology. Her I friendship made life quite interesting ... ... ... ... . .

My beautifid f+- the pillan of my post. They withstood all my mood swings while I was a

hennit in my mdy. Their love and sense of hurnor is what made getting up in the moming worth

lt.

My beloved Robert- we have shared so many of Me's beautiful experiences and have leamt to live

and share more.

Table of Contents

..................................................................................... 1 . 0 OVerview .................................................... 1.1 Cellular Metabolian of Homocysteine

............ 1.2 Initial Assoaarion between Hyperhomocyst(e)inemia and Atherosderosis ........................................... 1.3 Foms and Pathology of the Homocystinurias

.................................................... 1.3.1 Cyaathonine PSymhase Deficiency ..................................... 1.3.2 Methylenetetrahydro folate Reductase Deficiency

.................................................... 1.3.3 Methionine Synthase De fiumcy .................................................................... 1.4 Foms of Homocysteine

............................ 1.5 Evidence for the Homocysteine Theory and Atherogenesis ............................................................... 1.5.1 Studies on Animal Models

.................................................... 1.5.2 Studies of Human Cells and Tissues 1.22 2 S& As- ?&mbqs$e)memra to Pn,- T h ...................

............... 1.6 Podated Pathophysiologic Mechanisms of Hprhomocyst (e)inemia .............................................. 1.6.1 Reahve Cbygen Species as the Pathogen

........................................... 1.6.2 Homocysteine Thiolactone as the Pathogen ................................................... 1.7 Prevalence of Hyperhomocyst (e)inemia

................................................................... 1.7.1 Acquired Detenninants .................................................................... 1.7.2 Genetic Detemiinants

..................................... 1.7.21 M M W F R ~Gm,uyArteryDr'sPase

1.7.3 Role of Gene-Environment Intedons in Instigating Hyperhomocyst(e)inemia . ......................... 1.8 Mild Hjqxrhomocyst(e)inemia and Artend Occlusive Disease

........................ 1.8.1 Mild Hyperhomocyst(e)hernia znd Coronary Awy Disease ....................... 1.8.2 Mild ~rhomocyst(e)Li&a and m e r Occlusive Diseases

..................................................... i . 9 Monitoring Hyperhomocyst(e)inemia ....................................... 1.9.1 Quanutauon of Totd Homocysteine in Plasna

...................................................................................... 2.0 Rationde .................................................................................... 2.1 Hpthesis

........................................ 2.2 Objectives of the Studies Ourlined in this Thesis ...................................................................... 3 . 0 Matends and Methods ............................................................................. 3.1 C h c d Anaiysis ..................................................................................... 3.1.1 SM&

..................................................... 3 . 1 . 2 D g 5 w x w z ~ I m d R k C ~ ............................................................... 3.1.3 RepolasiZn Ho& Chamer

.......................................................................... 3.2 Labontory SN& es ........................................................................... 3.22 Ckmd Ama&w

3.2.2.1 PuMR for an Ideal Method of Quantitation of Plasma H(e) ............ ................... 3.2.2.2 Chmmatographic System. Gndirions and Sepamion

.................................... 3.2.2.3 pH and Physid Inspection of Buffers ....................................................... 3.2.2.4 Method Optimizauon

.......................... 3.2.2.4.1 Twid Sarn~le Prmaration Protocol ....... 3.2.2.5 Selection of an Optimal Analyt~d Column and Solvent System

.......................... 3.2.2.6 Assay Validation: Accuracy and Reproducibili ty ................................................................ 3.2.2.7 Stability Study

3.2.2.7.1 Stabilitv of H(e) in Plasma Stored at -200C and -WC ...... 32.2.7.2 Stabatv of Hlel in Plasma Stored at RT and 4-8ûC. ......... 3.2.2.7.3 Stabilitv of H(e) in Whole B l d Stored at RT and 4.8% ...

............................................. 3.2.2.8 Patient Plasma Sample A d y m 3.2.3 M&u& A d y s ...........................................................................

................................ 3.2.3.1 &T Mutation Anaysis of IklTHFR Gene .............................. 3.2.3.2 G1691A Mutation hdysis of Factor V Gene

.......................................................................................... 4.0 R d t s ................................................... 4.1 Qulntitation of H(e): An+d R e d ts

................................ ........................ 4.1.1 F-aeteriw- ... ............................................................... 4.1.2 HPLC MitWOpmtaation

...................................................... 4.1.2.1 Concentration of TnBP ............................................................. 4.1.2.2 Volume of TnBP

................................................... 4.1.2.3 Tme of TnBP Incubation ............................................... 4.1.2.4 Amount of SBD-F (lmg/rnL)

4.1.2.5 Tempe-rature and T i e with SBD-F Incubation ........................... ............................... 4.1.3 I n ; i p n n s n e z t o f H ( e l s e p C t r a t W n ~ ~ h ~

4.1.3.1 Selection of an Optimum HPLC Anayucal Column ...................... ..................................... 4.1.3.2 Optimizauon of HPLC Solvent System

4.1.4 HPLC Assay V d h m ..................................................................... .......................................................... 4.1.4.1 Accuracy Assessment

4.1.4.2 Reproduability Assessment: W~thin and Berween-D y VuiibiIity ........ 4.1.5 Stab.yS& ...............................................................................

4.1.5.1 Stabdity of H(e) in Plasma Storeci at - 2 K and -WC ..................... ......................... 4.1.5.2 Stability of H(e) in Plasma Stored at RT and 4-SOC

4.1 . 5.4 Stability of H(e) in Whole Blood Stored at RT and 4 . K .................. ................................................................ 4.1.6 P m i e n t ~ ~ A ~ ~

4.2 Trends and Redt s of Biochemic~ Moledar and &cal Analyses of MI Cohon .... .................................................. 4.2.1 F k q a q A g i l R a n g e d ~ ~

......................................................... 4.2.2 MTUm G&-- ...................................................................... 4.23 DamhmofH(e) Leudf

..................... 4.2.4 A.benuerH(e)La$tdàtsGarericandA@B- ........................................... 4.2.4.1 H(e)LwelanddKMFRGenotype ............................................ 4.2.4.2 H(e) Level and Factor V Genotype

................................................... 4.2.4.3a H(e) and RBC Folate Levels ................................................... 4.2.4.3b H(e) and Vitamin Bu Levels

................................... 4.2.4.4 RBC Folate Level and M E E R Genotype

.......................................... 4.2.7 Hfi) L.& md S L I I I I ? J S ~ U I ~ V O P Z - S ~ R e ................................ 4.2.8 IM7Hl% n>rl S ~ c ~ m - S u ~ ~ z l s r f U l R&~SZW.,

....................................................................................... 5.0 Discussion., ............................................................................... 5.1 Andynd Aspects

................................................. 5.2 CLLUcaI, BiocheMd and Mole& Aspects ....................................................................................... 6.0 Condusions

................................................................................ 7.0 Remmendations ......................................................................................... 8.0 References .......................................................................................... 9.0 Appendix

vii List of Tables

........................... Table 1.1 Some Important Deteminam of P h a H(e) Concentrations 17 Table 4.1.3 Major Factors Considercd in Choosing the Optimum HPLC Solvent Sy stem ............ 51 Table 4.1.4a A c m c y (94 Deviation) based on Duplicate +s of Standards Stored at -800C ... 52 Table4.1.4bAc~ncy(%Deviation)basedonI>uplicateAmtysisofQ0Storedat-80~C .......... 52 Table 4.1.4~ Accuracy (% Deviation) based on Duplicate A d y m of Standards Stored at -200C .... 52 Table 4.1 .4d Accuracy (% Deviarion) bved on Dupliate A+s of OG Stored at -2OOC .......... 52 Table 4.1.4e Intra-Day Vviability (CV%) based on Dupliure Analyss of Standards Stored at -800C . 53 Table 4.1.4f h - D a y Variability (eV%) based on Duplicate An*s of QG Stored at -80QC ....... 53 Table 4 .14 Intra-Day Variability (CV%) based on Duplicate A n h s of Standards Stored at -2OcC . 53 Table 4.1.4h h - D a y Variability (CVO!) based on Duplicate Analysis of OG Stored at -200C ....... 53 Table 4.1.4 Inter-Day Variability (CV%) based on hiplicate Analyns of Standards Stored at -80% . 54 Table 4.1.4j Inter-Day Variability (CV%) based on Dupliure Analysis of QG Stored at -80aC ....... 54 Table 4.1.4k Inter-Day Variability (W) based on Duplicate Analysis of Standards Stored at -200C . 54 Table 4.1.41 Inter-Day Variab'iity (W) based on Duplicate Anaysû of OG Stored at -200C ....... 54 Table 4 . lSa Average Change (%) within Any p(e)] in Standvds (stored at -8OOC) Constituting

Each Standard Curve Run During a 3 Month Stability Study .............................. 55 Table 4.1.5 b Average Change (%) wirhli Any m(e)] in Standards (stored at -200C) Consticuthg

Eadi Standd C w e Run DuMg a 3 Month Stability Study .............................. 55 Table4.1.5cFactorshdicativeofStuibrdsandQCsStabilityat-80~C ................................. 56 Table 4 . 1.5d Factors Indicative of Standards and QCs Stabiiity at - 2 0 C ............................... 56 Table 4.i .k Stabilty of H(e) in Plasma Stored at RT within 1 Working Day ........................... 57

..................................... Table 4.1.5f Srability of H(e) in Plasma Stored at +8OC for 1 Week 57 Table 4.1.5g Stabiliy of H(e) in Whole Blood Stored in a 'Standing Staten and on a 'Spin Miun at

RT within 1 Working Day ..................................................................... 58 Table 4.1.5h Stability of H(e) in Whole Blood Stored in a "Standing State" and on a "Spin ML( at

I

Table 4.2.2a Table 4.2.7

Table 4.2.8

~ 8 a C within 1 Working Day .................................................................. 59 rMTHFR Genotype and Gene Frequaicy Disuibution in MI Patient Cohort ........... 61 Disuibution of Successfd and Non-Succeufd Reperfusion Associated wnh W(e) and Non-HH(e) ................................................................................ 69 Distribution of Successfd and Non-Successful Reperfusion Associated with Each tMTHFR Genotype ........................................................................... 69

Coronary anery disease (CAD) is the leading cause of death and disabiliry in industr ikd nations.

Its moa common cause is a reduction in coronary artend blood supply due to atherosclerosis

(AS). Atherosderosis is the progressive build-up of plaque, which consists of a combination of

lipids, smooth muscle cels, inflammatory cells, and extracellular ma&, w i t h the intimai of a

vessel. As the plaque matures, the intimal cap that covers the atherosderotic material has a

potentiai to rupture and gives rise to thrombosis. Acute thrornbosis can then result in unaable

angina, myocardial infaraion, or death. Ahhough much is known about the pathologic process

whereby atherosderotic plaques develop, in many cases the underiymg etiology remains undear.

Certain nsk factors associated with the development of AS are wel defined, including diabetes

mellitus, hypertension, hyperlipidemia, tobacco abuse, and a positive fa& history. However,

these risk factors combined account for ody about 50% of the observed incidence of CAD

(Futterman and Lemberg, 1998). Additionally, these risk factors generally are only associations, and

the exact mechanism by which they may contribute to the development of AS in CAD is not

known. The srudy of lipid metabolism has dominated research into AS etiology for decades,

although now it is widely recognized that a larger number of people with syrnptomatic

atherosclerotic disease have no deteaable evidence of abnormai lipid metabolism. This fact has

been M e r reinforced by the inconstant inadence of AS in No& Amenca. Accordhg to the

National Health and Examination S w e y conducted by the Arnerican Hem Association in 1997,

death rate from MI has fallen by nearly hdf in the last 3 decades in North Amenca, but average

choleaerol levels have declined ody slighdy. Explmations for the fd in incidence of death rate

from rhis syndrome of CAD over the past 30 years indude irnproved medications, aitered life

styles, and a reduction in smoking. However, an alternative explanation is that abnormal lipid

metabolism may not be the only pathologic process in the development of AS and is a glaring

indication of the gaps in our knowledge regarding the etiology of CAD.

New risk factors rhat are emerging in an attempt to establish an etiology in the 509'0 of patients

with CAD that do not have any of the conventional risk factors include the most recendy reported

O&z infections (Bachmaier a al, 1999) and the " homocyst(e)ine theorf'. An elevated level of

total homocysteine {H(e)) in blood denotes hyperhomocyst(e)inemia {HH(e)} and is currendy

2 emerging as the nrongea and moa prevalent risk factor for atherosclerotic vascular disease in the

coronary, cerebral, and peripheral vessels, and for arteriai and venous thromboembolism. The

strong association between HH(e) and increased risk for vascular disease has been based on data

from 80 clinical and epidemiologid midies induding more than 10,000 patients (Refsum et al,

1998). Elevated H(e) confers a graded risk with apparently no threshold and is independent of, but

may enhance the effea of conventional nsk factors.

1.1 Cellular Metabolism of Homocysteine

Homocysteine stands at the intersection of two metabolic pathways: remethylation and

transsulfuration (Figure 1.1, pp.3). The remethylation pathway is favored in aates of excess

methionine, while the transdfwation pathway is favored in states of insufficient methionlie. In

remethylation, homocysteine acquires a methyl group from N-5-methyltetrahydrofolate

(CH3'THF), which is synthesized de novo when a carbon unit is tramferreci from a carbon source,

such as serine or glycine, to tetrahydrofolate 0 producing

methylenetetrahydrofolate(CH2THF), which is mbsequendy reduced by methylenetetrahydrofolate

reductase&lTHFR). Homocysteine is remethylated to methionine by the ubiquitous B,,-dependent

methionlie synthase ( M S ) or by betaine:homocysteine methyltransferase ( B W in the liver and

kidney .

A considerable proportion of methionine is activated by ATP to form S-adenosylmeduonine

(SAM), which serves primdy as a methyl donor (via methyltransferases) to a variety of acceptors,

induding nucleic acids, neurotransrnitters, phospholipids, and hormones. S-adenosylhomocysteine

( S m , the by-product of these merhyl transfer reactions, is hydrolyzed, thus regenerating

homocysteine which then becomes available to nart a new cyde of methyl-group transfer.

In the transsulhtion pathway, homocysteine condenses with serine to form cystathionine in an

Lrever sible r eaction cataiy zed by the py ridoxal-5' -p hosphate (PLP) dependent cy stadiionine p- sy nthase (C BS). Cy stathionine is hy droly zed by a second PLP dependen t enzyme, y -cy stathonase

to fotm cysteine and a-ketobutyrate Excess cysteine is oxidized to taurine and evenmally to

3 inorganic sulfates. Thus, in addition to the synthesis of cysteine, this transsulfuration pathway

effectively catabolizes potentialiy toxic homocysteine, which is not required for me&$ tmsfer.

Methionine - SAM

Homocysteine CBS 1 B'

Cy stathionine

Cycle

1 ---+ SAH

Figure 1.1: Biochemical Pathways in Homocysteine Metabolism

Biochemical pathways in homocysteine metabolism. Abbreviations: CBS, cynathionine P-synihase; CH,THF, methyltetrahydrofolate; CHJHF, methylenetetrahydrofolare; THF, tetrahydro folate; MS, methionine synthase; BHMT, beraine:homocysteine methyltransferase; DMG, dimethylglycine; S A M , S-adeonsyhethionine; SAI-E, S-adenosylhomocysteine.

Source: Jacobsen DW. Homocysteine and vitamins in cardiovascular disease. Clin Chem 441833, 1998.

4 1.2 Initial Association between Hyperhomocyst(e)inemia and Axteriosclerosis

A defiaency or defect in my of the three enzymes requked for the metabolism of homocysteine

cause different forms of homocystinwias (Mudd and Levy, 1978). Hyperhomoqst(e)inernia,

prernature vascular disease and thrombosis mentai retardation, ectopic lem, and skeletal

abnonnalities are all characteristics of any of the three forms of this amhoacidopathy.

Macrovascular and arteriosderotic changes in the f o m of homocystinuria caused most commonly

by a deficiency Li cystathionlie-fi-synthase (CBS) were weii described in the 1933 case record

(case 19471) of the Massachusetts Gened Hospital (Cabot and Painter, 1933) and in subsequent

cases reported sirnultaneously but independently from Northem Ireland (Carson and Neill, 1962)

and from Madison, Wisconsin (Gerritsen, Vau& and Waisman, 1962).

The form of homocystinuna caused by the abnormal enzyme, methionine synthase ( M S ) is much

mer. The first case in the world lirerature (Mudd, Levy and Abeles, 1969) of this form of

homocystùiuria was the key case that led to the discovery of the pathogenic effect of homocysteine

on anery wails (McCully, 1969). Due to its rarity, several years passed before macrovascular and

anenosclerotic changes in a second and third case were reported (Dyan and Ramsey, 1974;

Baumgartner et al, 1979).

The foxm of homocystinuna caused by the abnormal enzyme, 5,I O-methylenetetrahyciro folate

reducrase (MTHFR) was more frequent than the form of homocystiriuria caused by an abnormaiity

of MS. In this case initidy documented in Chicago, damage to the artenes and arteriosclerosis was

found to be WNaliy identical with that found in the other two types of homocystinuria (McCully,

1969). This independent confmation of the association between elevated blood homocyneine,

vascular darnage and arreriosderosis in a third ype of homocysllnuria was considered significanr

by Md=+ who derived that in al1 of the three principle types of inherited homocyainuk,

elevation of blood homocysteine caused artenal darnage and arteriosclerosis regardless of the

particular enzyme that was abnormai.

5 1.3 Fonns and Pathology of the Homocystinurias

13.1 Cystathionine PSynthase Deficiency

Severe hyperhomocyst(e)inemia {HH(e)) is usually the result of homozygous CBS deficiency. The

Liadence of homoygous deficiency is estimated to be approximately 1:200,000 (Fenton and

Rosenberg 1995). Patients develop dassic syndromes of homocystinuria, induding HH(e),

premature vascular disease and thrombosis, mental retudation, ectopic lem, and skeletal

abnomalities. Atherosclerosis and occlusion of major vessels such as myocardial, cerebral, rend

and pulrnonary artenes and veins may occur as early as the fust decade, often with fatal redts

(Mudd and Levy, 1978). To date, 17 mutations in the CBS gene have been reported (Fenton and

Rosenberg, 1995; Kluijtmans a al, 1996). The incidence of heterozygous CBS deficiency is

estimated to be between 0.5 to 1.5% in the generai population m e r , Evrovski and Cole, 1997).

The gene for CBS has been localized to chromosome 21q22.3 and is inherited in an autosomal

recessive fashion. Studies show conflicting data as to whether patients heterozygous for a CBS

mutation are at increased Nk for cardiovascdar or thrombotic disease (Mudd et ai, 1981; Boers et

al, 1985; Clarke et al, 1991; KIuijunans ad, 1996). Hence, the importance of mutations of the CBS

enzyme Li causing basai HH(e) remalis unclear. Possibly, in heteroygotes for CBS deficiency,

homocysteine metabolisrn is irnpaired but this impairment is manifested as HH(e) only after

ingestion of methionine, either done (post-methionine load test) or as a constituent of diet (Rees

and Rodgers, 1993).

Patients with CBS defiuency have elevated methionine levels. There is cIinical evidence that

methionine accumulation is unlikely to be important in the genesis of atherothrombotic lesions.

Several patients with HH(e) and low or nomal methionine levels have had vascular lesions very

similar to those found in patients with homocystkmia and hypermethioninemia (McCuiiy, 1969;

Lmy et al, 1970; Kanwar, Manaligod, and Wong, 1976; Baumgartner etd, 1979).

13.2 Methylai&ettahydrofoIate Reductase Deficiency

The form of homoqsllnuria clused by MïHFR deficiency is more frequent rhan the f o m caused

by MS, but is less frequent than homocystinuria caused by CBS deficiency. Located on

chromosome 1~36.3, eleven rare mutations for the MTHFR gene have been identified from

6 isolated cDNA (Goyette et al, 1994) of which two are common. These mutarions are th&.

Homozygous deficiency of themmtatje M l W R occurs in the general population at a rate of about

one-tenth that of CBS deficiency (Rees and Rodgers, 1993). Patients wiwirh homoygous deficiency

have no enymatic activity and develop HH(e) and a dinical syndrome consisting of neurologic

dysfunaion, psychomotor retardation, seizures, and ~enpheral neuropathy. Postmortem

examinations of homozygous patients have documented a high incidence (70%) of arterial and

venous thrombosis (Rees and Rodgers, 1993). Hererozygous deficiency of themnaabk MïHFR

renilrs in about 50% of normal enzyme activity thar appears to be adequate to protect against

neurologic deficirs. Unlike CBS deficiency, HH(e) is ununid in heterozygotes for th-

MTHFR deficiency.

1.3.4 Methionine Synthase Deficiency

The third ype of genetic defect in causing homocystinuria is deficiency of MS, which is quite rare.

There are no current estimates of the frequency of this defecrive gene in populations. The

pathology of this fonn of homocystinuria has not been documented but is suspected to be similar

to the pathology of homocystinuria caused by hlTHFR deficiency .

Due to similarities in the pathological manifestations of Marfan syndrome and homocystinuria,

homocystinuria has cornrnonly been misdiagnosed since it was first reported (Carson and Neill,

1962; Gemtsen, Vaugh and Vraisman, 1962). However, Carson et al hst captured recognition of

homocystinuria in patients with presumed Marfan syndrome only in 1965. Although ocular,

skeletal, and vascular changes in the homocystinurias simulate those of the Marfan syndrome,

osteoporosis and thrombosis are the distinguishhg features of homocystinuria Furchermore,

mental retardation and cutaneous flushing are found in homocystinuria but not in rhe Marfan

syndrome.

7 1.4 Forms of homocysteine

Vincent DuVigneaud coined the words "homocysteine" and "homocystine" in 1932. He

designated homy'iterjze the reduced (sulfhydryl) form and hmqstzw the oxidued (ciidfide) form of

the homologues cysteine and cystine. Nomai hurnan plasma contains a total concentration below

16 gM of homocysteine-denved moieties, in either sulfhydiyl or disulfide form (Mudd and Levy,

1995). Of these, ody about 2 percent occur as the sulfhydryl; the remaining 98 percent are

didfides i.e., homocystine itself or mixed disulfides combined with either free or protein cysteine

(ivlansoor et al, 1992). The composite of homocysteine-derived moieties existing in 2à0 should

concisely be descnbed by the tenn "homocyst(e)ine".

The distinction between this total quantity, homocyst(e)ine and homocysteine itself is of more than

triMd importance because many of the pathophysiologic effects of homocyst(e)ine depend on the

presence of the sulfhydryl group of homoqsteine (Mudd, Levy and Skovby, 1995). Plasma

homocysteine does not always account for the same fraction of plasma homocyst(e)ine. (Mudd and

Levy, 19%). In ~ b j e c t s with abnormally h t e d concentrations of plasma homocyst (e)ine, nich

as occur in the homocystinurias, the plasma homocysteine concentration increases exponentiaily as

a function of the homocyst(e)ine concentration, remaining as low as 2 to 5 percent untd the

homocyst(e)ine concentration exceeds 100 and reaching 10 to 25 percent at homocyst(e)ine

concentrations of 150 to 400 pM (Mudd and Levy, 1995). After methionine loading, the

concentration of plasma homocysteine reaches a peak severd hours before that of plasma

homocyst(e)ine (Mansoor et al, 1992). As efforts are made to determine the pathophysiology of

HH(e), ir is important to be specific about which forrns are elevated and to what extent.

Hwsteine (reduced form), bmylrtine (orridized form) of the homologues cysteine and cystine and

mixed disulfides involving homocysteine, homocysteine rhiolactone, free homocysteine, and

protein-bound homocysteine will colectively be referred to as homocyst(e)ine. Protein-bound (i.e.,

disulfide-linked) homocysteine accounts for 70 to 80 percent of the total pool (Ueiand, 1995).

Note: Since it is not known as to which fonn of homocysteine was measured in some of the carlier studies, occasionaiiy, when âiscussing these studies, hornocysteine wiU not be abbreviated.

8 1.5 Evidence for the Homocysteine Theory and Atherogenesis

The potentid atherogenic propehes of H(e) were evaluated by both i n h and in lao experiments.

Metabolic defects assumed to cause HH(e) were induced either directly or indirectly in the

experimentd system.

1.5.1 Studies on Animal Models

The fira in yaD experiment was done by subcutaneous injection of homocysteine in a dilute

solution of glucose and water into abbits (McCuiiy and Ragsdale, 1970). The thiolactone fonn of

homocysteine was injected because it is stable in solution and readdy converted to homocysteine

by normal enzymes of plasma and tissue. These fkt red ts showed early ~eriosclerotic plaques to

be found in the coronary arteries of yearling rabbits afrer only three weeks of twice-da* injections

of homoqsteine thiolactone. When young weanling rabbits were injected dady for five weeks, early

artenosclerotic plaques were found Li the coronary anenes, aom and arteries of the other organs.

If the animais were fed cholesterol and also injected with hornocysteine, the arteriosclerotic

plaques were found to contain fat deposits. If the animals were given a diet rhat was deficient in

vitarnin B6 and also injected with homocysteine, the plaques became more prominent and more

widespread. For the first t h e , arteriosclerosis had been produced by injection of an amino-acid,

reproducing many of the features of artenosderotic plaques found in children with

homocystinuria, both in rabbits fed choleaerol and in rabbits given a vitarnin B6-deficient diet.

The original experirnent with rabbits (McCdy and Ragsdale, 1970) was repeated with a fivefold

increase in dosage of homocysteine thiolactone in an attempt to overload the capaciry of the

rabbits' tissues to eliminate the amino acid (McCdy and Wilson, 1975). Afrer one month of

injections with high doses of homocyneine rhiolactone, several rabbits had died. Thrombosis in

veins of the legs and abdomen were found. Subsequent embolism caused bleedlig and necrotic

areas in the lungs. In animais given injections of vitamin B, as well as homocysteine, no evidence

of thrombosis was observed and the animals survived

Given the artificial nature of these experiments, more d e s followed whereby different groups of

rabbits had various foms of homocysteine incorporated into there dia (Md* and W&on,

9 1975; Kuzuya and Yoshimùie, 1978). Also, experirnenu that induced vitamli B, defiaency in

monkeys (Rmehart and Greenberg, 1956) were repeated (Kuzuya and Yoshimine, 1978), obsening

arterioxlerosis after prolonged periods of partiai vitamin deficiency. Vitamin B, therapy was

shown to reverse and regress arteriosclerosis. Not ody was vascuiar disease associated with

homocystinuria recreated, but the complications of thrombosis and pulmonaiy embolism were aiso

produced. Early midies showed that hyperhomoqrstinemia maintained in baboons over three

months produced susrallied endothelid cell loss in direct proportion to the level of plasma

homocystine when the concentration exceeded about 60 umol/L (Harker et al, 1976). In the last

15 years, in zRu, d e s using other animai models, including baboons (Harker et al, 1976), rats

(Hladovec, 1979) and mliipigs (Rolland et al, 1995; Srnolin et al, 1983) have all showed that

atwnosderosis and thrombosis are related to elevated blood homocysteine levels.

1.5.2 Studies of Human Celis and Tissues

One of the earliest atternpu to eluudate potential mechanisms by whidi elevated blood levels of

H(e) cause darnage and arteriosclerotic changes in the artenes involved growlig in in v h cukture,

ceils from the skin of children with homocystinuria. Since the CBS enzyme deficiency in

homocyninuria involves a genetic defect in all cells of the body, the cultured celis were dso found

to be deficient in this enzyme (McCully, 1970). McCdy found that cells cultured from two

unrelated CBS deficient individuals, synthesized abnormal proteoglycans that were granular,

aggregated and fiocculent. This abnormality renilred in marked distortion of the normal fibrillar

structure of proteoglycans. Furthemore, addition of homoqsteine to nomai ceii cultures caused

destruction of proteogiycan structure. McCully interpreted these r e d t s to indicate that elevated

endogenous or exogenous homocysteine levels produced pathological changes in the arteries and

other connecrive tissues by altering the state of aggregation and normal fibrillar structure of

proteoglycan molecules, consequently reducing its solubility. This effect was similar to the

observation that the aorta of cMdren with homocystinuria contained an extracellular mavix

component of reduced solubdity (Carson d, 1965).

Another feanire observecl in inzibo culture of cells from the skin of children with homocyainuria

was the distinctive pattern of growrh, which resembled that of cancer celI growth in culture. In the

10 homocystinUnas, smooth muscle cells of arteries grow in a s d a r pattern in early arteriosclerotic

plaques. It is not dear whether homocysteine causes smooth muscle cell proliferation directly or

indirectly. Since the injured endotheliurn produces growth factors that act on neighboring smooth

muscle cells to prornote their proliferation, previous studies of homocysteine-induced

atherosderosis have focused on the effect of homocyaeine on endothelial cells (Duciman et al,

1991~; Rodgen and Con., 1990; Lentz and Sadler, 1993; Hajjar, 1993; Stamler et al, 1993). In some

way, abnormal homocysteine production induces cells to lose control of growth processes, causing

growth of smooth muxle cells. Recent experiments have shown that homoqsteine damages

cultured endothelial cells and inaeases the growth of smooth muscle cells (Tsai etal, 1994). Tsai et

ai found chat although homocysteine alone induces quiescent rat aortic smoorh muscle cells

(MSMC) to re-enter the cell cycle and proliferate, hornocysteine also interacts with serurn (ferai

calf) in a vergistic manner to promote the proliferation of RASMC. Therefore, they concluded

that homocysteine likely interacts with other growth factors or cytokines present in atherosclerotic

lesions to promote the growth of smooth musde cells durlig atherogenesis. These diverse studies

with cells and tissues attempt to describe the pathogenic processes by which a build-up of

homocysteine in plasma, cells and tissues lead to aireria damage and anenosclerotic plaques.

1.5.2.1 Studies Associatirtg Hyperbomocy st(e)inemia to Prothrom botic Tendencies

The vascular mechanisms by which homocysteine induces a state of hypercoagulability are being

increasingly investigated. Harker et al (1976) showed that baboons continuously infused with

homocysteine for three months exhibited increased platelet retention to glass bead columns. This

suggested an association between homocyninuria and platelets aggregation. Later experiments

showed that of the several various chernical forms of homocysteine, only the free base of

homocysteine thiolactone caused primary platelet aggregation. Polar salts of homocysteine

thiolactone, homocystine, homocysteine, and homocysteic acid were inactive, while N-

homocysteine thiolactonyl retinamide and trans retinoic acid caused aggregation only at very hi&

concentrations. @IcCuUy and Carvaho, 1987). Studies on other disorders with a hi& inadence of

thrombotic complications suggest that normai hemostasis depends in part on the balance between

proaggregatory and prothrombotic platelet thromboxane (TXBJ and vascular prostacyclin (PGIJ,

which inhibits platelet aggregation and is thus antithrombotic (Moncada and Vane, 1979).

11 Therefore, an iiz lnbo study of the effens of homocysteine and related compounds (cystuie,

cysteine, or methionine) on platelet TXBz and vascuiv PGI, formation was undertaken by Graeber

ad (1982). They found that in the presence of 1mM homocystine or homocysteine, abnormalities

in platelet arachidonic acid metaboliun expeaed to favor thrombosis did occur. An increase in

platelet proaggregatory TXB,, induced by homocysteine or homocystine in the absence of

compensatory increase in antithrombotic tendency was observed. Graeber et al conduded that

these fmdings were compatible with the prothrombotic tendency seen in hurnan

hyperhomocystinemia. M i ~ o a al (1993) further reinforced these finduigs by observing

abnormally high urinas, excretion of II-dehydro-TXB,, a major enzymatic derivative of

thromboxane QXAJ in dl homocyainuric patients compared to controls. They concluded this

observation to likely reflect, at leas Li part, inziu, platelet activation, a finding also compatible with

the prothrombotic tendency seen in human hyperhomocystinemia.

The procoagulant protein, Factor V was the fîrst vascular endotheliai cell procoaguiant propelt). to

be identified as a potentid contributor to thrombosis in homocyninuria by Rodgers and Kane

(1986). They demonstrated that cultured bovine aortic and human umbilicai vein endothelid

(I-lUVE) cells exhibited enhanced Factor V activicy and increased prothrombin activation aker

treatment with homocysteine. Although their data did not address other potentid endothelid ce11

hemostatic mechanisms that might be affected by expowe to homocysteine, such as tissue factor

expression, Rodger and Kane showed that homocysteine-induced Factor V activity resulted from

activation of Factor V by an endothelial celi aaimtor. In another study, homocysteine-treated

artend and venous endothelial cells were shown to reduce protein C activation (Rodgers and

Corn, 1990). Investigation of the mechuiism(s) by which homocysteine reduced protein C

activation indicated that the metabolite did not induce an inhibitor to activated protein C, but in

low concentrations acted as a competitive inhibitor to thrombin. This data suggested to Rodgers

and Conn that perturbation of the vacular endothelial ce11 protein C mechanism by homocysteine

may contribute to the thrombotic tendency seen in patients with elevated levels of the metabolite.

In a recent study, homocysteine w u shown to initiate coagulation by the tissue factor

pathway through a mechanism involvhg the free thiol group of the amLio acid (Fryer et d, 1993).

The inability of methionine (with irs methylated sulfur group) and the marked ability of P-

12 mercaptoethanol and other potent reducing agents to stimulate TF activity were consistent with

the important role for the free thiol. However, although cysteine has a free thiol group, Fryer et al

showed it to induce less TF activity than homocysteine. Hence, they may not have captured other

additional requirements important for the induction of procoagulant aaivity.

1.6 Postulated Pathophysiologic Mechanisms of Hyperhornocyst(e)inemia

ANiough the exact mechanism(s) by which homocyst(e)ine adversely effens the macrovasculawe

is under intense investigation, there is growing evidence that H(e) exerts its effects by promoting

oxidative damage (Welch and L o s c h , 1998). However, another interesring pathophysiologic

mechanism involves homocysteine thiolactone as the primary initiator of arteriosclerosis, causing

oxidative modification of LDL (McCully, 1996). Regardless of the prirnary pathogen, an oxidative

mechanism is podated.

1.6.1 Reactive Oxygen Species as the Pathogen

Homocyst(e)ine is rapidly auto-oxidized when added to plasma, forming homocystine, mixed

didfides, and homocysceine thiolactone (Figure 1.2, pp.13). Potent reactive oxygen species (ROS),

induding superoxide and hydrogen peroxide (H20J, are produced during the auto-oxidation of

homocyst(e)lie, and H202 (dong with the hydroxyl radical), in partidar, has been implicated in

the vascular to~icicy of HH(e) (Welch, Upchurch and L o s c h , 1997). There is extensive evidence

thar homocyst(e)ine-liduced endothelid-ce1 injury indm is largely due to the generation of HQ,

(WaH et al, 1980; de Groot a al, 1983; Starkebaum and Harlan, 1986). It has been proposed that

homocyst(e)ine-induced endothelid-cell injus, mediated by H202 exposes the underlying matrix

and smooth-muscle ceus, which in turn proliferate and promote the activation of platelets and

leukocytes (Harker a al, 1974).

Auto-oxidation of homocya(e)ine produces other cytotoxic RQS, induchg the superoxide anion

radical and hydroxyl radcd (Msra, 1974; Rowley and HaiLweil, 1982). Superoxide-dependent

formation of the hydrox/l d c a l has been shown to initiate lipid peroxidation Rowley and

Halliwell, 1982), an effect that occurs at the level of the endothelial cell plasma membrane and

13 withùi lipoprotein partides (Heinecke, 1988; Loscalzo, 1996). Homocyst(e)ine auto-oxidation has

been shown to support the oxidation of low-density lipoprotein through the generation of the

superoxide anion radical (Heinecke, 1988; Heinecke et al, 1987). Oxidative modification of LDL

promotes the formation of foam cells, which in tum yield another source of ROS.

Figure 1.2: Podated Adverse Vascular Effects of Homocyst(e)ine Implicating Reactive Oxygen Species as the Pathogen

The posdated effens involve oxidative darnage to vasdar endotheliai ceus and increased proliferation of vascular smooth muscle ceiis after oxidative metabolism of homocysteine to homocystine and homocysteine thiolactone. Orridative modification of LDL promotes the fom&on of foam cells, which in tum yields another source of ROS.

Source: Gamer DA. Homocysteine vs cholesterol. Lab Med 29: 410,1998.

14 1.62 Homocysteine Thiolactone as the Pathogen

Another p o d a t e d mole& mechanism by which homocyst(e)ine causes endothelid dysfunction

is through direct damage to the vascular matrix, whereby biochernical and biosynthetic hct ions

of the vascular ceils are affeaed. Due to dietary, genetic, toxic or hormonal factors, decreased

remethylation or transdfuration of homocysteine leads to overproduction of homocysteine

thiolactone from methionine. Vitamin Btl and folate activate remethylation of homocysteine to

methonine. Vitamin B, activates irreversible uuissulfuration of homocysteine to cyneine and its

u r i n q metabolites. Excess homocysteine thiolactone, the higldy anhydrous byproduct of

homocysteine oxidation combines with LDL to from aggregates that are transporred in blood,

taken up by vascular macrophages of merid intima and incorporated into foam cels (Figure 1.3,

pp.15) widiLi nascent atheromatous plaques (Naniszewia etal, 1994). Foam cells release lipids and

cholesterol into fibrolipid plaques. Homocysteine thiolactone is also reieased from foam cells and

has been suggested to facilitate the conversion of rnitochondnd thiorethaco ozonide to thioco,

thereby promoting the proliferation and fibrosis of smooth musdes (MvlcCdy, 1971; Md*,

1994a; McC~dy,1994b). Consequentiy, rhis homocysteine thiolactone induced dimirbance leads to

overproduction of oxygen radicals, causes intimal damage, oxidizes thioretinamide to sulfate of

glycosaminoglycans, activates elastase, inates thrombogenesis, and increases calcium deposition,

forming the pathologicd features of artenosclerotic plaques (McCuüy, 1994~; McCuiiy, 1971).

1.7 Prevalence of Hyperhomocyst(e)inemia

Although severe HH(e) is rare, mild HH(e) o c m in approximately 5 to 7 percent of the general

population (We1di and L o s c h , 1998; McCuUy, 1996; Ueland and Refsurn, 1989). Reported

median or mean values from different laboratones Vary nearly twofold depending on sarnple

strategy, rnauix, analyucal methodology or the selection of subjects who are under the influence of

various factors that affect the concentration of p lasdsen im H(e) levels (Ueland et al, 1993).

Therefore, the quality of publihed d e s is dependent on the care taken in selection of cases and

controls. Currently, no aandardized reference range of H(e) levels has been ascetiaîned. However,

the consensus reference range in North Amenca appears to be 5- 15 pmoVL, while in Europe, the

Homocysttlne thf ohctont - + D L - W c y T aggregates

Figure 1.3: Postulated Adverse Vasculn Effeas of Homocyst(e)ine Implicating Homocysteine Thiolactone as the Pathogen

Postulated pathogenesis of arteriosclerosis. Homocyn(e)ine causes endothelid dysfunction through direct damage to the vascular ma&, thereby affecthg the biochemid and biosynthetic funaions of the vascular tells. Abbreviations: HcyT, homocysteine thiolactone; PAPS, phosphoadenosine phosphonilfate; GAG, glycosamuiogbrcans.

Source McCdy KS. Homocysteine and vascular dleare. Nat Med 2: 386,1996.

16 cut-off point is 5 12.07 pmol/L. Epideaiiological studies mon& suggea a graded response rather

than a threshold effect (Boushey et al, 1995). However, for practical reasons, m ( e ) is currendy

categorized as mild (16-24 pmol/L), moderate (25-100 pmoVL), or severe (> ICû pmoYL) in

North Arnerica In unselected populations, fasting H(e) values are not norrndy distrbuted but

show an upward or positive skew (Mmer, Cole, Stewart, 1996). This skew is consistent with the

presence of one or more sub-populations with elevated plasma H(e) levels (Figure 1.4).

Determinants responsible for the distribution of plasma H(e) levels in sub-population(s) are

complex and involve genetic, physiologic, acquired, pathologie or medicative factors. A lia of these

c h c a l factors is given in Table 1.1 (pp.17). What follows is a discussion of the moa relevant

acquired and genetic variables affecthg homocysteine metabolism. These variables have been

subjected to rigorous investigation by numerous researchers in the last decade.

H W n a m l r 13-25Irmd/L

Figure 1 A: Total Homocysteine Concentrations in the Circulation '

Shown here are the approlriamate reference intervals for free (reduced) homocysteine and total homocysteine, against a logarithmic d e . Also given are approximate reference intervals for H(e) in several group of disorders, including classical homocystinuriia, due to inheriteâ CBS activity and CO-factor deficiencies. The relatively n m w range of physiological HH(e) in indicated by the shaded bar.

Source: Miner SES, Evrovski J, and Cde EC. Chical chemistry and molecular biology of homocysteine metabolism: An update. CLin Biochem 30: 189, 1997.

Genetic:

Cysrathionine Bsynthase (CBS): heterozygote prevalence: 0.5- 1.5% Methionine synthase (MS) : rare MTHFR MiHFR heterozygosity approximateiy 50%

Pby siologic:

Age: H(e) increases with age Sex: pre- and postmenopausai wornen bave lower levels than men Diet: related to methionine, folate and vitamin CO-factor @6 and BIS

VitunLi defiùency: increased H(e) levels Rend disease: increased correlated with increashg semm creatinine Transplantation: Licreased Postnroke: transiently decreased Severe psoriasis: elevated

Medication s:

Oral conuaceptives/honnone replacement: decreased Corticosteroids: increased Cyclospo~e: increased Smoking: increased Methotrexate: increased Phenytoin: increased Carbemazapine: Licreased (possibly, but not cemin) Penicillarnine: decreased

Table 1.1: Some Important DetermLiants of Plasma Homocyst(e)ine Concentrations

This table outlines many of the determinants that are responsible for the distribution of plasma H(e) levels in unseleaed populations.

Source: Refsum H and Udand PM. &cal significance of phannacologicd moduiation of homocysteine metabolism. TIPS Review 1 l:4ll, 1990; Miner SES, Evrovski J, and Cole DEC. &d chemisuy and molecular biology of homocysteine metabolism. An update. Clin Biochem 30: 189, 1997.

18 1.7.1 Acquired D e t d a n t s

The coenzymes for CBS and MS and the produa of MTHFR (CH,THF) are dependent on dietary

consumption of vitamin B, , vitamin B,, and folate respectively. Experiments with a-ds (as

previously discussed) and human volunteers have shown that deficiencies of these rnicronutrients

cause HH(e) and, in the case of vitamin B, defiaency, arteriosderotic plaques in monkqrs. Since

homocysteine is derived metabolically ody from the essential amino acid methionine, diets

containing abundant methionine require sufficient levels of vitamin B, , Blz and folate to prevent

HH(e). Accordingly, populations with abundant dietary methionine from animal protein, coupled

with nutritional deficiencies of these vitamlis, are at increased risk of arteriosclerosis.

Nuvirional disorders that porentially lead to HH(e) are deficiencies of vitamin B,, , folate and

vitamin B,. As can be observed from Figure 1.1 (pp.3), the d e m synthesis of methionine methyl

groups requires both vitamin B,, and folate cofacton, whereas the synthesis of cystathionine

requires PLP (vira- BJ. While deficiencies of vitamin B,, and folate have generally been

demonstrated to be associated with increased plasma H(e) concentration, both Li humans and

animals, the relationship of homocysteine concentrations to vitamin B, status, however has been

less obvious (Jacobsen, 1998; Graham ai, 1997). A possible reason for this is that two rather

distinct foms of HH(e) have been hypothesized: fasthg and post-methionine loading (PML)

(Seihub and Miller, 1992). Animal experiments (Mder et al, 1 994), hurnan observations (Brattstrom

a al, 1990) and recent population-based data (Bostom B al, 1995) suppon this hypothesis.

Recently, the European Concerted Action Project (COMAC) found that approxhately 3ooh of

persons with HH(e) had isolated PML HH(e) with nomai fasting plasma H(e) levels, providing

further confirmation of this hypothesis (Graham a al, 1997). Pon methionine-loading test is a

cornparison between plasma H(e) concentrations before and after ingestion of a single oral dose of

methionine (IOOmg/kg-body weight). The large bolus of methionine leads to increased

homocysteine synthesis and consequendy tests the capacity of the homocysteine catabolic system.

Therefore, while basal elevated plasma H(e) levels are indicative of an acquired or genetic

Lnpaiment ro the remethyhon pathway, irdividuals with a ciysfwiaion in their transdfuration

pathway, eexhibit a larger and more sustaining increase in plasma H(e) concentrations following

PML.

In view of the very low prevdence of heterozygous CBS defiaency, esrimated to be between 0.5-

1.5% of the general population @Amer, Evrovski and Cole, 1997), and conflicring data as to

whether patients heterozygous for any of the 17 CBS mutations are at increased risk for

cardiovascular or thrombotic disease (Boers a al, 1985; Clarke et al, 199 1; Kluijtrnans et al, 1996),

much attention has been devoted to MTHFR deficiency, in the last decade.

Methylenetetrahydrofolate reductase is a regulating enzyme in folate dependent homocyneine

remethylation (Figure 1.1, pp.3); it catalyvs the reduaion of 5,lO-methylenetetrahydrofolace to 5-

methy1tetrahydrofolate. It is not the hmsiahk form of MTHFR, but the therm$lbile variant of

MTHFR that has received much recognition.

Thermoaability of enzymes reflects one of the moa fundamental characteristics of protein

structure. Many conditional mutant enymes in hurnans or microorganisms have been idenùfied

by vime of theii thermostability. Since it is well-known that enzyme activities in cultured cells are

highly variable (Kang et d, 1988a), moderate activity defiaency of some enzymes is demonsrable

most effectively by their instability at increased temperature. Evidence for allelic variation in

MTHFR deficiency was first niggested by thermostability studies of this enyme in 1977

(Rosenblatt and Erbe, 1977). However, it was a decade later that a "newly deteaed" variant of

MTHFR enzyme was discovered in two unrelated patients who had subnomal folate (Kang et al,

19884. Decreased MTEER activity ( ~ 5 0 % of the normal mean) was observed in lymphocyte

extracts obtained from both patients. However, Kang etal were unable to demonstrate a consistent

decrease of MïHFR aaiviy in cultured skin fibroblasts or lymphoblasts from the patients. It is

well known that enzyme activities in cultured cells are hi& variable (Fabbro et al, 1987). In

fibroblast dtures, ceil density and growth rate have a profound effect on MTHFR activity

(Rosenblatt and Erbe, 1977). In addition, enzyme activities are variable, dependhg on the growth

phase (Rosenblatt and Erbe, 1977). Therefore, according to Kang e ta l (19884, the observations

made on MTWR amivîties in culnued ceh from controls and from the patients were not

unexpected; they demonstrated the need, when cells in culture are studied, for biochemical

characterization of the enqme to define a mutam. Consequentiy, heat inactivation studies

provided svong evidence to suppoit the view that MTHFR in the two patients was a mutant

20 protein and that the alteration of the enzyme was not due to environmental factor(s) (Kang et al,

1988b). Kang et al observed thar specific enzyme a&+ after heat veaunent showed a àistlnct

difference bemeen the patients and the controls regardless of the source of enzyrne preparation.

Although both patients had sub-optimal serum cyanocobalamin, normal uptake and distribution of

~~anocobalarnin as well as normal methionine synthase activity in these patients exduded the

possibility of abnomal cyanocobalamin metabolism (Kang et al, 1988 b) . Thus, it was eaablished

that this "newly detected" variant had a specific MTHFR activity <50°/t of the normal mean and

possessed decreased thermolability after heat inactivation at 46OC (Kang etuf, 1988b, 1988a).

It is important ro reaiize that akhough the specific activicy of t M M m is <SûOh of the

normal mean, the mutation in this variant is distinct and different from the mutation that causes

severe h4THFR deficiency. In other words, subjects heterozygous for the t m M'IHFR

mutation are not heteroygotes for severe MTHFR deficiency ; the enzyme in heterozygotes for

severe MTHFR deficiency is thermostable.

The major dinicai feanves of severe MTHFR deficiency comprise moderate to profound

neurological abnormalities, mental retardation, and premature vascular disease (Kang et al, 1991).

Biochernical abnormalities include homocystinuria, HH(e), and sometirnes hypomethioninemia.

Methylenetetrahydrofolate activity of cultured fibroblasts from these patients varies from 6% to

12% of the normal mean (Kang a al, 1991). The severity of the clinical and biochemical

abnomalities appear to be correlated with the degree of enzyme deficiency. In contrast, these

clinicd and biochemical feanues are lacking in subjects with the thsmdabile variant of MTHFRt

except that increased plasma H(e) is observed in some cases (Kang etal, 1991).

On the basis of biochemical phenocype evaluation of IO subjeas with tM'IHFR and their f a d y

members, in 1991, Kang et al concluded that thermolability of MTHFR is inherited as an

autosomd recessive trait. In 1994, Goyette et al repoited the isolation of the human cDNA for

MTHFR and the assignrnent of the gene to chromosome 1~36.3. Genetic andysis in severe

MTHFR defiaency revealed nine mutations (Goyette et al, 1994, 1995). Recendy, this group

observed a common 677CJT transition in the MITER coding sequence, which changed a hi&

21 conserved alanine into a valine residue (A& (Frosst et al, 1995). This mutation has an dele

frequency of 0.35 (Wilcken etal, 1996; Schmitz ed, 1996) and introduces a novel HznFI restriction

endonudease site, thereby establishg a diagnostic test for evaluation of MTHFR thenalzbiq in

HH(e). Individuals homozygous for the mutation showed reduced specific MTHFR acitiviry,

inaeased thermolability, and elevated H(e) concentrations (Frosa et al, 1995). Eschd&z di

expression d e s with a mutagenized MTHFR dlNA demonstrated that this mutation was

responsible for the thermolabile phenotype (Frosst etd, 1995). To date, the 677C+T mutation is

the only one that has been found to render the MTWR enzyme t-. Most recently, a

second cornmon mutation in the same gene, the 1298A+C mutation, which changes a glutamate

into an alanine residue has been reported (van der Put et al, 1998). This mutation destroys an

M b U recognition site and has an allele frequency of 0.33. Though this mutation results in

decreased h4THFR activity, which is more pronounced in the homozygous than heterozygais

state, this mutation in the MIHFR gene does not r e d t in thermolability of the enzyme.

Frequency of themhhk MTHFR (thATEEX) may vary among ethnic groups; however,

approximately 10% of urban Canadian populations are homoygous for this variant, whiie nearly

50% cany at lean one copy of the gene m e r , Evrovski and Cole, 1997). In an unselected Italian

population, 15.1Y0 were found to be homozygous for this same variant, while 29% of those with

evidence for occlusive vascular disease were found to be homozygous (de Franchis R et ai, 1996).

1.7.2.1 Tbennolabile MTHFR and Coronary Artery Disease

In 1988(a), Kang er al were the finr to correlate presence of tM'IHFR to the development of

v d a r disease. In 1991, they reported a 17% frequency of t M ï H F R in patients with CAD and a

5% frequency in controls. In a subsequent study, the same group demonstrated a positive

association (independent of other known risk factors for CAD) between the severity of coronary

artery stenosis and the presence of tMiHFR (Kang et al, 1993). They found that the incidence of

severe CAD, defuied as at lem 70% aenosis in one or more coronary artery (CA) or >a% stenosis of the left main coronary artery (LMCA), in patients with the homozygous mutation for

MIHFR was significantly higher than in controls. Patients with moderate CAD (<70°/0 nenosis

in one or more CA or <50°h aenosis in the LMCA) had an incidence for homozygous t M T M

22 that was intermediate, and was found to independently increase the risk for CAD. They also

observed rhat patients heteroygous for the tIbVHFR gene did not appear to be at increased risk

for CAD.

In another study, 60 cardiovasdar patients and 11 1 controls were screened for genetic aberrations

in the CBS and MTHFR gene to determine whether these rwo mutations are Nk factors for

premature CAD (Kluijtmans et al, 1996). While Kluijtmans et ai observed heterozygosiry for the

833TjC mutation in the CBS gene in only one individual of the control group, they found

heteroygosicy for this mutation to be absent in patients with premature CAD. However,

homozygosity for the 677C+T mutation in the MTHFR gene was found in 15% (9/60) of

cardiovasdar patients and in only 5% (6/ 1 1 1) of control individuals (odds ratio: 3.1 (95% CI: 1 .O-

9.2)}. Thus, this group demonarated that the homozygous 677C+T mutation, the cause of

tMTHFR, is a risk factor for artenosclerotic diseue.

Recendy, there has been numerous midies suggening no association between the homozygous

gene mutation for t M l M X and premantre vascula. disease. In 1996, Schrnitz et al investigated

whether this point mutation influenced the nsk for Mi in 190 MI cases and 180 control subjects

denved from the Boston Area Health Study ( the larges study on this topic at the time). They

repoited genorype frequenaes to be 15.3% (+/+), 50.0% (-/-), and 34.7% (+/-) in the case group;

14.4% (+/+), 37.8% (4) and 47.8% (+ /-) in the control group. Schmitz et al calculated the

relative risk for MI associated with the +/+ genocype (cornpared with +/- and -/-) to be 1.1 (95%

CI: 0.6-1.9; p-0.8). PlasmaH(e) levels were 9.112.3 pmoVL in +/+ individuals, 9.912.7 p o V L

in -/- individuals and 10.6k3.8 pmol/L in +/- individuals; these levels were determined in an

unselected mbgroup of 68 cases and 59 control subjects ody (I>Imd=NS). Hence, this group

concluded that homozygosiry for did not represent a useful marker for increased

cardiovascular risk in Boston and simdar populations. Potential differences between ethnic groups

and the retrospective character of this mdy, were mo limitations of this investigation.

Most recendy, DUM et uf (1998) examined prevalence of the tM- in the homozygous state,

plasma folate statu and plasma H(e) levels in patients with early-onset CAD and cornpared them

23 to patients manifening CAD later in life. This study induded 300 patients with acute MI or angina

pectoris and angiographicaUy documented CAD. These patients were categorized into two groups:

group 1 (G1 = 150 patients) presented with these findings under age 50; while group 2 (G2- 150)

~resented for the hst M e over age 65 years. Higher H(e) levels were observed in the older age

group, whidi was expected because age is one of the known physiologie determinants of HH(e)

(Table 1.1, pp. 17). Dunn et al did not fnd any signifiant difference in the frequency of the

homozygous mutant (+ / +) genotype between the 2 groups (G 1: 1 1.3% vs G2: 1 1.3%). Hence,

Dunn a al conduded that the mutant MMFR genorype was not a determinhg factor in early-

onset CAD,

1.7.3 Role of Gene-Environment Interactions in Instigating Hyperhomocyst(e)inemia

The preceding midies advocate either an absolute la& of, or a direct association between

homozygosicy for t m and an increased risk of premature arterial occlusive disease.

However, other data promote the idea that genetic factors, induding homozygosity for tMTHFR

in combination with environmental (acquired) factors cause mild HH(e) and subsequent occlusive

disease. In one study, conuoi subjects and severd groups of vaxu1a.r patients wich mild HH(e)

and with non-HH(e) were examined (Engbersen et al, 1995). This investigation showed that in 28O/'0

of the HH(e) patients with prernature vascular disease, abnormal H(e) metabolism could be

attributed to MïHFR. In another major study, Ma et al (1996) assessed the MTHFR

polyrnorphism, H(e) and folate levels among 293 Physicians' Health Smdy participants who

developed MI during up to 8 years of follow-up and 290 control subjects. The frequency of the

three genotypes was 12O/0 +/+ (homozygous mutant); 47% -/- (homoygous normal); 41°/i +/- (heterozygous), with a similar distribution among both MI case patients and control subjects.

Compared with those with genotype (49, the relative risk (RR) of MI among those with (+ /-) was

1.1 (95% CI: 0.8-1.5), and it was 0.8 (95% CI: 0.5-1.4) for the (+/+) genotype; none of the RRs

were aatistically signifiant. However, those with genocype (+/+) had an increased mean H(e)

level (mean~SEM:12.6~0.5pmoVL), compared with those with genotype (4)

(meank~EM:10.6~O.3pmovL), (p~0.01). This difference was moa marked among men with low

folate level (the lowest quade distribution of the conuol subjects). Those with genotype (+/+)

had H(e) levels of 16.0&1.1 pmol/L, compared with 12.310.6 pmol/L @<.001) for genotype (4).

24 While Ma et al (1996) observed no assoaation between the MTHFR polynorphism and risk of MI,

there was an apparent association between the MTHFR polymorphism and elevated H(e) levels. In

a recent midy, Schwartz et al (1997) examineci the relationship between the risk of MI mong

young women and plasma H(e), folate, vitamin B,, levels and the MTHFR polymorphism. This

stuày induded 79 women <45 years old diagnosed with MI and 386 dern~~raphically s d a r

control abjects living in western Washington state between 1991 and 1995. Compared with

convol subjects, case patients had higher mean H(e) concentrations (1 3.41 5.2 vs. 1 1.1 4.4

pmol/L, p-0.0004) and lower mean plasma folate concentrations (12.4113.4 vs. 16.1112.2

nmol/L, p=0.018). There was no difference in vit& B,, concentrations between case patients

and control subjeas (346.8f188.4 vs. 349.71132.4 pmol/L, p-0.90). After adjusting for

cardiovasdar nsk factors, Schwartz a ai found that women with H(e)215.6 pmoVL were at

approximately mice the Nk of MI as women with H(e)< 10.0 umol/L (Odds Ratio (OR): 2.3; 95%

CI: 0.94 to 5.64). Women with folate28.39 nmol/L had a =50°/o Iowa risk of MI compared to

women with folatee5.27 nmol/L (OR: 0.54; 95% Ck0.23 to 1.28). There was no association with

vitamin B,, concentration. Among case patients, 10°h were +/+ for tMTHJ3, and there was no

association between homozygosity for tMïi-IFR with increased MI risk (OR: 0.90; 95% CI: 0.3 1 to

2.29). Among conuol subjects, 12.7% were +/+ for tMïHFR, and these women had higher

plasma H(e) and lower plasma folate than did women with other genocypes. Hence, Schwartz et al

conduded that although homozygosity for tMIHFR is related to increased plasma H(e) and low

plasma folate, this genetic chancteristic is not a risk factor for MI. They also showed that elevated

plasma H(e) and low plasma folate levels to be risk factors for MI among young women.

Like Engbersen et al (1995) and Ma a al (1996), Schwartz et al (1997) proposed that there was an

indirect association between genetic and biochemical interactions. It appeared that environmental

factors (folate, vitamin B, , vitamin B, , methionine, betaine and choline) influenced genetic

factors (MEER) in causing a biochemica abnomality {HH(e)}, and subsequent premature

vascular disease.

25 1.8 Hyperhomocyst(e)inemia and Arterid Occlusive Disease

Anenal occlusion) a consequence of arteriosclerotic àisease related to severely elevated

homocysteine levels in homocystinuria is well recognized. The relationship was fus suggested by

McCuiiy (1969), who observed autopsy evidence of precocious arterial thrombosis and

aiteriosclerosis in a homocystinuric patient with impaired cobalamin metabolism that was identical

to what had eariier been described in homocystinuric patients with CBS deficiency (Mudd et al,

1964). In an international collaborative midy, Mudd et al (1985) reported that 158 of 629

homocystinuric patients experienced a thrombotic event, induding MI or aroke, and in half of

these the event occurred by age 29 years. Mild HH(e) has been nrongly assouated with increased

nsk of CAD in 20-30% of patients (Genest JJ sd, 1990; Starnpfer etd, 1992; Arnesen et al, 1995;

Boushey CJ, 1995; Robinson K a d , 1995; Wilcken DEL et al, 1996).

1.8.1 Coronary Artery Disease

Patients with mild HH(e) have none of the dinical signs of severe HH(e) or horno~~stinuria and

are rypicaUy asymptomatic und the third or fourth decade of life when premature CAD develops,

as well as recurrent arterial or venous thrombosis (Welch and Loscalzo, 1998). The role of mild to

moderate HH(e) has been suspected for years. In 1976, Wilcken and Wiicken showed that the

concentration of homocysteine-cysteine Mxed disulfide after a methionine load was siightly higher

in CAD patients than in controls, thus pmviding the fus evidence of an association between rnild

FH(e) and vasdar disease. Mil* increased homocysteine levels have thus far been reported in

numerous CAD patients (Murphy-Cdorian et d, 1985; Kang et d, 1986; Israelsson, Brrattarom,

and Hultberg, 1988; Olszewski and Szostak, 1988; Malinow etal, 1990; Clarke et al, 1991; Ubbink et

d , 1991; Nygard etal, 1995; Graham &al, 1997; Robinson etd, 1998). However, und the last seven

years, reports irnplicating mild HH(e) as a rîsk factor were based solely on retrospective Nrveys or

s m d cross-sectional, case-control d e s (Mudd, Levy, Skovby, 1995). These cross-seaional

studies correlated concentrations of plasma H(e) with the extent of coronary disease determined by

coronary angiography. In 1991, Ubbink et al studied 163 male Caucasian patients 4 t h typical

angina without previous CHD. Hyperhomocyst(e)inemia was p r d e n t in a large proportion of

anglia patients wirhout apparent coronaiy artery stenosis; hirthermore, there was a linear trend of

increased p r d e n c e of m ( e ) associated with the number of coronary arteries with significant

aenosis. In a similar snidy of 199 CAD patients, the number of main coronary menes with > SOOh

26 stenosis Uicreased with higher HH(e) (Von Eckardstein et al, 1994). Both nudies suggested that

the extent of CAD was positively comlated with the concentrations of H(e). A major drawback of

retrospective d e s is that they cannot determine whether Licreases in H(e) are the cause or the

result of aiterial occlusive diseases. Prospective studies, though, do not have this limitation.

In 1992, the Physiuans Health S t d y was the fust prospective assessrnent of the risk of CAD

associated with elevated levels of plasma H(e). In this study, 14,916 maie physicians, aged 40 to 84

years, with no pnor MI or svoke provided plasma samples a baseline and were followed up for 5

years. Sarnples from 271 men who subsequentky developed MI were analyzed for H(e) levels

together with paireci conuols, matched by age and smoking. The cases had a slightly, but

aatistically significant, higher mean concentration of H(e) than the non-cases, i.e., 11.1 vs 10.5

pmol/L, respectively. Most of this difference was amibutable to an excess of high values arnong

the cases, but natistid power was not suffcienc to d e out a graded disuibution of risk. This

neaed case-control midy suggested that elevated H(e) levels were an independent risk factor for

the development of CAD with an estimated odds ratio of 3.2.

Prospective data are also available from >21,OOO individuals with blood samples collected from

1986 to 1987 (Amesen et al, 1993); 123 new cases of MI in this group had been confirmed by the

end of 1990. Each case was matched to controls. Mean H(e) level among cases was 12.07 pmoVL,

compared with 11.3 p o V L in the controls (p0.002). The distribution of values of H(e) was

shified towards higher concentrations among cases across the encire distribution of values of H(e).

There was a trend, as in the Physiuan's Health Study, for greater association among younger, as

opposed to older, subjects. The data provided important new evidence supporthg the hypothesis

that increased H(e) is an independent nsk factor for CAD. The data from this prospective study

are generally compatible with those observed arnong the US physicians.

In 1995, Boushey et al perfonned a meta-analyssis of 27 d e s relating HH(e) to artenosclerotic

vascular disease. They demonstrated that an elevation of 5 pmoVL in H(e) levels was equivalent

to the risk of GAD associated with an elevahn of 0.5 mmoVL (20 mg/&) in total cholesterol.

Conversely, an increment of 5 umoVL was found to be associated with odds ratio of

27 1.6 (95% CI 1.4 -1.7) and 1.8 (1.3 -1.9) for the development of CAD in men and women,

respectively. In addition, theù data suggested that incrernents of risk, for any given homocysteine

level, were linear, without obvious threshold effects.

Mon recently, the k i t case-controlled snidy to provide evidence of elevated homocysteine levels

as an important risk factor for MI (and strokes) in the elderly was documented (Bots et al, 1999).

Though the hidy was based on a relatively short follow-up period, the risk of M . (and nroke)

increved dire+ with totd homocysteine, aftu results were adjusted for age and sex. The linear

coefficient suggeaed a risk increase by 607% for every 1 urnol/L increase in total homocysteine.

1.8.2 Mild Hypahomocyst(e)inemia and 0th- Occlusive Diseases

Multiple clinid studies have reported an association between mild HH(e) and an increased risk of

cerebrovascdar disease (CVD) (Brattsuom, Hardebo and Hultberg, 1984; Boers et al, 1985; Ar&

et al, 1989; C o d et d, 1990; Clarke a al, 1991; Brattstrom et al, 1992; Robinson et al, 1998).

Robinson et al (1998) showed that low cirdating concentrations of folate and vitamin B, are

associated as risk factors for stroke (among other vascular diseases). AU of the midies, thus far,

have been based on populations that mody indude middle-aged subjects. However, the smdy

conducted by Bots et al (1999) was the first case-controlled midy to provide evidence of elevated

homocysteine levels as an important ri& factor for strokes (and MI) in the elderly.

With respect to evaluating H(e) as an independent risk factor for peripheral artenai disease (Pm), there have been numerou investigations involving more than 500 patients. However, only a

handful of major, welldesigned d e s evaluating homocysteine levels in patients with peripheral

vascular disease (PVD) have shown that an elevated H(e) level is an independent risk factor for

PAD, and may contribute to atherosderotic lesions in 28%-50% of patients with PAD, regardless

of whether the free fraction or totd homocysteine is measured, and whether or not PML is used

(Boers et al, 1985; M&ow a al, 1989; Brattstrom a ai, 1990; Clarke et al, 1991; Taylor et al, 1991;

Molgaard et d, 1992; Robinson et al, 1998). Furthennore, a higher percentage of women (42-50%)

than men appear to be affected. In fact, the meta-analysis by Boushey etal (1995) noted that every

28 inaement of homocysteine of 5 pmol/L was assoaated with odds ratio of 1.5 and 6.8 for CBD

and PVD, respecrively.

Although venous thromboembolism (VTD) accounts for MOh of the vascular complications of

homocystinuria (Mudd, Levy and Skovby, 1985), the association between mild HH(e) and VTD

remains controversial. m e , a handful of investigaton have found no significant association

between mild H H ) and VïD (Brattstrom er al, 1991; Amundsen et ai, 1995), other invenigators

have reported a positive association b ~ e e n HH(e) and VTD outcomes (Bienvenu et ai, 1993;

Falcon a/, 1994; Heijer et d, 1996).

In a cross-sectional 2-year evaluation of 157 consecutive unrelated patients with a hinory of

venous or arterial occlusive disease occvring before the age of 45 yean or at u n d sites, d d

HH(e) was detected in 13.1% (VTD) and 19.2% (PAD) of patients (Fermo et al, 1995). The

prevalence of HH(e) was almon twice as high when based on homocysteine measurements done

after oral methionine load as when based on fanuig levels. Deficiency of well-established risk

factors for thrombosis, were detected only in patients with VTD, with an overd prevdence of

18.7%.

Recendy, Paiareti et al (1996) analyzed the combined results of a total of 38 studies on

thrombovasdar disease and WH(e). The distribution of patients with total fanùig H(e) levels

above the 9Sh centile were 21.7% in CAD (17.9%), 26.6% in CBD (24.I0h), 32.8% in PVD

(28.6%) and 1 3.S0/t (25.5%) in VTD {bracketed numbers indicate PML HH(e) } .

Unlike many of the tndiUonal inherited dirombophilic defeas that are risk factors for

venous thrombosis, these data represent suong evidence nipporting the association of HH(e) with

the occurrence of thrombovascuIar events in both the venous and arterial circulations.

1.9.1 Quantitation of Total Homocysteine in Plasma

In the late 1960s, homocysteine w u considerd to be absent in the blood of non-homocystinuric

individds. However, technical advances have greatly facilitated the introduction of irnproved

techniques for measuring H(e) in plasma and senun. A m t e meanvements of low H(e) levels is

imperative becaux as discussed earlier, various revospenive and prospective case-conuol midies

have found mild HH(e) in 12-42O/0 of patients with coronuy, cerebrovasdar, or periphenl artenal

diseases. As to what consritutes a d H ( e ) level, however, remains to be defiied.

From a current labontory standpoint, problems exist in H(e) analysis. Extemal quality-assurance

prograrns for H(e) (profiaency tentig) are not available at present, and inter-laboratory correlation

of H(e) rneasuremenrs have not been evduated forrnllly (Eliason et al, 1999). Recendy, Eliason et al

evaluated the extent of variability attributable to inherent test imprecision as opposed to b i s

between laboratones. They pointed out that the signifiant observed bias between five U.S.

labontories created differences in reported results that could not be explained by randorn error

alone. They niggested multiple factors that could account for the inter-laboatory variability,

including different methodologies, different approaches to test calibration, la& of availabdity of

reference materials for the various form of homocysteine, and varying efficiencies for the

dissociation of homocysteinecontaining complexes. However, since high performance Liquid

chromatography WLC) with fluorescence detection and moa recently, fluorescence polarization

immunoassay are the only emerging methob of choice for H(e) analysis, it is suspected that the

factors responsible for causing inter-laboratory variability, as suggested by Eliason et al wiU

pduaily be cuttailed.

With the development of a fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole~

sulphonate, (SBD-F) (Imai, Toyo'oka and Watanabe, 1983), meanirement of biologically important

thiols has been made possible. U&e mvry other fluorogenic reagents which also denvatize

amines, SBD-F is a thiol-spedc reagent that $el& hi@ fluorescent derivatives. Unlike the

characteristic feanues of many fluorogenic reagents, un-reacted SBD-F does not fluoresce, the

thiol adducts are stable, and no fluorescent bydrolysis produas are foxmed. Their use renilu in

30 dean chromatograms with no reagent peaks. One dnwback of SBD-F is its low reactivity, which

may cause some problerns because of homocysteine re-oltidation. However, this problem cm be

avoided by inciuding EDTA in the reaction mixture, which chelates divalent metais and &bits re-

oxidation. Araki and Sako (1987) were the fm to demonstrate a simple but sensitive and selective

HPLC method for the detexmination of free and total homocysteine in plasma using SBD-F as the

pre-column labeling agent.

Most recently (December 1998), the U.S. Food and Drug Administration (FDA) approved Abbott

Laboratones' (Abbort Park, IL) homocysteine assay, making it the first automated test avdable to

quantitatively measure H(e) in human semm or plasma This assay is a fluorescence polarization

immunoassay (FPIA) which can be run on the Abbott Mc@ m e m . The biochernical prinaples

that are u n d e r h g this assay are quire simple. In brief, dithiothreitol reduces homocystine,

mixed-ciidfides and protein-bound homocyaeine. Total homocysteine is then converted ro

S-adenosylhomocysteine (SAH) by the use of SAH hydrolase and excess adenosine. The SAH chat

foms is incubated with a monoclonal anribody raised againn SAH and a fluorescentiy labeled

analog of SAH. Subsequently, SM and its analog compete for the sites on a monoclonal antibody

molecule. On passing polarized light dirough the sarnples, the degree of polarization is invenely

proportional to the arnount of label-bound antibody. Red t s are read automaacaliy and printed

out (Abbott Laboratories, Abbott Park, IL). One drawback, whidi may be faced by laboratones

that adopt this homocysteine immunoassay is a possible patenting issue if the "assay is used to

determine an elevated level of homocysteine with a correspondhg deficiency in folate or BI:

(AACCClinical Labontory Strategies, Decem ber 1998).

Elevated plasma homocyst(e)ine levels have been associated with a higher incidence of coronary

heart disease, but the pmgnostic value of measuring homocysteine levels in patients wirh

eaablished coronary mery disease, specifically myocardial infarction, has not been defined.

Endothelid dysfunction and a profound, localized hypercoagulable state are both inherent in

myocardial infarction. One of the theriapeutic goals is to achieve patency in the infarct-related

artery, which is ultimateiy dependent on the balance between procoagulant and anticoagulant

forces. Since mildly elevated homocyst(e)ine has been implicated in inducing a hypercoagulable

state, mild hyperhomocyst(e)inernia would be expected to have deleterious effects on clinicai

outcomes in myocardial infarction. Furthemore, the possible CO-existence of a very cornmon

cause of hypercoagulability, Factor V Leiden mutation could M e r worsen the prognosis of

myoçardial infarction.

2.1 Hypothesis

We investigated the hypotheses that hyperhomocyst(e)inemia could be an important prognostic

Lidicator in myocardial infarction and its outcomes. We aiso wished to test the hypothesis that the

concomitant occurrence of this biochernical imbalance with Factor V Leiden could further

intensify the detrimental outcomes of a myocardid infarction

2.2 Objectives of The Sîudies Outlined in t h i s Thesis

Before ascenaining the H(e) s t a t u of our MI parients and correlating it to their genetic or acquked

detenninants, it was fust necessary to ûnd an acceptable method for quantitation of plasma H(e)

levels. FoUowing optirnization, validation and stability wduation of the analyticai method of

choice, our main endeavor was to detennine the specific hequency of HH(e) and its relevant

genetic (MTHFR genotype) and acquiml (RBC folate and plasma vitamin BIJ detenninants in MI

patients admitted to the coronvy care unit at Sumybrodr He* Science Centre. FinaDy, we

wished to axertain the status of the Factor V gene in our patient cohort.

3.0 MATERIALS and METHODS

3.1 Clinka1 Analysis

3.1.1 Subjects

The biochemical and genetic status of ail our subjeas was assessed i n d . The study protocol was

reviewed and accepted by the ethics cornmittee at Sunnybrook Health Science Centre (SHSC).

Subjeas were partiapants in a pilot study (Sept 97'-Apr 98') consisting of 79 non-consecutive MI

patients recruited from the coronary a r e unit (CCU) at Sunnybrook Health Science Centre

(SHSC). Of this patient cohort, H(e) and genetic (tMTHFR and FVL) natus was available on 74

subjects; however, this data Licluding folate and vitamin B,, status was available on 66 subjeas.

Corn~Iete data induding clinical (ECG) a a w w u available on 53 subjects. Out of the 53 MI

patients, 37 expenenced an th hptai outcorne of death, congestive heart failure (CHF) or

recurrent ischemia Ody 22 out of the 53 patients were eligible for thrombolytic therapy {tissue

plasminogen activator ('ïPA)/streptokinase (SK)}. Dr. A. Moni, a Cardiology Fellow, enroiied di

MI patients in this cohort after ennuing that they satisfied the eligibility critena.

3.1.2 Definition of MI and Relevant Criteria

For the purpose of this audy, an acute MI was defied as chea pain lasting greater than 30

minutes and either :

a) elevation of creatine kinase (CK) to greater chan twîce the normal value with elevated CK-MB fraction

or - b) any one of the following electrocardiogram (ECG) changes:

9 New LBBB ii) Any ST Depression 2 Imm in two Contiguous Leads iii) A q ST Elevation 2 lrnm in cwo C ontiguous Leads iv) New Q-Waves in any two Contiguous Leads

The episode of chest pain musr have occurred w i h 72 houn of presentation to the hospital. The following cntena were in effect:

Inclusion

1) Patient able to give informed consent

Exclusion

1) Vitamin B,, or folate supplementation within the last 3 months 2) Severe psoriasis 3) Hernatologic malignanq 4) Rend failure requiring didysis 5) Patients taking ohhe bilowing medications:

a) methotrexate b) phenytoin c) carbemazapine ci) penidamhe

3.1.3 Reported In Hospital Outcornes

The following iz ho@ events were monitored:

1) Recurrent ischemia defmed as chest pain:

i) with 2 Imm of ST Ellevation or Depression ii) leadùig to CK Ne iii) requiring revaxularization in hospital

2) Occurrence of congestive heart failure (CFF) 3) Death

4) Success of reperfusion anallied by patients administered thrombolyuc therapy, induding TPA or SK as per treatment protocol. Success of reperfusion for patients given thrombolyncs was

i) complete resolution of chest pain at 3 hours ii) complete resolution of ST elevation (10.5mm in lunb leads or 5 lmm in

precoridal leads) at 3 hours.

3.2 Laboratory Studies

From consenting CCU MI patients following an ovemight fast, about 3 mL of whole blood was

collecteci by venipunaure into a Vacutainer Tube (Becton Dickinson, Rutherford, NJ) contaLiLig

EDTA. The tube was immediately refngerated. Within 4 hours, the tube was cenmfuged at 3500

34 rpdminute for 10 minutes. The plasma was separated and stored ai -80°C util H(e) analysis.

The buffy coat containing the white blood ceUs was used for DNA extraction, which was

subsequently anal@ to determine the patient's genotype for M.THFR and Factor V genes. Total

red blood cell (RBC) folate and serum vitamin B , were ordered on the same day and analyzed in

the Special Chernisuy Unir at SHSC. The technician in-charge determined CO-factor starus on ail

patients, using a Bdfolate radioassay (Quantaphase II, Bio-Rad Diagnostics Laboratones Ltd.,

Mississauga, ON).

Plasma was available on 66 MI patients. Tot4 homocysteine was determined using precolumn

denvatization, HPLC and fluorescence deteccion (FD) as described by h a k i and Sako (1987) with

modifications for optimization of readon conditions. All method optimization, assay validation

and stability studies were performed in duplicares (unless otherwise sated). A volume of 20 uL of

the relevant standard or sample was subjected to chromatographic analysis.

3.2.2 Chernical Analysis

3.2.2.1 Pursuit for an Ideu2 Metkod for Quuntitation of P h m u Homocyst(e)ine

Fluorescence detection using fluorescarnine, 4-p henylspiro[fÜran-2 (33, i '-p hthalanl-3-3 '-dione

was initially attempted for the fluoromeuic assay of plasma H(e). This non-fluorescent compound

reacts with primary amines (RNHJ to f o m pyrrolinones, which upon excitation at 390 nm emits

strong fluorescence at 475-490 nm. However, the reducing/releasing agent, sodium borohydride

(NaHB,) appeared to inhibit the denvatization reaction between fluorescarnine and H(e).

ElectrochemicaI detection was attempted, but there appeared to be non-selective separarion of

homocysteine, possibly due to detector limitations. Fluorescence deteaion was once again

employai but with SBD-F as the fluorogenic agent and TnBP as the reducing/releasing agent.

Good separation of H(e) was observed and the deiivatization reaction did not appear to be

3.2.22 Chromatogrupbic Sy stem, Conditions und Sepurution

A chomatographic separation was developed which allowed anaiysis of plasma H(e) and ensured

the separation of this amino acid from cysteine and other cornpounds. A + mobile phase

35 system (75% solvent A/25% solvent B) was used for assay validation. The composition of solvent

A w u 75% of O.1M acetate buffer (1M acetic aad and 1M sodium acetate purchased from Sigma

Chernical Co., St Louis MO) containing 1.5% methanol (MeOH) (Omnisolv, EM Science,

Toronto, ON), pH-4.1. The composition of solvent B was 25% 0.1M phosphate buffer (1M

dibasic and IM monobasic potassium phosphate purchased from Sigma Chernical Co., St Louis

MO) containing 5% MeOH, pH-6.3. The proportion of each solvent used during a 23-minute

chromatographic nui varied [rom 1.5% methanol to 5% methanol. However, we evennially

developed a shorter (7 minutes) and simpler kamatic solvent system. The i d c mobile phase

system, whereby the ratio of buffer to organic was held constant during a chromatognphic nin,

was used for method validation, srabiliry midies and patient sample analysis. The solvent

composition constituted 85% O.1M acetate buffer, pHm4.1 and 15% 0.1M phosphate buffer,

pH-6.3. Total MeOH composition in this solvent system was 2.025%. Both mobile phase

systems were pumped at i . 0dminu te through a 30 an x 3.9m.m C,, 5 p coiumn (Novapak,

Waters Limited, Mississauga, ON; part #: 11695) (assay validation) and through a 15cm x 4.6mrn

C,, 3 p n column (Supdcosil, S ipa -Ald r i ch Canada Limited, Oakde, ON; Cat # 5-8985)

(method validation, stability studies and patient sarnple analysis) using a liquid chromatographic

pump (SpectraSynem P4000, Thermo Separation Produas, Fremont CA) and a 712 WISP

autosampler (Waters Corporation, Mississauga, ON). An Aquapore RP- 18 ODS reverse-p hase

guard column (Chromatographie Specialties Incorporated, Mississauga, ON) was fitted between

the analyucal column and the autosampler. Using a F U O O O detector (SpectraSystem fluorescence

detector, Thetmo Separation Products, Fremont CA), a wavelengrh 6) spectral scan on eluting

peak(s) from a high DL-homocysteine standard and a plasma sample was run to determine the

optimum exatation and emission L maxima for the H(e) adduct. The detector signai was recorded,

and the peak area was quantified dire+ on cornputer using PC-1000 software m e r n o

Separation Produas, Freemont, CA). Using this isocratic chromatographic system, the separation of

cysteine and homocysteine was confirmed by anaiysis of both standards and plasma samples.

3.2.23 pH and Pbysical Inspection of Buffers

pH and physical inspection was performed on all solvent systems and reaction buffers upon

irnmediate preparation, and periodicaily thereafter. To prolong the longevity of our column, all

solvent systems were filtered prior to every chromatographic run using a micronsep cellulosic .22 p

36 fdter (Fisher Scientific, Unionville, ON). The reaction buffers were inspected visuaily for color and

cl* and filtered when necessary. The pH meter (Accumet-mode1 925; Fisher Scientific,

Unionville, ON) was equipped with a microprobe glas body electrode (Cat# 13-639-280; Fisher

Scientific, Unionville, ON) and was standardized with two cornmeraally available buffer solutions,

pnor to pH deterrnination.

3.2.2.4 Metbod Optirnization

To completely reduce thiols and to displace thern from plasma proteins, plasma sarnples were

hcubated with the reducing agent, tri-n-butylphosphine (TBP) in dimethyiformamide @MF) at

varying concentration, volume ranges and incubation tirnes. To ensure optimum derivatization of

H(e) by SBD-F, varying incubation tirnes, temperatures, volume and concentration ranges of

SBD-F were evaluaced. The chomatograms were vis* inspected for the appearance of any

additional peaks. The homocysteine peaks were aiso compared within each study day for changes

in retention Ume and peak shape.

3.2.2.4.1 Tvpical Samde Pmaration Protocol

A typical sample preparation protocol outline follows. Results of optimum reaction conditions for

the HPLC assay development of plasma H(e) will be diswsed in the appropnate section.

One part of plasma (500 uL) was treated with 70 uL of 10% (v/v) tri-n-butylphosphine (T'BP) in

âimethylformamide @MF) for 30 minutes at K. This solution was mixed with 500 uL of a

chilied solution of 10% tnchloroacetic aad (ICA) contaliing 1mM NaEDTA under vigorous

vortexing, followed by centrifugation at 3500 rpmhinute for 10 minutes. A 200 uL aliquot of the

clear supernatant was vigorously rnixed with 400 uL of 2.5M borate buffer (pH-10.5) containing

4mM N+EDTA and 250u.L of SBD-F (l.Omg/rnL) in 2.5M borate buffer (pHs9.5). The mùmue

was incubated in a water-bath for 105 minutes at 60°C. After temiinating the reaction, the solution

was cooled in crushed ice. An aliquot of 20 uL of the reaction mixture was ~bjected to the HPLC

anaysis.

Notc: di buffers prcparcd using chmiicais purchascd from Sigma Chernid Company, St Louis MO.

37 3.2.2.5 Selection of un Optimal Adytical Cohmn and Solvent System

In an attempt to improve separation of H(e) from other plasma compounds, numerous analytid

columns and a variation of solvent systems were evaluated respectively. The final choice of an

optimum analytd column was made between a short colurnn (15 cm x 4.6 mm LC- 18 Supelcosil,

3 p paxticle size, Cat #4 5-8985) and long column (3.9 mm x 300 mm C ,, Novapak, 5 p parude

size, Patt #: 1 1695).

3.2.2.6 Assay Vubdution: A c c u r . und Reproducibility

Validation of the method, with respect to accwacy and reproduubility was tested over a penod of

3 months in concurrence with the aability study. During this t h e course, using system niirability

criteria @eak tailing and retention t h e ) chromatograms were visually kpected H(e) to ensure

consistency between study days. Inter- and intra-day reproducibility was assessed using the

coefficient of variation (CV) of the peak area for samples deterrnined in duplicates. Accuracy was

assessed based on deviations from the known concentration of both standards and quality control

(QC) sarnples.

Guidelines adopted by the Health Protection Branch (Canada), FDA (U.S.) and published by Shah

a al (1992) were adopted for validating our HPLC assay for plasma H(e) quantitation. Precision

and accuracy of our method was considered to be & a+ iimm if mean concentration of

the standards on any given day of analysis was within r lSO/o of CV or deviation of the initial (day-

zero) concentration, except at limit of quantitation (LOQ), where it should not deviate by greater

than 120% of CV or deviation.

3.22.7 S&zbi&ty St~dy

3.2.2.7.1 Stabilitv of Homocvst~e)ine in Plasma Stored at -2OoC & -800C

A total of 5 spiked plasma standards, I plasma-blank and 3 QC stand&, stored at -80°C

and -20°C, were run periodicllly for a p e n d of 3 months. A stock solution of

approximately 4M DL-homocysteine war prepared and diluted with plasma to create the 5

standards induding 30,20,16,8 and 4 urnol/L and the 3 QCs including 24,11,6 umol/L. Liguid

chrornatographic analpis of the standards aored at -80°C were analyzed on days

1,3,9,21,24,28,60,79,92; aandards stored at -20°C were nibjected to analysis on days

38 1,2,7,10,21,32,57,99. Upon chromatographic analysis, a standard curve was conaructed on each

study day. Workhg buffered-stock standards of approxhately 40 urnoVL DL-homocysteine and

70 umo1L L-cysteine were also chromatognphically evaluated on each mdy day to ensure

optimum chromatographic performance.

3.23.7.2 Stabiiitv of Homocvst(e)ine in Plasma Stored at Raom Tem~erature k 4-8*C

To address the aability issue of patient samples aored at room temperature or refrigerated for 1

week pnor to analysis, plasma H(e) levels were measureà periodically over a specific tirne range.

3.2.2.7.3 Stabiiity of Hornocvst[e)ine in Whole Blood Stored at Rwm Temwnhw & 4-8OC

The rate of release of H(e) from erythrocytes in whole blood nored at room temperature and

4-8OC was evaluated over a time range, under nvo conditions: firstly, in a s&g sute and

secondly, on a spin-mix simulating the potentid rate of release during sample transpoxtation.

3.2.2.8 Putient Phsma Sumple A d y s i s

Following the fnst 2 weeks of stability studies, analysis of patient plasma samples were begun.

Patient plasma samples were analyzed for H(e) quantitation sirnultaneously within each stabiliry

study &y, or on ofitability midy days using the sarne reverse phase liquid chromatographic

separation described in section 3.2.2.2: Chromatographie System, Conditions and Separauon (pp.36-37).

On oflstability study days, 3 QC standards and working buffered-stock standards were always run

to confimi that the system suitability aiteria were being met. The average peak area of H(e) from

each of the two replicates run from every sample was subjected to leas squares linear regression;

the H(e) concentration within each patient plasma was interpolated from standard curves and

recorded.

3.2.3 Molecular Analyses

Genomic DNA was extracted from whole blood leukocytes using a standard phenol/chloroform

procedure (Fritsh and Maniatis, 1989).

39 3.23.1 CsnT Mutaiion Andysis of MTHFR gene

Genocyping for analysis of the CmT transition (alanine* vahe) in the MTHFR gene was

performed as describeci by Frosst a al (1995). Brie&, using the forward (5'-TGA-AGGAGA-

AGGTGTCTGCGG-GA-3') and reverse (5'-AGG-ACG-GTG-CGG-TGA-GAG-TG-3')

primers, a 198 base-pal (bp) fragment was amplified by the polymeme chain reaction (PCR).

Amplification was performed in a thermal d e r (model# PTC-200, MJ Research Inc.,

Massachusens, USA) with an initial denaturing s e p at 94OC for 3 minutes, followed by 35 +es of

denaniring at 940C at 45 seconds, primer annealing at 55OC for 30 seconds, and primer extension at

72OC for 90 seconds. A final primer extension at 72OC for 10 minutes completed the reaction. The

fragment was amplified in a reacrion volume of 50 pL using 35 pmol of each of the forward and

reverse primers and 1.25 U Taq DNA polymerase. Aliquots of the PCR produa were digested with

Hiyl (0.4 PL) endonudease restriction enzyme (RE) and the cleaved product was subsequently

electrophoresed on a 3% agarose gel, thereby revealing the mutationai aatus of the nibject. In the

presence of an alanine+valine substitution, the 198 bp fragment was digesced into 175 and 23 bp

fragments; the latter fragment is not usuaUy visible as it runs off the gel. Al1 three possible

genorypes are shown in Figure 9.1, kp.93).

3.23.2 Muration Anafysis of Factor Vgene

Genotyping for andysis of the G,,,A transition (arginine-iglutarnine) at Factor V residue 506

(Factor V Leiden) was performed as descnbed by Gudlenn e t d (1996). Briefly, using the forward

(5'-GTA-AGA-GCA-GAT-GTGG-AU-GTC-3 ') and reverse (5'-TCT-CTT-GAA-GGA-

AAT-GCCCCA-TIA-3') primers, a 137 bp fngment was amplified by PCR. Amplification was

performed in the thermal cyder with an initiai denaturing step at 95OC for 5 minutes, followed by

30 cycles of denaturing at 95OC at 1 minute, primer annealhg at 51°C for 1 minute, primer

extension at 72OC for 1 minute. A final primer extension at 72OC for 10 minutes completed the

reaction. The fragment wu amplified in a reanion volume of 50 PL using 50 pmol of each of the

fornard and reverse primers and 1.25 U Tq DNA poiymerase. Aliquots of the PCR product were

digested with TaqI (0.125 PL) endonudease RE, and subsequently electrophoresed on a 30h

agarose gel, thereby revealing the mutational natus of the subject. Digestion by TaqI RE genented

40 2 fragments (114 and 23 bp) in a wild-type allele, but did not cut PCR product from a mutant

G,,,A type allele. After gel electrophoresis, a homozygote wild-type (4) shows one band of 114

bp fragment (the smaller band of 23 bp fragment n0ma.ü~ nuis off the gel), a heterozygote (-/+)

for the mutation shows 2 bands of 137 and 114 bp fragments corresponding to the two alleles, and

a homozygote for the mutation (+/+) presents the single band of 137 bp fragment. AU three

possible genotypes c m be observed in Figure 9.2 (pp.94).

Note: Ali PCR amplification mixture reagents were purchased from GIBOBRL Life Technologies- (Burhgon, ON); RE digestion mixture reagents (EIPIFI and Ta$) were purchased from New England Biolabs (Massachusetts, USA)

3.2.4 Statistical Analyses

Ali red ts were cdculated as mean standard deviation for replicate anaiysis. An unpaired

Student's t-test, Anaiysis of variance, Fisher's exact test or Chi-Square test was used to determine

the two-tded p-value where appropriate. We considered a difference to be statistically significance

when pC0.05. Where necessary, the relationship between 2 variables was evduated by linear

regression and strength of an association was detemineci by the correlation coefficient.

4.1 Quantitation of Plasma Homocyst(e)ine: Andytkal Results

4.1.1 Fluorescence Deteftor Optimization

Upon runnuig a A spectrd scan on eluting peak(s) Pom a high DL-homocysteine standard and a

plasma sample, the optimum excitation (Figure 4.1.1 a) and emission (Figure 4.1 . l b, pp.42) A

maxima for H(e) was determined to be 240 nm and 508 nm respectively. While there appeared to

be a secondary exatation A maximum at 390 nm, the primat- exatation A at which there was

greater fluorescent intensity of Our addua was choxn as optimum.

O 100 200 300 400 500

Excitation (nm) W avelength

Figure 4.1.1~ Excitation Wavelength Optimization for H(e) on N O 0 0 Fluorescence Detcctor

Spectral scan depicting the exatation ?. at which derivatized H(e) o p q fluoresced, while die emission À

was held constant.

200 400 600

Emission Wavelength (nm)

Figure 4.1.h Emission Wavelength Optimization for H(e) on FU000 Fluorescence Detector

Spectral scan depicting the emission À at which denvarized H(e) optimaUy fluorexed, while the excitation i was heId constant.

4.12 HPU: Method Optimization

Note: Unless otherwise statcd, number vaiues adjacent to each plot point on graphs represent O/bCV.

4.1.2.1 Concentration of Tn BP

From a concentration range of 5 to 70%, there was greater replicarion error (CV: 8.7%) observed

when the concentration of TnBP was 556 as compared to a TnBP concentration of 10°/o (CV:

3.6%). The H(e) peak areas decreased with an increase in TnBP concentration thereafter.

Therefore, 10% TnBP was chosen to be o p W .

O 10 20 30 40 50 60 70 80

O/O TnBP (hqosition

Figure 4.1.h: Optimum Concentration of TnBP Required for Complete Reduction and Release of Bound Homocysteine in Plasma

44 4.122 Volume of TnBP

From a volume range of O to 110 PL, maximum H(e) peak area was observed when the volume of

10% TnBP was 70 PL. Although the replication error was IO%, this CV was within acceptable

fimits according to the HPB and FDA guidelines.

O 20 40 60 80 100 120 V o l m of TnBP Added (UL)

Figure 4.12b: Optimum Volume (pL) of 10 */O TnBP Required for Complae Reduction and Release of Bound Homocysteine in Plasma

45 4.123 Time of TnBP Incubation

From a thne span of O to 90 minutes, 30 minutes of incubation with 70 PL of (10%) TnBP was

found to be optimal. At this point, the H(e) peak area wu greatea and replication error (CV:l.OOh)

was within acceptable limits.

Figure 4.12~: Optimum Incubation Time (minutes) of 70 pL (101) TnBP Required for Complete Reduction and Release of Bound Homocysteine in Masma

46 4.1.2.4 Amount of S D F (Img/mL)

From a volume span of 100 to 750 yL, correspondlig to an SBD-F amount span of 0.10 mg to

0.75 mg, 250 pL (0.25 mg) of SBD-F was found to be optimal. At this point, the H(e) peak area

was marrimal.

4.1.2d: Optimum Amount of SBD-F (lmg/mL) Required for Complete Derivatization of Total H(e) in Plasma

Samples were not run in duplicates.

47 4.1.2.5 Tentperature und Tirne &th SBD-F Incubution

Incubation with SBD-F for a tirne span of 15 to 315 minutes at 3 temperatures depicted a

reasonably optimal incubation Ume to be 105 minutes at 61.5OC.

Figure 4.1.2f: An Evaluation of the Extent of Totai H(e) Derivatized at a Variation of Times and Temperatures

Optimai incubation Ume and temperature were selected based on 4 criteria:

i) maximum H(e) peak area ii) a point where changes in incubation Ume had minimal effect on H(e) peak area iii) minimum incubation time iv) acceptable CV%

48 4.1.3 Improvement of Homocyst(e)ine Separation h m 0th- Plasma Compounds

4.13.1 Selection of an Optimum HPLC Ancdyticd Column

Note: AU dvomatog~ms arc of the same plasma sampk.

Improved separation between H(e) and other plasma cornpounds was achieved upon replacing the

longer analyacal column {Figure 4.1.3a (i)), with a shorter analyucal column {Figure 4.1.3a (ii) }.

Due to the smaller partide size of the shorter column (3pm), the surface area was increased, and

the separation was more efficient.

Figure 4.13% Typical Chromatograms Generated h m HPLC Adysis of A Plasma Sample

49 4.132 Optimization of HPLC Solvent System

To achieve an ultimate optimal separation berneen the H(e) and other chrornatographic peaks, an

evaluation of varying compositions of the 2 buffers constituting the solvent synem was conducted,

keepiig track of the organic content (MeOH) in each composition. As c m be observed from

Figures 4.1.3b-f kp.49-50) and Table 4.1.3 (pp.51), an 850h solvent A/15Oh solvent B contMng

2.025% MeOH was found to yield the most immadate separation between H(e), cysteine and

other compounds eluting through the column. Furthemore, this change in solvent syscem enabled

employment of a simpler isanatic elution, compareci to the more complex initial gr&t ehion.

Note 1: Solvent A: 0.1M Acetate Buffa, pH=4.1 ; Solvmt B: 0.1M Phosphate Buffer, pHz6.3

Note 2: Boxed #s within each of the foilowing 5 chromatogrvns represent Oh by which the H(e) peak is separatcd from the 2 peaks immcdiately adjacent to it.

Figure 4.1.3b Figure 4.1.3~

Typical Chrornatograms Generated h m HPLC Aaalysis of a Plasma Sample Using the correspondhg Soivent System

s O l v e n t A t B + ~ / ~ % Me- f ; RT HCe): 3- Figure 4.1.3f

Typicai Chromatograms Generatcd h m HPLC Analysis of a Plasma Sample Using the Carrsponding Soivent System

85/ lS t.06 90/10 1.82 95/5 1.49 100/0 1.05

Note: R, : Column Resolution

Mce) Peak

1 .O5

Table 4.13: Major Factors Considercd In Choosing the Optimum HPLC Solvent System

An 8S0h solvent A/15% solvent B was chosen as the optimum solvent system due to excellent

separation of the H(e) peak from both chromatographie peaks irnmediately adjacent to it.

4.1.4 HPLC Assay Validation

4.1.4.1 Accuracy Assessrnent

Table 4.1.41: Accuracy (% Deviation) based on hpücate Analysis of Standards Stored at -80oC

The average deviation on duplicate analysis of ail standards and OG stored at -800C arar no greater than 4.30/0.

mandard Cuirs #

Table 4.1.4~: Accuracy (% Deviation) based on Duplicate Analysis of Standards Stored at -2OoC

~ C P r v a #

Table 4.1.4d: Accuracy (% Deviation) based on Duplicate Analysis of QCs Stored at -20oC

The average deviation on duplicate anaiyas of all standards and QG stored at - 2 0 9 ~ ws no greater dian 7%.

53 4.2.42 Reproducibility Asses sment: Wàthin- & Between-Duy Vmiability

The reproducibility of our method was considered to be wibm amPpabk tirnits according to

guidelines put fornard by the HPB (Canada), FDA (US) and Shah etai (1992).

Table 4.1.4e: htn-D~Y Variabiiity (CV%) based on Dupiicate Analysis of Standards Stored at -80oC

The average CV on duplicate analysis of ail standards and OG stored at -80OC was no greater than 2.Z0/o.

Table 4.1.4g: Intra-Day Variability (CV%) based on Duplicate Analysis of Standards Stored at -20oC

Table 4.1.4h: Intra-Dax Variability (CV%) based on Duplicate Analysis of OGs Stored at -2OoC The average CV on duplicate au+ of all standards and QOI stored at -2BC wu no greater than 3.1°/o.

1 3 1 6.0 ( 6 . 3 16.4 15 .2 15 .5 16.1 15.7 16.1 1 8.2 1 6.2 Table 4 . M : Inter-Dav Vanability (CVOh) based on Dupiicate Anaiysis of M Stmd a- The average CV on duplicate anaiysis of d stuidards and OG stored at -2VC was no grener than 14.7Oh.

Table 4.1.5a: Average Change (%) within Any [H(e)] in standards (stored at -8O0C) Constituting Each Standard Curve Run During a 3 Mon& Stability Study

Table 4.1.5b: Average Change (%) within Any [H(e)] (stored at -2OoC) in Each Standard Curve Run During a 3 Month Stabiüty Study

There was no significant effect @-0.56) of storage temperature (-20°C or -80°C) on

concentrations of plasma H(e) Li samples stored during the 3 month aability study.

Table 4.1.5~ Factors Indicative of Standards and QCI Stability at

Table 4.1.5d: Factors Indicative of Standards and QCs Stabüity a 1x1

Analysis of the standard curve dopes observed on each day as weii as visuai inspection of the H(e)

chrornatographic peak retention time over the 3 month stability study indicated that there was no

degree of H(e) degradation during this time period.

57 4.1.5.2 S~bility of Homocyst(e)ine in Plasma Stored ut Room Temperatare and 4-8CC

Total homocysteine was found to be "reasonably" stable in plasma within a day (0-8 hours) at

room temperature or a week (0-7 days) at 4-8%.

The absolute change in H(e) concentration varied between 0.2 ro 0.5 pmoVL in plasma samples

stored at room temperature within 1 working day and benveen 0.4 to 1.4 1rnoyL in plasma

samples stored at 4-8OC for 1 week.

4.1.5.4 Stubility of Homocyst(e)ine in m o l e Blood Stored crt Room Tempertzture and 4-80C

Room Temperature ( H o ~ s )

Table 4.1.5g: Stability of H(e) in Whole Blood Stored in a "Standing Stue" and on a "Spin Mix" at Room Temperature within 1 Working Day

Average Absolute Change in [H(e)] (pmol/L)

"Standing S&te"m of MW "Sph MWAw of Subiccts CID

1

The rate of release of H(e) from eryduocytes/hour was observed to be 0.51 umol/L in sarnples

positioned in a "standing aare" and 0.60 umol/L in sarnples positioned on a "spin miu".

SLOPE = 0.51 3 = 0.92

SLOPE = 0.60 8 = 0.97

59

Table 4.15h: Stability of H(e) in Whole Blood Stored in a "Standing State" and on a "Spin Mk" at 4 - 8 C within 1 Working Day

Room Temperaîure

(Hours)

SLOPE = 0.023 1 = 0.02

The rate of release of H(e) from erythrocytes/hour was observed to be 0.023 umol/L in samples

positioned in a "standing aate" and 0.093 umoVL in samples positioned on a "spin &.

Average Absolute Change in [H(e)] (pmol /L)

"Sl<rnding Stute"Aw .d W- "Spin M ~ A V ~ . of subicctc CID

SLOPE = 0.093 8 = 0.48

4.1.6 Patient P h m a Sampie Anaiysis

Totai homocysteine is an endogenous compound and plasma free of this amino-acid is impossible

to main. Spiking pooled plasma with 4-30 umolA synthetic DL- homocysteine creared the

standards and QCs. The standard cuve produced by this procedure yielded an intercept that was

proportional to the endogenous H(e) concentration in the pooled plasma. Slope of the standard

m e established the relationship between a change in H(e) concentration and a change in deteaor

response (fluorescence). Therefore, using the equation of a line &am+ b), the intercept aiways

had to be ignored when cdculating patient plasma H(e) concentrations. This allowed patient

samples with H(e) concentrations lower than the H(e) concentration in the pooled plasma to be

detemilied accurately. If the intercept was not ignored, H(e) concentrations in these patients

would have appeared negative.

60 4.2 Trends and Resuks of Biochemical, Molecular and Chical Analyses of MI Cohort

4.2.1 Frequency, Age-Ruige and Gender Distribution

Of the 79 MI patients admitteci to CCU at SHSC d u ~ g the course of our non-consecutive pilot

study (September 97'Apd 98'),70°h (55/79) were males.

Figure 4.2.1: Frequency of Distribution by Age Groups of Males and Females Admitted with MI to The CCU at SHSC during the Non-Consecutive Püot Study (Sept 97-Apr98)

The highest MI frequency in maies (13%) and femaies (10%) was found to occur within an age

range of 61-65 and 71-75 respectively.

61 4.2.2 Thamolabile MTHFR Gene Frequency

The relation between MTHFR ailele frequency and genosrpe frequency in our patient cohort was

detemhed by tesring for the Hardy-Weinberg equilibrium. Frequenues of homozygous mutants

(+/+), heterozygous (+/-) and wild-type (4) for tM'IHFR genotype were 11.4%, 3 0 . 4 O b and

58.2% respectively. These frequenàes were similar to the genotype frequencies in the urban

Canadian population (+ / + : 12%, + /- : 48% , -/- : 40%) @?mer, Evrovski and Cole, 1997; Rozen,

1997). The deiic frequency of the alanine to valine substitution in our patient cohofi was 26.696,

similar to recent midies conducted among normal subjects and CAD patients, reporting a

33.5-3596 gene frequency (Dunn ad, 1998; WiIcken etal, 1996; Schmitz et ai, 1996).

Val N a 1 (+ /+)

Table 4.2.2a: MTHFR Genotype and Gene Frequmcy Distribution in MI Patient Cohort

Total 79

Genoeype frequencies for tMIHFR did not deviate from the Hardy-Weinberg equilibrium

(x2 -1.59 , p-0.45).

9

I

Reminder : Of our MI patient cohort (n=79), 66 wae idcntificd with complete biochemicai and molecdar data. Thercforc, unless otherwise stated, from here on (und section 4.2.5, pp,68), ai1 trends and results are bosed on thesc 66 MI patients.

Note: Unless otherwisc statcd, the consensus Canadiau cutsff lcvel of H(e) (515.0 pmol/L) was used to define non-hyperhomocyst(e)indc subjects in dW study.

5.6 0.114

62 4.2.3 Distribution of Homocyst(e)ine Levels

Using the consensus Canadian cut-off level of H(e) (1 15.0 pmoVL), 36% (24/66) of our MI

patients were mildly hyperhomocyst(e)inemic. However, according to the European cut-off level

(512.07 pmol/L), d d HH(e) was prevdent in 58% (38/66) of our MI patients. Mean H(e) level

of the 66 MI patients wu 14.0 k 5.5 pmol/L. Mean H(e) level of the mil*

hyperhomocyst (e)inemic MI patients (24/66) was 19.9 ir 3.9f pmoVL.

- - - . - 5.8 9-12 - - - -

13-16 - 6 .- -- - 17-20 21-24 25-28 29-32

.H(e) 15 - - --- - - - - 27 26 20 8 2 3

Total Homocysteine Range (umol/L)

Figure 4.2.3 Frequencies of H(e) Level within MI Patient Cohort

The H(e) range in which the highest frequency (27%) of MI patients could be categorized was 9-12

pmol/L, followed dosely by 26% and 20% frequencies in the 13-16 and 17-20 pmoVL range

groups respectively.

63 4.2.4 Association between Hornocyst(e)ine Levels and its Genetic and A q u M Determinants

Note: Results in the next 2 sections ody (42.4.1 & 42.42) arc bascd on 74 MI subjects on whom complcte data with respect to MTHFR, Factor V Leiden gai- stmis and H(c) lcvd was avrilable.

4.2.4.1 Homocyst(e)ne Levef und MTHFR Genofype

Patients urrying the MTHFR homozygous mutant genotype had a slightty higher rnean H(e) level

(14.M 3.8 pol/L) than those with the normal genotype (13% 6.2 pnol/L). The mean H(e)

lwel of heterozygous carriers for t M ï H l 3 was 15.3I5.5 p o l / L . This difference amongst

genotypes was not of significance (p-0.66).

Figure 4.2.4.1: Distribution of H(e) Levels Associated with Each tMTHFR Genotype

In cornparison to 16.2% (12/74) of In/perhomoqst(e)inemic patients who were homozygous

normal for MIHFR, ody 2.7% (2/74) of MI patients were hyperhomocyst(e)inernic and

homozygous mutant for tMLHFR genotype. Hyperhomocyst(e)inemia and heterozygosity for

tMEFR genorype was prwalent in 20.3% (15/74) MI subjects.

64 4.2.4.2 Hom O cyst(e)ine Level and Factor V Genotype

None of the MI patients were homozygous for Factor V Leiden (FVL) mutation. Only 4% (3/74)

were heterozygous, of which only 1 subjea was mildly hyperhomocyst(e)liemic (1 8.0 pmol/L).

Nomial prevalence of FVL mutation is 3-79'0 of the white population @&er et al, 1997).

4.2.43a Homocyst(e)ine und RBC Fokate Levels

The reference range of RBC folate at SHSC is 255-1000 nrnoVL. A significant inverse correlation

was found between plasma H(e) and RBC folate Ievels (r 4 . 4 6 ; p=O.OOOI).

Figure 4.2.4.3a Correlation Between H(e) and Totd RBC Folate Staîus Among MI Patients

65 4.2.4Jb Homocyst(e)ine and Viiamin Bi2 Levels

The reference ange of vitamin B, at SHSC is 140-750 pmoVL. An inverse, but not significant

correlation wu found between plasma H(e) and vitamin B,, levels (r =-0.15; p-0.23).

Figure 4.2.4.3b: Correlation Between H(c) and Vitamin B12 $katus A m w g MI Patients

66 4.2.4.4 RBC Folate Level and tMTHFR Genotype

Mean RBC folate level iunong MI carriers of homozygous mutant tM?HFR genotype was

unexpectedly higher (794.81377.3 nrn01L) compared to levels in homozygous normal

(640.4G65.6 nmoVL) MTHFR genotypes (SHSC Reference Range: 2%- 1 O00 nrnol/L) . However, upon exclusion of IWO RBC folate levels that were potenuai outliers in the tMTHFR

homozygous mutant group, mean RBC folate levei was 563.8I 148.4 nmol/L. Mean RBC folate

level among subjects heterozygous for tMlHFR genotype was 673.7k231.2 nrnol/L. Difference in

mean RBC folate levels among carriers of the three tM?HFR genotypes was not of significance

Figure 4.2.4.4: Distribution of Mean RBC Folate Levels ktsociated with tMTHFR Genotype Stams in MI Patients

Reminder: Of our patient cohort (n=79), 53 wcrc idcntif~ed with complete clinical (KG) data; 37 were idcntificd with an in hospital composite outcorne and 22 wetc eligible for thrombolytic (TPA/SK) thmpy.

4.2.5 Homocyst(e)ine Leveis and b Hospital Composite Outcorne

An in hsprtnl composite outcome of death, renurent ischunia, or CHF wari observed in 43.21

(na 16) of the 37 patients d o m e n d with mild hyperhomocyst(e)inemia (mean H(e): 20.4rt5.7

pmoVL). Prevdence of HH(e) in this MI group that mffered a composite outcome was 6 to 9

times greater than the estimated 5 to 7 percent occurrence of rnild HH(e) in the general population

(Welch and Loscalzo, 1998; McCuUy, 1996; Ueland and Refsum, 1989).

4.2.61 Homocyst(e)iae Leveis and Successful Reperfùsion

Of the 22 MI abjects who received rhrombolytic therapy, 13 were successfully reperfused. Of

these 13 subjem, 76.9% were non-hyperhomocyst(e)inemic (mean H(e): 10.59.7 pmoVL)

compared to 23.1 % who were hyperhomocyst(e)ineMc (rnean H(e) : 19.W2.O pmoVL).

Figure 43.61: Distribution of SuccessM Rcpafusion Associated with H(e) Status

68 4 3 . 9 : Homocy st(e)ine Levels and Non-Successhil Repehsion

Of the 22 MI subjects recùving a thrombolytic dosage, 9 were n o n - s u c c e s s ~ reperhsed. Of

chose 9 patients, 44.4% were hyperhomocyst(e)inemic (mean H(e): 21 S16.2 prnol4J and 55.6%

were non-hyperhomoqst (e)inemic (mean H(e): 12.9k1.7 pmolL).

Figure 4.2.6b: Distribution of Non-Successful Reperfusion Associated with H(e) Status

69 4.2.7 Homocyst(e)ine Leveis and Successfd/Non-Successfd Reperhsion

A larger propomon of non-hyperhomocyst(e)inernic (1 O/S=GOh) compared t O

hyperhomocyst(e)inernic (3/4-43%) MI patients were successfuy. repehsed. However, this

difference was not statisticdy significant (p-0.38).

Table 42.7: Distribution of Successfd & Non-Successfd Rcpafusion Associated with HH(e) and Non-HH(e)

4.2.8 MTHFR Genoqpe and Successful/Non-Successfd Reperhtsion

Among successfuy. and n o n - s u c c e s s ~ reperfused subjeas, 69% and 78% were homoygous

nomai carriers for MTHFR genorype respectively; 23% and 22% were heterozygous respectively;

8Oh and 0% were homozygous mutants respeaively. There did not appear to be a significant

association between tMTHFR mutant genotype carriers and reperfusion (p=0.69).

Table 4.2.8: Distribution of Successfd (Ir Non-Successfd Rcpcrfusion Associated with Each M E E R Genotype

5.0 DISCUSSION

5.1 Analyticai Aspects

The HPLC method for the detennination of plasma H(e) describeci in this thesis was a

modification of the method described fint by Anki and Sako (1987). Both our method and the

one described by Anki and Sako involved pre-column derivatization of plasma H(e) with a thiol-

speafic fluorogenic reagent, SBD-F, and separation on a reversephase HPLC column. Compared

to the fluorescence spectrophotometer used by Araki and Sako, the fluorescence deteaor available

in the SHSC Pharmacy QC laboratory was superior, as it provided higher sensitivity and lower

baseline noise. Therefore, it was necessary to optimize the excitation and emission )c maxima for

H(e) on our detector, which was determined to be 240 nm and 508 nrn respectively. The proposed

method utilid a reduûng agent, TRBP, in order to reduce free oxidized homocysteine in the non-

protein bound fraction of plasma (ir., homocystine and cysteine-homocysteine mixed disulfide)

and to release homocysteine from plasma proteins. We observed complete reduaion and release of

protein-bound homocysteine by ueating plasma with 70 pL of 10% TnBP at 4OC for 30 minutes.

Protein precipitation was achieved wirh TCA and inclusion of Na2EDTA in die reaction mixture

enwed inhibition of homocysteine re-oxidation. Incubation of reducedheleased plasma

homocysteine with 250 PL (1 mg/mL) of SBD-F at 61S°C for 105 minutes ensured complete

derivatization of H(e). The sensitivity and specificity of our HPLC method was dependent on

optimization of these derivatization reaction conditions.

Improve separation between H(e) and other plasma adducts was achieved with a shoner analyticai

column (4.6 mm x 15 cm C, Supelcosil, 3 pm particle size) compared to the previous column (3.9

mm x 30 un C,, Novapak, 5 pm panide size). Smaller partide size of padrlig within the shorter

column yielded an inaeased surface area and consequendy more efficient separation. This

improved separation can be observed in the chromatograms depicted in Figure 4.1.3a(ii) (pp.48).

An ultimate optimal separation benveen H(e), cysteine and other plasma compounds was attained

with a HPLC solvent system of 85% 0.1M acetate buffer, pH=4.1/15% 0.M phosphate buffer,

pH-6.3), containing 2.025% MeOH. Furchermore, the H(e) chromatographic peak parameter,

peak-tailing (1.57) indicated an acceptable chromatographic perfomance with the chosen solvent

system. Ail of these parameters considered in dioosing the optimum HPLC solvent system can be

71 observed in Table 4.1.3 bp.51). Cornparison between a rypical chromatogram generated from

HPLC analysis of a plasma sample using the chosen solvent system compared to chromatograrns

using other vuylig compositions of the solvent systems can be observed in Figures 4.1.3b-4.1.3f

(pp.49-50). Change of solvent system also enabled employment of a simpler isocratic elution

compared to the more complex initial gradient elution. Thus, the solvent system was not only

modified, but also simplifieci to yield superior separation between H(e) and other

compounds.

Validation of Our method, with respect to accuracy and reproducibility was tested over a pet-iod of

3 months in concurrence with the aability study. The average deviation on duplicate analysis of ail

standards and QC samples stored at -80°C and -20°C was no greater than 4.3% and 7.2%

respectively. The average iwaday variability of aU standards and QC samples stored at -80°C and

-20'~ was no greater than 2.2% and 3.1% respectively; similarly, the average irrterday variabilicy

was no greater than 7.7% (-80°C) and 14.7% (-209. These analyses indicated that plasma H(e)

concentrations were measured accuntely and reproduably.

There was no significant effect (~10.56) of storage temperame (-80' or -20°C) on the aabiliry of

plasma H(e) samples. Statisticai adysis of plasma H(e) concenvation time data in this mdy was

b t e d to least squares linear regression, because demon~ration of a trend for the concentration to

decrease was considered more important than demonstrating a aatistical difference in

concentration between any two days (Waker EZ ai, 1990). In fact, according to Waker et al, the

random fluctuations in concentration wound a h e of 'best fit" are not of practical importance

and should be considered 'noise" or experimental error. The average slopes of standard curves

generated from standards and QC samples stored at -80°C and -20°C were 2,087 (range: 1,920 to

2,483) and 2,069 (range: 1,831 to 2,344) respeaively (hbles 4.1.5c/4.1.5d, pp.56). Clearly, these

slopes did not fluctuate substantially during the 3-month stability period. The average r-value for

di standard w e s generated from standards and QC samples aored at -80°C and -20°C were

0.9979 (range: 0.9939 to 0.9994) and 0.9980 (range: 0.9969 to 0.9990) respectively. This depicted

very good heu correlation within the standard curves. Our stabiliy d e s as well as visud

72 inspection of the H(e) chromatographie peak retention tirne indicated that there was no degree of

H(e) degradation during the 3-month period.

In an attempt to establish proper sarnpling conditions for H(e), stability of H(e) in plasma was

evduated at room temperature and 4-8OC during a Mcai day and week t h e period. The average

absolute change in H(e) concentration varied between 0.2 to 0.5 ymoVL in plasma samples stored

at rooin temperature/day and between 0.4 to 1.4 pmoVL in plasma samples aored at 4-8OC/week.

This showed plasma H(e) to be nasmddy stable at room temperanue and 4-8OC. To gain more

insight into the metabolisrn of homocysteine in blood after smpling and to establish proper

sampllig conditions for H(e), we assessed the stability of H(e) in whole blood stored at room

temperature and 4-8OC. We found that in whole blood stored at room temperanue, the rate of

release of H(e) from exythrocytes was observed to be 0.51 pnol/L/hour in sarnples positioned in

a "standing aaten and 0.60 pmol/L/hour in sarnples positioned on a "spin mix" (simularing

sarnple transpoxtation). In refrigerated (4-8OC) blood samples positioned in a "standing nate", the

rate of release of H(e) from eq&rocytes was observed to be 0.023 pmol/L/hour, in blood

samples positioned on a "spin mix", the rate of release of erythrocytes was 0.093 umol/L/hour.

ciearly, since plasma stored at room temperature for 1 working day or 1 working week did not

show any signifiant changes in H(e) concentration, the increase of plasma H(e) (0.51

pol/L/hour) after aorage of whole blood at room temperature mus have had a cellular origin.

In order to confirm that it was erythrocytes and not leukocytes responsible for the increase of

plasma H(e) in aored blood, Andersson a al (1992) demonstrated that tubes with depleted

leukocytes and unchanged eIythrocyte contenu showed the same plasma H(e) concentration as did the non-manipulated reference tubes, but that leukocytes-enriched and erythrocyte-depleted tubes

showed a smailer increase of plasma H(e) than did the reference tubes. Thus, Andersson et ai

proved that exythrocytes were mady responsible for the increase of plasma H(e) in stored blood.

Our data showed that there was an appreaable release of plasma H(e) in whole blood stored at

room temperature for 1 working day ("standing aaten: 0.51 pmoVL/hour; "spin mix": 0.60

pmol/L/hour), but chat this export from erythrocytes was subaantiaily retarded ("standing aare":

73 0.023 pmol/L/hour, "spin mixn: 0.093 pmol/L/hour) if blood samples were refngerated for upto

Although the HPLC method discussed here has been an optimization of the method previously

desuibeà by Anki and Sako (1983, improvements to this method have ensured a simple, but

prease, selective and sensitive determination of H(e) in patient plasma samples. Furthemore,

validation of this HPLC method, with respect to accuracy and reproducibility was not conduaed

by Araki and Sako or by any other investigators who have adopted this method for H(e)

quantitation. As asnitely pointed out by Shah et al (1992), any modification of an analyùd method

requires re-validation of the procedures. We may be the only investigators who have complied with

this dedaration. Our stability experiments provide us with confidence as to the integrity of our

plasma samples.

While the forthcoming homocysteine FPIA immunoassay may very well be equdly accurate to our

HPLC method for homocysteine quantitation, our completed method optknization, validation and

stability studies, provided us confidence in the validity of the HPLC homocysteine assay and

subsequently our patient H(e) redts.

74 5.2 Chical, Biochemical and Molecular Aspects

The r ed t s of rhis prospective pilot study confinneci that HH(e) was an important propostic

indifator in myocardial infaraion. This w u evident from our dinical data which showed that

43.2% of MI patients who had suffered an bz hopid composite outcome of death, rement

ischemia or CFF, were hyperhomocysr(e)inemic (mean H(e): 20.4I5.7 pmoVL). Prevaience of

HH(e) in this patient cohort was 6 to 9 times greater than the estimateci 5 to 7 percent prevalence

of mild HH(e) in the general population (Welch and Loscalzo, 1998; McCuily, 1996; Ueland and

Refsum, 1989). Furthemore, we found a decreased effectiveness of thrombolytic agents ('TFA/SK

as per treatment protocol) in hyperhomocyst(e)inemic MI patients. Of those subjeas successfuliy

reperfused (1 3/22), 76.9% were non-hyperhomocyst(e)inemic (mean H(e): 10.5k2.8 pmoVL)

compared to 23.1% of hyperhomocyn(e)kietnics (mean H(e): 19.0f2.0 pmoV L) (Figure 4.2.6a,

pp.67) Among the 9 patients who were not successfully reperfused folowing thrombolytic

therapy, 44.4% were hyperhomocyst(e)inernic (Figure 4.2.6b, pp.68). When we compared the

proportion of non- hyperhom~cyst(e)inemics (IO/ 15 = 67%) and hyperhomocyst (e)inemics

(3/7=43%) that were successfully reperfused, the difference was not found to be aatistically

significant (p-0.38) rable 4.2.7, pp.69). However, this may have been confounded by the limited

sample size and the higher nwnber of hyperhomocyst(e)inemics in this patient cohort than in the

general urban Canadian population. Confmation of a possible clinical and natistical significance

of rhis pilot mdy would require a larger sample size.

Our objectives induded an investigation of MiHFR and Factor V genes among our MI patient

cohorr, specifcaily screening for the C,T and G,,,A mutations, respeaively. Within the MI

groups niccessful.ly and non-successfdy reperfused, 69% and 78% were homozygous nomal

carriers for MIHFR genotype, respeaively; 23% and 22% were heterozygous, respectively; 8%

and O% were homozygous mutants, respectively Fable 4.2.8, pp.69). The observed la& of

association between thîTHFR statu and reperhsion (p-0.69) was not confounded in any way.

Upon screening our patients for Factor V Leiden, we were unable to detemine a relation between

reperhsion and this mutation. Only 4% of our MI subjects were bae>alsuc for FVL. Normal

prevalence of htmqpq for this mutation is 3-7% of the white population (Rdker et al, 1997).

Only 1 of the 3 MI subjects heteroygous for FVL had mild HH(e) (18 pmoVL), but no data on

75 reperfusion w u available as this subject was not eligible for thrombolytx therapy. A larger sample

size is necessary before die coexistence of HH(e), FVL or tMlHFR can be investigated.

We observed mild HE-I(e) in 36% (Canadian cutsff: 515.0 pmoVL) of MI patients, as cornpared

to an e h a t e d 5 to 7 percent of rnild HH(e) in the generai population (Welch and L o s c h , 1998;

McCuUy, 1996; Ueiand and Refsum, 1989). Our results were sirnilar to those obtained by Dunn et

al (1998), who found mild HH(e) in 33.5% of 150 Canadian patients with CAD. The most

comrnon H(e) range was 9-12 umoVL, observed at a frequency of 27% in our MI cohorr, folowed

closely by frequenâes of 26% and 20% in the 13-16 and 17-20 m o V L range, respectively (Figure

4.2.3, pp.62). This distribution combined with the finding that the mean H(e) level (14.Ckk5.5

pmol/L) was ody slightly lower than the Canadian cut-off level, supported suggestions made by a

handful of studies that a graded response rarher than a threshold effen of H(e) levels would be

more appropnate. In a meta-analysis of 27 snidies conducted by Boushey et ai (1995), it was

demonarated that a H(e) elevation of 5 pmol/L was equivalent to the nsk of CAD associared with

an elevation of 0.5 mmol/L in total cholesterol. In a prospective study, dy,gard et al (1997)

estimated the mortality ratio for an increase of 5 umol/L in the H(e) level among CAD patients to

be 1.6 between 10 and 15 pmol/L and 2.5 between 15 and 20 pmoVL. Most recently, Bots et al

(1999) suggested a MI nsk increase by 6-7O/0 for every 1 pmoVL increase in plasma H(e) levels.

In n o d homocysteine metabolism, the rnajority (slighdy > 50%) of homocysteine is recycled

into methionine by the transmethylation pathway. This anabolic pathwiy is dependent on

and the ubiquitous methionine synthase. Methionine synthase reqkes vitamin BI2 as a cofactor.

We found an inverse, but not signifiant correlation (r--0.15; p-0.23) berneen vitamin B,2 and

homocysteine concentrations (Figure 4.2.4.3b, pp.65). The mode of action by which folate plays its

role in maimainhg normal plasma H(e) levels has been poaulated by Landgren e t al (1995) as

follows. Ln remethylation, the prirnaty methyl donor for the vitamin B,, dependent conversion of

homocysteine to methionine is 5-methy1tevhydrofolare (CH3THF9, the prinaple f o m of

ciradatory folate. This p r h c t , S-CH,lHF is synthesized from 5,lO-methylenetetrahydrofolate

( 5 , l O - m by the enzyme MTHFR Increased CH,THF d l enhance the remethylation of

homocysteine to methionine (Bramwm a al, 1988; Brattstrom, Hultberg and Hardebo, 1985).

76 Subsequendy, this leads to Licreased levels of S-adenosyhethionine, an activator of homocysreine

catabolism (Seihub and Miller, 1992). However, the event of a mutation in MTHFR such as the

alanine-to-vahe substitution, renders the enzyme thermolabile and sustaining an activity <50°h of

the normal MTWR mean. This would result in inadequate folate availabilicy, thereby inhibiting

homocysteine removd due to decreased homocysteine remethylation, consequendy redting in

HH(e). As expected, we fomd a signifiant inverse correlation (r=-0.46; p=0.0001) between RBC

folate and H(e) concentrations (Figure 4.2.4.35 pp.6)). This fmding supported earlier studies

(Rozen, 1996; Verhoef et al, 1996 and Pancharuniti ad, 1994; Robinson etal, 1998).

We are not aware of the RBC folate and plasma vitamin BI, concentrations below which H(e)

levels begin to increase. However, one explmation for the aronger influence of RBC folate than

vitamin B,, concentration on the H(e) levels mong our MI subjects rnight be because the

threshold for inaeasing H(e) levels appear at a higher folate concentration than for vitamin B,,

levels. Supporting this possibility is the suggestion by Jacques et al (1996) that at equivaent folate

concentrations, patients homozygous for the thermolabile variant of MTHFR genorype have

higher H(e) levels compared to subjecu with the wild-type enzyme. Furrhemore, Sc- et al

(1996) suggested that the detrimenta effect of C,T mutation might depend on permissively low

levels of folate. In subjects replete of folate, and without other deficiencies of homocysteine-

metabolizing enzymes, the mutation may be tolerated without consequences on biochernid or

clinical phenotype. However, in individuals with insufficient folate intake (and in those with

concomitant other enymatic defitits of homocysteine metabolism) the mutation may contribute

to increased H(e) and heightened cardiovascular risk. Consequently, these midies suggea that

individuals with MIHFR rnay have a higher folate requirement for regdation of plasma H(e)

concentration, and more importandy, suggest a theriapeutic mtegy (folate supplementation) to

prevent fasthg m ( e ) in such persons.

In contrast, our data did not qulLfy the role of tMT'HFR as a detemiliant of fasting H(e) levels,

either independen* or with respect to the interaction between tM'IHFR genotype and folate

aatus. Among our MI subjects, frequencies of the three genotypes were 11.4% (+/+ ;

H(e):14.5&3.8 p o V L ) , 30.4% (+/- ; H(e):15.3f 5.5 pmoVL) and 58.2% (-/- ; H(e):13.9* 6 2

77 p o V L ) (Table 4.2.23 pp.61; Figure 4.2.4.1, pp.63), similar to fiequenaes reported in other

Canadian d e s by Miner a al (1997) (adult CaucWan), Dunn et al (1998) (selected CAD patients)

and Frosa et al (1995) (non-selected French Canadians). The allelic frequency of the alanine to

valine substitution in our patient cohort was 26.6% (Table 4.2.23 pp.61), similar to a recent report

of 150 selected (CAD) Canadian patients (33.5%) (Dm et al, 1998) and other investigations

conducted among both normal controls (35%) and CAD patients in the general North Arnerican

population (Wilcken et al, 1996; Schmitz ad, 1996). Although we observed mild HH(e) in 36% of

MI patients, as compared to an estirnated 5 to 7 percent of the general population (Welch and

Loscalzo, 1998; McCuliy, 1996; Ueland and Refsum, 1989), we did not find any difference in the

prevalence of mutant M'T'HFR between our patient cohort and the normal urban Canadian

populations [12% (+ /+), 48% (+ /-), 40% (4) ] Wner, Evrovski and Cole, 1997; Rozen, 1997),

nor did we observe a significant association bmeen homozygosity for tM?HFR and H(e) levels

@-0.66) (Figure 4.2.4.1, pp.63). Furthexmore, we did not observe any significant variation in mean

RBC folate levels arnong carriers of the tMIHFR genotypes @-0.42). In fact, mean RBC folate

level arnong MI carriers of homozygous mutant tMTi-ER genotype was unexpectedty higher

(794.8k3 77.3 nrnol/L) compared to the average level in homozygous n o d s (640.42265.6

nmol/L) and heterozygotes (673.7f231.2 nmoVL) (Figure 4.2.4.4, pp.66). One r r m n for an

unexpeaed elevation in the mean RBC folate concentration of mutant homozygotes was that it

might have been skewed due to two subjects having high levels (1207 and 1307 nrnol/L). Using

the reference range observed at SHSC (255-1000 nmol/L), folate deficiency was not correlated

with homozygosity for MïHFR Low folare natus, however may have been associated with this

mutation, afrer exdusion of the nvo potential outiiers, which yielded an average RBC folate level of

563k148.4 Nnom. Supporting the potentid significance of low folate aams is a recent

investigation by Robinson d (1998) who reported RBC folate levels to be lower in case subjects

wirh CBD, PVD and CAD than in control !abjects (81911.0 versus 87621.0 nmol/L; p-0.005).

Another reason for the unexpected elevation in mean RBC folate concenuarion of mutant

homozygotes ma/ be explained by the suggestion made by van der Put et al (1995) rhat

homozygosity for the C,T mutation redts in elevated H(e) levels and a redistribution of folates,

i.e., elevated RBC folate and lowered plasma folate levels. However, since RBC folate is recognized

as a superior indicaor of long-term folate stores as compared to plasma folate levels, which simply

78 reflect a folate flux, we chose to determine total RBC folate status in Our MI subjects.

Funhamore, lower RBC folate statu in subjects homozygous mutant for tMTHFR has been

previously reported (Zittoun ct al, 1998). In this moa recent midy, total and CH,THF, the

product of lVTIHFR were both lower in RBCs among carriers for tM'IHFR homozygous mutation.

79 6.0 Conclusions

Mildly elevated H(e) levels appeared to be of prognostic value in Ml patients when in

composite outcome of death, recurrent ischemia or CHF was used as a primary endpoint. We

obsemed a decreased effectiveness of thrombolyac t h e r a ~ in hyperhomocyst(e)inemic patients.

There was no association found between reperfùsion and CM= (p-0.69) or FVL natus.

Prevalence of FEI) in this MI group was 6 to 9 times greater than estimated mild HH(e) in the

general population. Distribution of H(e) levels among our subjects suggested that a graded

response rather than a threshold effen of H(e) levels would be more appropnate. A significant,

inverse correlation (r- -0.46; p- 0.000 1) between RBC folate and H(e) concentrations w u

observed. However, an inverse but insigdcant correlation (r- 4.15; p0.23) was found between

vitamin BI2 and H(e) concentrations. The variant MTHFR gene frequency (26.6%) and

hornozygous genocype frequency (1 1.4%) among Our MI patients was simiiar to that of normal

wban Canadian populations (variant gene frequency:3S0h; homozygous mutant genotype

frequency:120/o). There was no significant variation in mean RBC folate concentrations among

carriers of the MiHFR genorypes (p4.42); mean level among homozygous mutant carriers was

unexpectedly higher, but may have been obscureci by two RBC folate levels that were porentiai

outliers.

80 7.0 Recommendations

Although the results are not statiaically signifiant, our study shows that MI patients having

normal H(e) levels have about rhree times {non-HH(e): n-3; HH(e): n= 10) an improved chance

of successful reperfusion following thromboiym therapy compared to subjects with an elevated

H(e) level. This hding is in itself an important dinical endpoint with respect to cost and tirne and

warrants a subsequent, prospective study with a greater patient cohort. A larger number of subjects

eligible for thrombobc thenpy would be necessary to allow for discriminatory power to detect a

significant difference in the proportion of non-hyperhomocyst(e)inemics and

hyperhomocyst(e)inemics that are niccessfuy. reperfused. To attain this, it is recommended that in

a subsequent study, consecutive MI patients be recmited. Using the pmt hoc sample size caldation

for a didiotomous outcome variable (Stoiley and Suom, 1986), a sample size of approximately X X )

MI subjects, eligible for thrombolyric thenpy would be required. This sample size calculation was

based on r ed t s of our study. Furthemore, asniming a 50% reduction in the incidence of

composite outcome (deat h, recurrent ischernia, CHF) in non-h y perhomocyst (e) inemic patients

com~ared to hyperhomocyst(e)inemic patients, a sample size of 169 MI subjects should provide a

80°h power to detect a signifiant difference in composite outcome between the

hyperhomocyst(e)inemic (43Yt) and non-hyperhomocyst(e) inemic patients (2 1.59/0), based on a

dichotomous outcome variable (Stolley and Strom, 1986).

Although RBC folate is a supenor indicaror of longtemi folate stores as compared to plasma

folate levels which reflect a folate flux, it is recommended that plasma folate also be measured.

Since 80-90% of the circulatory form plasma folate is CH3THF, the product of MTHFR, abnormal

plasma folate in conjunction with abnormal RBC folate and plasma H(e) levels could more

persuasively suggest a defect in MTHFR.

It has been ascenained in the European Concerted Action Project (constituting 19 clinical centres

in 9 European counuies), that approximately 30% of persons with HH(e) had isolated poa-

methionine-loading (PML) HH(e), with normal faaing H(e) levels. These rwo distinct forms of

HH(e), PML and fasting are both associated with an increased inadence of CAD. If ail patients at

risk for mild HH(e) are to be identified, a PML t e r is necessary, but mry be logisticaly complex.

81 Altematively, impairment to the pyidoxal-5'-phosphatedependent transsulfuration pathway can

be monitored via vitamin B, statu, while impairment to the remethylation pathway can continually

be monitored via fasting H(e), folate and vitamin B,2 statu. Therefore, it is recornmended that

vitamin B, levels be determined in d MI patients recnited in a subsequent SU+.

If the dùiical importance of our pilot study were confimd by a subsequent nudy, a significant

impact on future management of MI would be made. Homocyst(e)ine determination could be

considered a STAT or priority test as it could be used for prognostic and therapeutic purposes.

Antioxidants could be administered to attenuate the adverse effects of HH(e). Funhermore,

loweruig H(e) levels with vitamin supplements could be used as a long-term thenpeutic maneuver.

However, most importantly, if thrombolyuc therapy was indeed ineffective in

hyperhomocyst(e)inemic MI patients, then the primary care physician could be encouraged to seek

other means of achieving patency in the infarct-related artes: such as angioplasty with or without

st enting.

82 8.0 References

Amundsen T, Ueland PM and Waage A. Plasma homocysteine levels in patients with deep vein thrombosis. Arteriosdev 7hd Vau Bid 15:1321, 1995.

Andersson A, hdgren A, Hultberg B. Effea of thid oxidation and thiol export from erythrocytes on determination of redox status of homocysteine and other thiols in plasma from healthy subjeas and patients with cerebral infaraion. Clin Chen 41:361,1995.

k a k i A and Sako Y. Determination of free and total homocysteine in human plasma by hi&- performance liquid chrornatography with fluorescence deteaion. J &mqy 422:43, 1987.

Araki A, Sako Y, Fukushima Y ad. Plasma sulphydryl-containing arnino acids in patients with cerebral infaraion and in hypertensive rats. A h - 79:139, 1989.

Arnesen E, Refsum H, Bonaa KH ad. The Troms~ Smdyserurn total homocysteine and rnyocardial infarction, a prospective midy [Abstraa]. 3rd Ita Gxfm E b d w Ckhbgy, June 27- Jdy 1, 1993, Oslo, Norway.

Arnesen E, Refsum H, Bonaa KH &al. S e m total homocysteine and coronary heart disease. Int J E p z a b d 24:704, 1995.

Bachmaier K, Neu Nikolaus, de la Maza UI ad. Cldzqdu infections and heart disease linked through antigenic mimiay. SMnre 283: 1335,1999.

Baumgartner ER, Ulick H, Maurer R et d. CongenitaI defea in intraceilular cobalaMn metabolism resuiting in homocystinuria and methylmaionic aciduria H$u?laca P& Acta 34465, 1979.

Bienvenu T, Ankn A, Chadefauz B ad Elevated totai plasma homocysteine, a risk factor for thrombosis; relation to coagulation and fibrinolyuc parameters. Th>rmbRes 70: 123, 1993

Boers GHJ, Smds AG, Trijbels FJ a al. Heterozygosity for homocystinuria in premature vascular, peripheral and cerebrai occlusive arterial disease. N Engl j Md 313:709, 1985.

Bonom AG, Jacques PF, Nadeau MR al. Post-methionine load in hyperhomocysteinemia in persons with normal fasting plasma homocysteine: hiriai resuits from the NHLBI Famdy Heart S e . A u 116:147,1995.

Bou ML, Launer LJ, Lindernans J etal. Homocysteine and short-texm risk of myocardid infarction and stroke in the elderty. A d hm Md 159:38, 1999.

Boushey CJ, Bereseford SA, Omenn GS ad. A ~uantitative wessment of plasma homocysteine as a risk factor for vascular disease: Probable benefits of inaeasing folk acid intakes. jAMA 274: 1049,1995.

83 Brattstrom L, Hardebo JE, and Hultberg B. Moderate homocysteinemia. A possible risk factor for arteriosclerotic cerebrovasdar disease. Smke 15: 101 2, 1984.

Bratunom L, Hultberg B, and Hardebo J. Folk acid responsive postmenopausal homocysteinemia. MekrEdzPn 34: 1073, 1985.

Brattstrom L, Israelsson B, Jeppsson JO et al. Folk acid - an innocuous means of reducing plasma homocysteine. Sad j Clin L.ub hnea 48:î 15, 1988.

Brattstrom L, Israelsson B, Norrving B et al. Impaired homocysteine metabolism in early-onset cerebral and peripheral occlusive anerial disease. A t b d i s 8 1 :5 1, 1990.

Brattstrom L, Tengbom L, Lagerstedt C ad. Plasma homocysteine in venous thromboembolism. Haenûnasis 2151, 1991.

Brattstrom L, hdgren A, Israelsson B a al. Hyperhomocysteinemia in stroke: Prevaience, cause, and relationships to type of aroke and suoke risk factors. EurJ Clin t i 222 14, 1992.

Cabot RC and Painter FM. Case Records of the Massachusetts General Hos~ital. Case 19471: Marked Cerebral symptorns followhg a limp of three months duration. N E& J ~ t d 209:1063, 1933.

Carson NAJ and Neill DW. Metabolic abnormalities detected in a survey of mentally backward individuals in Northem Ireland. A d DLc Child 37505, 1962.

Carson NAJ, Dent CE, Field CMB ad Homocystinuxia Clinical and pathological review of ten cases. jP& 66:565, 1965.

Clarke R, Daly L, Robinson K et al. Hyperhomocysteinemia: An independent nsk factor for vascdar disease. N Engl J Md 324: 1 149, 199 1.

Cod BM, MalLiow MR, Berner N a al. Elevated plasma homocysteine in acute strokr and TIA: A possible independent risk factor for aroke. Smke 21572, 1990

Cramer DA. Homocysteine vs Cholesterol. Lab Md 29:410,1998.

de Franchis R, Mancini FP, D'Ange10 A a al. Elevated total plasma homocysteine and 677C+T mutation of the 5,iO-rnethrienetetnliydrofolore reductase gene in thrombotic vascular disease. Am ]Hm Gbet 59:262, 1996.

de Groot PG, Wdems C, Boers GHJ i al. Endothelid cell dysfunction in homocystinuria Eur J C k haea 13:405, 1983.

Duciman NPB, Hicks C, Wang J a al. Human arterial endothelial cell detachment in vitro: Its promotion by homocysteine and cysteine. Athemsdercrris 91:77,1991.

84 Dunn J, Ti& LM, Bata 1, Johnstone DE ad. Rdation of a Common Mutation in Methylenetevahydrofolate Reduaase to Plasma Homaysteine and Eady Onset Coronary Anery Disease. Gb B& 3 l:%, 1998.

Dyan AD, Ramsey RB. An inborn error of vitamin B,, maabolism associated with a cellular defiuency of coenzyme f o m of the vitamin. J N d SQ 23:117,1974.

Eliason SC, Ritter D, Chung HD &al. Interlabontory variabili~ for H(e) andysis in plasma. Ch 0S8n 35:315,1999.

Engbersen W, Franken DG, Boers GHJ ad. Thenn01abile 5,lO-Methylenetetrahydrofolate Reductase as a cause of mild hyperhomocysteinemia. Am /Hm Ge2B 56:142,1995.

Fabbro D, Desnick RJ and Grabowski GA. Gaucher disease: genetic heterogeneity w i t h and among the subtypes detected by immunoblonlig. Am J Hm G ~ z 40: 15,1987.

Falcon CR, Cattaneo M, Panzeri D et al. High prevalence of hyperhomocysteinemia in patients with juven.de venous thrombosis. Artenb& 7bwzb 14:1080, 1994.

Fenton WA and Rosenberg LE. Inherited disorden of cobalamin transporr and metabolism. In: Scriver CR, Beaudet AL, Sb WS, Valie D, eds. 77%- hasis ofinhztddisazse. Th eâtion. New York: McGraw-Hill, 3 129,1995.

Fermo 1, Vigano'D'Angelo S, Paroni R ad. Prevaience of moderate hyperhomocysteinemia in patients with early-onset venous and atterial occlusive disease. Am Ihem Md 123:747, 1995.

Fritsh EF and Maniatis T. Mo& Gkmag: A L a h a w M d . 2nd Ed. Section 2.1,1989.

Frosst P, Blom HJ, Milos R &al. A candidate genetic risk factor for vascular disease: a cornmon mutation in methylenetetrahydrofolate reductase. Nat Ger 10: 11 1, 1995.

Fryer RH, Wilson BD, Gubler DB ad. Homocysteine, a nsk factor for premature vascular disease and thrombosis, induces tissue factor activity in endotheliai cells. Arteviosder %O726 13: 1327, 1993.

Futterman LG and Lemberg L. Fifty percent of patients with coronary artery disease do not have any of the conventional risk factors. Am J Cni GRe 7:240, 1998.

Genest JJ, McNarnara JR, Salem DN et al. Plasma homocyst(e)ine levels in men with premature coronary anery disease. /Am 6ü Gmüd 16: 1 114, 1990.

Gerritsen T, Vaughn JG and Waisman HA. The identification of homocystine in the urine. Bi& B&s Res Gnmun 9:493,1962.

Goyette P, Sumner JS, Milos R a al. Human methylenetnnhydrofolate reductase isolation of cDNA, mapping and mutation identification. Na Gmt 7:55l, 1994.

85 Goyette P, Fross P, Rosenblatt DS ad. Seven novel mutations in the methylentetrahydrofolate reduaase gene and genorype/phenorype correlations in severe merhylenetetrahydrofolate reductase defiaency. Am /Hm GBla 56: 1652, 1995.

Graeber JE, Slott JH, Ulane RE ad. Effen of homocysteine and homocystine on platelet and vascuiar arachidonic aad metabolism. P+ Ra 16~490, 1982.

Graham IM, Daly LE, Refsum HM a al. Plasma homocysteine as a nsk factor for vascular disease. The European Conmed Aaion Report. JAMA 277: 1775,1997.

Guiilerm C, Lellouche F, Daniige L ad. Rapid detection of the G1691A mutation of coagulation factor V by PCR-mediated site-directeci mutagenesis. Ch C b z 42:329, 1996.

Hajjar KA. Homocysteine-induced modulation of tissue plasmkiogen activator binding to its endothelial cell membrane receptor. J& I m ~ a 91:2873-79, 1993.

Heijer M, Koster T, Blom HJ et d. Ilrperhomocysteinernia as a risk factor for deep vein thrombosis. N Engl J Md 334:759, 1996.

Harker LA, Sliditer SJ, Scott CR ad. Homocystinerniix Vascular injury and arteriai thrombosis. N Engl jMed291:537,1974.

Harker LA, Ross R, Shichter SJ ad. Homocystine-induced artenosclerosis. The role of endothelid cell injury and platelet response to its genesis. j Ch ï' 58:73 1, 1976.

Heinecke JW. Superoxide-rnediated oxidation of low density lipoprotein by thiols. In: Ceuni PA, Fridovich 1, McCord JM, eds. C2xy-r- in mdgukr Mgy andpmbdogy. New York Alan R. Liss, 44357, 1988.

Heinecke JW, Rosen H, Suniki LA a d A. The role of dfur-containing amino a 0 6 in superoxide production and modification of low density lipoprotein by arterid smooth muscle cells. / Bd Cben 262: 10098, 1987.

Hladovec J. Experirnentd homocystinemia, endothelial lesions and thrombosis. Blad VesseIs 16:202, 1979.

Imai K, Toyo'oka T, Watanabe Y. A novel fluorogenic reagent for thiols: Ammonium 7- fluorobenzo-2-oxa- 1,3-diuole-4-sulfonate. Anal B z k h 128:471,1983.

Israelsson B, Brattstrom LE, and Hultberg BL. Homocysteinemia and myocardial infarction. A h & & 71:227, 1988.

Jacobsen DW. Homocystehe and vitamins Li cardiovascukr disease. Ch OSen #:1833, 1998.

86 Jaques PF, Bostom AG, Williams RR et al. Relation between fohe ams, a common mutation in methylenetnrahydrofolate reductase, and plasma homocysteine concentrations. C i d d ~ ~ 93:7, 1996.

Kang SS, Wong PWK and Becker N. Protein-bound homocyst(e)ine in nomul nibjecu and in patients with homocystinuria A d z t ~ Res 13: 1141,1979.

Kang SS, Wong PWK, Cook HY al. Protein-bound homocyst(e)ine. A possible ri& factor for coronaxy artery disease. J CYh linea 77: 1482 1986.

Kang SS, Wong PWK, Susmano A a ai. Thermolabile methylenetetrahydrofolate reductase in patients wirh coronary artery disease. MetabdLPn 37:6ll, 1988~.

Kang SS, Zhou J, Wong PWK a ai. Intermediate homgrsteinemia: a thermolabile variant of methylenetetrahydrofolate reductase. Am J Hlon GBla 43:4 14, 1988 b.

Kang SS, Wong PWK, Susmano A a al. Thermolabile methy~enetetrahydroofolate reductase: An inherited risk factor for coronary anev diseue. Cna$iKn 4 l:%, 199 1.

Kang SS, Passen EL, Ruggie N et al. Thermolabile defect of methylenetetrahydrofolate reductase in coronary mery disease. Ci& 88 1463,1993.

Kanwar VS, Manaligod JR, and Wong PWK. Morphologie midies in a patient with homocystinuria due to 5-10-meth$enetetain/drofolate reductase defickcy. P& Res 10:598, 1976.

Kluijtmans LAJ, van den Heuvel LPWJ ad. Molecular genetic andysis in rnild hyperhornocysteinemia: A cornmon mutation in the rnethy1enetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease. Am J Hm Geia 58:35, 1996.

Kuzuya F and YoshLnLie N. Homocysteine theory of axteriosclerosis. Dtmykzhka @oumal of Japan Atherosclerosis Sociq) 6: 135, 1978.

Landgren F, Israelsson B, Lindgren A a d Plasma homocysteine in acute myocardid infarction: Homocysteine-IoweMg effect of folic acid. J Ihem Md 237: 38 1, 1995.

Lentz SR and Sadler JE. Inhibition of thrombomodulin d a c e expression and protein C activation by the thrombogenic agent homocysteine. J CYm I' 88: 1906,199 1.

Lentz SR and Sadler JE. Homocysteine inhibits von Wrûebrand factor processing and secretion by preventing vansport fiom the endoplasmic retidum. Blad 81:683,1993.

Loiy HL, Mudd SH, Schulman JD a al. A derangement in B,, metabolism associated with hornocystkiemia, cystathioninemia, hypomethioninemia, and methyhalonic aciduria. Am j Mai 48:390, 1970.

87 Ma J, Stampfer MJ, Hemekens CH, Frosst P ad. Methylenetetrahydrofolate Reductase Polymorphism, Plasma folate, homocysteine, and Ri& of Myoçardial Infarction in US Physicians. Ci'& 94:2410, 1996.

Malinow MR, Kang SS, Taylor LM et d. Prevalence of hyperhomocysteinemia in patients with penpherai attend occlusive disease. C 3 m h ~ ~ 79: 1180, 1989.

Mansoor MA, Svardal AM, Schneede J et al. Dynamic relation bween reduced, oxidized, and protein-bound homocysteine and other thiol components in plasma during methionine loading in heaithy men. C b 38: 13 16,1992.

M&dy KS: V d a r pathology of homocysteinemia: Implications for the pathogenesis of artenosc~erosis. Am J P& 56: 1 1 1, 1969

McCuUy KS and Ragsdale BD. Production of arteriosderosis by homocysteinemia. Am /Pa& 61:1, 1970.

McC* KS. Importance of homoqsteine-induced abnormalities of proteogiycan structure in arteriosderosis. Am/ Pathd 59: 18 1, 1970.

McCuUy KS. Homocysteine metabolism in scurvy, growth and arteriosclerosis. Nature 23 1 :39 1, 1971,

M&dy KS and Wilson RB. Homocysteine theory of arteriosclerosis. Absderasis 22:215, 1975.

McCuiiy KS. Chernical pathology of homocysteine. II. Carcinogenesis and homocysteine rhiolactone metabolism. A m Om Lab Sci 24:27, 1994a.

McCUiiy KS. Chemicai pathology of homqsteine. III. Cellular function and aging. Ann ûm Lab Sci 24:134-152, 1994b.

McCdy KS and Carvalho ACA. Homocysteine thiolactone, N-homocyneine thiolactonyl retinamide and platelet activation. Res Chmn OSen PaoSd f b m d 56:349, 1987.

McCuUy KS. Homocysteine and vasdar diseue. Nat Md 2:3 86, 1996.

Malinow MR, Sexron G, Averbuch M ad. Homocyst(e)inemia in dady practice: Levels in coronary artery disease. Can Amy Lhs 1:215, 1990.

Miller JW, Nadeau MR, Smith D et al. Vitamin B-6 deficiency vs folate deficiency: Comparison of responses to methionine loading in rats. Am /Ch Niar 59:1033-9,1994.

Miner SES, Cole DEC and Stewart DI. Homocysteine and Vascuiar Disease. Cardiology Rounds (StMchae17s Hospital) 1: 1, 1996.

88 Miner SES, Evrovski J and Cole DEC. Chical chemistry and m o l e d u biology of homocysteine metabolism: An update. Ch B& 30: 189, 1997.

Minno DG, Dari G, Margaghone M ad. Abnomially hi& thromboxane biosynthesis in homozygous homacystinuria. Evidence for platelet involvernent and probucol-sensitive mechanism. / C h hneo 92:1400,1993.

Misra HP. Generation of superoxide free radical during the autooxidation of thiols. / Bid C h 249:2151, 1974.

Molgaard J, Maiinow MR, Lassvik C a d Hjprhomocyst(e)inernia: An independent nsk factor for intermittent claudication. j lntem Md 23 1:273,1992.

Monada S and Vane JR. Arachidonic aud metabolites and the interactions between platelets and blood vesse1 wds. N Engl J Md 300: 1 142, 1979.

Mudd SH, Finkelaein JD, Irreverre F a al. Homocystinuria: An enzymatic defect. S c i m 143: 1443, 1964.

Mudd SH, Levy HL and Abeles R H A derangement in BI2 meraboiism leading to homocystinernia, cystathioninemia and merhylmalonic auduria. BiooS8n B&s Ra Chmm 35121, 1969.

Mudd SH and Levy HL: Disorders of transsulfiiration. In: Stanbury, J.B., Wyngaarden, J.B. and Fredrickson, D.S. Eds.: 7k M e &ûü c y V n h d k , 458 (4'h edition, McGraw-HiIl, New York, 1978)

Mudd SH, Havlik R, Levy HL, McKusick VA, Feinleib M. A study of cardiovascular risk in heterozygotes for homocystinuria. Am j H m Gem 33:883, 1981.

Mudd SH, Levy HL, and Skovby F. The n a d hiaory of homocystinuria due to cynathionlie P- synthase defiaency. Am J H m Gimt 37: l, 1985.

Mudd SH, Levy HL and Skovby F: Disorders of transsulfuration. In: Scriber CR, Beaudet AL, Sly WS, Valle D, eds. %m~Edicmdmdazrlm bves $Wh. ed. Vol. 1. New York: McGraw-Hill: 1279,1995.

Mudd SH and Levy HL. Plasma homocyst(e)ine or homocysteine? N Engl j Med 333:325,1995.

Murphy-Cuthorian DR, Waanan M P, Grieco AJ &al. Methionine intolerance: A possible risk factor for coronary artery disease. /Am Gd ûniid 6:725, 1985.

Naniszewicz M, MVkiewia E, Olszewski AJ ad. Thiolation of low-density lipoprotein by homocysteine thiolaaone causes inmased aggregation and altered interaction with cdtured macrophages. Nurr Merab chn&uucDXc 4:70,1994.

89 Nygard O, Volset SE, Refsurn H ct al. Total Plasma Homocysteine and C a r d i o ~ s d a r Risk Profile: The Hordaand Homocysteine Study. JAMA 274: 1526,1995.

Oiszewski AJ, and Szoaak WB. Homocysteine content of plasma proteins in ischernic hem disease. Athmdmu 69: 109, 1988.

Palareti G, Legnani C, and Cocchen S. Hyperhomocyaeinemia and vascular disease. In: @pmqp%k S m , Segatchian MJ, Samama MM and Hecker SP, Eds. CRC Press: 395,1996.

Pancharuniti N, Lewis Ch, Sauberiich HE d al. Pksma homocys(e)ine, folate, and viramin BI2 concentrations and risk for early-onset coronary vtery disease. Am J Clm NW 59~940, 1994.

Rees MM and Rodgers GM Homocysteinemia: Association of a metabolic disorder with vascular disease and thrombosis. ?'hm Res 71:337, 1993.

Refsurn H and Ueland PM. Cltiical significance of pharmacologid modulation of homocysteine metabolism. 77"s Raz& 1 Mil, 1990.

Refnim H, Ueland PM, Nygard O & al. Homocyneine and cardiovaxular disease. Anm Ra, Md 49~31, 1998.

Ridker PM, Hemekens CH, Selhub J ad. Interrelation of hyperhomocysteinemia, factor V Leiden and risks of f m r e thrombosis. Cbczhtm 951777, 1997.

Rinehart JF and Greenberg LD. VitamLi B, defxiency in the Rhesus monkey with particular reference to the occurrence of atherosderosis, dental caries and hepatic cirrhosis. Am /Clin N m 4:318, 1956.

Robinson K, Mayer EL, Miller DP et al. Yrperhornocysteinernia and low pyridoxd phosphate: cornmon and independent reversibie risk factors for coronary artery disease. Ci& 92:2825, 2995.

Robinson K, Arheart D, Refsum H etal. Low circularing folate and vitamin B, concentrations: Risk factors for stroke, penpherai vascular disease, and coronaiy artery disease. C u 97: 437, 1998.

Rocigers GM and Kane W Activation of endogenous factor V by a hornocysteine-induced vasdar endothelial ce1 activator. J CYm aaet 77: 1909,1986.

Rodgers GM and Corn MT. Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial ceus. B U 75:895,1990.

Rolland P, Friggi A, Barlatier A d. Ylperhomocysteinemia-induced vasdar damage to the mhipig. Ci'& 9l:ll6l, 1995.

90 Rosenblatt DS and Erbe RW. Methylenetetrahydrofolate reductase in cultured human ceils. II. Genetic and biochemid studies of me&ylenetetrahydrofolate reduaase deficiency. Pe& Ra i1:1141, isn.

Rozen R Molecdar genetic aspects of hyperhomocysteinemia and its relation to folk acid. Clk Z m Md 19:171, 1996.

Rozen R Genetic predisposition to hyperhomocysteinemia: Defiuency of methy1enetetahydrofolate reductase @dTWR). Hiunast 78523, 1997.

Rowley DA and Halliwell B. Superoxide-dependent formation of hydroxyl radicals in the presence of hi01 compounds. FEBS Lett 138:33,1982.

Schmitz C, Lindpaintner K, Verhoef P ad Genetic polymorphism of methylenetetrahydrofolate reductase and myocardid Infamion: A Case-Gntrol Study. C m 94: 1812,1996.

Schwartz SM, Siscovick DS, Malinow R Myocardial infarction in yaing wornen in relation to plasma rotai homocysteine, folate, and a common variant in the methylenetetrahydrofolate reductase gene. Ci& 96:412, 1997.

Selhub J and Miller JW. The pathogenesis of homocysteinernia: Interruption of the cooràinate regulation by S-adenosyImethionine of the remetliylation and transdfuration of homocysteine. Am J Clin Na* 55: 13 1,1992.

Shah VP, Midha KK, Dighe S ad. Analyucal methods validation: Bioavailability, bioequivalence, and phmacoklietic midies.JPhmm Si 81:309, 1992.

Srnolin LA, Crenshaw TD, K u r t y a D and Benevenga NJ. Homocysteine accumulation in pigs fed diets deficient in vitamin B,: Relationship to atherosderosis. J N w 113:2122, 1983.

Stamler JS, Osborne JA, Jaraki O et al. Adverse vascdar effects of homocysteine are modulated by endotheliumdenved relaxhg factor and related oxides of niuogen. J Clin i m 91:308, 1993.

Stampfer MJ, Malinow R, WiUet WC ~ d . A prospective study of plasma homocyst(e)ine and nsk of myocardial infarction in US physiaans. JAMA 268:877, 1992.

Starkebaum G and Harian JM. Endothelid cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. / C h liaea 7: 1370,1986.

Stolley PD and Strom BL. Sample size cdculations for clinical phannacological d e s . ClhPhmm I h P i 39:489, 1986.

Taylor LM, De Frang RD, Harris EJ Jr etal. The assoaation of elevated plasma homocyst(e)ine with progression of symptomaric penpheral arterial disease. J Varc Srng 13: 128,199 1.

91 Tsai JC, Perreila MA, Yoshiauni M ttd Promotion of vascular smooth-musde growth by homocysteine: A link to atherogenesis. Aar Nd AdSri US A 91:6369, 1994.

Ubbink JG, Vemiack WJH, Bennett JM ft 4. The predence of homocysteinernia and hypercholesterolemia in angiographically defined coronaxy artery disease. Kh WoPS8inhr 69527, 1991.

Ueland PM and Refsum H. Plasma homocysteine, a risk factor for vascular disease: Plasma levels in health, disease, and h g therapy. J lab Clin Mai 1 14473,1989.

Ueiand PM, Refsum H, Stabler SP et ai. Total homocysteine in plasma or serum: methods and clinid applications. & Chm 39: 1764, 1993.

Ueland PM. Homocysteine speaes as components of plasma redox thiol status. Ch Chm 41:340, 1995.

van der Put, RPM Steegers-Theunissen, Frosa P et al. Mutated methylenetetnhydrofolate reductase as a risk factor for spina bifida. Lanra 346: 1071, 1995.

van der Put NMJ, Gabreels F, Stevens EMB etd. A Second Comrnon Mutation in the Methylenetetrahydrofolate Reduaase Gene: An Additional Risk Factor for Neural-Tube Defects? Am ]Hm Geret. 62:1044,1998.

Verhoef P, Starnpfer M and Buring JE. Homocysteine metabolkm and risk of myocardial infardon: Relation with vitarnins B,, B,, and folate. Am/ E p W 143:845, 1996.

Von Eckardstein A, Malinow h4R, Upson B &al. Effects of age, lipoproteins and hemostatic parameters on the role of hyperhomocyaeinemia as a cardiovasdar risk factor in men. A& M 14: 460, 1994.

Walker SE, Lau DWC, DeAngelis C ad. Epinibicin aability in syringes and glass viais and evaluation of chemical conramination. Guz /HOT Prt/am, 6:265,1990.

Wall RT, Harlan JM, Harker LA and Striker GE. Homocysteine-induced endotheliai ceil injury in ziao. a mode1 for the study of vascuiar disease. IhmnbRes 18: I13,198O.

Wekh GN, Upchwch GR Jr and Los& J. Hyperhomocyst(e)inemia and atherothrombosis. Ann N Y A d Ski 8 1 l:48,1997.

Weldi GN and L o s c b J. Homocysteine and atherothrombosis. N Engl J M d 338: 1042, 1998.

Wilcken DEL and Wilcken Bridget. The pathogenesis of coronaxy artery disease: A possible role for methionine metabolism. J Clin hest 57: 1079, 1976.

Wddcen DEL, Wang XL and Sim AS. Diaribution in healthy and methyienetetrahydrofolate reductase (MTHFR) C 6 m mutation. 16:878, 1996.

92 coronary populations of the Avterjosder lhmnb V a Res

Zittoun J, Tonetti C, Bories D a al. Plasma homocysteine levels related to interactions between folate status and methylenetetrahydrofolate reductase: A mtdy in 52 healthy subjecu. Metabdkm 47: 1413, 1998.

Molecular Marker (M)

Figure 9.1: Typical Redction Endonudease Aiulysis of the GnT Substitution (alanine + valine) in the MTHFR Gaie

A marker 0 ladder is shown in lane 1. Lanes 5, 9 and 12 respecrively show PCR-amplified and

Hi$ digested fragments from nomals ( O / - ) , heterozygotes (+/-) and homozygotes for the

M I H F R (+/+) dele (175 bp venus 198 bp).

Figure 9.2: Typicai Restriction Endonudese Anaiysis of the GIMIA Substitution (argi.nine+ glutamine) at Factor V Residue 506 (Factor V Leiden)

A Marker (M) ladder is shown in lane 1. Lanes 5,13 and 16 respectiveiy show PCR-amplifid and TaqI

digested fragments from heteroygoies (+/-), nomals (4) and homozygotes for the FVL (+ /+) alleb (137 bp vernis 1 14 bp).