Total porphyrin and coproporphyrin and uroporphyrin fractions in urine measured by second-derivative spectroscopy.

A second-derivative scan of an acidified urine sample allows the amplitude of deflection (delta A) and the minimum wavelength of the trough (lambda min) to determine the correct porphyrin concentration and the coproporphyrin:uroporphyrin (copro:uro) ratio, respectively, from a nomogram constructed from calibrator solutions. We measured 24 urine samples for total porphyrin as coproporphyrin equivalents and adjusted the results with factors from the nomogram. The adjusted results (x) (mean +/- SE, 501 +/- 57 nmol/L) compared favorably with the expected results (y) (514 +/- 57). The regression equation and correlation coefficient were: y = 0.993x - 8.9 (r = 0.998, S(y/x) = 16.2). Results of the copro:uro ratio derived by second-derivative spectroscopy and HPLC showed no significant difference (chi 2-test) from samples with various copro:uro ratios. Recovery studies on four urine samples supplemented with known proportions of coproporphyrins and uroporphyrins gave good agreement between the measured and the expected porphyrin ratios. The overall imprecision (CV) of the assay ranged from 3.6% to 6.0% for coproporphyrin and from 3.2% to 9.1% for uroporphyrin.

A sensitive method of screening for urinary porphobilinogen.

In this screening method for urinary porphobilinogen (PBG), urine is added to Dowex 2 resin under alkaline conditions in a test tube and mixed. The supernate is removed and the adsorbed PBG is eluted with acid and reacted with Ehrlich's reagent. We compared results with those by the Watson-Schwartz screening method, using urine samples from normal people with and without added PBG. At a PBG concentration of about five times the upper limit of normal, the resin method gave a sensitivity of 100%; the Watson-Schwartz method gave a sensitivity of 51%. At lower PBG concentrations of just over and twice the upper limit of normal, the sensitivity by the resin method was respectively 97% and 100%. With normal urine samples, the resin method gave negative results for all samples (100% specificity) and the Watson-Schwartz had 95% specificity. Our data indicate that the resin method is sensitive, specific, and reliable and is superior to the Watson-Schwartz method.

A simple enzymatic method for the measurement of abnormal levels of formate in plasma.

A simple and practical method designed to measure abnormal concentrations of plasma formate is described. The method uses formate dehydrogenase and a color reagent to produce a stable formazan color. The method requires no deproteinization and has a one-point standard calibration. The precision at 1.0 and 5.0 mmol/L formate is 2.9% and 1.7% within-day and 5.5% and 2.3% between-day. Recovery averages 100% for formate concentrations of 2.0 to 10.0 mmol/L. The proposed method is inexpensive, robust, and suitable for routine use and shares the color reagent used for the assay of plasma lactate and 3-hydroxybutyrate, both important analytes in metabolic acidosis.

Colorimetric measurement of plasma lactate.

A rapid and simple enzymatic method for plasma lactate is proposed using stable reagents to produce the formazan color. This method agrees well with a reference kit method, r = 0.955 and the regression equation is y = 0.99x + 0.09. The over-all recovery averages 100%, with a precision ranging from 0.6 to 3.3%. No interferences have been shown with the formazan reaction. The proposed method is inexpensive, ideal for batch analyses, and is an attractive method for the busy clinical laboratory.

A scheme for determining the correct activity of the kinetic angiotensin-converting enzyme.

The absorbance difference measured when angiotensin-converting enzyme (EC 3.4.15.1) hydrolyzes the substrate N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine is the basis for measuring its activity. We show this difference to be instrument dependent, and describe a method for deriving it that is applicable to manual or automated procedures.

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 measurement of erythrocyte ammonia using the Hyland Ammonia kit.

We modified the Hyland Ammonia kit for plasma to measure blood ammonia from which the erythrocyte ammonia is calculated. Our modified method gave good recoveries and its precision based on replicate assays was excellent (CV less than 3.0%). The within-day and day-to-day precision was determined from pooled blood and aqueous ammonia solution respectively. The precision calculated from duplicate results was not as good but agreed with other published values. A critical examination of Hyland's method showed the efficiency of resin adsorption to be 78%, and that the resin caused a 16% reduction in the Berthelot reaction, while 4 mol/l NaCl increased the reaction by about 11%. Blood specimens for ammonia can be frozen but specimen instability occurred during the thawing process. Measurement of ammonia directly on frozen specimens overcomes this problem. The reference range for erythrocyte ammonia was 14.5-46.1 (mean 30.1, SD 7.9) mumol/l.

A note of caution on the use of the LD ratio in myocardial infarction.

Different LD1/LD2 ratios for both normal and pathological samples were obtained with two different reagent kits. These ratio differences are due to the substrate formulation and not to the electrophoresis separation technique. In 45 patients with abnormal serum "cardiac" enzymes, results obtained by the two kit methods showed 17% and 50% "flipped ratio" abnormalities as against 78% and 67% abnormalities when the ratios were compared against appropriate reference ranges. The improper use of the LD ratio test is probably the main cause for its diminished diagnostic usefulness.