Behaviour of urinary dipeptidase in patients with chronic renal failure.

Renal dipeptidase (EC 3.4.13.19) activity in serum and urine from healthy volunteers (n = 20), patients with diabetes (n = 18) and patients with chronic renal failure (n = 5) was measured using glycyl-D-alanine as substrate. The assay was highly specific for the enzyme and was not affected by the various aminopeptidases present in serum and urine. No difference in serum renal dipeptidase activity was observed between the groups. The enzyme activity (U/L) in urine was higher than that in serum, irrespective of the group, suggesting the urine concentration was not affected by the serum concentration. The mean renal dipeptidase activities in urine were 2.56, 2.46 and 0.78 U/mol creatinine for healthy subjects, patients with diabetes and patients with chronic renal failure, respectively. The renal dipeptidase activity was significantly lower in the chronic renal failure group. The urinary excretion of dipeptidase (U/mmol creatinine) showed significant inverse correlations with that of beta 2-microglobulin, albumin and alpha 1-microglobulin, and with serum concentrations of creatinine, beta 2-microglobulin and alpha 1-microglobulin. We suggest that urine dipeptidase may be a useful marker of renal diseases.

Fully enzymatic method for determining 1,5-anhydro-D-glucitol in serum.

We have developed a fully enzymatic method to measure 1,5 anhydro-D-glucitol (1,5-AG) in serum through use of pyranose oxidase (PROD: EC 1.1.3.10), glucokinase (EC 2.7.1.2), and an ATP-regenerating system. In a previous report (Clin Chem 1989;35:2039-43) the glucose interfering with the measurement of 1,5-AG was removed with a minicolumn. In the method used here, glucokinase and an ATP-regenerating system efficiently convert glucose to the unreactive compound, glucose 6-phosphate, making the method selective for 1,5-AG. The hydrogen peroxide produced in the oxidation of 1,5-AG by PROD is detected with a standard enzymatic color-developing system. The within-run and day-to-day precision (CV) of this method was 0.52-1.29% and 1.17-4.48%, respectively. The correlation (r) between the results obtained with our proposed method (y) and those obtained with the mini-column method (x) was 0.998 (y = 1.007x + 0.493 mg/L; n = 100; Sy/x = 0.641 mg/L). This newly developed method allows quicker and easier measurement of serum 1,5-AG than previously described methods.

[Clinical markers in endocrine-metabolic diseases].

We attempted to reevaluate the clinical significance of two parameters, 1,5-anhydroglucitol (AG) and type IV collagen, which are widely available in the fields of clinical activity. 1) 1,5 AG 1,5 AG was measured as a marker of glycemic control for diabetic patients by means of a column-enzymatic test (Nippon Kayaku Co., Ltd). Serum 1, 5 AG levels in diabetic patients (3.0 +/- 5.8 micrograms/ml, mean +/- SD) were significantly lower than in normal subjects (22.4 +/- 6.9 micrograms/ml). 75 g OGTT was performed on 428 subjects with urinary glucose detected on previous medical examination. According to the selectivity index (sensitivity value x specificity value) and the receiver operating characteristic curve (ROC) for diabetes, glycosylated hemoglobin (HbA1c) was slightly superior to 1,5 AG and fructosamine for diabetes screening. Furthermore, unexpectedly high levels of 1, 5 AG were obtained from the plasma of diabetic patients with this kit, when the patients were given a drip infusion containing maltose. We found that the maltose contained in the assay system interfered with measurement of 1, 5 AG. Nevertheless, 1, 5 AG measurements are thought to be useful in the diagnosis and screening of diabetes mellitus because of its wideranging fluctuations under relatively good glycemic control, as suggested by Yamanouchi, et al. 2) type IV collagen The type IV collagen peptide is known as a useful marker of progressive liver diseases and early stages of diabetic nephropathy. Type IV collagen was measured by one step sandwich enzyme immunoassay (EIA) using monoclonal antibodies (Panaassay IV CL; Fuji Chem. Ind., Ltd).(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphotungstate as a useful eluent for antithrombin III purification by heparin-agarose affinity chromatography.

We have recently found that phosphotungstate (PTA) has a heparin-like anticoagulant effect. In the present paper, we studied whether PTA is useful as an eluent of antithrombin III (ATIII) on heparin-Sepharose affinity chromatography. Human ATIII adsorbed on the heparin-Sepharose gel was eluted with NaCl buffer at the NaCl level of 1M. Whereas, PTA could elute ATIII at the level of less than 1mM and thus obtained ATIII fraction contained less impurities than such a fraction eluted with NaCl. Residual PTA in the eluate was easily decomposed by alkalization, being convenient for subsequent studies.