CLINICALCHEMISTRY,Vl. 40, No. 6, 1994 873 UN. CHEM. 40/6, 873-881 (1994) #{149} General Clinical Chemistry Rapid HPLC Determination of Total Homocysteine and Other Thiols in Serum and Plasma: Sex Differences and Correlation with Cobalamin and Folate Concentrations in Healthy Subjects Donald W. Jacobsen,”2’7 Vytenis J. Gatautis,’ Ralph Green,”2 Killian Robinson,3 Susan R.. Savon,2 Michelle Secic,4 Ji Ji,5 Joanne M. Otto,6 and Lloyd M. Taylor, Jr.6 High-performance liquid chromatography with fluores- cence detection has been utilized for the rapid determi- nation of total homocysteine, cysteine, and cysteinyigly- cine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;1 78:208), which used monobromo- bimane to specifically derivatize thiols, has been exten- sively modified to allow for rapid processing of samples. As a result, >80 samples a day can be assayed for total homocysteine, cysteine, and cysteinylglycine. The method is sensitive (lower limitof detection 4 pmol in the assay) and precise (intra- and interassay CV for homo- cysteine, 3.31% and 4.85%, respectively). Mean total ho- mocysteine concentrations in plasma and serum were significantlydifferent, both from healthy male donors (9.26 and 12.30 mol/L, respectively: P <0.001) and healthy female donors (7.85 and 10.34 j.mol/L, respectively; P <0.001). The differences in total homocysteine between sexes were also significant (P = 0.002 for both plasma and serum). Similar differences were found for cysteine and cysteinylglycine. We found a significant inverse cor- relation between serum cobalamin and total homocys- teine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocys- teine in both sexes. Indexing Terms: hypethomoysteinemla/cysteine/cy’stein}dgtydne/ fluorometry/chromatography, reve,sed-phase/monobromobimane/sex- related differences Hereditary cystathionine (3-synthase deficiency and certain inborn errors of cobalamin (B12) and folic acid transport and metabolism can result in severe hyper- homocysteinemia and homocystinuria (1-3). An early recognizable clinical manifestation of homocystinuria is an unusually high incidence of premature cardiovascular disease (4, 5), often the cause of patient mortality (1). Recently, several clinical studies (reviewed in 6-10) re- ‘Department of Clinical Pathology, Division of Pathology and LaboratoryMedicine,2 Department of Cell Biology, The Research Institute, and 3Department of Cardiology, Division of Medicine, and4 Department of Biostatistics andEpidemiology,The Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 41195. 5Department of Chemistry, Cleveland State University, Cleve- land, OH 44115. Current address: Department of Laboratory Med- icine and Pathology, Mayo Clinic, Rochester, MN 55905. 6Division of Vascular Surgery, Oregon Health Sciences Univer- si7, Portland, OR 97201. Address for correspondence: Department of Cell Biology, FF4- 05, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Fax 216/445-5480; Internetjacobsd@ccsmtp.ccf.org. Received October 26, 1993; accepted February 22, 1994. ported an association between milder degrees of hyperh- omocysteinemia and coronary artery disease (11, 12), cerebrovascular disease (13,14), and peripheral arterial occlusive disease (14, 15). In many of these studies, hy- perhomocysteinemia was an independent risk factor for the cardiovascular disease (11, 14, 15). The etiologr of mild to moderate hyperhomocysteinemia-in the ab- sence of frank deficiency of cobalamin, folic acid, pyridox- me (B6)-in patients with cardiovascular disease has not been established with certainty. Evidence suggests that some of these individuals are heterozygous for cys- tathionine $-synthase deficiency (11, 16). However, the phenotypic determinants currently used to establish heterozygosity show considerable overlap with normal individuals (17). Thermolabile methylenetetrahydrofo- late reductase, an inherited enzyme defect that also results in hyperhomocysteinemia (18), has recently been reported to be an independent risk factor for coro- nary artery disease (19). The roles played by homocys- teine in atherogenesis and thrombogenesis and the effect of therapeutic lowering of plasma homocysteine on car- diovascular disease are unknown. Accurate determina- tion of serum and plasma concentrations of homocysteine is essential for understanding the role of homocysteine in the pathogenesis of vascular disease. Because plasma homocysteine concentrations can bi lowered by adinin- istration of folic acid (20-22) or obalamin (23, 24), assessment of homocysteine status in subjects involved in dietary modification or vitamin s ipplementation pro- grams, as well as in cardiovascular disease patients at large, will require rapid and reproducible assays. Early studies of plasma from patients with homocys- tinuria reported an assortment of at normal sulfur-bear- ing amino acids, including homoc3stine and homocys- teine-cysteine mixed disulfide, whi(h, at the time, were undetectable in normal plasma (2.5). Only later were these metabolites also found in not mal plasma at very low concentrations (26-28). It is now known that 75- 90% of the homocysteine in normal lasma is covalently bound to plasma proteins by disulfide bonds (29-31). To determine total plasma homocysteine-the sum of all protein-bound forms, oxidized low-molecular-mass forms, and free reduced homocysteu Le-it is necessary to reduce disulfide bonds. An early radioenzymatic method for total plasma ho- mocysteine included dithioerythriol as the reducing agent to break disulfide bonds (30). More recent meth- ods for determining total plasma homocysteine have utilized 2-mercaptoethanol as the reducing agent and