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.

Assessment and optimization of kinetic methods for angiotensin-converting enzyme in plasma.

In the kinetic angiotensin-converting enzyme (ACE) method, a practical and optimal buffer is 80 mmol/L borate buffer at pH 8.2 (37 degrees C). A lag phase is detected in the reaction, and a 5-min incubation of substrate and plasma is suggested before the kinetic measurement. The substrate, N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG), concentration is maximized at 1.0 mmol/L and the measurement wavelength is at 345 nm to ensure linearity of measurement. The proposed procedure uses a 1:9 plasma-to-reagent volume ratio. The linear range of the assay extends to approximately 170 U/L, representing a 25% substrate hydrolysis. The FAPGG absorptivity is determined by measuring the difference in absorbance between 1.0 mmol/L FAPGG and the product solutions. The wavelength fidelity is checked by noting the expected absorbance value of the FAPGG solution, and a 1.0-nm deviation from 345 nm alters the absorbance by 15.5%. The precision of ACE assays at approximately 60 and 100 U/L is 3.5% and 2.4% within batch and 2.9% and 2.6% between batch, respectively. The reference interval (2.5th to 97.5th percentiles) is 41-139 U/L, and there is no difference between values for men and women.

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.

Measurement of porphobilinogen in urine by a simple resin method with use of a surrogate standard.

In this method for urinary porphobilinogen (PBG), urine is added to Bio-Rad AG1-X2 (200-400 mesh) acetate resin under alkaline conditions and mixed. After two water washes, the adsorbed PBG is eluted with acid and reacted with Ehrlich's reagent (p-dimethylaminobenzaldehyde). Quantification is by comparison of the color developed with that of a PBG standard similarly treated or of a calibrated methyl red solution. The precision of assay of PBG at 20 and 50 mumol/L is 1.4% and 0.9% within-batch and 4% and 3.9% between-batch, respectively. The analytical recovery is about 90%, a proportion that appears to be inherent in the resin method. The proposed method (y) agreed well with the method of Mauzerall and Granick (x; J Biol Chem 1956;19:435-46), yielding the regression equation y = 0.957x + 2.9 (r = 0.993, n = 26). Good agreement was achieved with results determined from the PBG and the methyl red standards. The proposed method is inexpensive, simple to perform, and suitable for routine and emergency use.

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

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. 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 mumol/L) added to 12 patient urine samples averaged 99% and the overall imprecision for the assay was then less than 5%. Modifications to the original hydrolysis and colour development procedures allow linearity to 1500 mumol/L. Details of our procedure are given.

Reliability of the porphobilinogen screening assay.

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.

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.