This is a kinetic assay for measuring serum Na+ concentration based on determination of Na+-dependent beta-galactosidase (EC 3.2.1.23) activity. The method, sufficiently sensitive to measure sub-millimolar concentrations of Na+, was modified by including a Na+-binding agent (cryptand) to provide a linear assay for serum Na+ concentrations between 110 and 160 mmol/L. The assay was developed with and evaluated in the Cobas Fara centrifugal analyzer (and has been used in other kinetic analyzers). Within-run and between-run CVs were less than 1%. The reaction rate for normal serum samples (0.20 delta A/min) is about 10-fold that of the reagent blank. Results correlated well with flame photometry. Interference from bilirubin, hemoglobin, lipemia, heparin, and other cations was negligible. The method offers a practical alternative to the use of ion-selective electrodes and flame photometry for measuring serum Na+ in high-throughput or "stat" biochemical analyzers.
This is a kinetic assay for measuring K+ in serum, based on the activation of pyruvate kinase (EC 2.7.1.40) by K+. We eliminated interference from Na+ and NH4+ ions, which also activate this enzyme, by including Na+-binding and NH4+-consuming reagents in the reaction mixture. The assay was developed with and evaluated in the Cobas Fara centrifugal analyzer (and has been used in other kinetic analyzers). Within-run and between-run CVs were less than 1.4% and less than 1.6%, respectively. The reaction rate per millimole of K+ per liter (0.05 delta A/min) was more than double that of the reagent blank (0.02 delta A/min). Results correlated well with those by flame photometry, and interference from bilirubin, hemoglobin, lipids, heparin, and other cations was negligible. This method, in conjunction with a previous method we have reported in which beta-galactosidase is used for measuring Na+ in serum, offers a practical alternative to the use of ion-selective electrodes and flame photometry for measuring these clinically important monovalent cations in high-throughput or "stat" biochemical analyzers.
SUMMARY Intestinal alkaline phosphatase activity was measured using levamisole inhibition, and results were compared with a previously reported method using L-phenylalanine. Sixty two per cent intestinal, 39% placental, and 1-3% of either bone or liver alkaline phosphatase activity remained when alkaline phosphatase activity was inhibited in a 2-amino-2-methyl-l-propanol (AMP) buffer reagent system with 10 mmol/l levamisole (final assay concentration 8-1 mmol/l). The assay imprecision (SD) was 0-6 U/l compared with 3-9 U/l using L-phenylalanine for specimens with total alkaline phosphatase activity less than 250 U/l (reference range 30-120 U/l). In serum pools with raised total alkaline phosphatase activity errors in recovered intestinal activity were small (usually less than 3 U/1) when intestinal alkaline phosphatase was added. Much larger errors and many underestimated results were found using L-phenylalanine. For non-haemolysed specimens it is concluded that an assay based on levamisole inhibition provides a better measure of intestinal alkaline phosphatase activity than L-phenylalanine.A raised serum alkaline phosphatase activity [EC 3.1.3.1] is a common finding in the clinical chemistry laboratory. In a proportion of such cases either the tissue source of the enzyme activity is not identifiable by the clinician, or the contribution from one or more pathological processes is masked.' A method for separately quantifying bone, liver, intestinal and placental alkaline phosphatase isoenzyme activities has, however, recently been described.2This method determines the residual serum alkaline phosphatase activity following various inhibitory treatments, with individual isoenzymes being affected in a predictable manner. Each isoenzyme activity present in the original specimen is then quantified by insertion of the residual activities into an appropriate algorithm. Because four separate activity measurements must be made, it is important that the precision and accuracy of each quantitative step is optimised so that the cumulative error on the calculated isoenzyme activities is minimised. One of the inhibitors used is L-phenylalanine, a non-competitive inhibitor of intestinal alkaline phosphatase. The activity of the intestinal component is computed from activities remaining in the presence and absence of L-
SUMMARY.A number of colorimetric methods, particularly enzyme activity assays, are usually standardised using calculation factors based on the molar absorptivity of a principle reactant or product. Such methods are subject to long-term variation. The relationship between long-term variation in results and instrument variables affecting calculation factors has not been quantitated. In this study, we have shown that, on a centrifugal analyser having a within-run coefficient of variation of less than 1%, instrument variables affecting calculation factor alone could result in changes in results of up to 8·5% over 75 days. We therefore advocate daily use of a solution of potassium dichromate to monitor instrument variables that can independently affect calculation factors and within-run imprecision. This procedure is useful for maintaining long-term performance and for differentiating problems of instrumental or chemical origin.We have previously examined standardisation techniques for colorimetric analytical procedures!: 2 and have demonstrated experimentally that the mode of standardisationvariable, using a standard in each analytical batch, and constant, using a long-term relationship between concentration and absorbance-must be objectively selected for each method.In current practice, a number of analyses, particularly plasma or serum enzyme activity assays, are most often standardised using the constant calibration mode. For example, for enzyme activity assays, at a given temperature, the calculation involves: enzyme activity = calculation factor x absorbance change per minute.The absorbance change is corrected to a 1 em light path and the factor is derived from the formula:total reaction volume factor = b .. sample volume x umolar a sorptrvity Although this approach circumvents the very real difficulties of primary standardisation," a number of analyses, particularly enzyme activity assays, are often poorly performed at both inter-and intra-laboratory levels."There are a number of reasons for analytical problems; these have been reviewed by Lott.? Models for determining total system random error have been published;" 7 models for determining the effect of instrument variables on calculation factors have not.We therefore examined the reliability of calculation factors for analyses such as enzyme activity assays by making multiple daily absorbance measurements of a concentrated solution of a stable moiety, acid potassium dichromate, in order to monitor the principle variables of the above equation. Use of this solution, as previously recommended," has significant advantages over the use of less stable solutions of NADH 9 or of quality control materials. The importance of optimisation of the standardisation procedure in improving the standard of performance of all types of assay has previously been stressed. III This study has, therefore, the aim of assessing the relationship between long-term changes in results and the mode of standardisation. Materials and methods REAGENTApproximately 4 g of potassium dichromate...
Platelets reportedly inhibit lactate dehydrogenase activity in plasma under reaction conditions of low osmolality. We describe observations inconsistent with these reports, and we attribute this "inhibition" to optical interference by platelets during the course of a reaction. We conclude that when platelet lysis is prevented and the optical interference of platelets corrected, platelet-rich plasma, platelet-poor plasma, and serum show essentially the same lactate dehydrogenase activity. Furthermore, platelet contamination can cause unexpected problems when lactate dehydrogenase is assayed with centrifugal analyzers. Results can be high or low, depending on the volume of diluent pipetted with the sample, and extreme within-run variations in activity are possible. When plasma is used instead of serum for routine analyses, regular checks for platelet contamination should be performed as a quality-control procedure, especially by laboratories separating plasma with bench-top centrifuges. Platelets can also interfere optically with assay of other enzymes and metabolites.
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