Immunoturbidimetric method for routine determinations of apolipoproteins A-I, A-II, and B in normo- and hyperlipemic sera compared with immunonephelometry.

We describe a method for routine immunoturbidimetry of apolipoproteins (apo) A-I, A-II, and B in both normo- and hyperlipemic sera. A special antiserum reagent, consisting of a highly concentrated mixture of nonionic and anionic detergents (final concentration in the assay, 36 g/L), rapidly removes intrinsic turbidities of even strongly lipemic sera without interfering with the antigen-antibody precipitation reaction. The method has good precision, and obviates the need for special sample pretreatment, extended incubation periods, and measurment of sample blanks. A comparison with established immunoephelometric assays generally showed close agreement for analytical recoveries of the three apolipoproteins. However, in samples containing greater than or equal to 18 g of triglycerides per liter, the nephelometric assays yielded about two- to threefold higher values for apo A-II and B than did the turbidimetric procedure. To elucidate this discrepancy, we used the turbidimetric methods to assay sera with and without enzymatic lipolytic pretreatment. Even for samples with triglyceride concentrations up to 60 g/L, complete enzymatic lipolysis (as evidenced by thin-layer chromatography) did not significantly alter the recoveries of apo A-II and B from those obtained with the untreated specimens. Thus the immunoturbidimetric methods yield reliable results for apo A-I, A-II, and B, not only in normo- but also in hyperlipemic sera.

Precipitation methods for the determination of LDL-cholesterol.

The selective precipitation of low-density lipoproteins (LDL) with polyvinyl sulfate (PVS), and the immunoprecipitation of high-density lipoproteins (HDL) and very-low-density lipoproteins (VLDL) with an anti-HDL antibody, can both be used to establish simple methods for the determination of LDL cholesterol. Whereas the PVS method requires the calculation of LDL cholesterol as the difference of total and supernatant cholesterol, the immunoprecipitation method allows the direct measurement of LDL cholesterol in the supernatant. As a first step, both methods were optimized to yield accurate values for normolipemic and slightly hyperlipemic serum samples. Moreover, the determination of LDL-cholesterol in lipemic sera can be achieved by a combination of immunoprecipitation and polyanion precipitation.

A new approach to photometry of glycated hemoglobin in human blood.

This new approach to measurement of glycated hemoglobin (Hb A1) is a three-step procedure involving (a) conversion of oxyhemoglobin into NO-hemoglobin by sodium dithionite and sodium nitrite in the presence of inositol hexaphosphate; (b) incubation with haptoglobin, which preferentially binds glycated hemoglobin; and (c) determination of the hemoglobin/haptoglobin complex by its peroxidase activity in acid buffer. The procedure is suitable for automated analysis with instruments that are capable of adding a starting reagent. We describe preliminary adaptations for the Abbott VP and Hitachi 705 analyzers and demonstrate the correlation of results with Hb A1 concentrations determined by ion-exchange chromatography.

Kinetic enzymic method for automated determination of total cholesterol in serum.

We describe a rapid, kinetic, fixed-time method for determining serum total cholesterol by use of cholesterol esterase, cholesterol oxidase, and the indicator reaction with peroxidase, 4-aminophenazone, and phenol. On addition of the competitive inhibitor 3,4-dichlorophenol the Michaelis constant of cholesterol oxidase is apparently increased, which extends the linear relation between absorbance change and cholesterol concentration to 20.7 to 25.9 mmol/L, depending on the analyzer being used. For calibration, a single standard is used. Total analysis time is in the range of 80 to 210 s. Incubation temperature is 25 degrees C or 37 degrees C. The single-reagent procedure has been adapted to three different centrifugal analyzers and to the Eppendorf ACP 5040 analyzer. It yields precise and accurate results and is insensitive to potential interferences.

Reagent for the enzymatic determination of serum total cholesterol with improved lipolytic efficiency.

We describe a sensitive method for quantifying the extent of cholesterol ester cleavage during enzymatic assay of total cholesterol in serum. Lipids are extracted from the assay mixture with chloroform/methanol (1/1 by vol), concentrated, then quantified by "high-performance" thin-layer chromatography. Although with conventional enzymatic reagents for determination of serum total cholesterol the hydrolysis of the cholesterol esters may be incomplete, a new enzymatic cholesterol reagent (Monotest Cholesterol, High Performance, Boehringer Mannheim) gives virtually complete cholesterol ester cleavage (i.e., greater than or equal to 99.5%). Use of this reagent with its improved lipolytic efficiency yields results for serum total cholesterol that are identical to those measured with a candidate reference procedure involving alkaline cholesterol ester saponification.

Turbidimetric kinetic method for serum low density lipoprotein quantitation.

A turbidimetric kinetic fixed-time method for the rapid determination of serum low density lipoprotein (LDL) has been developed. It is based on specifically precipitating this lipoprotein with the use of a combination of heparin, Ca2+, EDTA and lipase. The method has been adapted to the Eppendorf spectrum line photometer and to the ENI Gemsaec centrifugal analyzer. It yielded satisfactory results with regard to linearity and precision. The values agreed closely to those obtained by conventional procedures for LDL-cholesterol and LDL-apolipoprotein B. The predictive value of this new method for the assessment of the individual risk of vascular disease remains to be proven by clinical and epidemiological studies.

Determination of the biological activity of heparin by use of a chromogenic substrate.

We describe a new method for determining the biological activity of heparin in plasma with use of thrombin and the substrate Tos-Gly-Pro-Arg-pNA. The procedure is based on the photometric determination of the inactivation of thrombin after incubation with plasma in the presence of endogenous antithrombin III (At III). The method allows the specific determination of heparin concentrations from 0.02 USP to 0.8 USP/ml of plasma in the presence of normal At III levels. It has been carried out manually by use of an Eppendorf spectrum line photometer or automatically by use of a Vitatron Akes analyzer. For evaluation, the results were compared with two standard samples which contained heparin in the low and high therapeutic range, respectively.

Kinetic determination of serum glucose by use of the hexokinase/glucose-6-phosphate dehydrogenase method.

We have developed a kinetic fixed-time approach for the quantitative determination of serum glucose by use of the hexokinase/glucose-6-phosphate dehydrogenase method. To achieve a large measuring range, we have apparently increased the Michaelis constant of glucose-6-phosphate dehydrogenase through addition of the competitive inhibitor ATP. By this means, serum samples with glucose concentrations up to 55.5 mmol/l could be analyzed without pre-dilution. The method has been adapted to the ENI GEMSAEC analyzer and to the LKB 2086 Mark II analyzer. It yielded satisfactory results with regard to precision. A comparison of the kinetic procedure with the manual end-point method showed good agreement. No interferences from hemoglobin, bilirubin, or lipemia have been observed.

Improved automated kinetic determination of uric acid in serum by use of uricase/catalase/aldehyde dehydrogenase.

The enzymatic determination of serum uric acid by use of uricase, catalase, and aldehyde dehydrogenase according to Haeckel [J. Clin. Chem. Clin Biochem. 14, 101 (1976)] showed interferences from ethanol-converting enzymes, which are present in some patients' sera. We have identified these enzymes as alcohol dehydrogenase isoenzymes. Among other substances, a mixture of pyrazole and oxalate can be used to eliminate these interferences. This inhibitor system gives good results when used in the automated kinetic uric acid determination, as is shown by a comparison with the manual assay for uric acid according to Kageyama [Clin. Chim. Acta 31, 421 (1971)].

Kinetic enzymatic method for automated determination of glucose in blood and serum.

Studies are reported on the reaction kinetics of the glucose assay according to Trinder which involves the specific oxidation of glucose by glucose oxidase and the determination of the hydrogen peroxide released by means of phenol and 4-aminophenazone in the presence of peroxidase. The results have been used to develop a general kinetic fixed-time method for the analysis of glucose in whole blood and serum. The single reagent method has been adapted to the ENI GEMSAEC centrifugal analyzer and to the Abbott ABA-100 analyzer. The procedures exhibited excellent precision and the results correlated well with those obtained by the hexokinase method, Linearity was achieved from 3 to 64 mmol/1 glucose for the GEMSAEC method, and from 3 to 33 mmol/1 glucose for the ABA-100 method. Reagent or sample blank corrections were not necessary. There were no interferences from various drugs, hemoglobin, bilirubin, or lipemia.

Molar absorptivities of beta-NADH and beta-NADPH.

Re-investigating the accuracy of the commonly used values for molar absorptivities (epsilon) of beta-NADH and beta-NADPH at Hg 334, Hg 365, or 340 nm, we obtained the following results: The maximum of absorbance of NADH is shifted from about 340 nm at 0 degrees C to about 338.5 nm at 38 degrees C; the corresponding maxima of NADPH are located at about 0.5-nm longer wavelengths. In addition, the absorption curves of both coenzymes broaden with increasing temperature. For these reasons, the epsilon-values of NADH and NADPH are generally different from each other, and are temperature-dependent. Only at 334 nm are they almost identical and nearly independent of temperature. Therefore this wavelength is recommended for precise measurements. The epsilon-values of these coenzymes are influenced by ionic strength and pH. To determine the absolute values of the molar absorptivities, we performed the glutamate dehydrogenase or lactate dehydrogenase assay with carefully purified 2-oxoglutaric acid or pyruvic acid in the presence of excess coenzyme. The purity of the substrates was checked through differential scanning calorimetry, moisture analysis, gas-liquid chromatography, gas chromatography in combination with mass spectrometry, and nuclear magnetic resonance spectroscopy. The epsilon-values observed under the various conditions are about 1-7% higher than those currently used.

Improved method for enzymatic determination of serum triglycerides.

I describe an enzymatic method for determing serum triglycerides (triacylglycerols). The triglycerides are hydrolyzed by a mixture of lipase and esterase. The glycerol released is determined by kinetic fixed-time analysis, with use of glycerol kinase, pyruvate kinase, and lactate dehydrogenase. Through addition of the competitive inhibitor ATP the Michaelis constant of pyruvate kinase is apparently increased, considerably extending the linearity of the assay. There is no need for serum blanks or reagent blanks. The method has been adapted to a centrifugal analyzer (the ENI GEMSAEC). It yields satisfactory results with regard to precision, accuracy, and insensitivity to interferences.

Molar absorptivities of beta-NADH and beta-NAD at 260 nm.

To determine the molar absorptivities of beta-NADH and beta-NAD at 260 nm, we purified the reduced form of the coenzyme by repeated anion-exchange chromatography. A NADH preparation was so obtained for which the 260 nm/340 nm absorbance ratio was 2.265. When calculated with epsilon 340 beta-NADH = 6.22 times 10-3 for beta-NADH at 260 nm and 25 degrees C, a molar absorptivity of 14.1 times 10-3 liter - mol minus 1 - cm minus 1 resulted from this quotient. By use of the lactate dehydrogenase or glycerol-3-phosphate dehydrogenase assay, respectively, and referring to the new absorption coefficient for NADH at 260 nm, the molar absorptivity of beta-NAD at 260 nm and 25 degrees C was established to be 17.4 times 10-3 liter - mol minus 1 - cm minus 1. The values observed are lower than those reported thus far.

[Ezymatic method for the automated determination of total serum cholesterol (author's transl)]. / Enzymatische Bestimmung des Gesamt-Cholesterins im Serum mit Analysenautomaten

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.