We developed a method to determine rat serum bone specific alkaline phosphatase (ALP) by gel electrophoresis. The method is based on the densitometric scan using polyacrylamide disc gel electrophoresis. The coefficients of variation for bone specific ALP was less than 7% for within-and between-run precisions, respectively, and the detection limit was 10 IU/I. A group of twelve-week-old female rats were ovariectomized and an other group were given sham operations. The bone specific ALP in serum, 6 months after the operation, showed five times higher activity in the ovariectomized groups compared with the sham-operated group. The determination of bone specific ALP will be helpful in animal models of bone disease and the evaluation on the effects of therapeutic agents.

View full abstract

We describe the enzymatic determination of urinary methylguanidine with methylguanidine amidinohydrolase (EC 3.5. 3.16) and methylamine oxidase. In the first step, urinary methylguanidine was hydrolyzed by methylguanidine amidinohydrolase to methylamine and urea. Methylamine was subsequently oxidized by methylamine oxidase to formaldehyde and hydrogen peroxide. The formaldehyde reacting with methyl 3-aminocrotonate gave a fluorescence product. The fluorescence intensity was measured at an excitation wavelength of 375 nm and an emission wavelength of 465 nm. Good linearity was obtained in a wide range of methylguanidine concentrations (0.02-8.00 mg/l). The analytical recovery of methylguanidine added to urine ranged from 96.5 to 98%. The results obtained by the present method correlated well (r=0.984) with those obtained by the high-performance liquid chromatographic method. The proposed method is accurate and simple. The mean methylguanidine concentration in healthy spot urine samples was 560, μg/l. We determined the concentration of methylguanidine in 24-h urine samples by the above method, and found that the urinary excretion of methylguanidine increased markedly in samples of inpatients with chronic renal failure.

View full abstract

A simple and rapid high-performance liquid chromatographic method for identification and quantitation of several benzodiazepines in human serum is described. Benzodiazepines extracted from serum sample using a solvent extraction were separated on a C18 column and detected at 223 nm. The resultant peak was identified by UV photodiode array detection. Six benzodiazepines were separated within 20 min by an isocratic elution with wateracetonitrile-methanol as a salt-free mobile phase. The detection limits of the benzodiazepines spiked ranged from 8 to 28 ng per ml of serum. The within-day and day-to-day precisions for serum sample gave relative standard deviations of less than 3.1% and 8.6%, respectively.

View full abstract

We developed a simple immunological method for detection of urinary morphine, which does not contain narcotics and radioactive substances. The structure of naloxone is similar to that of morphine. Anti-morphine antibody-Protein A-Gold (particles with orange pink color of colloidal gold which adsorbed Protein A) was reacted with naloxone-bovine serum albumin (BSA) bound to a nylon membrane. BSA-naloxone-anti-morphine antibody-Protein A-Gold complexes then formed on the surface of the nylon membrane (Biodyne R membrane). This Biodyne R membrane was layered as follows: poly-tetrafluoroethylene (PTFE) membrane, Biodyne R membrane, cellophane membrane with a hole 1 cm in diameter, nylon membrane and an absorption mat. Units of these layers were packed in a plastic box with a hole in the top. A urine sample containing morphine when applied to the hole in the box, sequentially passed through the membranes to the absorption mat. The PTFE membrane filtered the urine sample. On the Biodyne R membrane, urinary morphine was replaced with naloxone and bound to the anti-morphine antibody-Protein A-Gold. This morphine bound complex was liberated from the Biodyne R membrane and moved to the lower layer. Finally, the morphine bound complex nonspecifically bound to the nylon membrane. As the concentration of urinary morphine increased, the color of the Biodyne R membrane decreased, whereas that of the nylon membrane was increased. The assay was completed within 1 h and the minimal amount of morphine detected was 25 ng/ml. Naloxone is not a narcotic drug, so this method may be used during criminal investigation to identify individuals who consume morphine.

View full abstract

Various kinds of purification methods for the first complement component, Cl, have already been introduced, but most of them used gel-filtration or affinity chromatography after salting in and the Cl activity of the purified fractions were usually monitored by a hemolysis method. This paper describes an alternative method instead of a hemolytic method. For the activity assay, a synthetic substrate specific for Clr and Cls was used. In the purification steps, diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, inhibitors for serine proteases were added for gel-filtration. The former showed strong inhibitory effects on the natural activation of Cl during the purification steps, but the latter showed little effect. The biochemical characters of purified products were the same as those previously reported. Efficient utilization of proteases and synthetic substrates could provide good separation of inactive and active Cl components from the pooled plasma.

View full abstract

HDL-cholesterol (HDL-C) levels of human sera (n=70) from normolipidemic and hyperlipidemic subjects have been measured by the improved HPLC method using a gel filtration column (TSKgel Lipopropak, 7.5 mm×600 mm/TOSOH Co. Tokyo) and an eluent (TSKeluent LP1). HDL-C levels by the HPLC method were compared with three methods: (1) one-step ultracentrifugation using a Beckman Lp-4²⁺Ti Rotor,(²⁺) the precipitation method with heparin/Ca²⁺+, Ni²⁺+, and (3) the direct measurement method with polethylene glycolmodified enzymes and sulfated α-cyclodextrin. Although correlations between the HPLC method and the other three methods were high (r=0.97²⁺-0.995, n=70), HDL-C levels by the ultracentrifugation were significantly higher than those by the HPLC method in subjects with low HDL-C levels, and were lower in subjects with high HDL-C levels, Peaks of lipoproteins larger than HDL were observed in the HPLC patterns of HDL fraction separated by the ultracentrifugation for many subjects with low HDL-C levels. HDL-C levels obtained by the precipitation method and the direct method were slightly lower than these obtained by the HPLC method. The HDL-C levels obtained by the precipitation method were markedly lower in subjects with high HDL-C levels.

View full abstract