Colorimetric assay method for determination of the tannin acyl hydrolase (EC 3.1.1.20) activity

A new colorimetric method of tannase (tannin acyl hydrolase, EC 3.1.1.20) assay has been developed using its specific substrate tannic acid. It is based on the changes in optical density of substrate tannic acid after enzymatic reaction at 530 nm. The residual tannic acid was measured by a modified BSA precipitation method. This assay is very simple, reproducible, and very convenient, and with it tannase activity can be measured in relation to the growth of the organism.     

On the genetics of the human serum paraoxonase (EC 3.1.1.2)

Summary Incubation of the sera of 799 nonrelated persons with paraoxon led to varying degrees of inhibition of the serum cholinesterase (EC 3.1.1.8) with residual activity between 0% and 67.4% of the initial activity. This is the result of a differing paraoxonase (EC 3.1.1.2) activity. The residual activities show a trimodal distribution. The results of studies of 99 families with children show that an autosomal dominant heredity factor is most likely. Consideration of the constellations of the activity values within the families can thus yield a stochastic external criterion. This, together with the shape of the distribution of the individual values, gives good statistical estimates for the distributions and frequencies of the three groups obtained by an iteration technique. Tests of association that take account of group membership show that residual activity does not depend on the blood groups A, B, 0, and Rh, or on age. A conclusive argument for our assumption of three activity groups is that the resulting group frequencies are consistent with the Hardy-Weinberg rule.     

A completely enzymatic method for the determination of glycerophospholipids in human serum.

A simple method is reported for the determination of GPL in serum, requiring small amount of sample and adequate for screening of large population groups. The method is based on the release of glycerol portion of the phospholipid molecule by the combined action of phospholipase C and lipase. The glycerol is then determined by well established methods. The importance of screening of lecithin, that accounts for more than 84% of the glycerophospholipids of serum lipoproteins, is discussed in view of lipoprotein function and structure and in view of interaction between lipoproteins and plasma membranes.     

A high-precision selective determination method for free N-acetylneuraminic acid in human serum

We established a high-precision selective determination method for free N-acetylneuraminic acid in human serum, involving capillary gas chromatography-mass spectrometry with selected ion monitoring and specific separation on a Sep Pak silica cartridge. In contrast to the value of 800 ng/ml of N-acetylneuraminic acid previously reported by Haverkamp et al. (J. Haverkamp, R. Schauer, and M. Wember (1976) Hoppe-Seyler's Z. Physiol. Chem. 357, 1699), who used the thiobarbituric acid method, the present method gave a value of 194 +/- 96 ng/ml for 22 serum samples from normal Japanese male volunteers aged between 20 and 30 years. The mass fragmentogram of serum showed a good signal/noise ratio, and the measurement was very specific, accurate, and reproducible.     

Liquid chromatographic method for the determination of uridine in human serum

A validated gas chromatography (GC)-mass spectrometric (MS) method for the analysis of hydroxyproline in rat femur is reported. Hydroxyproline in bone hydrolysates was extracted with an anion exchange resin and the N(O)-tert-butyldimethylsilyl derivatives analyzed by GC-MS. The hydroxyproline concentration was estimated relative to pipecolic acid, 3,4-dehydroproline and n-tetracosane as internal standards. The mass-to-charge ratios (m/z) for the ions used for quantitation by single ion monitoring were 314 m/z for hydroxyproline, 198 m/z for pipecolic acid, 256 m/z for dehydroproline and 57 m/z for n-tetracosane. A coefficient of variation of 5.8% was achieved and the limit of detection was calculated to be 0.233 micromol/l bone hydrolysate.     

An LC-MS-MS method for the determination of cyclizine in human serum

Cyclizine is a piperazine derivative with anti-emetic activity that is useful in the prevention and treatment of nausea and vomiting associated with motion sickness. A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is presented for the quantitation of cyclizine in serum. Sample pretreatment involved liquid-liquid extraction of 200 microl of serum with dichloromethane after the addition of 100 microl each of ammonium hydroxide and internal standard solutions. The extracts were analyzed by HPLC on a Luna C18 reversed-phase column and an ion-trap mass spectrometer with an electrospray interface. A limit of detection of 1 ng/ml was determined which allowed for the reliable measurement of cyclizine in the serum of human subjects. The method was found to be linear over the calibration range of 2.5-100 ng/ml. The applicability of this method was demonstrated by the analysis of serum obtained from a human volunteer following administration of a single 50 mg cyclizine hydrochloride tablet. The reported method was observed to have the necessary sensitivity, selectivity, precision and accuracy for monitoring cyclizine concentrations in human subjects following oral administration.     

Candidate definitive method for the determination of cortisol in human serum

As a substantial part in the development of a reference material in clinical chemistry, a highly accurate and precise isotope dilution mass spectrometric method has been worked out for the determination of cortisol in human serum. The candidate definitive method consists of addition of 4-[14C]cortisol and, following equilibration, solvent extraction of cortisol and its internal standard from the serum matrix. After conversion into methoxime-trimethylsilyl derivatives, the extract is purified by gel chromatography. Measurements are made by combined capillary gas chromatography mass spectrometry. The ratio of peak heights at m/z 605 and 607 for each sample and calibration mixtures is used for quantification. To ensure maximum accuracy each set of calibration mixtures was composed from two stock solutions and closely bracketed the anticipated serum concentration. An analysis of variance, involving comparison of within-run and between-run variability, gave a total coefficient of variation (CV) of 0.38% for a serum pool containing 90.79 ng cortisol ml-1 serum.     

Concerning the question of individual constancy and individual specificity of three serum enzyme activities (serum cholinesterase EC 3.1.1.8, ceruloplasmin EC 1.10.3.2, and alkaline phosphatase EC 3.1.3.1)

Summary Several assays of the enzymatic activities of serum cholinesterase, ceruloplasmin and alkaline phosphatase of 30 healthy students during a 9 months period revealed quite constant enzyme levels for each subject. Further, it was observed that most of the subjects maintained a specific enzyme activity value and that the individual range of scatter was characteristic for each one of them.According to our studies it seems probable that quantitative enzyme assays in adults can also be rated as biochemical parameters of individuality.Supported by the Deutsche Forschungsgemeinschaft.     

The effect of doxycycline on the intestinal alkaline phosphatase (EC 3.1.3.1) activity in rat. A quantitative study

The doxycycline effect on the alkaline phosphatase activity in the small intestinal glandular epithelium in rat was examined, using the interferometric technique. The antibiotic caused a statistically significant fall of the alkaline phosphatase activity and the enzyme was found to decrease with consecutive doses of the drug.     

A simple and sensitive HPLC method for determination of gliclazide in human serum

A simple, rapid and specific method for analysis of gliclazide in serum by a sensitive high-performance liquid chromatographic method is described. Only 100 microl of serum and a little sample work-up is required. A simple procedure of extraction by toluene followed by evaporation to dryness under a gentle stream of air and dissolving the dried residue in mobile was used. The gliclazide peak was separated from endogenous peaks on a C(8) column by a mobile phase of acetonitrile-water (45:55, v/v), pH 3. Gliclazide and internal standard (phenytoin) were eluted at 6.8 and 3.8 min, respectively. The limit of quantitation (LOQ) for gliclazide in serum was 75 ng/ml at 230 nm. The method was linear over the range of 75-10,000 ng/ml with r(2) of 0.999. Mean recovery for gliclazide and internal standard was 84.5 and 87%, respectively.     

A method for determination of the specific activity of receptor-bound human chorionic gonadotropin (hCG).

Precise knowledge of the specific activity (S.A., muc/mug) of the receptor bound radiolabelled hormone is required for study of the stoichiometry of peptide hormone-receptor interactios. A radioligand receptor assay using 131-I-hCG as tracer and transplantable mouse luteoma homogenates (Biol. Reprod. 8:550, 1973) as a source of receptor was used as a model to determine the specific activity of receptor bound 125-I-hCG. Progressive saturation of the gonadotropin receptor by 125-I-hCG suggests the presence of a high affinity-low capacity binding event (saturating between 14 and 37 ng/100 mg homogenate) that does not distinguish between non-radioactive hCG and 125-I-hCG, and a low affinity-high capacity binding event (saturating between 240 and 270 ng/100 mg homogenate) that shows a preference for non-radioactive hCG over 125-I-hCG. Parallelism between bound 125-I-hCG and non-radioactive hCG in terms of competition with tracer 131-I-hCG could only be demonstrated for the high affinity event.