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In an in-depth examination to better define the renal effects of mild hypertension, we used urinary proteins to indicate damage to the glomerulus (albumin), tubular reabsorption capability (retinol-binding protein), and turnover of tubular tissue (alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase) in a group of 18 people with mild hypertension not associated with diabetes and a control group (n = 12). The participants' activity was controlled on a high normal salt diet for 3 days followed by a low salt diet for 4 days. Two distinct patterns of albumin excretion were evident in the hypertensive group: 22% had elevated, highly variable excretion patterns, and the rest had tightly grouped values below 16 mg/g creatinine, 16 micrograms/min, or 16 mg/L, with the lowest within-person biological variability given by albumin calculated as a ratio to creatinine. Albumin and NAG excretion primarily correlated with systolic blood pressure and the best correlations were given by ratios to creatinine. A marked decrease in salt excretion of 71% (to 50.8 mEq/day) resulted in significant (P < .0005) decreases in systolic (13.9 mm Hg), diastolic (6.4 mm Hg), and mean arterial pressures (8.9 mm Hg) only in the group with mild hypertension. However, albumin excretion did not decrease when dietary salt content was lowered. The group with hypertension also had higher urinary excretion of lysosomal N-acetyl-beta-D-glucosaminidase (P < .01), and whites in the group had a higher excretion of retinol-binding protein than did whites in the control group (P < .02). Retinol-binding protein values, however, were within the normal range, indicating that the elevated albumin values were the result of changes in selectivity of the glomerulus.

We measured sensitive indicators of renal damage in three different populations occupationally exposed to cadmium, and examined the degree of variation in damage and the relative sensitivity of different types of indicators. The three studies included (1) men exposed in a cadmium recovery plant, (2) men exposed in a nickel/cadmium battery plant, and (3) women exposed in the latter plant. The indicators of renal damage were urinary proteins in three categories: (1) the high molecular weight enzymes alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG), (2) the intermediate molecular weight protein albumin (ALB), and (3) the low molecular weight proteins retinol-binding protein (RBP) and beta 2-microglobulin (B2M). These tests indicate that exposed groups with higher urine cadmium levels had varying degrees of renal damage. All exposed groups showed evidence of renal damage when compared with their respective control groups. A higher percentage of elevated protein levels was noted in the exposed group of Study 1 than in the exposed groups of Studies 2 and 3. In Study 1, the means of all five protein levels and ALB, RBP, and B2M fractional clearances were significantly elevated in the group with higher urine cadmium concentrations when compared with the groups with lower urine cadmium concentrations. Highly significant dose-response relationships for all of the urinary protein tests, including fractional clearances, were found. All of the tests were more sensitive in detecting evidence of subclinical renal damage than serum creatinine, a commonly used indicator of renal function. The order of test sensitivity in men was determined by considering three factors: (1) the magnitude of the correlation coefficient between the test and the urine cadmium concentration in the study with the most advanced damage, (2) the relative cadmium level predicted by the dose-response model at which there is a 10% chance of observing an elevated test value, and (3) the ability of the tests to detect renal effects in the population with less advanced damage. The tests in order of decreasing sensitivity in men are ALB, AAP, NAG, RBP approximately B2M. The women with higher urine cadmium levels in Study 3 had a higher percentage of elevated AAP and NAG values when compared with the control group.

The measurement of small but abnormal amounts of albumin in urine is important in evaluating kidney disease in people with diabetes mellitus, hypertension, or possible adverse health effects from exposure to nephrotoxins. Routine laboratory methods for measuring albumin are not sensitive enough to measure the amounts that are significant in urine (less than 30 mg/L). In our laboratory we used three immunoassays for measuring urinary albumin: enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), and immunoturbidimetric assay (IT). We calculated the CVs of the three methods, investigated potential interfering substances at three times their normal concentrations, and stored urine under different conditions to find the best way to protect the sample until assay. The potential interferents we checked were transferrin, urea, beta 2-microglobulin, retinol-binding protein, creatinine, kappa and lambda light chains, IgG, hemoglobin, ketone, and glucose. The stability study involved two study temperatures (-20 and -70 degrees C) and four treatments (centrifuging or filtering, before or after storage). We found the following: the RIA had the lowest CV; the results from the interference study showed no interference from normal physiological concentrations of the substances investigated; storage at -70 degrees C regardless of the treatment should be adequate to prevent loss of albumin immunoreactivity.

Because of increased interest in the assay of albumin in urine and the sensitivity required to quantify concentrations associated with (a) increased risk of developing end-stage renal disease and cardiovascular disease among people with diabetes and (b) renal damage caused by exposure to nephrotoxic substances, we conducted a pilot study of the variation of these measurements within and among five laboratories that use various immunoassays. These assays included two different enzyme immunoassays, two different immunoturbidimetric assays, a fluorescent immunoassay, and a zone immunoelectrophoresis assay. The results indicate considerable variation both within and among laboratories for measurements at or near the normal range. Variability is equally attributable to the precision of individual immunoassays and to the variation of the mean values obtained by each laboratory. Individual laboratory CVs ranged from 5.8% to 18.2% for mid- and high-concentration samples treated with preservative and from 8.4% to 23.6% for mid- and high-concentration samples containing no preservative. The relative bias of individual laboratory means ranged from -56.4% to 20.5% for the two preserved materials and from -32.6% to 0.8% for the two materials containing no preservative. To reduce the chance of misdiagnosing the risk associated with above-normal albumin concentrations in urine, we need to address the problems contributing to imprecision and inaccuracy, particularly laboratory-to-laboratory variability.

We evaluated the ability of three enzymes--N-acetyl-beta-D-glucosaminidase (NAG; EC 3.2.1.30), alanine aminopeptidase (AAP; microsomal aminopeptidase, EC 3.4.11.2), and gamma-glutamyltransferase (GGT; EC 2.3.2.2)--and adenosine deaminase binding protein (ABP) in urine to predict or confirm renal-transplant rejection in patients treated with cyclosporine. We measured the enzymes daily during the early post-transplant hospital stay of 104 renal-transplant recipients (72 men and 32 women). We also measured ABP in 32 of these patients. We analyzed the data by calculating the activity ratio of each day's test value to the previous day's result and optimized the sensitivity (SN) and specificity (SP) to determine the optimal ratio for each test. The results indicate that cyclosporine treatment reduces the optimal sensitivity and specificity of these tests. Three comparable tests (ABP, GGT, and AAP) yield the best optimal values (SN = 0.77, 0.69, 0.77; and SP = 0.71, 0.74, 0.63, respectively), and the NAG test yields the lowest combination of sensitivity and specificity (SN = 0.62, SP = 0.66). All four tests were less sensitive and specific than the plasma creatinine test (optimal day-to-day difference = 5 mg/L). However, the ABP and AAP tests gave indications of rejection at least 24 h before clinical diagnosis for 50% of the patients experiencing rejection, while early plasma creatinine increases of 5 mg/L occurred in only 19% of this group.

We describe the development of a stable reference material for prostatic acid phosphatase, derived from human prostatic tissue and human seminal fluid. The enzyme was purified by an L-tartramic acid affinity-chromatography technique. Two-dimensional electrophoresis revealed essentially no contaminating proteins, and specific tests revealed no contaminating enzymes. The preparations, in a matrix containing 30 g of human serum albumin and 0.1 mol of sodium acetate per liter, pH 6.0, were studied with respect to stability of both catalytic activity and immunological identity. We conclude that the preparations from either source are satisfactorily stable, and that either is acceptable for use in preparing clinical reference materials. These materials will be used in developing a reference method.

Urea inhibition of urinary N-acetyl-beta-D-glucosaminidase (NAG) was examined with human isoenzymes and total enzyme in urine. The fluorometric substrate 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide (MUNAG) and the colorimetric substrate m-cresolsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide (MCPNAG) were used to determine substrate kinetics and apparent urea inhibition kinetics. Apparent KmS of 0.47 (MUNAG) and 0.35 mmol/L (MCPNAG) for human NAG isoenzyme A and 0.46 (MUNAG) and 0.30 mmol/L (MCPNAG) for human NAG isoenzyme B were determined. Apparent Kis of approximately 100 mmol/L for urea inhibition of both isoenzymes in either substrate and apparent Kis of 79 (MUNAG) and 91 mmol/L (MCPNAG) for NAG in dilute urine samples were found. The potential for urea inhibition at physiological concentrations of urea and at normal assay dilutions therefore exists. Inhibition at these dilutions was minimal when substrate concentrations of 2 x Km or greater were used, but the substrate MUNAG at 1/2 x Km gave the highest sensitivity and lower blanks and allowed better quantitation of low activity samples. Within-run CVs of 3.2% with MUNAG and 10% with MCPNAG were found for low activity samples.

We describe the preparation and characterization of materials containing human pancreatic and salivary alpha-amylase (EC 3.2.1.1) and examine their relationship to endogenous amylase in human serum. Amylase was purified from human pancreas and saliva by solvent- and salt-fractionation and column chromatography to specific activities of 63 and 279 kU/g, respectively. Four liquid pools, differing only in activity, were prepared from each source of amylase, each in a matrix containing, per liter: 30 g of human albumin, 50 mmol of sodium chloride, 1 mmol of calcium chloride, and 50 mmol of Tris hydrochloride buffer, pH 7.4. Characterization of the pools showed that the amylase activity in the materials was stable for at least six months at 25 degrees C; among-vial variability of amylase activity was less than or equal to 0.5% (2 CV); and the pools were free from eight possible contaminating enzymes. Plots of salivary vs pancreatic amylase activity measure in our materials with eight commercially available methods showed least-squares slopes ranging from 0.51 to 1.0. The intermethod "commutability" of the materials (i.e., how closely they mimic endogenous serum amylase) was examined in relationship to approximately 100 human sera.

The most commonly used method for quantifying D-glucaric acid, the enzyme inhibition method, is a two-step procedure. In the first step of the present study, urine was boiled at acidic pH to convert glucaric acid to 1,4-glucarolactone, which inhibits beta-glucuronidase activity. In the second step, inhibition of beta-glucuronidase by the prepared urine was measured. This is an indirect measure of the D-glucaric acid concentration. The second step was adapted to a Cobas-Bio centrifugal analyzer. Values on a reference population obtained by the manual method were highly correlated with values obtained by the semiautomated method described here (r = 0.976, p = 0.25), and precision was comparable by the two methods. The throughput increased from 8 to 20 samples per day when the automated method was used.

More than 300 laboratories participated in an interlaboratory survey of creatine kinase (CK, EC 2.7.3.2) determinations in which they analyzed seven lyophilized samples for total CK and CK isoenzymes and furnished information about their methodology. The samples were not necessarily intended to mimic typical patients' specimens but rather to determine the analytical ability of the laboratories to distinguish isoenzyme fraction CK-MB from CK-BB and to detect small but abnormal amounts of CK-BB. For total CK measurement, most laboratories used an NADP+ reduction method monitored at 340 nm (89%), and reported results in units per liter (U/L) (99%) at either 30 degrees C (34%) or 37 degrees C (60%). Despite the variety of analytical conditions, most laboratories (89%) correctly reported results within their normal range for all samples. The 287 laboratories that reported isoenzyme distributions in the samples used either cellulose acetate (37%) or agarose (44%) electrophoresis, ion-exchange chromatography (9%), or immunoinhibition (7%). Results from laboratories that used nonspecific CK-MB immunoinhibition techniques were biased when a significant amount of CK-BB isoenzyme was present.

An enzyme-inhibition assay was evaluated and modified to quantify D-glucaric acid in a population exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The modified assay combined improvements described separately in previous reports, including pH adjustment by standard addition of buffers rather than by titration, an optimum pH of 2.3 for converting D-glucaric acid to 1,4-glucarolactone, and the use of the relation reciprocal of absorbance versus concentration for calculating unknowns. Reference limits for adult males were 0.06-5.90 mmol D-glucaric acid/mol creatinine and for adult females 0.87-6.23 mmol D-glucaric acid/mol creatinine. Children under the age of 15 yr had a reference range of 0-8.34 mmol D-glucaric acid/mol creatinine. Persons on anticonvulsant therapy excreted from 3 to 10 times the upper reference limits of D-glucaric acid. Urinary concentrations of D-glucaric acid in persons identified as being at high risk for exposure to 2,3,7,8-TCDD in Times Beach, Mo., were not significantly different from concentrations in those identified as being at low risk for such exposure.

In developing a Reference Material for alkaline phosphatase, we studied the stability, kinetic properties, and commutability of separate preparations of the purified enzyme from human liver, intestine, bone, and placenta. The Michaelis constants (Km) for the preparations from liver, bone, and intestine agreed well with the Km values we obtained for five human serum specimens, whereas that for the placental isoenzyme differed significantly. The first three isoenzymes exhibited nearly identical response-surface patterns, which closely paralleled those observed for 12 human serum specimens (commutability), but not that of the placental isoenzyme. Thus, we believe that a reference material could equally well consist of either the bone, intestinal, or liver isoenzyme. All four isoenzymes were satisfactorily stable in temperature-accelerated degradation studies. We chose the liver isoenzyme as an appropriate reference material because liver tissue is easier to obtain than bone or intestine and the isoenzyme is abundant in liver, is easy to extract, and is the one most commonly increased in human serum. This material is stable at -20 degrees C, is free of interfering and degradative enzymes and, being of human origin, is commutable with the enzyme in human serum.

Studies were conducted to determine the effect of temperature and wavelength on the absorbance of alkaline solutions of picric acid in the presence and absence of creatinine. Absorbance values of an alkaline solution of picric acid were found to be influenced by temperature. At wavelength settings between 475 and 520 nm, absorbance values increased as the temperature increased. The magnitude of the thermochromic response (temperature-induced increase in absorbance) was found to be a function of wavelength: At 490 nm, the response was about three times greater than it was at 500 nm and about fifteen times greater than it was at 520 nm. Other experiments demonstrated that the response was: quantitatively related to picric acid concentration, reversible, rapid, and independent of creatinine concentration.