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.

High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinylglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;178:208), which used monobromobimane to specifically derivatize thiols, has been extensively 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 limit of detection < or = 4 pmol in the assay) and precise (intra- and interassay CV for homocysteine, 3.31% and 4.85%, respectively). Mean total homocysteine concentrations in plasma and serum were significantly different, both from healthy male donors (9.26 and 12.30 mumol/L, respectively; P < 0.001) and healthy female donors (7.85 and 10.34 mumol/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 correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocysteine in both sexes.

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection.

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.

Separation of plasma carotenoids and quantitation of beta-carotene using HPLC.

A method was developed for the extraction and separation of human plasma carotenoids and quantitation of beta-carotene. Carotenoids were extracted from plasma with ethanol: hexane and separated by C18 reversed phase HPLC using spherical 3 micron packing. beta-Carotene was identified and quantitated using an external standard. The within-run precision of three different plasma pools ranged from 3.53-5.72% relative standard deviation (RSD). The between-run precision was 7.34% RSD. The method was linear to 500 micrograms/l with a statistical detection limit of 3.80 micrograms/l. Recovery of added beta-carotene was from 90.41-100.37%. This method was compared to a spectrophotometric 'total carotene' method. The mean plasma concentrations of 25 male and 25 female human volunteers for the 'total carotene' were 1,549 micrograms/l for all samples, 1,487 micrograms/l for males and 1,611 micrograms/l for females. The corresponding true beta-carotene concentrations obtained by HPLC analysis were 134.8, 115.9 and 153.7 micrograms/l, respectively. The true beta-carotene concentrations were on the average only 8.76% (8.07% for males and 9.46% for females) of the concentrations obtained by the spectrophotometric 'total carotene' method. Correlation between the methods had an r = 0.6107. The poor correlation is due to the difference in the measured components. Total carotene methods measure all solvent extractable moieties having absorbance in the 430-460 nm region, while the HPLC method quantitates true beta-carotene after chromatographic separation from other carotenoids. Reference intervals were established for plasma beta-carotene using REFVAL, an IFCC computer program for determining statistical reference intervals. The reference interval for all samples is 40 to 344 micrograms/l.

Liquid-chromatographic determination of urinary riboflavin.

We describe a method for determining urinary riboflavin by "high-pressure" liquid chromatography, with fluorometric detection. An aliquot of a 24-h urine specimen is injected directly into the chromatograph and the natural fluorescence of riboflavin is measured as the compound is eluted. Interference from other fluorophores is obviated because of differing retention times. Urinary components other than riboflavin and its analogs or degradation products exhibit no significant fluorescence at the wavelengths used (450 nm excitation, 530 nm emission). Analytical recovery of added riboflavin was 96.5 (SD 1.1)%. The CV within-run was 0.7%, between-run it was 4.3%. Concentrations of riboflavin as low as 10 micrograms/L are readily detected, with a linear relation of response to concentration to at least 2000 micrograms/L.