[Determination of serotonin in whole blood and platelet rich plasma by high-performance liquid chromatography with fluorometric detection].

An improved high-performance liquid chromatographic method with fluorometric detection (Ex 285nm, Em 345nm) for the assay of serotonin in whole blood and platelet rich plasma was developed for the routine clinical examination. Clinical sample was successfully deproteinized with perchloric acid in the presence of a reducing agent (ascorbic acid). Precision (CV < 5.0%), accuracy (recovery > 95%), lower assay limit (20ng/ml) and assay time (15min/sample) were allowable for the routine clinical examination. The level of serotonin (ng/ml, mean +/- SD) in whole blood and platelet rich plasma from normal volunteers (n = 53) was 143 +/- 47 and 180 +/- 60, respectively.

Determination of glycated proteins in biological samples based on colorimetry of 2-keto-glucose released with hydrazine.

We developed a novel method for measuring glycated (glc) proteins in biological samples, based on the colorimetry of 2-keto-glucose which is released from the glc protein (ketoamine) on heating with hydrazine. The ketoamine-induced coloration remained constant at room temperature (25-27 degrees C) for 1 h. The method gave reliable precision and accuracy. However, high concentrations of serum pigments caused positive interference, suggesting that hemolytic or hyperbilirubinemic serum would give false-positive results. The concentration of glc protein in clinical serum samples measured by the present method (y) correlated well with those (fructosamine values, x) measured by the nitroblue tetrazolium-reducing method: y = 1.27x-1.69 (r = 0.92, n = 93). The concentrations (microM, mean +/- S.D.) of glc protein in sera from normal and diabetic subjects were 275 +/- 37 (n = 32) and 403 +/- 98 (n = 32), respectively, and the concentrations (nmol/mg hair, mean +/- S.D.) of glc protein in back hairs from non-diabetic and diabetic rats were 3.7 +/- 0.3 (n = 10) and 8.6 +/- 1.5 (n = 10), respectively. Thus, the technique gave reasonable concentrations of glc proteins in humans and rats with diabetes mellitus, indicating it to be reliable and diagnostically useful.

A novel colorimetric method for determination of glycated protein based on 2-keto-glucose release with hydrazine.

We tried to measure glycated proteins by a novel method based on colorimetry of 2-keto-glucose which is released from the glycated protein (ketoamine) on heating with hydrazine. Reaction conditions were optimized with glycated human serum albumin (glc HSA) as a model compound. Ketoamine reacted quantitatively with hydrazine on heating at 100 degrees C for 0.5 h, followed by heating with phenylhydrazine at 60 degrees C for 1 h. Glucose interference with the assay was eliminated by preincubation of the sample with glucose oxidase at 37 degrees C for 0.5 h. Time courses for the coloration of glc HSA and human serum showed a profile similar to that of N-p-tolyl-D-isoglucosamine under optimized reaction conditions. The lower limit for the assay of glc HSA was 0.7 microM. The serum level of glycated proteins measured by the present method correlated well with that (fructoamine value, microM) measured by the conventional method (nitroblue tetrazolium-reducing method) (r = 0.92, n = 35). In conclusion, the present method is a novel, highly sensitive and reliable one for measuring glycated proteins in biological samples.

Deglycation of glycated proteins with hydrazine analogues.

Glycated human serum albumin (glc HSA) and glc proteins in back hairs from diabetic rats were deglycated by incubation with hydrazine or aminoguanidine (guanylhydrazine) at physiological temperature (37 degrees C). Glc HSA decreased by 35% with 0.2 M hydrazine at day 20 and by 56% with 0.1 M aminoguanidine at day 10. The glc proteins in hairs decreased by 40-50% and 70-77% with hydrazine and aminoguanidine at more than 0.1 M by day 20, respectively. The deglycation of glc protein with aminoguanidine was slower than that with hydrazine. The present result suggests that the inhibitory effects by aminoguanidine on the late Maillard reaction may include a decrease in the level of glc proteins (Amadori compounds) due to deglycation.

Pharmacokinetics and hemostasis following administration of a new, injectable oxacephem (6315-S, flomoxef) in volunteers and in patients with renal insufficiency.

Flomoxef is a new oxacephem of broad antibacterial activity. The compound is mainly excreted through the kidneys. Two dose finding studies in patients with various degrees of renal insufficiency revealed that the dosage of flomoxef has to be reduced exactly according to the renal function. Although the N-methylthiotetrazole group has been replaced by a hydroxyethyl group, an inhibitory effect of flomoxef on vitamin K metabolism persisted. This effect was, however, less pronounced than with latamoxef. Only patients with low vitamin K stores are endangered. For those in whom low vitamin K stores are suspected repeated controls of prothrombin time are advised during the treatment. In contrast to latamoxef the platelet system was not affected by flomoxef. With the exception of loose stools in some patients no other clinical side effects during treatment were observed.

Measurement of K vitamins in animal tissues by high-performance liquid chromatography with fluorimetric detection.

A highly sensitive method for measuring endogenous phylloquinone and menaquinones in animal tissues was developed, based on high-performance liquid chromatography with coulometric reduction and fluorimetric detection, following extraction from tissue homogenate and purification on a Sep-Pak silica cartridge followed by thin-layer chromatography. The detection limits of phylloquinone, menaquinone-4, -6, -10 and -13 were 40, 40, 50, 70 and 80 pg/g in rat liver, respectively.

Studies on oxacephem antibiotics: comparison of the effects of 1-oxa and 1-thia cephalosporins on blood coagulation activities and vitamin K metabolism in rats.

Oxacephem antibiotics have been developed to increase the antibacterial activity of cephem antibiotics, but the effect of 1-oxygen replacement of cephem antibiotics on blood coagulation activities is not yet known. Therefore, latamoxef (LMOX), flomoxef (FMOX) and their 1-S congeners were examined for their effects on prothrombin time, activated partial thromboplastin time, plasma prothrombin and Factor VII levels, plasma and liver descarboxyprothrombin (PIVKA-II) levels, and liver microsomal vitamin K epoxide reductase activities in rats kept on a vitamin K-deficient diet. Under the vitamin-deficient states, LMOX, FMOX and their 1-S congeners inhibited the vitamin K epoxide reductase, although the effect of FMOX or its congener was much less than that of LMOX, and they decreased the blood clotting activities in rats fed a vitamin K-deficient diet. However, no difference was found in these effects between LMOX and its 1-S congener or between FMOX and its 1-S congener. This result suggests that the 1-oxygen replacement of cephem antibiotics is not responsible for the hypoprothrombinemic effect of the antibiotics.

Simultaneous determination of vitamin K1, vitamin K1 2,3-epoxide and menaquinone-4 in human plasma by high-performance liquid chromatography with fluorimetric detection.

A highly sensitive method for measuring endogenous vitamin K1, menaquinone-4 (which is one of the K2 vitamins) and vitamin K1 2,3-epoxide in human plasma was developed, based on high-performance liquid chromatography with coulometric reduction and fluorimetric detection, following extraction from plasma and purification on a Sep-Pak silica cartridge. The detection limits of vitamin K1, menaquinone-4 and vitamin K1 2,3-epoxide were 5, 5 and 8 pg per injection for the standard substances and 30, 30 and 50 pg/ml in human plasma, respectively.

Menaquinone production and utilization in germ-free rats after inoculation with specific organisms.

Although menaquinones are biologically active forms of vitamin K, factors that influence their production by bacteria or their absorption from the gut are not well understood. Germ-free rats were inoculated with four different strains of organisms and fecal and tissue menaquinone concentrations were determined. No menaquinones were detected in the tissues or feces of rats colonized with Bifidobacterium longum or Clostridium ramosum, two organisms that have not been reported to produce menaquinones when grown in pure cultures. Rats colonized with Bacteroides vulgatus had high levels of fecal MK-10 with significant amounts of MK-9 and MK-11, whereas rats colonized with Escherichia coli had high levels of fecal MK-8 and small amounts of MK-7. The same menaquinones are produced in pure cultures of these organisms. The predominant fecal menaquinones were also detected in liver and were present in higher concentrations in the liver of those rats not maintained in coprophagy-preventing cages.