Improvement of the quantitative method for glucose determination using hexokinase and glucose 6-phosphate dehydrogenase.

This paper has two aims. The first one is to point out the shortcomings of Food and Drug Administration's (FDA's) reference method for the measurement of glucose. We found that the quantity of enzyme used in the method recommended by the FDA was more than the exact quantity needed for accurate measurement. The use of exact quantity of enzyme is important to minimize the negative effects due to impurity and side reactions of enzymes. The second aim is to simulate the coupling enzyme reaction using computer. We have successfully established the exact quantity of enzyme needed in the assay through the computer simulation. The quantity of the enzyme was lesser than the that recommended by FDA, but the reaction ended at the same time as in the FDA method. In addition, optimum conditions and inhibitory effects of various reagents have also been successfully analyzed using computer. In conclusion, we suggest a reference method using computer simulation to determine the exact quantity of the coupling enzyme needed in the assay.

Effects of renal sorbitol accumulation on urinary excretion of enzymes in hyperglycaemic rats.

We studied the effects of epalrestat, a specific inhibitor of aldose reductase, on renal sorbitol accumulation and the resulting urinary enzyme excretion in hyperglycaemic rats. The activities of proximal tubule-derived enzymes such as N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), gamma-glutamyltranspeptidase (GGT) and dipeptidyl aminopeptidase IV (DAPIV) in urine were determined in five groups of male Wistar rats (each n = 7): (a) 0.9% saline-loaded, (b) 10% glucose-loaded, (c) 10% glucose-loaded with epalrestat pretreatment, (d) 10% mannitol-loaded and (e) 10% mannitol-loaded with epalrestat pretreatment. Epalrestat was given mixed in chow at a dose of 50 mg/kg body weight. Urinary NAG, AAP, GGT and DAPIV activities were significantly increased (P<0.005, P<0.05, P<0.01, P<0.01, respectively) by the induction of hyperglycaemia. In contrast, enzyme excretion was not increased in the mannitol- or saline-loaded groups. Pre-treatment with epalrestat completely prevented the increased urinary excretion of NAG, AAP and GGT. At the end of the infusion study, renal cortical glucose concentrations of the glucose-loaded groups with and without epalrestat pretreatment were approximately fivefold higher than those of the mannitol- or saline-loaded groups (P<0.005 each). Renal cortical sorbitol concentrations of the glucose-loaded group was also approximately twofold higher than those of the mannitol- or saline-loaded groups (P<0.01 each). However, in the group that received both glucose and epalrestat, renal cortical sorbitol concentration was not increased. These results suggest that accumulation of intracellular sorbitol leads to proximal tubular cell dysfunction and abnormal enzymuria.

[Amylase].

alpha-Amylase in human serum consists of at least two isozymes originating from either the pancreas or salivary glands. These isozymes can be determined by three methods, electrophoresis, inhibitor method and antibody method. The committee on enzymes of the International Federation of Clinical Chemistry recommended the approved method for the catalytic concentration of alpha-amylase using 4,6-ethylidene-4-nitrophenyl-alpha-1,4-D-maltoheptaoside as a substrate. However, the assay method using 2-chloro-4-nitrophenyl-alpha-1,4-D-maltotorioside as a substrate is the IFCC method. On the other hand, the committee on enzymes of the Japan Society of Clinical Chemistry recommends an other method using maltoheptaose as a substrate. Various other methods are used as the routine method in clinical laboratories.

A kinetic model of mitochondrial aspartate aminotransferase transmigration in hepatobiliary disorders.

A hypothetical kinetic model of transmigration of mitochondrial aspartate aminotransferase (m-AST) from liver to blood and its elimination from blood was constructed and assessed for prediction by computer simulation. The elimination of m-AST from plasma in healthy rats followed first-order kinetics. Depletion of m-AST from the liver after induction of hepatobiliary disorders in rats with alpha-naphthylisothiocyanate (ANIT) also followed first-order kinetics. In contrast, the changes in metabolic rate in the liver after ANIT administration, as estimated from the plasma indocyanine green clearance, approximated to a second-order time function. Based on these data, the time-dependent change in plasma m-AST activity after ANIT administration was simulated by computer according to a hypothetical kinetic model. The result of computer simulation of the change in plasma m-AST coincided well with that obtained experimentally, suggesting that continuous measurements of m-AST may be helpful to the clinical diagnosis of liver injury.

Glucose loading induces DNA fragmentation in rat proximal tubular cells.

A 10% glucose, 10% mannitol, or 0.9% saline solution was infused in male Wistar rats for 300 minutes via the left cervical vein. Glomerular filtration rates (GFRs) were not significantly altered in any of the three groups. DNA was extracted from isolated proximal tubular cells at the end of each infusion. Electrophoresis on agarose gels showed a distinct ladder pattern of DNA fragmentation in 10% glucose-loaded rats, but no such pattern in 10% mannitol- or 0.9% saline-loaded rats. After infusion for 300 minutes, the plasma glucose level of the 10% glucose-loaded group was higher than that of the other two groups (each P < .005). These results suggest that hyperglycemia led to DNA fragmentation in the DNA of proximal tubular cells, similar to the process of programmed cell death known as apoptosis. DNA fragmentation may be associated with renal proximal tubular damage in the early stages of diabetic nephropathy.

Diagnostic significance of urinary enzymes for diabetes mellitus and hypertension.

In order to evaluate tubular damage in diabetic patients, the activity of renal proximal tubule derived enzymes excreted in 24-hour urine were recorded in 5 groups as follows: (i) 48 noninsulin-independent diabetic patients with normal renal function and a urinary albumin excretion rate within the normal range; (ii) 45 noninsulin-dependent diabetic patients with normal renal function and a high urinary albumin level; (iii) 26 noninsulin-dependent diabetic patients with renal failure; (iv) 40 patients with essential hypertension and normal renal function, and (v) 48 normal control subjects. Regardless of whether cases were noninsulin-dependent diabetics with normal or high urinary albumin excretion rate or cases with renal dysfunction, urinary dipeptidyl aminopeptidase IV and N-acetyl-beta-D-glucosaminidase excretions were significantly higher than in healthy subjects, and urinary gamma-glutamyl transpeptidase excretion was significantly lower than in healthy subjects. No significant changes in urinary enzyme excretions showed specific variations in the essential hypertensive patients. These results suggest that there is tubular damage in the early stages of noninsulin-dependent diabetic patients with normal renal function and normal urinary albumin excretion rate. Detection of urinary excretion of dipeptidyl aminopeptidase IV, N-acetyl-beta-D-glucosaminidase and gamma-glutamyl transpeptidase may be especially useful for the early diagnosis of diabetic nephropathy.

Effect of proximal tubular glucose transport blockade on urinary enzyme excretions in hyperglycemic rats.

To investigate proximal tubular dysfunction under hyperglycemic status, we infused 10% glucose solution into male Wistar rats with and without 0.16% phloridzin (a specific inhibitor of proximal tubular glucose transportation) and measured the urinary excretion rates of enzymes that are derived predominantly from proximal tubules. We used 10% mannitol solution and 0.9% saline as controls. Urinary excretion levels of N-acetyl-ss-D-glucosaminidase, alanine aminopeptidase, gamma-glutamyl transpeptidase and dipeptidyl aminopeptidase IV were significantly increased in the 10% glucose-loaded group. In contrast, these increased enzyme excretions were not observed in the 10% mannitol or 0.9% saline-loaded group. Moreover, addition of 0.16% phloridzin to 10% glucose solution completely prevented these increases in N-acetyl-beta-D-glucosaminidase, alanine aminopeptidase and gamma-glutamyl transpeptidase excretion, and slightly decreased dipeptidyl aminopeptidase IV excretion. At the end of the infusion study, a rise in renal cortical sorbitol concentration of the 10% glucose-loaded group was about 2 times higher than the 0.9% saline or 10% mannitol-loaded group. However, in the group that received both glucose and phloridzin, elevation of renal cortical sorbitol concentration was not observed. This study showed that glucose-load results in both abnormal enzymuria and renal cortical sorbitol accumulation; they were completely prevented by phloridzin.

Effects of sodium and chloride ions on blood pressure in deoxycorticosterone acetate-treated rats.

The effects of sodium (Na+) and chloride ions (Cl-) on blood pressure were studied in rats treated with deoxycorticosterone acetate (DOCA). Four groups were prepared, each consisting of male Wistar rats that underwent heminephrectomy and administration of DOCA: the control group was maintained with tap water, the NaCl group with tap water containing 1% sodium chloride, the NaCit group with tap water containing 1.67% sodium citrate (including an equivalent dose of Na+ to 1% NaCl), and the ChoCl group with tap water containing 1.15% choline chloride (including an equivalent dose of Cl- to 1% NaCl). The time-course of systolic blood pressure showed only slight change in blood pressure in the control and ChoCl groups, and in the NaCl and NaCit groups. The rotational correlation time, an index of the fluidity of erythrocyte membrane, with spin-labeling of 16-doxyl-stearic acid, was significantly (p < 0.05) higher in the NaCl and NaCit groups than in the control group, indicating an increase in the membrane fluidity, i.e., membrane fragility. The sodium, potassium ions-activated adenosine triphosphatase (Na+,K(+)-ATPase) activity of the erythrocyte membrane was decreased to 22% (P < 0.01) and 24% (P < 0.01) in the NaCl and NaCit groups, respectively, compared with the control groups; this activity was decreased to 43% in the ChoCl group (P < 0.05). The Ca(2+)-ATPase activity showed similar changes. In contrast, there were no marked differences in the erythrocyte electrolyte level between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Isopropylidine maltoheptosyl fructofuranoside, doubly blocked substrate for determination of endoamylase activity.

We synthesized o-(4,6-o-isopropylidene-alpha-D-glucopyranosyl)-(1----4)- [o-alpha-D-glucopyranosyl-(1----4])5-o-alpha-D-glucopyranosyl-(1----2)- alpha-D-fructofuranoside (IPG7F) and developed an assay for determining the activity of amylase in human serum and urine by using this substrate. Glucoamylase, alpha-glucosidase, and mannitol dehydrogenase are used as coupling enzymes. The coupled reactions are monitored by continuously measuring the oxidation rate of NADH. In this procedure, various substances in the test specimens do not interfere with the detection of amylase activity. Exactly one molecule of NADH is oxidized by one attack of amylase on the substrate, although four products can be produced in the reaction. The within-assay coefficient of variation (CV) ranged from 1.0% to 4.1% and the between-assay CV ranged from 2.6% to 5.3%. The results of our new assay correlate well with those of the amylase assay involving p-nitrophenol maltoheptaoside as substrate (r = 0.978) and with those of the amylase assay involving maltopentaose (r = 0.987).

Na+, K(+)-ATPase inhibitory activity of fractionated urine during changes in dietary sodium intake in man.

Na+, K(+)-ATPase inhibitory activity in urine fractionated by HPLC was quantified in 7 normotensive male subjects during changes in dietary sodium intake. Subjects were studied on free sodium intake for 2 days, on low sodium intake (2 g/day) for 3 days, on high sodium intake (22 g/day) for 4 days and subsequently on normal sodium intake (6 g/day) for 2 days. Na+, K(+)-ATPase inhibitory activity in fraction 10 eluted with 17% acetonitrile by reverse-phase HPLC was 12.3 +/- 5.2% (mean +/- S.D.) on free sodium intake, 8.7 +/- 9.8% on the 3rd day of low sodium intake, 61.2 +/- 6.6% on the 4th day of high sodium intake, and 20.5% +/- 0.7% on the 2nd day of the normal sodium intake. Changes in Na+, K(+)-ATPase inhibitory activity of fraction 10 were closely associated with those in urinary sodium excretion. These results suggest that an endogenous Na+, K(+)-ATPase inhibitor(s) which plays a physiological role in the control of sodium and water balance may exist in this particular fraction.

Effect of chlorpromazine on transmigration of mitochondrial aspartate aminotransferase in rat liver.

Cholestatic liver injury was experimentally induced in rats by administration of chlorpromazine (CPZ). The peak activity of mitochondrial aspartate aminotransferase (AST) released in serum was found to precede the peak of total AST activity. The liver mitochondria obtained from rats treated with CPZ was fractionated into two subfractions: one containing the intermembrane space, and the other containing the matrix and the membranes. As a results, the relative activity of AST in the intermembrane space was significantly increased at 12 h after CPZ administration. This result suggests that mitochondrial AST, which is dominantly located in the mitochondrial matrix, transmigrated to the intermembrane space via the inner membrane under the effect of CPZ administration.

Circadian rhythm of plasma corticosteroid in adult female rats: chronological shifts in abnormal lighting regimens and connection with oestrous cycle.

The circadian rhythm of plasma corticosteroid (CS) levels in adult female rats was studied chronologically under the following conditions: normal light-dark (LD), inverted light-dark (DL), constant dark (DD) and constant light (LL). Animals were accustomed to LD condition for 7 days before exposure to each abnormal lighting regimen. Normal circadian rhythm established under LD condition was clearly inverted on the third day of DL regimen, and the inverted rhythm persisted thereafter under DL condition. The circadian CS rhythm persisted essentially unchanged throughout DD condition, but lost its regular periodicity showing "free running" and changed day by day under LL condition. The average CS levels over a 24 h period were higher under LL than under DD condition. Plasma CS levels in each lighting regimen exhibited diurnal variations regardless of the vaginal smear patterns of autopsied animals. Exposure of rats to LL for 21 days made the circadian CS rhythm flat, but induced persistent oestrus in only a few animals. The data suggest that (1) an unexpectedly rapid inversion of the circadian CS rhythm occurs if animals are exposed to inverted light-dark environment; (2) constant darkness seems to be a near-natural environment for rats, and changes little of the pre-established circadian CS rhythm; (3) constant light, on the contrary, is assumed to be a stress for rats, and disrupts the circadian CS rhythm and elevates CS levels; (4) the change in circadian CS rhythm in adult female rats is not mediated by a change in gonadal function and the two conditions may not be connected directly with each other.