CLINICAL CHEMISTRY
Cardiovascular Disorders (Risk Assessment) Donald W.Jacobsen Departments of Cell Biology and Clinical Pathology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Coronary artery disease, cerebrovascular disease, and peripheral occlusive vascular disease are the most common causes of mortality and morhidity in the IJnited States and many other countries today. Although there has been a declining mortality associated with cardiovascular disorders (CVD)over the past 40 years. in 1988there were nearly I 000 OOO deaths attributed to CVD in the United States alone: over S O $ oft hese deaths were due to coronary artery disease (CADI ( A f J .The declining mortality rate in CVD is related to our ability to identify and manage risk factors associated with the disease process. The three major risk fartors associated with CAI) that have been identified to date are dyslipidemia. smoking. and hypertension r A f , A2). These risk factiirsare. toa largeextent,modifiable. Othercommon risk factorsthat arealniassnciated with CAD includephysiral inactivity. ohesity, diabetes, strws, gender (CAD occurs less frequently in premtnquiusal women than in age-matrhed men,, and alcohol consumption. This review will forus on recent work involving two independent risk factors for CVD: lipoprotein (ai [I.p(ail and homocysteine. The serum levels of I.p(a,. an unusual lipoprotein consisting of a LDL particle covalently linked to apolipuprotein (ai lapo(ail, are under genetic rontrol. Approximately :W-50$ of individuals in different ethnic populations express higher levels of Lp(aJ and may beat greaterrisk forde\,elopingCVI). l’lasmalevels of homocysteine are, tu some extent, under genetic control. but other factors. such a9 nutritional or pathologiral status. may cause gross alteratiims. Slight to moderatr elevations in plasma homocysteine are now thought to tw assoriated withaninrreasedriskforCVD. Thereview willcover material that. for the most part, has appeared from 1987 ti! the end of 1992.
LIPOPROTEIN (a) Lipoprotein (a)was discovered just over three decades ago by the Norwegian geneticist Kare Berg. who observed that elevated levels of this unusual lipoprotein were associated with an increased risk for CVI) (A3i. However, only within the past five years has the importance of this unique lipoprotein as an independent risk fartur for cardiovascular diseases been widely accepted. This was due in large part to the discovery in 1987 that aporai, one of the two protein constituents in Lprai, bore a striking resemblanre to plasminogen based on partial amino arid sequence data from purified apoIaJ (A4). From the amino arid sequenre data. oligonucleotide probes were prepared and used for screening a human liver cDNA lihrary from which the nucleotide sequenceof thestructural gene for apo(ai wasohtained (A.51. It wasshownin 1999that Lp(a) competesfortheplasminogen binding sites on blood cells and vascular endothelium (Afi, AT). Although I.p(a) has kringle domains that have high sequenre homology to the fourth kringle domain of plasminogen,it cannot beconvertedtoanacti\,eenzyme. It therefore lacksthecatalytir activityof plasmin, the major thrombolytic enqmein the body. Highlevelsofplasmal.prai mayenhance the prothromhotirand procoagulant propertiesof the vascular endothelium and rirrulating hlvud cells by competing with plasminogen for hinding sites. Whrther this hypothesis arrounts for the atherogenic effects nt high plasma Lptai levels is not known, but i t is an area of active investigation. Several general reviewson I.p(ai have recentlyappeared lA8,4121 as well as more specialized reviews concerning Lp(aJas a risk factor I A f 3 - A f 8 J . A monograph on Lprai ha3 also heen published ( A f 9 ) . Thissectionwillreviewthp 11)genetics and hiochemistry of Lp(a,. (21 methuddogies for the determination of plasma Lp(al, (3) resultsol recent clinicalstudies on Lotai. and 141 future directions of Lo(ai research. &ne& and Biochemistry. Thegeies for human apo(a) and for plasminogen are located on the long arm of chromosome 6 r.420. .-l’2f). Linkage analysis has demonstrated that the genes are in close proximity ( A W . The genetic size polymorphism observed in human Lp(a) appears to he due to the codominant expression of different alleles at the apo(a) structural gene locus (A23). At least six different isoforms of apo(a),designated LpF, LpB, LpS1, LpS2, LpS3, and LpS4,
Donald W. Jacobson Is Dhector of iha
Laboratoryof Developmental Hematology lnthe Departmentof CllnicalPathologyand a member of the staffin the Department of Cell Biology, The Research Institute of the Cleveland Clinic Foundation. He isalso an Adjunct Associate Professor In the Department of Biology at Cleveland State University. He received his B.A. degree in chemistry and biology from the Unlversily of Pennsylvania in 1961 and his M.S. and Ph.D. degrees in biochemistry and cell biology from Oregon State University In 1965and 1967. respectively. Allerdolng postdoctoral training in enzymology at
1,
i
’
ScrippsClinicandResearchFoundationtrom196710 1971,heremfined
at Scrlpps as an assistant member of the staff. In 1984 he became a Staffmember at the Cleveland Clinic Foundation wlth appointments In The Research lnstiule and the Division of Pathology and Laboratory Medicine. Dr.Jawbsen IsamemberoftheAmericanChemlcalSoclety. the American Society for Biochemistry and Molecular Biology. the American Society ofHematology.the American Federatlon for Cllnlcal Research, and the Protein Society. He served as Chairman of the Cleveland Section of the American Chemical Society In 1992. HIS research Interests include cellular and intracellular transport of cobalamin, biosynthesis and mechanism of action of cobalamln coenzymes. hyperhomocysteinemia and the mechanism of homccystelnslnduced atherosclerosis.and the pathogenesis and prevention of sulfie hypersensltivity. have been described, ranging in molecular weight (M,) from 300 to greater than 800 kDa. The designations F (faster), B (equal to), and S (slower) for apo(a) isoforms were based on their electrophoretic mobilities (sodium dodecyl sulfatepolyacrylamide gels; SDS-PAGE) relative to the mobility of B-100 (A24). The size heterogeneity of apo(a) isoforms is due totandem repeatsofthe plasminogen-like kringle 4 region in the apo(a) structural gene (A%). The number of kringle 4 repeats ranges from 15 to 40. There is an inverse relation between apo(a) isoform size and plasma concentration (A26, A27). Thus, homozygous individuals with the B phenotype (Lp(a), 460 kDa) have relatively high concentrations (mean =64 mgidL) of plasma Lp(a) while homozygous individuals with the S4 phenotype have low concentrations (mean =6 mgldL) (A27, A28). Additional isoforms of Lp(a) may be present in plasma and recent report8 of 10 and 11 isoforms have appeared (A29, A30). Running SDS gels with lower acrylamide content appears to give better resolution of Lp(a) containing high M,forms of apo(a) (A29, A30). At least 19 alleles for apo(a) have been identified in genomic DNA from 102unrelated Caucasians (A31). Heterozygotes have plasma concentrations of Lp(a) that appear to represent the sum of the contributions from the individualalleles (A32). Although it is not clear how the apo(a) gene is regulated, it has been suggested that a methylated DNA binding protein may recognize sequence motifs within the tandem repeats of the kringle-4 region (A33). Apolipoprotein (a) is synthesized in the liver, where it is presumably assemhled into Lp(a) by combining with apolipoprotein 8-100, the apolipoprotein of low-density lipoprotein (LDL) (A34). Messenger RNA for apo(a) has been found in rhesus monkey brain and testis (A35),but the contribution of overall Lp(a) production by these other organs is unknown. Acquisition oflipidhytheapo(a)-apoB-l00complexprobably occurs within the hepatoeytesince thesecreted products have densities resembling plasma Lp(a) (A34). However, since the apo(a)-apo B-100 complex is amphipathic (i.e., has both hydrophobic and hydrophilic properties), it can bind to a variety of triglyceride-rich and cholesteryl ester-rich particles (A36). The M , polymorphism of Lp(a) is due to size heterogeneity associated with the apo(a) component. The deduced amino acid sequence of apo(a) is strikingly similar to that of plasminogen (A5). Thus, beginning with the N-terminus, the followingplasminogen-like domainsare found in apo(a): signal peptide with 100% sequence homology; multiple tandem repeats of kringle 4 with 75-85% sequence homology; a single kringle 5 with 91 % sequence homology; ANALYTICAL CHEMISTRY. VOL. 65. NO. 12, JUNE
15. 1993
387R
CLINICAL CHEMISTRY
Each a protease domain with 94% sequence homolo krmgle 4 domain of apo(a)contains SIXcysteine resiges which occupy sequence positions that are identical to the krin le 4 positions of lasminogen. These residues appear to form t k e e intrachain $sulfide bonds which are important in conferrin the kringle-like (named after the appearance of a Danis! pretzel) conformation found in plasminogen. The short regions (36 amino acids) linking the kringle domains of apo(a) have six 0-linked sites and, unlike plasminogen, are highly glycosylated (A37). Approximate1 30% of the maas of apo(a) is carbohydrate. This may partial& explain the amphipathic nature of the apo(a)-apo B com lex, since apo B alone tends to be insoluble. There is an afditional cysteine residue in kringle 36 which is thou ht to form a disulfide link with a cysteine residue on apo 8.100. The physiolo ical function of Lp(a) is unknown. Although LDL and Lp(a)%avefractional catabolicrates that are within the same order of magnitude (A38), the role played by the LDL receptor pathway for removal of Lp(a) from circulation has been somewhat equivocal (A39-A42). Also unknown is how Lp(a) reci itates or participates in the atherogenic rocess, alttougg it may compete with lasmino en for ginding sites on endothelial cell surfaces. #he intro8uction of an a o(a) transgenic mouse model may provide answers to a numEer of these questions (A43, A44). Determination of Lp(a). Most of the practical assays for plasma LP(a) are immunologically based (for a review, see ref A45). Durin the 1970s quantitative immunoelectrophoresis was intro8uced and became the method of choice for the determination of Lp(a) ( A N ,A47). The establishment of more ra id and sensitive immunological-basedassays with high samppe throughput has only recently been realized. The problems encountered by earlier investigators included the following: (1)anti-apo(a) antibodies, both monoclonal and polyclonal, that cross reacted with plasminogen; (2) antiLp(a) antibodies that cross reacted with apo B-100; (3) antiapo(a) antibodies that failed to react with all of the isoforms of apo(a); (4) lack of sensitivity. Although a double antibody radioimmunoassay with relatively hi h sensitivity was developed in the late 19709, it failed to jetect Lp(a) in approximately 9% of human plasma samples (A#). An ELISAbased assay was introduced by Fless et al. (A49)in 1989that eliminated many of these problems. In this assay immunoaffinity-purified rabbit anti-human apo(a)-antibody was used to ca tule serum or plasma Lp(a). Goat anti-human apoB antiKod was then added to react with the LDL com onent of Ep(a). A third antibody, rabbit anti-goat IgG anti& conju ated with alkaline phosphatase, was then added to comJete the sandwich. Enzyme substrate @nitrophenyl phosphate) was added and developed, and after sodium hydroxide was added, the product was determined spectrophotometrically at 410 nm with a microtiter late reader. In this study human Lp(a) was purified Ky a combination of rate zonal and density gradient ultracentrifu ations (A241 and by FPLC anion-exchange chromatograpty. Apo(a) was prepared from purified Lp(a) by reduction, carboxymethylation, and rate zonal ultracentrifugation (A50) and by gradient gel electrophoresis before antiserum roduction. Rabbit anti-apo(a) antibodies were purifiea by passage through tandem columns of LDL-Sepharose and Lp(a)-Sepharose. The LDL-Sepharose column was included to remove any traces of anti-human apo B antibodies. After removal of nonspecifically bound protein, rabbit anti-apo(a) I G was eluted from the Lp(a)-Sepharose column at pH 2.5. ?he ELISA assay was insensitive to plasminogen; hi h plasma levels of apo B or triglyceride did not interfere. $he lower limit of detection wasO.O30mg/dLLp(a)protein. The affinity of the capture antibody for Lp(a) containing high MIisoforms of apo(a) was approximately 20% less that for the smaller M, isoforms (the ca ture antibod was produced usin small isoform apo(a)). Eowever,this Jfference was renderechar ely insignificant by expressing the data on a molar basis. t h e mean concentration of Lp(a) protein in 84 individuals was 3.2m /dL witha range of 0.045-13.3 mg/dL. The distribution was stewed with one-third of the individuals having less than 1mg/dL Lp(a) protein. Conversion of Lp(a) protein values to Lp(a) lipoprotein, as re orted by most other investigators, ave a mean of 13.3 mg/df: with a ran e of 0.19-55.3 mg/dL. hean Lp(a) lipoproteinvalues re orted by other investi ators range are similar (A48, A 5 0 . A) monoclonal-based #LISA 3881 ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
for Lp(a) has recently been described (A52). BALB/c mice were immunized with purified human Lp(a) intraperitoneall over a 5-month period. Spleen cella were then fused w i d mouse myeloma cells,.and antibody- roducin hybrids were identified with an antiserum screen ELISA. bositive hybridomas were cloned twice by limiting dilution and then grown as ascites in BALB/c mice. The selected monoclonals were purified by affinity chromatography using protein ASepharose. After further specificity testing, one monoclonal antibody was selected as the capture antibody, and a second monoclonal was selected as the detection antibody and conjugated to horseradish peroxidase. The assa was valifor Lp(a) dated by comparison to a polyclonal-based (A53). It was very sensitive, with a lower limit of detection approaching 20 pg of Lp(a) protein. The assay range is 0.5 -180 ng/mL Lp(a) protein, and up to 80 5-pL plasma sam les can be processed on a single microtiter plate. Finally, a t&rd variation of the ELISA has been described that utilizes a monoclonal capture antibody and a sheep anti-human apo B polyclonal conjugated to horseradish peroxidase as the detection antibody (A54). The monoclonalcapture antibody in this assay recognizes six isoforms of apo(a) and shows no cross reactivity to plasminogen, apo B, or LDL. Since the capture antibody recognizes all six isoformsand the detection antibody is directed toward apo B, the assay is reported to give accurate molar assessments of Lp(a) no matter what isoform of apo(a) is present. The standard curve is linear over the concentration range of 3-100 mg/mL Lp(a) lipoprotein. There is still disagreement on how to report plasma Lp(a) values. It has been pointed out that Lp(a) lipoprotein mass values are subject to error due to the variability (-30 5% ) in lipid composition (A49),and it therefore recommended that plasma concentrations be reported in Lp(a) protein only (A49, A52). Data on the levels of Lp(a) lipoprotein in different ethnic populations and age groups have been accumulated fairly recently (reviewed in ref A55). The availability of rapid and sensitive ELISAs for plasma Lp(a) has made it possible to determine Lp(a) concentrations in relatively large groups of normal individuals. Helmhold et al. (A29)have observed significantdifferencesin Lp(a) lip0 rotein levels amongethnic groups. Using an affinit purifiefrabbit anti-Lp(a) ca ture antibody and a biotin-lagiled anti-Lp(a) detection antgody (followedby biotinylated streptavidin peroxidase conjugate), the mean (fSD) concentrations of Lp(a) lipoprotein in Germans (n = 1661, Ghanaians (n = 1901, San (n = 671, and Chinese (n = 88)were 18.7 f 23.1, 36.2 f 31.5, 21.1 f 19.3 and 22.9 f 18.3 m /dL, respectively. There were differences between sexes in tk, German, Ghanaian, and San populations (onlymen were studied in the Chinese). In this study samples were also run on SDS-PAGE gels (5.28% acrylamide) to identify MI isoforms. Ten M,isoforms were identified with increasing M,from 1to 10. Isoforms 6-10 were in all four populations, but isoforms 1-5 were relatively rare and not detected in all population groups. The investigators concluded that "differences in a o(a) allele frequencies are not primarily responsible for difirences in Lp(a) levels between populations" and 'the greatest ethnic variation is observed in plasma Lp(a) concentrations associated with the high molecular weight apo(a) polymor ha". Taddei-Peters et al. (A56),using the monoclonal-basexcapture ELISA described above (A54), found that the eometric mean of Lp(a) lipoprotein concentration in a aucasian population (n = 233) was 6.82 f 25.8 mg/dL with no sex difference (114males, = 6.29 f 23.6 mg/dL; 119 females, 7.36 f 27.7 mg/dL). In contrast, the geometric mean in a Black population (n= 158) was 16.1 f 18.0 mg/dL and the data s ested a possible sex difference (135 males 17.8 f 17.7 mg?& 23 females 8.90 f 19.0 mg/dL). In a more recent study by the same grou ,the er geometric mean ofLp(a) lipoprotein concentration in a Black population (n = 312) was 16.7 f 17 mg/dL (170 mdes 17.9 f 18 mg/dL; 142 females 15.0 f 16 mg/dL) Approximately 27% of the Caucasian population had Lp(a) lipoprotein levels of 225 m dL while 50 % of the Black population F As reported by other oups, there had levels of 125 mg/ C L. was an inverse correlation between apo(a) isogrm M,and Lp(a) concentration in the Caucasian opulation. However, no correlation was observed in Blacks M,and Lp(a) levels. There was no correlation between Lp(a) levels and apo A l , A2, or B in either population.
ELISX
E
P,
.
8,
CLINICAL CHEMISTRY
Clinical Studies. Recent clinical studies have provided additional evidence that elevated Lp(a) is an inde endent risk factor for CVD. In a study involving 103 midxle-aged normotensive men who were hypercholesterolemic, atherosclerotic plaques were found at carotid, aortic, and femoral sites in 36 7%,51%,and 53% of the subjects,res ctively (A57). Lp(a) lipoprotein levels were significantlyhiggr in the group w t h carotid plaques than in the group without. In patients with high LDL values, Lp(a) was associated with carotid and aortic pla ues. Earlier studies have reported an association of elevate1 plasma Lp(a) and cerebrovascular disease and stroke (A58-A62). In a study involvin 100randomlyselected men with intermittent claudication an%100randomly selected healthy controls, plasma Lp(a) levels were significantlyhigher inpatients (20.12 mg/dL) than controls (11.11mg/dL) (A63), and isoforms of apo(a) with low M,were more prevalent in patients than controls. The association of elevated plasma Lp(a) was independent of other known risk factors. In earlier studies, Lp(a) did not appear to be associated with venous thrombosis (A64) and peri heral vascular disease (A65). Hemodialysis patients, in wlom there is a hi h incidence of CVD, have elevated levels of Lp(a) (A66). 8ressman et al. ( A 6 9 found that elevated Lp(a), or a previous clinical event, were the only independent contributors to the risk of a CVDrelated clinical event occurring during a 48-month follow-up period. Recent studies have found an association between elevated Lp(a) and an increased risk for development of coronaryartery disease (A58, A68-A71), but other studies have not found this association (A72, A73). Lp(a) levels may also have predictive value for the risk of myocardial infarction (A74A76) with the possible exception of the Black opulation (A77). Sandholzer et al. (A781 have provide$ evidence suggestingthat alleles at the apo(a) locus, which regulate the synthesis of apo(a) isoforms (and thus serum levels to a large extent) determine the risk for CVD. The same group found that in a multipopulation study (Tyrol, German, Welsh, Israeli, etc.) the B, S1, and S2 isoforms were found more frequently in patients with CVD (A79). Although it has been reported that lasma Lp(a) levels decrease dramaticall after heart t r a n s p k t s (A80), Lp(a) may .be associate2 with accelerated coronary artery disease in heart transplant reci ients (A81) and is also associated with restenosis of sapfenous vein bypass grafts (A82, A83). Based on many o ulation studies, usually in Caucasians, it is generally e ieved that individuals with plasma Lp(a) lipoprotein concentrations of >30 m /dL have a 2-3 times greater risk The risk in individuals with for the development of concentrations of >48 mg/dL is 3-4 times greater. Elevated plasma levels of Lp(a) are present in almost 30% of the Caucasian population and ap roximately 50% of the Black population; however, the o v e r 8 incidenceof CVD in the Black opulation is similar to that found in the Caucasian popuPation. Future Concerns. Basic and clinical research on Lp(a) will continue to provide useful and practical information on the predictive value of plasma L (a) levels in CVD risk assessment. There will undoubte8y be a greater effort to standardize the many different types of assays that are available for measuring plasma L (a) concentration. Only recently have assays become avaigble that recognize all of the known isoforms of Lp(a). Perhaps in the future there will be clinical assays that will rapidly identif and quantitate isoformsthat are particularly atherogenic. Tgere is still much to be done in understanding how plasma levels are controlled. Thus, transcriptional and posttranscriptional levels of regulation are yet to be worked out as well as the catabolic pathway(s) involved in L (a) degradation. There have been only a limited number o studies on reducing plasma Lp(a) levels through therapeutic intervention. “Genetic surgery” may be a new approach in the treatment of this genetic trait.
EP
8VD.
P
HOMOCYSTEINE Durin the past five years, several clinical studies have a pearedi supporting the hypothesis that mild to moderate egvations of serum homocysteine,a metabolite of methionine metabolism, are associated with the development of CVD including coronary artery disease, cerebrovascular disease, and peripheral vascular occlusive disease. In many of these
reports, the association suggests that homocysteinemia is an independent risk factor for cardiovascular diaeases. These studies have utilized methodolo ies for the determination of “total”serum homocysteine. is section will address the following: (1)the association between homocystinuria and vascular disease; (2) the biochemistry of homocysteine; (3) methodological approaches for determining homocysteine; (4) recent clinical studies on mild homocysteinemia and vascular disease; (5) future directions in homocysteine research. The subject has been recently reviewed (A84-A88). Homocystinuriaand Vascular Disease. The excretion of excessive levels of homocystine in the urine identifies the condition known as homocystinuria. Under normal conditions the intracellular concentration of homocysteine is kept low as a result of remethylation reactions and catabolism via the transsulfuration pathway (see below). However, several hereditary and acquired conditions can alter normal homocysteine metabolism. This results in an increased secretion of homocysteine into circulation producing homocysteinemia and homocystinuria. Clinical homocystinuria was first described in 1962 by Carson and Neill (A891 and Gerriteen et al. (AM). The vascular complications of homocystinuria, which included venous and arterial thrombosis and premature atherosclerosis,were soon recognized. Vascular com lications were often the cause of death in these patients. dreditary homocystinuriasare most commonly caused by inborn errors of metabolism affecting the p idoxal phos hate-dependent enzyme cystathionine fl-syntrase (A91). $his enzyme catalyzes the first step of the transsulfuration pathway. The pathogenesis of homocysteine-inducedCVD in these patients is poorly understood. It is estimated that homocystinuria occurs with a fre uency of 1:200 OOO worldwide. However, there are geograp%ical regions where the incidence is much higher (e.g., 1:lO OOO in Ireland and 1:60 OOO in New South Wales) (A87). Recent interest has now shifted to individuals with only slightto moderate homocysteinemia. The incidence of this form of homocysteinemiais much higher in the general PO ulation. For example, it is estimated that the incidence of Reterozygosity for cystathionine &synthase in the eneral population is 1:70 (A91). Whether heterozygosity for this enzymatic defect results in elevated lasma homocysteine in all carriers has not been firmly estatlished. Homocystinurias are also associated with inherited disorders of folate transport and metabolism (A921 and cobalamin (B12) transport and metabolism (A93). McCully’s ”homocysteine theory of arteriosclerosis”was, in fact, based on the vascular disease observed in a patient with an inborn error of cobalamin metabolism (AM, A95). Functional cobalamin and folate deficienciesdue to dietary insufficiency, autoimmune disease, acquired defects in vitamin transport or metabolism,(e.g., HIV infection),or inborn errors of vitamin transport and metabolism will usually produce elevations in plasma homocysteine that range from mild to severe. For example, in the autoimmune disease pernicious anemia, antiintrinsic factor antibodies block normal absorption of cobalamin causing a functional deficiency of this vitamin. Serum levels of homocysteine become grossly elevated in untreated cobalamin deficiency (A%-A98). Biochemistry of Homocysteine. Homocysteine is an intermediate in the methionine cycle and the substrate for cystathionine ,%synthase, the enzyme that initiates the transsulfuration pathway (reviewed in ref A99). The other intermediates in the methionine cycle are S-adenosylhomocysteine and S-adenosylmethionine. The latter compound is the major methyl oup donor in the body as well as the substrate (after decarfoxylation) for polyamine biosynthesis. The transsulfuration athway which leads to the formation of cysteine is initiate$ by diverting homocysteine from the methionine cycle. There are four known enzymatic reactions which utilize homocysteine as a substrate. The cobalamindependent enzyme methionine synthase (bmethyltetrahydrofo1ate:homocysteine methyltransferase; EC 2.1.1.13) catalyzes the synthesis of methionine and tetrahydrofolate from homocysteine and 5-methyltetrahydrofolate (reaction1).This
A
-
homocysteine + methyltetrahydrofolate methionine + tetrahydrofolate (1) cytosolic enzyme, which re uires methylcobalamin as coenzyme, plays two critical ro?es in intermediary metabolism: ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
3691
CLINICAL CHEMISTRY
(1)the remethylation of homocysteine to form methionine; (2) the conversion of methyltetrahydrofolate to tetrahydrofolate, the coenzymatically active form of folic acid. Methionine is converted to S-adenosylmethionine by methionine adenosyltransferase (EC 2.5.1.6). S-Adenosylmethionine then serves as substrate for over 100 different methyltransferases in mammalian cells. The methyl group acce tor substrates include proteins, lipids, and nucleic acids. S - A ienosylhomocysteine, the product of methyltransferase activity, is hydrolyzed by S-adenos lhomocysteine hydrolase (EC 3.3.1.1; reaction 2). Although tKis is a reversible reaction,
*
homocysteine + adenosine S-adenosylhomocysteine + H,O (2) the equilibrium constant strongly favors S-adenosylhomocysteine formation. Under normal metabolic conditions, the intracellular concentrations of homocysteine and adenosine are very low, allowin the hydrolysis of S-adenosylhomocyskine to proceed. $he low intracellular concentration of homocysteine is due primarily to the enzymatic activities of methionine synthase and cystathionine &synthase (EC 4.2.1.22). This p idoxal 5-phosphate-dependent enzyme catalyzes the congnsation of homocysteine with serine to form cystathionine (reaction 3) (A91). Approximately 50 % homocysteine
+ serine
-
cystathionine + H,O
(3)
of the homocysteineformed in the methionine cycle is diverted into the transsulfuration pathway. Both pathways a pear to be regulated by the intracellular concentration of ladenosylmethionine (A99, A100). Homocysteine can also be remethylated to methionine by the enzyme betaine:homocysteine methyltransferase (EC 2.1.1.5; reaction 4) which has
-
homocysteine + betaine methionine + dimethylglycine (4) been found only in liver tissue. This enzyme, perhaps due to its limited tissue distribution, is incapable of handling excess homocysteine accumulation in pathological conditions that result in homocysteinemia. Approximately 80-90 5% of the homocysteine in the plasma (or serum) of normal individuals is bound to protein by a covalent mixed disulfide bond (A101,A102). The remaining 10-20% consists of other oxidized forms including the homodisulfide homocystine and the heterodisulfide homocystein lcysteine. Trace amounts of homocysteine-mixed disulfiim with reduced lutathione and cysteinylglycinemay also be present in normafplasma Free reduced homocysteine makes up lessthan 5 % (7.5 mM. Lentz and Sadler (A1221 found that 5 mM D,L-homocysteine increased thrombomodulin mRNA and protein synthesis in HUVEC and CVl(18A) cells without affecting viability. However, thrombomodulin was not expressed on the surface of these cells, suggesting that homocysteine interfered with the secretory process by partially blocking gycosylation and sulfation of the protein. The authors also reported that homocysteine and other thiols blocked cell-free protein C activation by irreversibl inactivating protein C and thrombomodulin but not thromgin. Although these are perhaps the most interesting (and provocative) of all of the in vitro studies to date, their significance should be questioned in view of the relative1 high levelsof homocysteine used in the study. Using HUVE6 cells, Hayashi et al. (A123)found that 10 mM homocysteine (isomer not specified) inactivated the cofactor activity of thrombomodulin, essentially confirming the results of Lentz and Sadler (A122). Ha”ar (A1241 recently reported that 1-5 mM D,L-homocysteine$locked tissue plasminogen activator (t-PA), but not plasminogen, binding to human endothelial cells (type not specified) in a time- and dose-dependent fashion. Interestingly the blockade appeared to be specific. L-Cysteine did not block t-PA bindin but did reverse homocysteine-associated reduction in t- A binding. Determination of Total Plasma Homocysteine. Durin the past five years, several laboratories have introduce! methods to determine total plasma homocysteine. A feature common to all of these methods is the inclusion of a chemical reducing step to liberate free homocysteine from the predominant oxidized forms present in plasma. Stabler et al. (A125)introduced one of the first methods for the determination of total homocysteine in 1987 using ca illary gas chromatyaphy/mass spectrometry (GC/MS). In &s method the samp e (100 pL of serum) is reduced with 2-mercaptoethanol for 15min at 100 OC. After protein precipitation and centrifugation, sequential cation-exchange and anion-exchange chromatograph derivatization with N-methyl-N(tert-butyldimethylsily$trifluoroacetamide, the sample is analyzed by GC/MS. The method is sensitive (1nmoVmL for homocysteine), accurate (due to the use of deuterated internal standards), and capable of determining other sulfur amino acids (e.g., cysteine,methionine, and cystathionine) in asingle run. The mean total serum homocysteine concentration in
#
CLINICAL CHEMISTRY
50 human subjects was 13.0 nmol/mL; the ran e (mean f 2 SD after log transformation) was 7.2-21.7 nmo&mL. Araki and Sako (A1261 introduced a method for total plasma homocysteine based on high-performanceli uid chromatography with fluorescence detection (HPL8-FD) in 1987. Sample (500 pL of plasma) preparation included reduction of disulfide bonds with tri-n-butylphosphine (30 min at 4 "C) rotein precipitation and centrifugation, derivatization of s&ydryl groupswith the thiol-specific reagent ammonium 7-fluorobenzo-2-oxa-l,3-diazole-4-sulphonate (SBD-F) at 60 OC for 60 min, and HPLC-FD using a (2-18 reversed-phase column. The concentration of total plasma homocysteinein 35 normal subjects was 6.18 f 1.19 nmol/mL (mean f SD), somewhat lower than reported by Stabler et al (AI%). Ubbink et al. (A127), using the same sample preparation method, modified the HPLC chromatographic conditions and were able to resolve SBD-conjugatesof cysteine, cysteinylglycine, homocysteine, and glutathione in human serum. With this assay the mean total plasma homocysteine concentration in a group of rural South African Black males (n = 52) from the Venda area of South Africa was 9.72 nmol/mL; the range (mean 1 2 SD) was 3.14-16.3 nmol/mL (A128). This population, which has not been exposed to a Westernized diet, has little or no CVD. In the same study, the mean total lasma homocysteine concentration in a group of South Lrican Caucasian males (n = 34) with no angiographic evidence of narrowin of any of the three major coronary arteries was 13.7 nmo&mL (range 4.1-23.3 nmol/mL). Male Caucasian patients with narrowingin one (n= 46))two (n= 391, or three (n = 44) coronary arteries had mean homocysteine levels of 15.8 f 7.6,18.1* 7.5 (p
