Joseph E. Buttery

A Stable Reagent Mixture for the Whole Blood Transketolase Assay

(TIM = triosephosphate isomerase; GDH = glycerol 3-phosphate dehydrogenase). The combined reagent mixture for the TK assay consisting of Tris buffer, ribose 5phosphate, NADH and GDH/TIM is prepared freshly prior to assay. As there is no information in the literature concerning the stability of this reagent mixture we investigated the effects of storage. Preliminary investigation showed it was stable for 10 days at 4°C; beyond this time stability could be maintained by the addition of NADH and GDH/TIM. These results suggested that reagent stability could be prolonged by increasing the concentrations of NADH and GDH/TIM.

STABILITY OF THE CK-MB ISOENZYME ON ROUTINE STORAGE

Unlike earlier studies on the stability of the CK-MB isoenzyme carried out on control sera and on semi-purified and purified CK isoenzymes, we have studied the stability of CK-MB measured electrophoretically in patient sera under different laboratory storage conditions. The values obtained if the test was done immediately were significantly higher than those done on stored samples. There was no difference between specimens stored at -20 degrees C overnight or kept at room temperature (RT) for a few hours, but values were significantly lower (p less than 0.005) in specimens left at RT for 6 h and then stored overnight at 4 degrees C. To determine the effects of longer storage, further specimens stored either at -20 degrees C or at 4 degrees C for up to 4 days were also tested for CK-MB stability by electrophoresis and by immunoinhibition and immunoenzymetric methods. The immunological methods were included in the study to assess method dependency of CK-MB stability. CK-MB was stable at -20 degrees C by all methods, but at 4 degrees C, CK-MB was stable only by immunological and not by electrophoretic (p less than 0.005) measurement. Specimens stored under adverse conditions (4-6 days at RT) showed 50% deterioration of CK-MB when measured electrophoretically but only about 20% when measured immunologically.

Colorimetric measurement of D(-)lactate in plasma

We describe a method for the direct assay of D(-)lactate in plasma using D(-)lactate dehydrogenase and a color reagent to produce a formazan color measured at 510 nm. This method agrees well with a modified lactate UV end-point method r = 0.998 and the regression equation y = 0.974x + 0.13. The precision for low and high concentrations of D(-)lactate is 4.8% and 3.6% within-batch and 8.9% and 1.7% between-batch. Recoveries average 105% and 98% for D(-)lactate at concentrations of 1 and 5 mmol/L, respectively. The proposed method is inexpensive and suitable for routine and emergency use, and shares reagents used for the assay of L(+)lactate and 3-hydroxybutyrate.

Studies on zinc and angiotensin-converting enzyme

Angiotensin-converting enzyme (ACE, EC 3.4.15. I) is a zinc-requiring metalloenzyme which is responsible for the conversion of angiotensin 1 to angiotensin If. It is a membrane-bound glycoprotein localised mainly in the endothelial cells of pulmonary capillaries. Its main clinical application is the diagnosis and management of satcoidosis. Plasma ACE activity is elevated with active sarcoidosis but about a third of patients with this disease have plasma ACE activity within the reference range. High ACE activities are also found in Gauchers disease, hyperthyroidism, silicosis, asbestosis, alcoholic liver disease and primary biliary cirrhosis. EDTA is a powerful inhibitor of ACE and zinc has been reported to restore the ACE activity [1,2]. There are conflicting reports of zinc causing either activation [3] or inhibition [1,4] of plasma ACE activity or having no effect [5]. We have therefore readdressed this problem by examining the effect of added zinc on ACE activity in heparinised plasma samples and also on EDTA blood samples.

Reliability of the porphobilinogen screening assay

&NA; A survey conducted by the Australian Association of Clinical Biochemists Porphyrin Working Party on urinary porphobilinogen screening showed good sensitivity (75–97.5%). This is contrary to reports in the literature and to our own observations. We therefore assessed a widely used screening method (Watson‐Schwartz) and found poor sensitivity (40–69%), and even less sensitivity (28–53%) when the urine samples were normally coloured or concentrated. Thus the results obtained by the Working Party might mislead one to infer that the Watson‐Schwartz method is reliable.

Atypical LD isoenzymes with five diffuse bands and evidence for two types of binding

We describe a plasma LD isoenzyme pattern in which all five bands are unusually diffuse and of retarded mobility, an appearance not previously reported. This was shown to be due to binding to immunoglobulin G from which the enzyme could be displaced by an antiserum to gamma chains. Protein A-Sepharose removed most of the LD activity from plasma but did not dissociate the complex, indicating that the Fab end of the Ig molecule was involved in binding. Antiserum to kappa chains displaced LD5 from the complex but had no effect on the anodal bands. By contrast NAD displaced the anodal isoenzymes but not LD5. We suggest that two types of binding are involved. The LD5 complex appears to represent a true antigen-antibody reaction whereas the binding to the faster isoenzymes does not. We also suggest that immunoprecipitation is more reliable than immunoelectrophoresis or immunodiffusion for demonstrating IgG in such complexes and may be of value in defining the nature of binding.

Correction for the suppressive effect of haemoglobin on NADH absorbance in the transketolase assay.

The kinetic transketolase assay measures the absorbance change of NADH in the presence of haemoglobin. Haemoglobin was shown to suppress NADH absorbance which resulted in an apparently lower enzyme activity. To minimise this suppression, the transketolase assay was performed on samples with haemoglobin concentration of 30 g/l. At this level the absorbance of NADH in the presence of haemoglobin averaged 90.5% of that in Tris buffer. It is proposed that the calculation of the enzyme activity should be corrected for this suppression.

Urine hydroxyproline: A potential error in quantification and a proposed procedure for its measurement

&NA; A modification of the colorimetric method of Bergman and Loxley for the measurement of urinary hydroxyproline using external aqueous hydroxyproline standards instead of individual internal standards is described.1 We show that this modification leads to an underestimation (average 32%) of hydroxyproline because suppression of colour development occurs in urine samples but not in aqueous standards. Use of an internal standard for each urine test corrects for this suppression. Recovery of hydroxyproline (250 μmol/L) added to 12 patient urine samples averaged 99% and the overall imprecision for the assay was then <5%. Modifications to the original hydrolysis and colour development procedures allow linearity to 1500 nmol/L Details of our procedure are given.

Erythrocyte Ammonia in Liver Disease

This study reports the relevance of plasma and erythrocyte ammonia concentrations in patients with liver disease. Three groups of subjects were studied: group 1, 47 normal subjects; group 2, 73 patients with liver disease; and group 3, 14 patients with portal-systemic encephalopathy (PSE). The difference in plasma ammonia concentrations between groups 1 and 2 was not significant, but for erythrocyte ammonia this was significant (p less than 0.05). Group 3 subjects had significantly elevated plasma (p less than 0.001) and erythrocyte ammonia (p less than 0.001) compared with the other two groups (Mann-Whitney U-test). In group 3, two patients had plasma ammonia values within the reference range, whereas six patients had values within the range of group 2 subjects. However, none of group 3 subjects had erythrocyte ammonia concentrations within the range of either group 1 or 2. A cut-off level of 65 mumol/l was assigned to differentiate group 3 from group 2 subjects. We conclude that erythrocyte ammonia measurement is a better biochemical index of PSE than plasma ammonia.

The effect of pre-incubation and direct procedure for the alanine transaminase assay in two kits compared

The kinetic measurement of serum alanine transaminase was carried out on 2 enzyme kits (Roche and Calbiochem), done as a direct method and a pre-incubation method. Variable results from commercial quality control sera and patient sera by these 4 procedures were obtained. Pre-incubation enzyme values were generally lower than the direct values (mean difference 10%), but there were marked differences between the results from the 2 kits. This finding has important significance especially for laboratories participating in external Q.C. programmes. A frequent precision check of the method and a local reference range are therefore necessary.