Enzymic assay of creatinine in serum and urine with creatinine iminohydrolase and glutamate dehydrogenase.

We describe an assay for creatinine in which it is converted by creatinine iminohydrolase (EC 3.5.4.21) into ammonia and N-methylhydantoin. The ammonia is subsequently assayed by use of alpha-ketoglutarate and glutamate dehydrogenase (EC 1.4.1.3). Use of NADPH as coenzyme eliminates all interferences from endogenous reactions. Endogenous ammonia in the sample is eliminated during a preincubation. The reaction reaches the endpoint in 15 min at working temperatures of 20-37 degrees C. No sample blank or reagent blank is needed. The standard curve is linear at least to 884 mumol (100 mg) of creatinine per liter. Average analytical recovery of creatinine in serum and urine is 99%. Within-run and between-run CVs are less than or equal to 2% and less than or equal to 6% for creatinine values of 335 mumol/L (38 mg/L) and 80 mumol/L (0 mg/L), respectively. Results by the described method (y) compare well with those by Jaffé's kinetic test (y = 1.01x -- 12.8), Berthelot/AutoAnalyzer method after treatment with immobilized creatinine iminohydrolase (y = 0.987x -- 13.2), Jaffé's test run on the SMA 12/60 (y = 1.011x -- 5.8), the Wahlefeld method (y = 1.014x -- 0.88), and Jaffé's test after deproteinization and absorption on fuller's earth (y = 0.985x -- 3.08). The method may be suitable for discrete, including centrifugal, automation.

Improved method for determination of high-density-lipoprotein cholesterol I. Isolation of high-density lipoproteins by use of polyethylene glycol 6000.

We describe a modified method of precipitating low- and very-low-density lipoproteins with polyethylene glycol, for quantitation of high-density lipoprotein cholesterol. A 100 g/L concentration of polyethylene glycol 6000 in a buffered (pH 10) solution is used. Under these conditions precipitation of beta-lipoproteins is complete. Values for HDL cholesterol as measured with this method fully correspond to those obtained on separating the lipoproteins by ultracentrifugation or with heparin-Mn2+, 46 mmol/L; are slightly higher than those obtained with heparin-Mn2+, 92 mmol/L; and are significantly higher than those determined after precipitation with polyethylene glycol, 100 g/L at pH 8.0. Comparison with results by the ultracentrifugation method showed excellent correlation (r = 0.957) with a good correspondence of results (slope 1.065 and y-intercept -22.231); comparison with the heparin-Mn2+, 46 mmol/L, method gave r = 0.998 with slope of 0.985 and y-intercept 2.963. The method is unaffected by precipitation and centrifugation time and temperature (up to 24 h with 4-22 degrees C temperature for precipitation and between 10 and 30 min with 4-22 degrees C temperature for centrifugation). This extremely simple method is particularly suitable for routine analyses of many samples, even in small laboratories.

Improved method for determination of high-density-lipoprotein cholesterol II. Enzymic determination of cholesterol in high-density lipoprotein fractions with a sensitive reagent.

A reagent is described for the colorimetric enzymic determination of high-density-lipoprotein (HDL) cholesterol. The reagent can be used with HDL fractions isolated by the various methods of precipitation of low- and very-low-density lipoproteins we investigated. The considerable sensitivity obtained by use of Barham-Trinder's reaction allows the sample/reagent volume ratio to be decreased to 1:80, and major interferences thus eliminated. The response is linear from 100 to 2000 mg of HDL cholesterol per liter. The maximum CV obtained in precision tests was approximately 1% within series and approximately 3% between series. Most of the bilirubin interference is eliminated by adopting a reaction pH of 6.1. Because of its sensitivity, the reagent is particularly suitable for use with HDL fractions isolated after precipitation with polyethylene glycol 6000, which are characterized by a marked dilution. HDL cholesterol determination with the proposed reagent is accurate and precise. Values obtained are in line with those provided for by the Abell-Kendall method. The method can easily be automated.

Immobilized enzymes in continuous-flow analysis.

Glucose oxidase, uricase, and urease were immobilized on the interior surface of activated polyamide tubing. The shelf-life of such enzyme bearing tubes was at least six months. The tubes were used for continuous-flow analysis of glucose, uric acid, and urea with conventional systems and with hybrid micro-scale systems in which modules of different manufacture were combined. The length of enzyme-bearing tube required for each system was ascertained empirically. Each tube could be used for several thousand assays, but glucose oxidase-bearing tubes were more stable than urease- or uricase-bearing tubes. Results for patients' samples correlated well with results obtained by accepted methods.