[Enzyme spectrophotometry determination of sodium, potassium and chloride ions in serum and urine]. / Enzymatische spektrophotometrische Bestimmung von Natrium-, Kalium- und Chloridionen im Serum und Urin.

The principles and main features of enzymatic methods for the measurement of sodium, potassium, and chloride are reviewed and their performance compared with current procedures. Each method makes use of a relatively specific enzyme, catalysing a reaction whose rate is sensitive to the ion to be determined. Where the (S)0.5 of the enzyme for the ion is much lower than the assay concentration, the ion concentration may be reduced by a binding agent. Alternatively, a competitive inhibitor may be used to raise the (S)0.5 of the enzyme. In the case of chloride determination with amylase the (S)0.5 of the enzyme is raised by limiting the concentration of free calcium. In the measurement of potassium, interfering ions such as sodium are removed by binding with Kryptofix 221 and improvement in performance is also achieved by use of a bacterial pyruvate kinase less sensitive to sodium. The enzymatic methods are applicable to measurement of sodium, potassium and chloride in blood or urine with good precision, accuracy, and specificity. They can be used on mechanized or manual instruments. There appears to be minimal interferences from compounds found in normal or pathological serum or urine.

Enzymatic determination of potassium in serum.

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.

Enzymatic determination of sodium in serum.

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.

Quantitative method for determining serum alkaline phosphatase isoenzyme activity: estimation of intestinal component.

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-1-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/l) 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.

Laboratory evaluation of the Beckman Synchron CX3 clinical chemistry analyzer.

In this evaluation of the Beckman Synchron CX3, the multi-analyte clinical chemistry analyzer exhibited high precision, good linearity, and no carryover for each of the eight analytes measured. Results obtained correlated well with those produced by our routine instrumentation (Beckman Astra, Varian atomic absorption spectrophotometer). The instrument can process up to 75 samples per hour (600 tests per hour if all tests available are requested) and, after calibration, can provide urgent results for the complete panel of tests within 2 1/2 min. The performance characteristics of this instrument make it ideal as a routine or a "stat" analyzer for commonly requested tests in the clinical chemistry laboratory.

Immunoturbidimetric measurement of transferrin.

This paper describes the development of an automated immunoturbidimetric assay for transferrin on a centrifugal analyser. Regression analysis of transferrin values measured immunoturbidimetrically demonstrates good agreement with data obtained by radial immunodiffusion (y = 0.997 + 0.024 g/l, r = 0.980, n = 50). The assay has a detection limit of 1.0 g/l and working range of approximately 1.0 to 6.0 g/l of transferrin. Day-to-day coefficient of variation is less than 3.5%. Immunoturbidimetric transferrin (g/l) and total iron binding capacity values (mumol/l) were compared using an established total iron binding capacity method (y = 0.050 X - 0.030 g/l, r = 0.967, n = 50). Minimal interference was found for lipaemic, haemolysed or icteric samples. Transferrin reference values with a mean of 3.05 g/l and 95% limits from 2.45 to 3.65 g/l were derived using serum from 300 apparently healthy subjects (150 males, 150 females). We conclude that the proposed transferrin method is more reliable and easier to perform than presently available total iron binding capacity methods.

Quantitative method for determining serum alkaline phosphatase isoenzyme activity I. Guanidine hydrochloride: new reagent for selectively inhibiting major serum isoenzymes of alkaline phosphatase.

The potential use of the protein denaturant guanidine hydrochloride to inhibit selectively the enzyme activity of serum alkaline phosphatase isoenzymes from liver, bone, intestine, and placenta was investigated. Inhibition of each isoenzyme was shown to be dependent on time and concentration of inhibitor. In the presence of 0.3 mol/l (28.7 g/l) guanidine hydrochloride for 170 seconds 14%, 47%, and 90% of the total alkaline phosphatase activity remained in samples of bone, liver, and intestinal origins, respectively. In contrast, the activity of the placental isoenzyme increased by 24%. The degree of inhibition was shown to be independent of total alkaline phosphatase activity. Investigations were performed at 37 degrees C using the Cobas Bio centrifugal analyser. We conclude that this reagent has several practical advantages over urea as a selective inhibitor of alkaline phosphatase isoenzymes, including a faster and more reproducible inhibition at a much lower reagent concentration.

Quantitative method for determining serum alkaline phosphatase isoenzyme activity II. Development and clinical application of method for measuring four serum alkaline phosphatase isoenzymes.

A method for quantitating the liver, bone, intestinal and placental alkaline phosphatase activity of serum, using an algorithm for converting selective inactivation by guanidine hydrochloride, L-phenylalanine, and heat into equivalent isoenzyme activity is described. The method can individually quantify mixtures of isoenzymes to within a margin of 3%; it has acceptable reproducibility and has been used to develop both age and sex related reference ranges. Analysis time is about 30 minutes. The clinical reliability of this method has been shown in a study of 101 patients, in 79% of whom isoenzyme results were compatible with the final clinical diagnosis; in 10% a clinical diagnosis resulted from isoenzyme analysis, and in a further 11% the source of the increased alkaline phosphatase activity was identified and supported by electrophoresis, with a definite clinical diagnosis yet to be made.

Direct ultraviolet method for enzymatic determination of uric acid, with equilibrium and kinetic data-processing options.

We developed and evaluated a direct ultraviolet method for the enzymatic determination of uric acid in serum, plasma, or urine, without deproteinization of sera and plasma. Equilibrium and nonlinear curve-fitting kinetic options are evaluated and compared, and results of the proposed method are compared with those of a candidate Reference Method. All data-processing options yield a linear relation for absorbance and concentration of uric acid between 0.1 and 2 mmol/L; with the equilibrium option, results are linear to 5 mmol/L. For 100 plasma samples, results correlate well between the proposed method (y) and a reference method (x): y = 0.99x - 0.002 mmol/L. Between-run imprecision is about 2.3%, and interference by hemolyzed, icteric, or lipemic specimens or specimens containing high concentrations of xanthine or paraproteins is minimal. The kinetic option with a data-processing range of 100 s or longer yields results that correlate well with the equilibrium method: y = 1.006x + 0.002 mmol/L (n = 118). For 20 urine samples processed by the proposed (y) and a reference (x) methods, y = 1.04x + 0.038 mmol/L.

The effect of instrument variables on calculation factors for standardisation of colorimetric analyses.

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.

Mechanism of platelet interference with measurement of lactate dehydrogenase activity in plasma.

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.

Determination of inorganic sulfate in plasma with a centrifugal analyzer.

Turbidimetry of inorganic sulfate, after precipitation with barium sulfate, can be done simply in a Cobas Bio centrifugal analyzer. Polyethylene glycol is used as the precipitate-stabilizing agent. Reproducibility of precipitation is enhanced by the presence of BaSO4 particles, which function as seed nuclei. There is no interference by normal or above-normal concentrations of phosphate, heparin, bilirubin, hemoglobin, or erythrocyte contents, or by lipemia (triglyceride concentrations up to 6.5 mmol/L). Analytical recovery of added inorganic sulfate was found to be quantitative. Precision is similar to that for other methods for inorganic sulfate in plasma. This method is suitable for the rapid, routine analysis of plasma inorganic sulfate, and it is simple and less expensive to perform than alternative methods.

Theoretical constraints in the measurement of serum bilirubin binding capacity.

The large majority of methods for measuring serum unoccupied bilirubin binding capacity involve adding an exogenous ligand to a diluted serum sample. If the added ligand binds specifically to bilirubin binding sites, the extrapolation is made that the amount of ligand which becomes bound represents the previously unoccupied bilirubin binding capacity of the original sample. This simple theory ignores the labile nature of the equilibrium reactions between bilirubin binding sites and the ligands with which they interact. The present theoretical and experimental study of these equilibrium reactions shows that (a) sample dilution alone results in changes in the proportional occupancy of bilirubin binding sites by bilirubin and other endogenous ligands, so that the extent of vacancy of those binding sites becomes exaggerated; (b) addition of an exogenous ligand to the diluted sample results is further displacement of native bilirubin and other endogenous ligands from bilirubin binding sites. The amount of added ligand which becomes bound is thus likely to represent a greater extent of vacancy of bilirubin binding sites than was present in the original sample. Results from such methods can therefore only overestimate the unoccupied bilirubin binding capacity of blood plasma in vivo. In order to avoid these analytical problems, current methods must be redesigned. It is suggested from this work that the ability of a serum to sequester safety additional bilirubin may best be assessed from measurement of its bilirubin buffering capacity. This parameter is different from the total or unoccupied binding capacity currently attempted, and its measurement is within the capability of published methods for measuring free (= unbound) bilirubin, modified to analyse minimally diluted samples.

Comparison of standardization techniques for colorimetric analyses on a centrifugal analyzer.

The influences of the mode of standardization and the type of standard on the precision of four mechanized methods performed on a centrifugal analyzer are described. Experimental results show that the mode of standardization -- variable, using a standard in each analytical batch, and constant, using a direct relationship between concentration and absorbance -- must be objectively selected. If the variable mode is chosen, the type and level of standard must be carefully chosen and absorbance of assays of the standard must be carefully monitored for good quality control. It is recommended that the optimum standardization technique and standard, where applicable, should be assessed and subsequently documented in evaluations of methods, kits and instruments.