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 (original) (raw)
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High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinyiglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;1 78:208), 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 limitof detection 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 significantlydifferent, bothfrom 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 correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174).
Homocysteine and other thiols in plasma and urine: automated determination and sample stability
Clinical Chemistry, 1993
We have developed a modified version of our fully automated column-switching HPLC method for determining total plasma homocysteine based on single-column (reversed-phase) separation. Homocysteine, cysteine, and cysteinylglycine in plasma (total concentrations), acid-precipitated plasma (non-protein-bound concentrations), and urine can be determined. The derivatization and chromatography were performed automatically by a sample processor. The successful separation of all thiol species (within 15 min) was accomplished by accurate adjustment of the pH of the mobile phase to 3.65 (plasma) or 3.50 (acid-precipitated plasma, urine). Maximal fluorescence yield of cysteine, cysteinylglycine, and, to a lesser degree, homocysteine was dependent on optimal concentrations of EDTA and dithioerythritol during reduction (with NaBH4) and derivatization (with monobromobimane). The method is sensitive (detection limit approximately 0.05 pmol) and has a high degree of precision (CV < 5%). The sampl...
Rapid measurement of total plasma homocysteine by HPLC
Clinica Chimica Acta, 2003
Background: Determination of plasma homocysteine has gained increasing interest during the past few years. Several HPLC methods for determination of homocysteine are available. Based on these methods, we developed a new HPLC assay for rapid and sensitive measurement of total plasma homocysteine. Methods: As a reducing reagent tris-(2-carboxylethyl)-phosphine is used, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate serves as the derivatization agent. Separation is performed by reversed-phase HPLC using a precolumn and a 55-mm RP 18 cartridge; mobile phase: 0.1 mol/l KH 2 PO 4 with 5% methanol, adjusted to pH 2.7 with ortho-phosphoric acid, flow-rate 1.1 ml/min. Results: Homocysteine is clearly separated from other thiols, the retention time being 2.2 min, total analysis time is 6 min. Tests for linearity, recovery and precision are satisfactory, as well as the comparison with a commercial available assay method. Detection limit of the method is 0.5 Amol/l, it could be further enhanced for measurements of even lower homocysteine concentrations in, e.g., cell culture supernatants. Conclusions: The described method is well suited for analysis of thiols in blood specimens. It is more convenient and more rapid than methods described earlier. D
Assessment of homocysteine status
Journal of inherited metabolic disease, 1997
Plasma total homocysteine (tHcy) determination is used in the diagnosis of homocystinuria, in cobalamin and folate deficiency and in cardiovascular risk assessment. However, determination of tHcy includes many pitfalls which complicate the assessment of homocysteine status. In the present article, we review basic knowledge for a rational use of plasma tHcy in diagnostic as well as scientific work. The subjects dealt with are procedures for sample handling and processing, the principles of tHcy analyses, and genetic and acquired determinants of the plasma tHcy concentration.
Total homocysteine in plasma or serum: methods and clinical applications
Clinical chemistry, 1993
Total homocysteine is defined as the sum of all homocysteine species in plasma/serum, including free and protein-bound forms. In the present review, we compare and evaluate several techniques for the determination of total homocysteine. Because these assays include the conversion of all forms into a single species by reduction, the redistribution between free and protein-bound homocysteine through disulfide interchange does not affect the results, and total homocysteine can be measured in stored samples. Total homocysteine in whole blood increases at room temperature because of a continuous production and release of homocysteine from blood cells, but artificial increase is low if the blood sample is centrifuged within 1 h of collection or placed on ice. Different methods correlate well, and values between 5 and 15 mumol/L in fasting subjects are considered normal. Total homocysteine in serum/plasma is increased markedly in patients with cobalamin or folate deficiency, and decreases ...
Clinical Chemistry
Total homocysteine is defined as the sum of all homocysteine species in plasma/serum, including free and proteinbound forms. In the present review, we compare and evaluate several techniques forthe determination of total homocystelne. Because these assays include the conversion of all forms into a single species by reduction, the redistribution between free and protein-bound homocysteine through disulfide interchange does not affect the results, and total homocysteine can be measured in stored samples. Total homocysteine in whole blood increases at room temperature because of a continuous production and release of homocysteine from blood cells, but artificial increase is low if the blood sample is centrifuged within 1 h of collection or placed on ice. Different methods correlate well, and values between 5 and 15 mol/L in fasting subjects are considered normal. Total homocysteine in serum/plasma is increased markedly in patients with cobalamin or folate deficiency, and decreases only when they are treated with the deficient vitamin. Total homocysteine is therefore of value for the diagnosis and follow-up of these deficiency states and may compensate for weaknesses of the traditional laboratory tests. In addition, total homocysteine is an independent risk factor for premature cardiovascular diseases. These disorders justify introduction of the total homocysteine assay in the routine clinical chemistry laboratory.
Clinical chemistry, 1998
We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine--cysteine, homocysteine, cysteinylglycine, and glutathione--as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97-107%, with little difference between plasma and urine samples. The detection limit was approximately 50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (a...
Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion
Clinical Chemistry, 2004
Background: Measurement of plasma total homocysteine has become common as new methods have been introduced. A wide range of disorders are associated with increased concentrations of total homocysteine. The purpose of this review is to provide an international expert opinion on the practical aspects of total homocysteine determinations in clinical practice and in the research setting and on the relevance of total homocysteine measurements as diagnostic or screening tests in several target populations. Methods: Published data available on Medline were used as the basis for the recommendations. Drafts of the recommendations were critically discussed at meetings over a period of 3 years. Outcome: This review is divided into two sections: (a) determination of homocysteine (methods and their performance, sample collection and handling, biological determinants, reference intervals, within-person variability, and methionine loading test); and (b) risk assessment and disease diagnosis (homocystinuria, folate and cobalamin deficiencies, cardiovascular disease, renal failure, psychiatric disorders and cognitive impairment, pregnancy complications and birth defects, and screening of elderly and newborns). Each of these subsections concludes with a separate series of recommendations to assist the clinician and the research scientist in making informed decisions. The review concludes with a list of unresolved questions.
Biomedical Chromatography, 2009
A semi-micro column HPLC-fluorescence method for routine determination of thiol derivatives such as homocysteine (Hcy), cysteine (Cys) and cysteamine (CA) is described. The thiol derivatives labeled with ammonium-7fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) were isocratically separated within 12 min on a semi-micro ODS column (Daisopak-SP-120-5-ODS-BP) with a mixture of 25 mM acetate buffer (pH 2.00) and CH 3 CN as a mobile phase. The purity and similarity of SBD-thiols by a multi-wavelength fluorescence detector were more than 92.3 and 96.7%. The detection limits of Hcy, Cys and CA at a signal-to-noise ratio of 3 were 0.16, 0.47 and 0.03 mM, respectively. Furthermore validation parameters such as accuracy, precision and robustness of the proposed method showed satisfactory results. Almost 850 plasma sample injections (range 572-1076, n = 3) for a column could be performed without differences in retention time and peak heights of labels. As an application of the proposed method, the determination of thiol derivatives in normal human plasma (n = 103) was demonstrated. The correlation coefficients between Hcy vs Cys and Hcy vs CA were 0.38 and −0.35, respectively.