Determination of HDL phosphatidyl choline by an enzymatic method

A routine method is described for the enzymatic determination of phosphatidyl choline in the apolipoprotein B-free supernatant after precipitation of blood sera with phosphotungstic acid/MgCl2. The principle of this method is based on the specific cleavage of phosphatidyl choline by purified phospholipase C from B. cereus, and the enzymatic determination of choline by choline kinase after hydrolysis of phosphoryl-choline. The enzymatic method provides HDL phosphatidyl choline values which coincide with those of the conventional chemical method. Furthermore, the values obtained with the enzymatic method for the HDL fraction isolated by ultracentrifugation (1.063--1.21 kg/l) also closely coincide with those of the apolipoprotein B-free supernatant fraction. The precision, linearity and sample stability were also checked. The findings obtained show that the enzymatic assay introduced here is suitable for the routine determination of phosphatidyl choline in the apolipoprotein B-free supernatant.     

Determination of urinary myo-inositol concentration by an improved enzymatic cycling method using myo-inositol dehydrogenase from Flavobacterium sp

BACKGROUND: To determine myo-inositol more accurately, we improved the enzymatic cycling method. METHODS: We screened myo-inositol dehydrogenase (MIDH; EC.1.1.1.18) from Flavobacterium sp., which was highly specific to myo-inositol. We measured urinary myo-inositol/creatinine ratio 2 h after 75-g oral glucose tolerance test (2 h MI) of 71 volunteers, and investigated the relationship between diabetes and urinary myo-inositol concentration. RESULTS: The calibration curve was linear (r = 1.00) up to 2000 micromol/l, and the detection limit was 10 micromol/l. Within-run and between-run CVs were 0.5-1.1% and 0.4-1.3%, respectively. The 2 h MI of impaired fasting glycemia (IFG; 65.1 +/- 46.6 mg/g Cr, P < 0.005), impaired glucose tolerance (IGT; 85.0 +/- 73.7 mg/g Cr, P < 0.001) and diabetes (163.4 +/- 73.7 mg/g Cr, P < 0.0001) increased significantly compared with that of normal glucose tolerance (NGT; 24.0 +/- 14.4 mg/g Cr). From receiver operating characteristic analyses on 2 h MI, with 50 mg/g Cr as a tentative cutoff value to detect diabetes, the sensitivity and specificity were 100% and 77%, respectively. With 40 mg/g Cr as a tentative cutoff value to detect NGT, the sensitivity and specificity were 74% and 85%, respectively. CONCLUSIONS: The myo-inositol measurement method demonstrated high specificity and yielded accurate results. The results of clinical trials suggested that 2 h MI could not only determine diabetes but also distinguish IFG and IGT from NGT.     

Determination of propranolol in plasma by radial compression liquid chromatography with fluorometric detection

This radial compression liquid-chromatographic assay for propranolol in plasma is rapid, reproducible, and suitable for use in routine monitoring. A 10-micron particle, 8 mm X 10 cm CN cartridge is used in conjunction with a radial compression separation system. The mobile phase is monobasic sodium phosphate (pH 3) solution/methanol/acetonitrile (760/84/156 by vol), the flow rate 6 mL/min. Propranolol was detected by use of a spectrofluorometer equipped with a 20-microL flow-through cell, at excitation and emission wavelengths of 250 and 336 nm. The retention times for propranolol and metoprolol (the internal standard) are 3.13 and 1.42 min, respectively. A one-step extraction with chloroform yields "clean" chromatograms, with greater than 90% of the drug being analytically accounted for. Under these conditions, results are precise and accurate. Currently we are using this method to monitor propranolol in hypertensive neonates. Data on changes in the concentrations of propranolol in plasma with time are presented for one such patient.     

Determination of serum triglycerides by an accurate enzymatic method not affected by free glycerol

In this automated single-run enzymatic procedure for specific determination of triglycerides in serum, free glycerol is removed from the reaction mixture by pre-incubation with glycerol phosphate oxidase and peroxidase. The subsequent addition of lipase and the chromogen, 4-aminoantipyrine, results in the formation of color proportional to the amount of triglycerides in serum. Standards containing triolein in aqueous detergent are used to calibrate the method. For serum pools from the Centers for Disease Control with target values of 0.74, 1.41, and 2.63 mmol/L, the method produced biases of +0.01, -0.05, and 0.00 mmol/L, respectively (mean: -0.01 mmol/L or -0.4%). The mean coefficient of variation was 1.4% within and 2.5% between days; the combined CV, 2.9%. Ninety 6-microL serum samples can be analyzed per hour. The method is more accurate and precise than one based on an NADH-coupled enzyme system with separate addition of lipase.     

Dextran determination in human serum with the aid of an enzymatic glucose method

A method is presented for the determination of dextran concentrations. Following acid hydrolysis and partial neutralization, the resulting glucose is determined with the aid of glucose oxidase. Specificity is better than that of previously applied methods. Mean recovery is 100.2 +/- 1.6% (SD) for concentrations from 180 mg.l-1 to 6000 mg.l-1. Coefficients of variation are 2.2% for 240 mg.l-1, 1.2% for 600 mg.l-1 and 1.8% for 1200 mg.l-1. When used for volume measurements, the accuracy is 99.3 +/- 2.1% (SD) for volumes of 2.5 to 3 liters.     

Urinary D-glucaric acid assay by an improved enzymatic procedure

Modifications to Marsh's procedure for measuring urinary d-glucaric acid are described, which rapidly and accurately provide optimum conditions for the assay.Using the modified technique, normal ranges for 24 h urinary output of d-glucaric acid have been established, the mean output in men being significantly higher than in women.     

改良酶学方法获得异种血管脱细胞支架

背景：扩大脱细胞基质的孔径与孔隙率,保证其作为移植物的力学特性,是目前血管组织工程研究的热点之一。目的：观察改良的酶学方法处理异种血管的力学性能和组织相容性。方法：取猪的颈动脉作为基质,采用传统胰蛋白酶-EDTA加1%TritonX-100和0.1%氨水顺序脱细胞方案,并做了适当的方法改进,延长胰酶处理的时间,分别为胰酶处理4h,5h,6h。结果与结论：经组织学分析,脱细胞基质中无细胞成分,脱细胞的支架结构完整,随着胰酶处理时间的延长,其基质内弹力板破坏明显,孔径和孔隙率逐渐增大,缝合强度与爆破强度相对于自然血管虽略有降低,但差异无显著性意义。组织成分胶原蛋白含量也有所下降,尤其胰酶延长至6h组,其胶原蛋白含量明显低于正常未处理的自然血管（P〈0.05）。结果显示延长胰蛋白酶处理时间后得到的脱细胞血管基质,具有良好的孔隙率、生物力学性能和组织相容性。     

改良酶学方法获得异种血管脱细胞支架

背景:扩大脱细胞基质的孔径与孔隙率,保证其作为移植物的力学特性,是目前血管组织工程研究的热点之一。目的:观察改良的酶学方法处理异种血管的力学性能和组织相容性。方法:取猪的颈动脉作为基质,采用传统胰蛋白酶-EDTA加1%TritonX-100和0.1%氨水顺序脱细胞方案,并做了适当的方法改进,延长胰酶处理的时间,分别为胰酶处理4h,5h,6h。结果与结论:经组织学分析,脱细胞基质中无细胞成分,脱细胞的支架结构完整,随着胰酶处理时间的延长,其基质内弹力板破坏明显,孔径和孔隙率逐渐增大,缝合强度与爆破强度相对于自然血管虽略有降低,但差异无显著性意义。组织成分胶原蛋白含量也有所下降,尤其胰酶延长至6h组,其胶原蛋白含量明显低于正常未处理的自然血管(P<0.05)。结果显示延长胰蛋白酶处理时间后得到的脱细胞血管基质,具有良好的孔隙率、生物力学性能和组织相容性。     

Determination of phospholipids in biological samples by an improved densitometric method on thin-layer chromatograms

The quantitative determination of the different phospholipids present in samples of bile, liver or plasma has been performed by densitometric scanning of high-performance thin-layer chromatography plates. These have been developed as follows: pre-treatment with sulphuric acid-ethanol mixture, staining with ethanol-modified molybdenum blue reagent and post-treatments with water and ethanol added with a small quantity of sulphuric acid. The sequential treatment, which introduces some modifications in a previously described procedure, allowed a linear and stable colour response for phosphorous-containing lipids on uniform and colourless background. The calibration of each plate with standard mixture was required for obtaining results in good agreement with other routine methods of determination.     

Analysis for potassium in human erythrocytes by use of a standard-additions method and an ion-selective electrode

A method based on standard additions to the sample was used to measure potassium (K+) in human erythocytes with an ion-selective electrode. A computer program was developed for rapid analysis of data obtained with the electrode. The analysis takes into account factors such as temperature that may affect electrode behavior. Results obtained by the method agree with those obtained by flame-emission spectroscopy. The coefficient of correlation between the two methods is 0.96. Our method is simple and rapid. The computer program is applicable to other analyses with ion-selective electrodes.     

一种改良的人DNA微量快速检验技术

目的 探索一种以口腔粘膜脱落细胞为材料的安全、高效、低成本、敏感性高的人DNA微量快速检测方法。方法 采用从漱口液或棉签擦拭口腔粘膜获取脱落细胞,应用混合树脂（Chelex100）煮沸沉淀法提取DNA;用PCR分别对线粒体特异性DNA片断和基因组中的特定基因进行扩增检测。结果 通过对线粒体DNA中440 bp的非编码片段和染色体基因组中220 bp的乙醛脱氢酶DNA片段进行扩增,结果显示,从漱口液脱落细胞提取的DNA中可以稳定地扩增出上述两种DNA片断。结论 建立了一种改良的人口腔粘膜脱落细胞DNA微量快速检验技术。该法取样方便,DNA样品获取量较大,一次取样可同时进行多项线粒体和基因组标志DNA片段的快速PCR检测。     

A simple one-step enzymatic fluorometric method for the determination of glycerol in 20 microliters of plasma

An assay for the determination of glycerol concentration in blood or other biological materials is described. The method is based on the conversion of glycerol to dihydroxyacetone in the presence of NAD, the reaction being catalysed by the enzyme glycerol dehydrogenase. The NADH which is formed in stoichiometric quantities during the reaction is estimated fluorometrically. In the presence of the ketone-trapping agent hydrazine the reaction can be made to go to completion above above pH 9.0. Using the method described, glycerol can be measured routinely in a 20-microliters sample of serum or plasma. Although the enzyme is known to react with sorbitol and ethanol, the addition of these substances to the reaction mixture had no significant effect on the determination of glycerol.     

Determination of 5-hydroxytryptophan in plasma by high-performance liquid chromatography and fluorometric detection after phthaldialdehyde reaction.

We describe the sensitive and precise determination 5-hydroxytryptophan in plasma from patients treated with this compound. The method is based on solvent extraction of plasma, high-performance liquid chromatography and continuous fluorometric detection after reaction with phthaldialdehyde in strong acid. The combined time for chromatography and reaction is 25 min. The sensitivity of the method is adequate to quantitate 0.05 mumol of 5-hydroxytryptophan per liter, in 750 microliter of plasma. No interference by other hydroxyindole compounds was found.