Multicenter Analytical Evaluation of an Enzymatic Method for the Measurement of Plasma Homocysteine and Comparison with HPLC and Immunochemistry

Abstract

trations for EtG. The peak values for both compounds were obtained in the 4-h collection, and both compounds were still measurable in the sample collected at 29 h, but not at 32 h.Among 54 clinical urine samples selected at random from those sent to the laboratory for routine testing of recent alcohol consumption, all 31 samples with a detectable EtG (mean, 427 mol/L; range, 1.7-3162 mol/L) were also positive for EtS (mean, 257 mol/L; range, 1.1-2095 mol/L), and 2 others were positive only for EtS (0.6 and 2.4 mol/L, respectively). There was a good correlation between EtS and EtG (r 2 ϭ 0.839; P Ͻ0.0001) with somewhat higher mean concentrations for EtG (mean EtG:EtS ratio, 1.5; range, 0.3-3.0). The remaining 21 samples were negative for both EtS and EtG. No EtS was detected in 25 urines collected on separate days from two healthy individuals who had abstained from ethanol for several days before sampling, according to self-reports.These results demonstrate that sulfate conjugation is a metabolic pathway for ethanol in humans and that EtS is a common constituent in the urine after alcohol intake. Sulfotransferases constitute an important inactivation and detoxification enzyme system for xenobiotics and small endogenous molecules (13 ). However, based on comparison with previous data on the relative importance of EtG to overall ethanol metabolism (1 ), it appears that only a very small fraction (Ͻ0.1%) of the ethanol ingested undergoes sulfate conjugation in humans. Being a direct derivative of ethanol, EtS appears to be a specific indicator of recent alcohol consumption and, as for EtG, also shows a much longer window of detection than the parent compound, implying a higher sensitivity. This means that urinary EtS could be a new promising candidate marker to disclose recent alcohol consumption even when ethanol is no longer measurable in body fluids. Whether there is any advantage in measuring EtS instead of the other markers of acute alcohol consumption remains to be elucidated. Potential applications include verification of abstinence or detection of relapse drinking during outpatient treatment of alcohol-dependent individuals and in forensic toxicology to determine whether the ethanol identified originates from alcohol ingestion before death or sampling or was generated artifactually (14 ).The study was supported in part by funds from the Karolinska Institutet. Homocysteine (Hcy) is a sulfur amino acid and a metabolite of the amino acid methionine. Hcy is very reactive because it contains a free sulfhydryl (thiol) group, and it readily oxidizes to form various disulfides. The fraction of free Hcy in plasma is therefore Ͻ2% of total plasma Hcy (tHcy) (1 ).Increased Hcy has been associated with cardiovascular, cerebrovascular, and peripheral vascular disease (2-4 ) and is recognized as an independent risk factor. The need for a simple automated assay is therefore increasing, and various analytical methods have become available. Currently the two most widely used techniques are HPLC and immunochemistry.We implem...

Hyperhomocysteinemia and thrombophilia

“…All immunoassays provide results with small bias compared to chromatographic assays (55,56,60,62). As for chromatographic and immunoassays, the first step in all enzymatic assays involves the reduction of all tHcy forms to homocysteine, followed by an enzymatic conversion (64,65). Values obtained from different laboratories that use the same commercial assay can be compared directly (54 In the last five years colorimetric enzymatic assays have become available that can be run on routine clinical chemistry analyzers.…”

“…Also, they are accurate, reproducible, simple to perform and do not require skilled technical staff (54,63). Although their total imprecision does not meet the criteria for desirable performance at all concentrations -based on the within-subject coefficient of variation -enzymatic methods seem to perform better than chromatographic methods (64). As for chromatographic and immunoassays, the first step in all enzymatic assays involves the reduction of all tHcy forms to homocysteine, followed by an enzymatic conversion (64,65).…”

Dosage de l’homocystéine plasmatique, comparaison de deux méthodes : CLHP versus immunonéphélémétrie

“…Ces méthodes nécessitent également une centrifugation du sang le plus rapidement possible après le prélèvement ainsi que le respect strict de conditions pré-analytiques contraignantes [10]. La méthode de dosage de l'Hcy prise comme référence est la CLHP [1,13,[19][20][21], avec une valeur de seuil de normalité haute et décisionnel clinique fixée à 15 M. L'Hcy peut également être dosée par les techniques immunoenzymatiques avec détection en polarisation de fluorescence, enzyme linked immunosorbent assay (Elisa) [2], en chimiluminescence [14], en chromatographie échangeuse d'ions [15], en électrophorèse capillaire [16], en chromatographie gazeuse couplée à un spectromètre de masse [17,18] et enfin en immunonéphélémétrie [11,12].…”

Study of Homocysteine Concentration in Coronary Heart Disease Patients and Comparison of Two Determination Methods

“…The reference values of tHCY are within the range [5][6][7][8][9][10][11][12][13][14][15] mol/l, but the recommendable, i.e. safe levels should be kept in the range 9-10 mol/l [7] .…”

“…Moreover, different techniques, i.e. chromatographic, enzymatic, and immunochemical, have become available for the HCY measurement [14][15][16][17] . Among the immunochemical methods, some are designed to be performed on multiparametric appliances, leading to a greater accessibility of the test for most of the clinical laboratories.…”