Zeitschrift fur klinische Chemie und klinische Biochemie最新文献

The incubation of urine with beta-glucuronidase/arylsuphatase from Helix pomatia results in the incomplete hydrolysis of urinary steroid conjugates, because inhibitors of these enzymes are present in urine; The inhibitors can be separated from the steroid conjugates by chromatography of the urine on a column of Amberlite XAD-2. By this method a complete enzymatic hydrolysis of urinary steroid conjugates is possible in 24 hours at 37 degrees C or in 2 hours at 55 degrees C.

This paper deals with a new method for the determination of serum nucleotidase (EC 3.1.3.5). The assay is performed with cytidine-5'-monophosphate as substrate, followed by deamination of the generated cytidine. The principle of the method and the determination of the liberated ammonia by the Berthelot indophenol-reaction are comparable to the Persijn--van der Slik method in which adenosine-5'-monophosphate is used as substrate. The correlation between the results obtained with these two methods was found to be good; the new method has the advantage of higher sensitivity.

In order to establish normal values, samples of cerebrospinal fluid from 109 healthy children between the ages of 0 and 13 years were analyzed for total protein, different protein fractions after electrophoretic separation, and IgG. Total protein was determined by the biuret method after precipitation with trichloroacetic acid. For the determination of the different protein fractions, the fluid was concentrated by high pressure filtration; the fractions were separated by microzone elektrophoresis on cellulose acetate membranes, then assayed photometrically after staining with amido black B. IgG was determined by radial immunodiffusion.

A simple kinetic method for the automated determination of total serum cholesterol was developed using cholesterol esterase, cholesterol oxidase, catalase and the color reaction according to Hantzsch. The procedure has been adapted to the Braun SysteMatik, Greiner GSA II, Eppendorf 5032, Lars Ljungberg Autolab, Perkin-Elmer C4 and Perkin-Elmer C4B analyzers. The variants of the method yielded satisfactory results with regard of precision, accuracy and sensitivity to interferences.

A kinetic measurement of the eluates from the column purification of T4 is described. The choice of cation exchange chromatography to reduce interfering X-ray contrast substances, and the influence of protein in the reaction have been evaluated. Data for precision, inter and intra assay reproducibility, detection limit and normal values are reported. Using sample volumes of 0.5 ml and an analysis time of one minute, the proposed method compares favourably with published methods.

Radial immunodiffusion, which is a presently accepted reference method, was compared with the mechanized immunoprecipitation. Each of these specific methods was compared with two normal clinical-chemical methods for the determination of albumin and transferrin, and an enzymic method for the determination of ceruloplasmin. The correlation coefficients, the mean slope for regression, and the percentage variation of the average values from those of the average values of the reference methods are presented. In the determination of albumin and ceruloplasmin, radial immunodiffusion and mechanized immunoprecipitation give the same results. Surprisingly, these two specific methods give different results in the determination of transferrin. In as far as there is no doubt concerning the accuracy of the mechanized immunoprecipitation, this method is recommended, since it also has the advantage of being less demanding in cost and time.

A continuous flow method is described for the determination of uric acid in serum using uricase and peroxidase, with omicron-dianisidine as the chromogen. The method can be used with single channel systems and with the multichannel system SMA 12/60 (Technicon). The new method was compared with six other manual and mechanized methods for the dosage of uric acid, and the results were analyzed statistically. The results from all the enzymic methods showed a good correlation, whereas the "chemical oxidation" methods showed systematic deviations.

Reaction conditions were optimized for the determination of serum glutathione reductase, which has not yet been investigated systematically. Imidazole was found to be the most suitable buffer material; the highest glutathione reductase activity in serum was always obtained with imidazole/HCl buffer, which, in contrast to all other tested buffers, also resulted in the maximal enzyme activity without preincubation. In imidazole buffer, the pH-activity curve of serum glutathione reductase shows a broad optimum between pH 6.5 and 6.9. A GSSG concentration of 2 mmol/l and a NADPH concentration of 0.43 mmol/l gave maximal enzyme activity and a linear reaction over 10 min up to 20 U/l test solution. An investigation of serum glutathione reductase activity from 100 clinically healthy probands gave values between 20 and 50 U/l. In the optimized assay system the glutathione reductase in the serum reacts specifically with GSSG and NADPH.

The isolation of glucose dehydrogenase from Bacillus megaterium M 1286 is outlined. Data on the specificity of the enzyme towards carbohydrates are given. A specific method for glucose determination using this enzyme was developed. Methods and results of four variants of this glucose determination are presented: End point determination in the UV range, determination with formazan as reaction product, kinetic determination in the UV range, and continuous flow analysis in the UV range (AutoAnalyzer method).