A manual spectrophotometric method for the measurement of serum sodium and potassium by enzyme activation.

Manual procedures suitable for use on standard benchtop spectrophotometers have been developed for the enzymatic determination of Na+ and K+ in serum. Both assays require only minimal modification of reagents already available for BM/Hitachi analyzers and are performed in an endpoint mode, allowing up to 20 assays per run. The addition of a stop reagent is required--dipotassium EDTA for the Na+ assay and sodium dodecyl sulphate for the K+ assay. The most important criterion for achieving good assay performance is the precise pipetting of sample and reagent. Within-run imprecision is < 1% for Na+ and K+, and between-run imprecision < 1.5%, for both assays at all but the lowest concentrations of K+. Enzymatic electrolyte results compare well with flame photometry, however the assays are more prone to interference by very high concentrations of bilirubin or triacylglycerols than those performed on automated, dual-wavelength kinetic analyzers. It is possible to correct for most interferences by inclusion of appropriate sample and reagent blanks.

[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.

Urinary risk factors in calcium oxalate stone disease: comparison of men and women.

The daily excretion of calcium, oxalate, uric acid and glycosaminoglycans, the 24-h urinary pH and volume, and the inhibitory effects of the urines on calcium oxalate crystal growth and aggregation, were measured in 44 normal women, 41 normal men, 32 female stone formers and 63 male stone formers. No significant differences could be found between the normal men and women, the male and female stone formers, or between the patients and their normal controls with regard to the excretion of oxalate and glycosaminoglycans, and the urinary pH. The normal women exhibited significantly lower urinary volumes and excreted less calcium per day than did the other subject groups. The excretion of calcium by the female stone formers was indistinguishable from that of both groups of men. The male and female stone formers did not differ from their corresponding control groups with regard to the excretion of urate, but both groups of male subjects had significantly higher daily urate excretions than did either female category. This was attributed to the greater body weights of the men. There were no discernible differences between any of the subject groups with regard to the inhibitory effects of their urines on calcium oxalate crystal growth, but urines from both groups of female subjects demonstrated a significantly greater inhibitory influence on crystal aggregation than did those of the men. It would appear that the relatively low incidence of uninfected calcium oxalate urolithiasis in women compared with men may be attributable to (a) a lower daily calcium excretion and (b) a higher inhibitory activity of their urines towards crystal aggregation.

The effect of serum on the crystallization of calcium oxalate in whole human urine: inhibition disguised as apparent promotion.

The effect of serum on calcium oxalate crystallization was studied in whole human urine. At concentrations between 0.005% to 0.10% (v/v), serum had no effect on the metastable limit of urine as detected by the Coulter Counter. However, serum caused a marked increase in the number and volume of calcium oxalate crystals deposited in response to an oxalate load. That this increase in volume reflected true deposition of calcium oxalate was confirmed by determining calcium concentration and by including control urines containing added serum but no exogenous oxalate. Analysis of particle size distributions showed that the crystals deposited in the presence of serum were smaller than those occurring in the control. Since serum did not affect the limit of metastability, the enhancement of crystal volume is unlikely to be a result of heterogeneous nucleation. We hypothesize that the apparent promotion effect of serum is due to its inhibition of crystal aggregation which increases the crystal surface area available for calcium oxalate deposition.