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.

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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.

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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.

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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.

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補体の第一成分であるC1の精製は数多く紹介されている。その多くは塩入操作後, カラムによるゲル濾過あるいはアフニティークロマトグラフィーなどによって分離されており, その溶出液中のC1の分画は赤血球溶血反応によりC1活性の認められた分画を集めている。今回我々は, C1活性の確認を溶血反応を用いずに, C1の構成成分のCIrと Clsの酵素特性を利用し開発された特異的合成基質を用いて行った。結果, 過去に報告されているものとほぼ同一のものが精製された。更に, 精製過程にセリンプロテアーゼの一般的な活性インヒビターであるdiisopropy目f-uorophosphateあるいはphenylmethyl sulfonyl fluorideを共存させたが, DFPでは非活性型C1 (前酵素) として血清中に存在する状態のC1が得られ, PMSFでは精製過程で活性化されて直ちに酵素活性を示す活性型C1が得られた。以上の結果からセリンプロテアーゼインヒビターの効果が, 単に酵素活性を阻止するだけでなく, 精製過程においてこれらのインヒビターが非活性型C1 (前酵素) を保護しながら精製できる効果の有するものと有しないものがあることがわかった。この原理で阻害剤を利用して精製したC1を更に溶血反応を用いずに活性型と非活性型を確認しながらの精製法は, 今後のC1の研究手段として役立つものと考えられる。

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健常者血清を含む70例について, HDL-コレステロール (HDL-C) をHPLC法で定量し, その結果を超遠心法, 結合沈澱法, 直接法と比較した。いずれもHPLC法と他法の間に高い相関 (r=0.972～0.995) が得られたが, 超遠心法ではHDL-C低値検体の場合はHPLC法より高値で, HDL-C高値検体の場合は低値となった。超遠心法のHDL分画にHDLより大きいリボ蛋白が含まれることが, d>1.063分画のHPLC分析により確認された。沈澱法や直接法によるHDL-CはHPLC法より若干低値であった。沈澱法では HDL-C濃度の増加に比例してHPLC法との差が増加する傾向がみられたが, 直接法ではそのような傾向はみられず, 直接法がHPLC法と最も高い相関を示した。

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