No abstract

Monoclonal antibodies were obtained against the membrane‐bound lysosomal enzyme β‐glucocerebrosidase (acid β‐glucosidase), which is deficient in Gaucher's disease. BALB/c mice were immunized with homogeneous enzyme protein extracted from a sodium dodecyl sulphate/polyacrylamide gel. The mice were subsequently hyperimmunized with partially purified enzyme prior to fusion of spleen cells with myeloma cells. After fusion, 32 primary hybrid cell populations were obtained which continued to produce antibodies against β‐glucocerebrosidase after prolonged time of culture. All antibodies reacted with both native and denatured enzyme. Four primary cell populations were subcloned and the antibodies produced were characterized. The antibodies were all four of the IgG1 subclass. Three of these antibodies bind to protein A whereas one does not. The results of binding assays indicated that three of the antibodies react with the same antigenic domain (epitope 1), but the fourth with a different one (epitope 2). Probably two antigenic determinants are present in epitope 1 since one of the antibodies with specificity for epitope 1 is inactivated after iodination by the chloramine‐T procedure whereas a second one is not.

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...

The different analytical methods for measuring vitamin B6 produce different results when whole blood patient samples are measured. The recognition of a reference method or the development of suitable reference materials and quality control materials might serve as a first step towards improved standardization or harmonization of the whole blood vitamin B6 assay.

Tris and HEPES were systematically compared äs buffers for the enzymatic assay of L-carnitine. The deproteinization methods preceding the assay were also compared. The following conclusions were drawn.1. Both Tris and HEPES act on the catalytic site of the enzyme, acetylCoA: carnitine O-acetyltransferase (EC 2.3.1.7), which is used for the conversion of L-carnitine to acetylcarnitine. HEPES is a competitive inhibitor, and no acetylated product of HEPES is formed. In the presence of Tris a limited amount of acetylTris is formed, and an appropriate blank corrects for this effect.2. The incubation time of the assay is strongly influenced by the preceding deproteinization method. The enzyme is influenced by inorganic salt, which acts äs a competitive inhibitor.3. If Tris is used in place of HEPES in end-point assays, optimal conditions and shorter assay times are achieved with less enzyme and less acetylCoA, provided more elaborate deproteinization methods are used.4. The HEPES System is more costly, but preferable for the determination of both total and free L-carnitine in combination with a matched deproteinization method.