CLINICAL CHEMISTRY Review Article. 1985, Boers 12 described accelerated vascular disease associmature

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1 CLINICAL CHEMISTRY Review Article H y p e r h o m o c y s t e i n e m i a An Emerging and Important Risk Factor for Thromboembolic and Cardiovascular Disease SUSAN C. GUBA, MD, 1 " 3 LOUIS M. FINK, MD," AND VIVIAN FONSECA, MD 1 ' 3 Homocysteine is an important contributing factor to thrombosis, vascular injury, and vascular disease. Mechanisms for homocysteine-induced vascular disease include alterations in coagulation as well as endothelial cell and vessel wall injury. Hyperhomocysteinemia (HH e ) can occur when homocysteine metabolism is altered by mutations in enzymes responsible for homocysteine metabolism. Characterization Manuscript received June 6, 1996; revision accepted August 9, Address reprint requests to Dr. Fink: Pathology and Laboratory Medicine Service (113/LR), John L. McClellan Veterans Hospital, 4300 West 7th Street, Little Rock, AR of these mutations identifies patient groups at risk for vascular disease. Treatment of 1111(e) consists of vitamins and raises the possibility that some forms of vascular disease may be easily, safely, and inexpensively treated. (Key words: Homocysteine; Cardiovascular disease; Methionine; Homocystinuria; Vitamins; Folate; Thromboembolism) Am J Clin Pathol 1996;105: Homocysteine is a thiol-containing amino acid metabolized by remethylation to methionine or by transsulfura- to 10% to 25% of the total H(e). 3 Thus, plasma measure centage of the total H(e) concentration, and can increase tion to cysteine. Elevated homocysteine levels may occur ments of H(e) should include both the reduced and oxidized forms of the amino acid. as a result of inherited disorders that alter enzyme activity in the transsulfuration and remethylation pathways. Homocystinuria is an inherited disorder characterized by Alternatively, nutritional deficiencies of cobalamin (vitamin B 12 ), folate, or pyridoxine (vitamin B 6 ), can result tion. Homocystinuric children are known to develop pre severely elevated plasma H(e) and resultant urinary excre in blockade of homocysteine metabolic pathways, because activity of these enzymes depends on these vita 1985, Boers 12 described accelerated vascular disease associmature vascular disease involving all major blood vessels. In mins as cofactors. Clinically, elevated homocysteine levels are significant because they may indicate cobalamin, out the other manifestations of homocystinuria. Since then, ated with moderate elevation in plasma homocysteine, with pyridoxine, or folate deficiency. More importantly, elevated homocysteine levels are an independent risk factor factor for coronary artery disease, stroke, and peripheral vas there has been considerable interest in mild HH(e) as a risk for thrombosis and vascular disease. 1 "". cular disease. The understanding of the different etiologies Homocysteine is the reduced (sulfhydryl) form and of HH(e) is changing because of the ability to discriminate homocystine the oxidized (disulfide) form of the homologues cysteine and cystine. 312 For the purpose of this ders, the ability to distinguish between homozygous and het between the types of mutations present in inherited disor review, both forms of "homocysteine" will be referred erozygous mutations, and a better understanding of factors to as H(e) and hyperhomocyst(e)inemia as HH(e). In patients in whom H(e) levels are normal, only about 2% which relate vascular disease and HH(e) is depicted in Figure that modify H(e) metabolism. A paradigm of the variables of the total concentration occurs as the sulfhydryl form. 1. H(e) plasma concentration increases as a function of age, With elevated H(e) levels, the percentage of homocysteine in the sulfhydryl form represents an increasing per- tus (renal insufficiency) and is an independent risk factor in nutritional deficiency status (folate, B 12 or B 6 ), or disease sta vascular disease. This review will discuss methionine metabolism, H(e) measurements, genetics of HH(e) and mechanisms of vascular disease in HH(e). From the 'Department of Medicine and 2 Department of Pathology. University of Arkansas for Medical Sciences and i John L. McClellan Memorial Veterans Hospital. Little Rock. Arkansas. METHIONINE METABOLISM The metabolism of methionine 1314 is illustrated in Figure 2. In the transsulfuration reaction, methionine is converted to homocysteine which irreversibly condenses with serine to 709

2 710 CLINICAL CHEMISTRY Review Article FIG. 1. The genetic and epigenetic factors influencing H(e) metabolism. form cystathionine (reaction 1). This reaction is catalyzed by the B 6 -dependent enzyme cystathionine /3-synthase (CBS). Cystathionine is hydrolyzed to cysteine by the enzyme y- cystathionase (reaction 2). Alternatively, in the remethylation pathway, methionine reforms from homocysteine when a methyl group is donated by N-5-methyltetrahydrofolate (reaction 3b) in a reaction catalyzed by the enzyme methylene tetrahydrofolate reductase (MTHFR) (reaction 3a). Alternatively, the methyl group may be donated by Betaine (reaction 4). METHODS OF MEASUREMENT OF PLASMA HOMOCYSTEINE Measurement of total H(e) should include both the reduced or sulfhydryl and oxidized or disulfide forms of H(e) because the relative percentage of the sulfhydryl form increases in HH(e). 315 This may be accomplished 5,10-METHYLENE TETRAHYDROFOLATE REMETHYLATION METHIONINE LOAD TEST The methionine load test (MLT) is essential in the comprehensive assessment of HH(e) because heterozy- TETRAHYDRO FOLATE 5-METHYL- TETRAHYDROFOLATE METHYL-B,, SERINE PROTEINS r by conversion of all forms to a single species by reduction. Measurement further depends on proper handling of blood samples: blood cells continuously produce and release H(e), which can lead to a false elevation in total H(e) levels if cells are not promptly separated from plasma H(e) levels are age and gender specific and reference intervals should be established accordingly. 17 H(e) levels are also dependent on the state of vitamin repletion. As folic acid supplements are added to foods, as recently recommended by the Food and Drug Administration, reference intervals are likely to decrease. 17 " 19 Methods to assay plasma H(e) include gas chromatographymass spectroscopy, HPLC, with or without fluorescence detection, HPLC and electrochemical detection, amino acid analyzer detection, and an antibody-fluorescence polarization technique. 15,20 " 25 A summary of methods to quantitate H(e) are presented in Table 1. Readers are referred to an excellent review by Ueland and colleagues 15 for a more detailed summary of methods. Elevation of basal H(e) levels has been defined as mild (15-24 mm), moderate (25-100), or severe (>100 mm). 13 However, these values bear no direct relationship to the development of vascular disease. Normal values vary slightly between laboratories and with the age, race, and gender of the patient population. 15 A better definition of H(e) level ranges must correlate with the absence or presence of clinically significant vascular disease. More recently, correlative studies of vascular disease and H(e) levels suggest that the upper limit of normal should be reduced to 12mM. 7 TRANSMETHYLATION METHIONINE fc. 5-ADENOSYLMETHIONINE DIMETHYLGLYCINE m BETAINE. HOMOCYSTEINE -^ 5-ADENOSYLHOMOCYSTEINE -^ 1 CBS + B, [T FIG. 2. H(e) metabolic pathways. Reaction 1 is catalyzed by choline oxidase; reaction 2, betaine-homocysteine methyltransferase; reaction 3, 5-methyltetrahydrofolate-homocysteine methyltransferase; reaction 4, phosphatidylethanolamine methyltransferase; reaction 5, guanidoacetate methyltransferase; reaction 6, glycine methyltransferase; reaction 7, cystathionine B synthase; reaction 8, 7-cystathionase. CYSTATHIONINE 1 jtj g-cystathionase CYSTEINE TRANSSULFURATION A.J.C.P. December 1996

3 Method of Detection GC-MS HPLC, with fluorescence detection (method has several variations) HPLC with electrochemical detection HPLC with pulsed integrated amperometry Amino acid analyzer (several variations) Antibody fluorescence polarization immunoassay (IMx analyzer [Abbott; Abbott Park, IL]) Reduction Step Required Yes (DTT) Yes (DTT) Yes(NaBH4) Yes(NaBH4) TABLE 1. HOMOCYSTEINE MEASUREMENT METHODS Yes Derivatization Required Advantages of Method Disadvantages of Method References Yes (SBD-F), precolumn No No Yes (DTT or /3ME) Yes, postcolumn Yes (DTT) Anti-SAH monoclonal antibody and fluoresceinated analog SAH Distinguishes cystathionine, methionine, cysteine, A'-methylglycine, betaine, methyl malonic acid Sensitive and specific, no interfering reagent peaks, distinguishes between cysteine, glutathione, gamma glutamyl cysteine, cysteinylglycine and H(e) Resolves cysteine, homocysteine, and cysteinylglycine; method is sensitive, specific, and rapid Eliminates electrode fouling by alternating between oxidative cleaning and reductive reactivation potentials; is sensitive and selective Measures homocysteine, cystathionine, methionine (cysteine, cysteinylglycine, glutathione, and glutamylcysteine) SAH hydrolate converts H(e) to SAH; shortens assay time; assay has high selectivity, and calibration curve is stable for 2 weeks Time-consuming 15 Long reaction time, some variations 15,20-22 complicated by dissolution of silica column matrix due to low ph required (2,1), high column temperature (60 C) Gold electrode required; electrode 15,23 fouling and flow cell contamination occur Gold electrode required 23 Less sensitive than other methods, 15,24 nonselective for sulfur amino acids Monoclonal based assay (requires 25 antibody raised to SAH GC-MS = gas chromatography-mass spectrometry; HPLC - high-performance liquid chromatography; DTT = dithiothreitol; /3ME = mercaptoethanol; SBD-F = sulfonylbenzofurazans;sah = S-adenosyl-L-homocysteine; H(e) = homocysteine. I 5- o 3 1-

4 712 CLINICAL CHEMISTRY Review Article gotes for CBS deficiency have abnormal methionine load test results in the setting of normal fasting H(e) levels The MLT is performed after an overnight fast. Blood samples are collected immediately before and 2, 4, 6, 8, (12 and 24 hours) after a 100 mg/kg methionine load. In normal subjects, an oral load of methionine induces a transient plasma increase in free and protein bound homocysteine peaking between 4 and 8 hours. 22 An abnormal load test results in a plasma homocysteine level more than 2 standard deviations above normal control levels. GENETICS OF HYPERHOMOCYSTEINEMIA Enzyme mutations most commonly associated with HH(e) are point mutations of cystathionine /3-synthase (CBS) (reaction 1) and 5, 10-methylenetetrahydrofolate reductase (MTHFR) (reaction 3a). One point mutation of MTHFR produces a thermolabile variant. Another reported mutation present in HH(e) includes mutation of 7-cystathionase (reaction 2).' 4 Another candidate, for which discrete mutations have not yet been described is 5-methyltetrahydrofolate-homocysteine methyltransferase (reaction 3b) (methionine synthase) (Fig. 2 and Table 2). 28 Mutations of the cobalamin coenzyme synthesis enzymes (Cb C, D, E, F or G) 14 are also rarely involved in HH(e), but are not discussed here. A comprehensive review of cobalamin coenzyme synthesis enzyme mutations is available elsewhere. 28 The characteristics of CBS and MTHFR mutations are summarized in Table 3. Cystathionine ^-Synthase Deficiency Homozygous CBS mutations are the most common cause of homocystinuria. CBS catalyzes the B 6 dependent conversion of homocysteine to cystathionine (A), 14 as well as cysteine sulfhydration (B): A. Homocysteine + serine -» cystathionine + H 2 0 B. H 2 S + serine -»- cysteine + H 2 0 Reaction (A) is reversible under conditions of homocysteine deficiency, but normally proceeds in the direction shown. Mutations of the CBS gene can result in an enzyme with decreased affinity for pyridoxal phosphate as well as serine and homocysteine, and an enzyme that is more heat labile than wild type enzyme. Also in contrast to the wild type enzyme, activity varies as a function of homocysteine concentration. This may be due to steric abnormalities in the hybrid normal-mutant molecule. For these reasons, heterozygotes have been found to have less than the 50% expected CBS activity. 14 These changes explain the variable activity of CBS seen be- tween patients with both homozygous and heterozygous mutations. The CBS gene has been assigned to the subtelomeric region of band 21q22.3 of chromosome 21. CBS deficiency is inherited in an autosomal recessive pattern, resulting in homozygous (homocystinuria) and heterozygous (hyperhomocysteinemia) carriers. 12 Sequencing of the cdna for the CBS gene has already shown 17-point mutations. I,29 ' 3 Tsai and colleagues 31 have characterized three of the more common mutations as either a G 9 i 9 A, a T 833 C, or a C 3 4,T transition. Different populations demonstrate differing mutation frequencies, with the G 9 9 A transition occurring in 70% of an Irish cohort tested, whereas the T 833 C transition occurred in 50% of a Dutch population tested.' Early studies 12,32 initially reported that patients heterozygous for the CBS mutation had <50% of the expected CBS activity and were at increased risk for cardiovascular disease. However, because molecular genetics analyses of CBS zygosity has become available and enzymatic activity data have been repeated, one study has suggested that heterozygous CBS mutation is not associated with an increased risk of cardiovascular or thrombotic disease in these patients.' However, the importance of mutations of the CBS enzyme in causing HH(e) is therefore unclear. It is possible that they may be responsible in only post methionine load HH(e). In contrast, the CBS homozygote is at markedly increased risk for vascular thrombosis and arteriosclerosis. It is possible that some mutations in CBS affecting pyridoxine binding may have some proclivity for vascular disease. Perhaps this may relate to the observations that chronic pyridoxine deficiency has been shown to induce vascular lesions in some monkeys and dogs. Lower pyridoxal-5'-phosphate levels have also been observed in patients with vascular dis- Methylene Tetrahydrofolate Reductase Deficiency Homocysteine may either be transsulfurated to cysteine by CBS (Fig. 2, reactions 1 and 2) or remethylated to methionine (reaction 3a and 3b). Methionine production requires the co-factor 5-methyltetrahydrofolate. MTHFR catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (reaction 3a) with folate as a cofactor. 5,10-MTHF 5-CH3THF + homocysteine -* methionine. The thermolabile variant of MTHFR is inherited as an autosomal recessive trait and is the most commonly A.J.C.P.' 1996

5 TABLE 2. ENZYME MUTATIONS ASSOCIATED WITH HYPERHOMOCYSTEINEMIA Enzyme Deficiency (references) Mode of Inheritance/Incidence Chromosome Synthetic Block* Enzyme Cofactor CBS(1,13,14,28,29,31) 5,10 MTHFR, thermostable (1,34,35,36) MTHFR, thermolabile (1,2,37,38,39) 7-Cystathionase (14) 5-Methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase) (14) AR; 1:200,000 in homozygotes and 1:70-1:200 in heterozygotes AR Homozygotes: 12-15% among French Canadians Europeans, Japanese, Middle East population, about 5% in Dutch and Finnish populations, 1.4% in African-Americans AR 57 patients reported Not identified 21 q 22.3 (17 point mutations identified) lp 36.3 (nine mutations identified) lp 36.3(1 mutation identified) a 3b 2 3b B6 (must be folate replete) B 2, folate B12, folate B6 B,2 AR = autosomal recessive; CBS = cystathionine ^-synthase; MTHFR = methylene tetrahydrofolate reductase. * Refer to Figure 2. f ^ as

6 TABLE 3. CHARACTERISTICS OF CBS AND MTHFR MUTATIONS Defects in Remethylation MTHFR Mutation Defects in Transulfuration CBS Mutation Thermostable Thermolabile Homozygous Heterozygous Homozygous Heterozygous Homozygous Heterozygous Plasma H(e) level Commonly identified mutations Common phenotypes Risk of vascular disease/cad Response of H(e) level tob6, B 2, or folate References Severely elevated (fasting) T833C G919A C341T Ectopia lentis, myopia, osteoporosis, dolichostenomelia, pectuscarinatum, pes cavus, genu valgum mental retardation, psychiatric disturbances, seizures, thromboembolism Premature arteriosclerosis and thrombosis 50% respond to B6 ( mg daily); patients must be folate replete 1,13,14,29, Normal to mild elevation (fasting), mild to moderate elevations post methionine load Normal No increased risk of vascular disease in true heterozygote; early studies reported increased risk Severely elevated (fasting) C599T G482A Infancy: delayed psychomotor development, psychiatric disturbances, mental retardation In adults: marfanoid habitus, severe atherosclerotic disease and thromboembolism Increased Not reported 1,34-36 Normal Normal Not reported Not reported Normal to moderately elevated No neurologic abnormalities Increased risk of premature CAD Responds to folic acid C677 1,2,37 r Normal Normal Not increased -39 CAD = coronary artery disease. See Table 2 for other abbreviations.

7 GUBA, FINK, AND FONSECA 715 Hyperhim inherited disorder of folic acid metabolism. Located on chromosome lp36.3, nine different mutations for the MTHFR gene have been identified from isolated cdna (GenBank U09806) Two other common mutations of MTHFR are a C559T transition that converts an arginine codon to a termination codon and a G 482 A transition that converts an arginine to a glutamine residue. 34 These mutations are thermostable in contradistinction to the thermolabile mutation discussed below. Clinical symptoms vary as a function of residual enzyme activity. Homocysteine levels seen in MTHFR deficiency are usually lower than those in CBS deficiency. 34,36 Unlike CBS deficiency, HH(e) is unusual in heterozygotes for MTHFR deficiency. A third mutation in the MTHFR gene results in a thermolabile variant. 37 " 39 In the homozygous state, the gene frequency has varied between different populations studied: 5% of white controls, 12% among French Canadians, and 12% to 15% of European, Middle Eastern and Japanese populations. In contradistinction, only 5.4% of Finnish, 5.2% of Dutch, and 1.4% of black populations are homozygotes for the C 677 T mutation. 2 This mutation is autosomal recessive. Homozygous thermolabile MTHFR deficiency is characterized by the absence of neurologic abnormalities and an enzyme activity of about 50% of normal. Heat inactivation at 46 C distinguishes the mutant from the normal MTHFR enzyme. The enzyme is considered thermolabile when there is <20% residual activity after heating. HH(e) is not a consistent finding in patients with the thermolabile variant of this enzyme. In homozygotes with folic acid levels above the median, H(e) levels are normal. 2 ' 37 " 39 Thus, thermolabile enzyme activity, at least in part, depends up the availability of folate. The thermolabile mutation has been identified as a C 677 T transition that creates a Hinf I restriction site and substitutes an alanine for a valine residue in the MTHFR protein. 39 Patients with the mutation for the thermolabile variant of MTHFR have an increased incidence of coronary artery disease. The incidence of severe CAD (at least 70% stenosis in one or more coronary artery or >50% stenosis of the LMCA) in patients with the homozygous mutation for thermolabile MTHFR was significantly higher than in controls. Patients with moderate CAD (<70% stenosis in one or more CA or <50% stenosis in the LMCA) had an incidence for homozygous thermolabile MTHFR which was intermediate, and was found to independently increase the risk for CAD. Treatment with B 2 and folate reduces H(e) levels. Patients heterozygous for the thermolabile mutation of MTHFR do not appear to be at increased risk for CAD PREMATURE VASCULAR DISEASE AND HYPERHOMOCYSTEINEMIA Children who have homozygous homocystinuria develop premature and severe vascular disease. In a survey, 158 of 629 such patients had 253 thromboembolic events. Of these, 51 % were venous thromboses, 32% were cerebrovascular accidents, 11 % were occlusive peripheral arterial disease, and 4% had myocardial infarctions. These patients are prone to sudden death in young adulthood or even in childhood. These data prompted the study of moderately elevated H(e) levels as a risk factor for occlusive arterial disease. 12 Epidemiologic and Prospective Studies In the first prospective epidemiologic study of homocysteine levels as a cardiovascular risk factor, 14,916 male physicians with no prior vascular disease provided plasma samples at baseline and were followed for 5 years. 40 Plasma homocysteine levels in 271 physicians who developed an MI were significantly higher than in paired controls. The relative risk for MI in those physicians in the 95th percentile for homocysteine levels was three times greater than those in the 10th percentile, even after adjustment for other known risk factors for vascular disease. In contrast, a prospective study in Finland of 7,424 healthy subjects at baseline, showed development of stroke in 265 subjects over a 9-year period. 41 The fact that the affected subjects did not have an elevated serum homocysteine level has been explained by the exceptionally low C 677 T mutation frequency predisposing to hyperhomocysteinemia in Finland. Determination of ethnic variations of gene frequency predisposing to HH(e) are important. For example, in Black South Africans, plasma H(e) levels are low and the incidence of vascular disease is also low despite a high prevalence of obesity, hypertension, and smoking. 42 Studies in Patients with Established Vascular Disease Several studies have attempted to establish the prevalence of HH(e) in patients with premature and accelerated vascular disease. These studies are summarized in Table 4. In some studies, fasting H(e) was elevated, whereas in others only the post load H(e) was elevated. Some patients have both fasting and post load HH(e). Interest in this field was triggered by the first report by Boers and colleagues 12 who found elevated plasma H(e) following a methionine load in 28% of patients with peripheral vascular and cerebrovascular disease. Vol. No. 6

8 716 CLINICAL CHEMISTRY Review Article Author Boers Malinow Stampfer Clarke den Heijer Alfthan TABLE 4. EPIDEMIOLOGIC STUDIES AND STUDIES TO DETERMINE THE PREVALENCE OF HHe IN VASCULAR DISEASE Population Early onset arterial disease Healthy and vascular disease Male physicians (prospective) Early vascular disease Venous thrombosis Finnish epidemiology study Fermo Venous and arterial disease (age <45 years) Falcon Juvenile venous thrombosis Selhub Elderly in Framingham study Munshi Diabeteswith vascular disease age <60 years Bostom End-stage renal disease Basal versus Post Load HU(e) Findings Reference Post load Basal Basal Post load Basal Basal Basal and post load Basal and post load Basal Post load HH(e) = hyperhomocyst(e)inemia; H(e) = homocysteine; MI = myocardial infarction;dm = diabetes mellitus. In a meta-analysis of 27 studies of H(e) in atherosclerotic vascular disease, Boushey and colleagues 7 concluded that elevations of H(e) were an independent risk factor for arteriosclerosis. In addition, approximately 10% of the populations' coronary artery disease risk appears attributable to homocysteine. The odds ratio for development of coronary artery disease from increased plasma H(e) at a level of 5 mmol/l above normal is 1.6 for men and 1.8 for women. Even after adjustment for other risk factors, fasting plasma H(e) has been found to be significantly higher in patients with peripheral vascular disease compared with healthy individuals. 43 Elevations in peak H(e) following a methionine load occur in 28% to 42% of patients with vascular disease but rarely if ever in normal subjects. 9,44 Perhaps the methionine load test delineates the at risk population better than basal levels. Some studies have found an association with vascular disease in patients with fasting HH(e), whereas other have needed a methionine load test to demonstrate such a relationship. Fermo and colleagues 45 studied patients below the age of 45 with both venous thrombosis and arterial occlusive disease, and found moderate HH(e) in 13.1% and 19.2% of patients, respectively. The prevalence of HH(e) was almost twice as high following a methionine load than when based on fasting levels. Furthermore, patients with HH(e) have a higher rate of recurrence of venous thrombosis. 45 Other studies have confirmed the high prevalence of post-methionine load HH(e) in juvenile 46 and recurrent venous thrombosis. 10 HH(e) seems to be more common in patients with stroke and carotid disease when compared to patients HH(e) in 28% of patients High H(e) in patients High H(e) in physicians who had an MI High H(e) in 28-42% of patients High risk of thrombosis if H(e) >90th percentile No association between high H(e) and vascular disease Post load detected higher incidence of H(e) in patients 19% of patients had HH(e) Twofold increase in carotid disease in subjects with highest H(e) 42% DM with vascular disease had high H(e) Predialysis High H(e) with other forms of vascular disease. This finding was highlighted in a cross-sectional analysis of 1,041 elderly subjects in the Framingham Heart Study. A two-fold increase in the incidence of carotid disease was seen in patients with the highest plasma homocysteine concentrations when compared to those with the lowest concentrations. 5 Furthermore, plasma concentrations of folate and pyridoxal-5'-phosphate were also inversely associated with carotid artery stenosis. As plasma H(e) concentrations change acutely following a stroke, 47 measurements are best performed in stable patients HYPERHOMOCYSTEINEMIA IN HIGH-RISK POPULATIONS AND INTERACTION WITH OTHER RISK FACTORS Early studies of HH(e) as a risk factor for vascular disease were controlled for other traditional cardiovascular risk factors. Recently however, a number of studies have looked for synergism between HH(e) and other risk factors such as chronic renal failure, diabetes, hyperlipidemia and age. In the Hordaland H(e) study, elevated plasma H(e) was associated with male gender, increasing age, smoking, hypertension, elevated cholesterol, and lack of exercise. 48 In a multivariate analysis, Malinow and colleagues 49 demonstrated that systolic blood pressure, plasma uric acid, and hematocrit were predictors of concentrations of plasma homocysteine in men who did not have a history of atherosclerotic disease. The rise in H(e) levels following menopause may partly explain the sharp rise in cardiovascular disease that occurs in this age group, and its attenuation by hor- 44 A.J.C.P.- December 1996

9 GUBA, FINK, AND FONSECA 717 Hyperhomocysteinemia mone replacement therapy. The lower incidence of vascular disease in premenopausal women may be related to the lower concentrations of total homocysteine and to the lower peak plasma H(e) concentration after a methionine load. In 482 patients already at high risk for atherosclerotic vascular disease by virtue of hyperlipidemia, 3.7% had high plasma homocysteine. 50 In hyperlipidemic patients, the relative risk of atherosclerotic events for the 80th percentile of plasma homocysteine was 2.8 times greater than that seen for the 20th percentile. Furthermore, it was possible to reduce H(e) concentrations in this hyperlipidemic population with vitamins, suggesting a possible therapeutic approach for multiple risk factor intervention. There have been several reports of HH(e) in chronic renal failure. 51 Although some of this elevation may be due to decreased clearance of homocysteine, other mechanisms may also be responsible. 52 Intact kidneys have considerable H(e) metabolizing capacity, 51 impairment of which may be an important determinant of the marked HH(e) frequently observed in end-stage renal disease. High dose multiple vitamin treatment has been shown to lower plasma H(e) in dialysis patients. Hyperhomocysteinemia has also been demonstrated following successful renal transplantation 53 and may be exacerbated by cyclosporin. 54 Other conditions have also been associated with HH(e). Two studies have demonstrated an increased frequency of HH(e) in patients with noninsulin dependent diabetes (NIDDM) and vascular disease. 9,44 Elevated plasma H(e) levels have been demonstrated following coronary bypass surgery 55 as well as following cardiac transplantation. 56 Plasma H(e) concentrations increase with age and remain an independent risk factor for vascular disease in the elderly. 56 The marginal vitamin deficiency known to be common in the elderly, is likely to be a contributing factor to HH(e). 57,58 POSSIBLE MECHANISMS OF ACCELERATED VASCULAR DISEASE IN HOMOCYSTEINEMIA Putative mechanisms of thrombosis in hyperhomocystinemia include endothelial cell injury, increased platelet adhesiveness, enhanced LDL deposition in the arterial wall, and direct activation of the coagulation cascade. The vascular changes in hyperhomocysteinemia may be multifactorial. Platelet Dysfunction Platelets from patients with HH(e) have increased adhesiveness that is corrected by pyridoxine. 60 Treatment with pyridoxine also restores the decreased platelet survival seen in some patients. Homocysteine alters arachidonic acid metabolism in platelets, with increased release of proaggregatory thromboxane A Coagulation Abnormalities Activation of the coagulation cascade by homocysteine may also contribute to vascular disease. Homocysteine activates Factor XII and induces arterial endothelial cell Factor V activation. 62,63 In addition, high concentrations of homocysteine may inhibit thrombomodulin. 64,65 Because the binding of thrombin to thrombomodulin enhances formation of the anticoagulant activated protein C and inhibits thrombin activation of fibrinogen, a deficit of thrombomodulin enhances fibrin formation. All of these events effectively change the balance between procoagulation/anticoagulation and enhance the risk of thrombosis. In patients with the more severe condition of homocystinuria, there is activation and hyperconsumption of Factor VII, Factor X, and consumption of antithrombin III. 65 " 68 In homocystinuria, levels of coagulation factors are reduced. Markers of activation of coagulation, such as Fl + 2, are elevated and are correctable with treatment. 69 Studies on human umbilical vein endothelial cells demonstrate that H(e) increases tissue factor activity in a dose-dependent fashion, by increasing the rate of synthesis of tissue factor RNA. 70 Effects on the Endothelium Homocysteine has cytotoxic effects on the endothelium. Endothelial cells from CBS heterozygotes are deficient in CBS and are more susceptible to homocysteinemediated injury. 71 There has been considerable interest in HH(e) causing endothelial damage by increasing free radical production and subsequent lipid peroxidation. 72 However, we have observed that in subjects with elevated free radical activity, such as patients with diabetes and vascular disease, the presence of HH(e) does not lead to a further increase in plasma concentrations of thiobarbituric acid reactive substances, an in vivo index of lipid peroxidation (unpublished observations). Despite several studies, the exact role of free radicals in HH(e) induced endothelial damage remains unclear. In addition to the increase in tissue factor, a number of other endothelial changes have been described. Normal endothelial cells modulate the adverse effects of homocysteine by facilitating the formation of the endothelium-derived relaxing factor (EDRF) adduct, S-nitro- Vol. 106-No. 6

10 718 CLINICAL CHEMISTRY Review Article sohomocysteine. 73 The toxic effects of homocysteine may result from inability of the endothelium to sustain adequate production of EDRF. 74 Celermajer and colleagues 75 demonstrated that children with homozygous homocystinuria had impaired endothelial function and vascular reactivity. In contrast, endothelial function assessed by similar methodology is preserved in heterozygous adults. Anticoagulation and fibrinolysis are critical for blood flow. Several studies have been concerned with measuring the endothelial-derived proteins critical for these processes. Van den Berg and colleagues 76 assessed endothelial function by measuring plasma concentrations of endothelium-derived proteins such as von Willenbrand factor (vwf), thrombomodulin (TM) and tissue-type plasminogen activator (tpa). vwf and TM were elevated whereas tpa was normal in patients with HH(e). Following treatment with pyridoxine and folic acid, vwf and TM levels decreased and tpa was unchanged. Tissue plasminogen activator inhibitor (PAI-1) antigen has been shown to correlate with total plasma H(e) concentrations and may also be a marker of impaired fibrolytic activity and endothelial function. 77 H(e) has also been shown to suppress anticoagulant heparin sulfate expression in cultured porcine aortic endothelial cells and may thus contribute to thrombogenesis. 78 Finally, H(e) inhibits cyclo-oxygenase activity in human endothelial cells, decreasing prostacyclin production. 79 Effect of Hyperhomocysteinemia on the Arterial Wall Tsai and colleagues 80 studied the effect of H(e) on the growth of vascular smooth muscle cells and endothelial cells. H(e) causes a 25% increase in DNA synthesis in rat aortic smooth muscle cells. In contrast, in human umbilical vein endothelial cells, H(e) leads to a decrease in DNA synthesis in a dose dependent manner. These findings suggest that H(e) has a growth promoting effect on vascular smooth muscle cells along with an inhibitory effect on endothelial cell growth. This combination could lead to atherosclerosis. 80 Minipigs fed on a methionine-rich casein-based diet develop HH(e). These animals developed aberrations in the elastic lamina with hypertrophy of smooth muscle. 81 Co-inheritance of Factor V Leiden in Homocystinuria Seligsohn" recently reported co-inheritance of homocystinuria and Factor V Leiden mutation (activated protein C resistance), which has been found to have an association with thrombophilia. Because only one-third of patients with homocystinuria develop venous or arterial thromboses, a search was made for other contributing factors in patients who developed thrombosis. A mutation in the gene coding for Factor V replaces glutamine for arginine at position 506, increasing a patient's risk for thrombosis by altering the first cleavage site involved in the activation of Factor V. This suggests that in patients with homocystinemia, the risk of venous or arterial thromboembolic disease may be exacerbated by the presence of other concomitant etiologies of thrombophilia. MANAGEMENT OF HYPERHOMOCYSTEINEMIA Evaluation of Patients with Accelerated Vascular Disease We believe that measurement of a fasting level of H(e) should be included in the evaluation of early onset atherosclerosis. Whether a methionine load test is required in patients with a normal fasting H(e) is not clear. As the test is cumbersome, it may require some modification (standardization of a shorter test or a higher dose of an IV load) before it will be widely used. It is possible that genetic screening for mutations in enzymes may decrease the need for the test. Other tests that should be done include: lupus-anticoagulant/phospholipid antibodies, activated protein C (APC) resistance, protein C, protein S, antithrombin III (ATIII), fibrinogen, plasminogen activated inhibitor (PAI), and serum lipid and lipoprotein analysis. In patients in whom the H(e) is high, measurement of B 2, folate, and possibly methyl malonate should be performed. Selected cases should have DNA analysis for determination of MTHFR and CBS alteration. Treatment A number of agents are known to decrease plasma H(e) concentrations in patients with both HH(e) and homocystinuria. Some of these, such as folate and pyridoxine, are inexpensive, and safe. Ongoing clinical trials have been initiated that may demonstrate their efficacy in preventing or halting the progression of vascular disease. The impact of increased nutritional supplementation with folic acid that has been recently recommended on homocysteine levels in the general population also needs to be evaluated. 18 The exact dose of vitamins needed to treat mild HH(e) has not been determined. Different doses have A.J.C.P

11 GUBA, FINK, AND FONSECA 719 Hyperhomocysteinemia been used in several, short-term studies. Combination therapy with different vitamins may be necessary to achieve adequate suppression of H(e) levels in many patients. For example, Franken and colleagues 82 treated mildly HH(e) patients with vitamin B 6, 250 mg daily, for 6 weeks following which the post-load H(e) concentration decreased in 56% of patients. Further treatment with the addition of folic acid and/or betaine resulted in normalization of H(e) levels in 95% of the remaining patients. In three patients with homocystinuria, Palareti and colleagues 83 demonstrated not only reduction in H(e) levels but also correction of a number of abnormalities in blood coagulation. It is important to determine whether correction of coagulation and other vascular risk factors occurs in patients with mild HH(e) following treatment. Pyridoxine Only half of patients with CBS deficiency respond to pyridoxine. This may be because some nonresponders may have folate deficiency 84 (which can block the response to pyridoxine until folate is replenished 14 ) or decreased affinity of the mutant enzyme for the cofactor. 85 Alterations that affect other structure functions, such as mutation in the active site of the enzyme, may also be reason for non-responsiveness. Van den Berg and colleagues 76 have also demonstrated correction of mild HH(e) in a subset of young patients with cardiovascular disease (below the age of 50). Brattstrom and associates 33 demonstrated that pyridoxine 240 mg per day plus folic acid 10 mg per day reduced fasting homocysteine levels by a mean of 53% and a post-methionine load homocysteine by a mean of 39%. Folate Folic acid deficiency has been noted in a number of CBS deficient patients. 14 Some of these patients respond to folate alone whereas others require combination therapy. Brattstrom and associates 86 showed that folic acid administration in normal individuals who were not folate deficient, also reduces the plasma H(e) both fasting as well as after a methionine load. Folic acid has also been shown to be effective in reducing the acute elevation in H(e) that occurs following myocardial infarction. 87 Betaine Betaine is a methyl group donor involved in the metabolism of methionine and has been suggested as a possible treatment for HH(e). Betaine lowers plasma H(e) concentration but raises methionine levels, the significance of which is not clear Betaine has been found to be ineffective in lowering H(e) in patients on hemodialysis. 90 Other Possible Treatments The above treatment strategies may not apply to patients with very advanced aggressive vascular disease or patients with chronic renal failure and/or diabetes mellitus, where other factors may elevate H(e) levels. These patients may require much higher doses of vitamin replacement therapy. 52 Other possible treatments include fish oil, 91 tamoxifen, 92 and the chellating agent D-Penicillamine. 93 Pregnancy or estrogen replacement in post-menopausal women lowers H(e) levels, 94 and in the latter case an additional benefit of hormone therapy. Cardiovascular risk rises following menopause and recognition and correction of yet another risk factor for cardiovascular disease by hormone replacement therapy is important. SUMMARY A more precise definition of patient groups is developing from the rapidly accumulating identification of new mutations found in the CBS and MTHFR genes. While the genotype is an important predictor of clinical risk for vascular disease, CBS and MTHFR enzyme activity is variable. This phenotypic variability suggests that epigenetic and environmental factors contribute to H(e) levels and metabolism. Currently, vitamin therapy is effective in treating HH(e). Recognition of HH(e) is therefore important: HH(e) represents an easily, safely and inexpensively treatable risk factor for vascular disease. Further study is required to determine whether treatment of HH(e) with vitamins will prevent vascular disease. Acknowledgments. The authors thank Diane Sawyer, Mike Blanton, and Lisa Marr for their contributions in the preparation of this manuscript. REFERENCES 1. Kluijtmans LAJ, van den Heuvel LPWJ, Boers GHJ, et al. Molecular genetic analysis in mild hyperhomocysteinemia: A common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease. Am J Hum Genet 1996;58: Motulsky AG. Nutritional Ecogenetics: Homocysteine-related arteriosclerotic vascular disease, neural tube defects, and folic acid. Am J Hum Genet 1996; 58: Vol. 106-No. 6

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