Homocysteine assay

TABLE 16.2. Intraassay and Interassay Variation Coefficients for Calibration Data from the Plasma Homocysteine Assay"... 

Pernet P, Lasnier E, VaubourdoUe M. Evaluation of the AxSYM homocysteine assay and comparison with the IMx homocysteine assay. Clin Chem 2000 46 1440-1. 

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

A coupled enyzmatic assay makes use of S-adenosylhomocysteine hydrolase (SAHH) which hydrolyzes the methyltransfer product SAH to homocysteine and adenosine. The homocysteine concentration can be determined by conjugation of its free sulfhydryl moiety to a thiol-sensitive fluorophore [61]. This could of course also be used for arginine methyltransferases. 

The range of recovery and the inter- and intra-laboratory imprecision in some assays is listed in Table 1.2.2. It is salutary to note that when measuring homocysteine in a patient whose true concentration may be 64.0 pmol/1, within the same laboratory the results obtained may range from 55 pmol/1 to 73 pmol/1 and that in different centres the same patient could be given results ranging from 50 to 78 pmol/1. This perfor-... 

Special assays (urine and plasma) lactate, orotidine, thiosulphate, carnitine, succinylacetone, hydroxyproline, urate, orotate, sialic acid, MPS, guanidino-acetate, HVA, pyroglutamate, 5H1AA, pipecolate, pyruvate, 3-hydroxybutyrate, phytanate, VLCFA, homocysteine, 7-dehydrocholesterol, phenylalanine ERNDIM 8 148... 

Ubbink JB, Hayward Vermaak WJ, Bissbort S (1991) Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 565 441-446... 

On the day of admission, the patient had developed a deep venous thrombosis in his right calf, a site not involved in the injury. In investigating the underlying cause of the deep venous thrombosis, serum homocysteine was measured and found to be 17.4 pmol/L (normal is < 14 pmol/I.).To distinguish between folic acid and vitamin B12 deficiencies, a serum methylmalonic acid (MMA) assay was performed it yielded a result of 0.59 pmol/I. MMA (normal is < 0.30 pmol/L). This confirmed the presence of vitamin B12 deficiency, despite a serum B12 concentration that was within the normal range. 

The enzyme also known as methionine synthetase catalyzes the conversion of homocysteine to methionine using a folate derivative as the methyl donor. The assay of Garras et al. (1991) is based on quantitation of the o-phthaldehyde derivative of methionine. 

The incubation mixture contained in a Anal volume of 100 /iL 400 fiM DL-homocysteine, 500 (iM ( )-L-A/5-methyltetrahydrofolate, 50 fiM cyanocobalamin, 300 fiM 5-adenosylmethionine, 125 mM 2-mercaptoethanol, 20 fiM L-norvaline, 50 mM potassium phosphate buffer (pH 7.4), and 50 /xL of liver or cell extract. The incubation mixture was immediately flushed with nitrogen and overlayered with 50 /tL of bis(3,5,5-trimethylcyclohexyl)-phthalate. The incubation, carried out at 37°C in the dark, was stopped by the addition of 10 / L of 4 TV perchloric acid. The acid was then neutralized by addition of 10 / L of 4 TV KOH containing 3.3 M potassium bicarbonate. After centrifugation, 90 /iL of supernate was mixed with 175 fiL of o-phthaldialdehyde reagent (prepared by mixing 1 mL of 56 mM o-phthaldialdehyde in methanol with 9 mL of 0.1 M sodium borate buffer, pH 9.5, then adding 40 fiL of 2-mercaptoethanol). After 2 minutes at 23°C, 220 /xL of this mixture was used for HPLC analysis. The assay is linear for at least 2 hours. 

Many different applications for gold cysteine nanoclusters have been proposed. One application is the use of citrate capped nanoparticles as sensors for the detection of thiol containing stractures. This application rehes on the fact that thiols can easily displace oxygen coordinated moieties. Zhong et al. have studied this process between cysteine and homocysteine to develop an assay for the homocysteine biomarker, a correlate to cardiovascular disease. When citrate stabilized particles of 13 run were incubated with either thiolate structure, the solution color changed from a dark red to a deep blue. The displacement of citrate by these moieties resulted... 

Elevated concentrations of plasma homocysteine (HCY) are related to an increased risk of cardiovascular disease, which exists in numerous forms in plasma, with the main form existing as a disulfide with itself, cysteine, or albumin. Therefore, the first step in the measurement involves treatment with a reducing agent, in this case dithiothreitol (DTT), to obtain HCY in its free form (Eq. 16.34). Some amino acids (e.g., L-cysteine and L-methionine) are present in human plasma at higher molar concentrations than HCY and may interfere with this assay. To avoid this possible interference, the highly selective enzymatic conversion of HCY to S-adenosyl-L-homocysteine (SAH), as shown in Eq. 16.34, is used. Both reactions (reduction and conjugation) are accomplished in 30 min at 34 °C. 

Nexo E, Engbaek F, Ueland PM, Westby C, O Gorman P, et al. Evaluation of novel assays in clinical chemistry quantification of plasma total homocysteine. Chn Chem 2000 46 1150-6. 

Both direct and indirect (ftmctional) methods are available for assessing vitamin B status. The indirect tests include assays for urinary and serum concentrations of methylmalonic acid, plasma homocysteine, the deoxyuridine suppression test, and the vitamin B12 absorption test. Cyto-chemical staining of red blood cell (RBC) precursors and the test for IF blocking antibodies are also ancillary methods for assessing vitamin B12 status. 

Pastore A, Massoud R, Motti C, et al. FuUy automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine and 2-mercaptopropionylglycine in plasma and urine. Clin Chem, 1998 44 825-832. 

The diagnosis of homocystinuria is based on the recognition of the clinical phenotype in conjunction with the identification of an elevated total plasma homocysteine and elevated plasma methionine concentrations (via quantitative plasma amino acid analysis). Low cystine and low cystathionine are also seen (Box 14.3). In addition, increased urinary excretion of homocysteine as well as cysteine-homocysteine disulfide can be identified on urine amino acid analysis. Confirmation of the diagnosis can be done via enzyme assay, typically performed on cultured skin fibroblasts, lymphocytes, or liver tissue, or via molecular studies. 

Major vitamin Bi2-dependent metabolic processes include the formation of methionine from homocysteine, and the formation of succinyl coenzyme A from methylmalonyl coenzyme A. Thus, apart from directly determining vitamin B12 concentration in serum, elevated levels of both methylmalonic acid and homocysteine may indicate a vitamin B12 deficiency. Serum cobalamine concentration is often determined by automated immunoassays using an intrinsic factor as binding agent. These assays have mainly replaced the microbiological methods. Literature data about vitamin B12 concentration in serum varies. Values <110-150pmoll are considered to reflect deficiency, whereas values >150-200pmoll represents an adequate status. 

This enzyme contains tightly bound cyanocobalamin which imparts a salmon pink colour. It catalyses one of the reactions which generate methionine from homocystein, and is thus involved indirectly in transmethylation. It may be assayed radiochemically. 

Since the oxidation of thiols at mercury electrodes is facile and specific, it should be adaptable to HPLC detection. Rabenstein and Saetre" first reported this and so gave rise to a significant improvement in the assay of thiols in complex mixtures. Cysteine and other amino-thiols were separated on a cation-exchange column and detected at a mercury-pool EC detector (-1-0.05 V vs SCE) with a sensitivity of < 1 nmol for most of the thiols tested. Sample preparation consisted of little more than diluting a protein-free extract of a biolluid. These authors subsequently detailed many applications of this assay, e.g. D-penicillamine in blood and urine, cysteine and homocysteine in plasma and urine" and cysteine and glutathione in... 

E. Causse, C. Issac, P. Malatray, C. Bayle, P. Valdiguie, R. Salvayre, et ai. Assays for total homocysteine and other thiols by capillary electrophoresis-laser-induced fluorescence detection, I. Preanalytical condition studies, J. Chromatogr. A, 2000, 895, 173-178. 

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