Activity of lysosomal exoglycosidases in the urine of healthy normotensive and pre-hypertensive children.

PURPOSE: We investigated the relationship between blood pressure (BP) and the activity of lysosomal exoglycosidases: N-acetyl-ß-hexosaminidase (HEX), its isoenzymes A (HEX A) and B (HEX B), α-fucosidase (FUC), ß-galactosidase (GAL), ß-glucuronidase (GLU) and α-mannosidase (MAN) in pre-hypertensive (high normal blood pressure - HNBP) and normal blood pressure (NBP) children. MATERIAL AND METHODS: The study was carried out with urine samples collected from 176 children, aged 6-17.9 years, divided into 2 groups: 42 HNBP and 134 NBP subjects. The children were stratified depending on systolic and diastolic BP (SBP; DBP): HNBP (SBP and/or DBP greater than or equal to the 90th percentile, but less than the 95th percentile) for sex, age, and height; and NBP (SBP and DBP less than the 90th centile). The activities of lysosomal exoglycosidases were determined by the colorimetric method, and expressed in pKat/mL and pKat/µgCr. RESULTS: The activity of urinary HEX A in HNBP group was significantly higher than in NBP (p < 0.05). The HNBP group showed significant positive correlation between HEX, HEX A (pKat/mL) and SBP. AUC for HEX A was 0.616, cut-off value -29.351 pKat/mL (sensitivity 51.2%, specificity 71.8%), and 0.589, cut-off value -0.054 pKat/µgCr (sensitivity 31.7%, specificity 86.3%). CONCLUSIONS: This is the first report of the relationship between BP and the activity of urinary lysosomal exoglycosidases: HEX, HEX A and HEX B, FUC, GAL, GLU, and MAN in healthy children and adolescents. It seems that HEX A (pKat/mL) can be used as a useful tool in identifying children with HNBP.

Urinary exoglycosidases, reference values in healthy children.

PURPOSE: The purpose of the study was to determine the effect of age on lysosomal exoglycosidase activities: α-fucosidase, ß-galactosidase, ß-glucuronidase and α-mannosidase in healthy children and adolescents. MATERIAL AND METHODS: Urine samples were collected from 203 healthy children and adolescents (girls = 99, boys = 104), aged six months to 17.9 years. The activities of α-fucosidase, ß-galactosidase, ß-glucuronidase and α-mannosidase were determined by colorimetric method and expressed in pKat/µg of creatine (pKat/µg Cr.). RESULTS: Urinary α-fucosidase, ß-galactosidase, ß-glucuronidase and α-mannosidase activities (pKat/µg Cr.) were the highest in children below 3 years of age in comparison to the remaining age groups. There was a statistically significant negative correlation between urinary α-fucosidase, ß-galactosidase, ß-glucuronidase and α-mannosidase (pKat/µg Cr.) and age (r = -0.36; r = -0.36; r = -0.35; r = -0.35; at p < 0.0001, respectively). In addition, we constructed the reference values for urinary activity of α-fucosidase, ß-galactosidase, ß-glucuronidase and α-mannosidase (pKat/µg Cr.) in percentiles according to age in 3-year intervals. CONCLUSIONS: Our study is the first to show reference values for urinary α-fucosidase, ß-galactosidase, ß-glucuronidase and α-mannosidase in children and adolescents.

Indirect Detection of Glycosidases Using Amperometry.

Glycosidases are essential enzymes that cleave glycoside bonds. The presence of glycosidases have been widely used to detect pathogens, label cells/tissues, and report specific diseases. We have developed a rapid electrochemical assay to detect glycosidases. Exposure of electrochemically inactive substrates to glycosidases releases glucose, which can be measured easily using an electrochemical cell. Five different glycosidases were detected rapidly within 1 h using disposable electrodes. This assay could readily be incorporated into repurposed glucose meters to rapidly detect glycosidases, which in turn could be useful to report the presence of a pathogen or illness.

Urine exoglycosidases are potential markers of renal tubular injury in children with ureteropelvic junction obstruction.

AIM: Hydronephrosis caused by ureteropelvic junction obstruction (UPJO) is an important problem in children and young adults. The aim of this pilot study was to determine the urine profiles of a number of lysosomal exoglycosidases to see whether they indicated tubular renal damage in children with UPJO. METHODS: We measured lysosomal exoglycosidases urine activities in 32 patients with UPJO, dividing them into three groups. The surgical group comprised 16 children with severe hydronephrosis who required surgery, the nonsurgical group comprised 16 patients with mild hydronephrosis, and the reference group comprised 42 healthy children. The following indicators were measured: N-acetyl-ß-hexosaminidase and its A and B isoenzymes, α-fucosidase, ß-galactosidase, α-mannosidase and ß-glucuronidase. RESULTS: The urine activities of all exoglycosidases were significantly higher in children with UPJO than children in the reference group (p < 0.01). A strong positive correlation was also found between most of the urine exoglycosidases and the urine albumin/creatinine ratio (p < 0.01). CONCLUSION: Our findings demonstrated that children with UPJO showed increased renal activities of assessed exoglycosidases, which correlated positively with the urine albumin/creatinine ratio. A larger multicentre study is required to confirm the clinical applications of these observations.

Lysosomal exoglycosidases in serum and urine of patients with pancreatic adenocarcinoma.

Lysosomal exoglycosidases: N-acetyl-ß-D-hexosaminidase (HEX), ß-D-galactosidase (GAL), α-L-fucosidase (FUC) and α-D-mannosidase (MAN) modify oligosaccharide chains of glycoconjugates in endoplasmatic reticulum and/or Golgi apparatus and degrade them in lysosomes. In acid environment of lysosome, exoglycosidases degrade oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans by eliminating single sugars from the edges of oligosaccharide chains. Neoplasms change biochemical processes in tissues and may significantly change the activity of many enzymes including the activity of lysosomal exoglycosidasses in serum and urine of persons with neoplasmatic diseases. The aim of the present paper was evaluation the activity of HEX, GAL, FUC and MAN in serum and urine of patients with pancreatic adenocarcinoma. Serum and urine samples were collected from 15 patients with adenocarcinoma of the pancreas and 15 healthy persons. The activity of lysosomal exoglycosidases was determined by the method of Marciniak et al. adapted to serum and urine of patients with adenocarcinoma of the pancreas. Our results indicate significant decrease in activity of GAL (p=0.037) in serum of patients with pancreatic adenocarcinoma, significant increase in activity of HEX (p<0.001) and FUC (p=0.027) in serum, and HEX (p=0.003) in urine, as well as significant decrease of FUC (p=0.016) and MAN (p=0.029) in urine o patients with adenocarcinoma of the pancreas, in comparison to the control group. Increase in activity of some lysosomal enzymes in serum and urine of pancreatic adenocarcinoma patients, may indicate on destruction of pancreatic tissue by pancreatic adenocarcinoma. Determination of the HEX, GAL, FUC and MAN in serum and urine may be useful in diagnostics of pancreatic adenocarcinoma.

alpha-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine.

Most newly synthesized lysosomal enzymes contain a transient carbohydrate modification, mannose 6-phosphate (Man-6-P), which signals their vesicular transport from the Golgi to the lysosome via Man-6-P receptors (MPRs). We have examined Man-6-P glycoproteins in human urine by using a purified soluble fragment of the soluble cation-independent MPR (sCI-MPR) as a preparative and analytical affinity reagent. In a survey of urine samples from seven healthy subjects, the pattern of Man-6-P glycoproteins detected with iodinated sCI-MPR as a probe in a blotting assay was essentially identical in each, regardless of sex or age. Two bands of approx. 100 and 110 kDa were particularly prominent. Man-6-P glycoproteins in human urine were purified by affinity chromatography on immobilized sCI-MPR. Seven distinct bands revealed by SDS/PAGE and Coomassie Blue staining were subjected to N-terminal sequence analysis. The prominent 100 and 110 kDa Man-6-P glycoproteins were identified as N-acetylglucosamine-6-sulphatase and alpha-glucosidase respectively. This identification was confirmed by molecular mass determinations on the two major bands after deglycosylation. Sequence analysis revealed arylsulphatase A and several previously unidentified proteins as minor species. Man-6-P glycoproteins were also purified on an analytical scale to determine the proportion of a number of lysosomal enzyme activities represented by the mannose-6-phosphorylated forms. The lysosomal enzymes in urine containing the highest proportion of mannose-6-phosphorylated form were beta-mannosidase (82%), hexosaminidase (27%) and alpha-glucosidase (24%). The profiles of Man-6-P glycoproteins detected by blotting in urine and plasma were not similar, suggesting that the urinary species are not derived from the bloodstream.

Prospective study of the enzymatic activities in urine of N-acetyl-beta-D-glucosaminidase, alpha-D-mannosidase, alpha- and beta-D-glucosidases, alpha-L- and beta-D-fucosidases, and beta-D-galactosidase in type I diabetes mellitus with early nephropathy.

Different surveys have been carried out on the plasma activities of different glycosidases in patients with insulin-dependent diabetes mellitus, but research on urinary glycosidases in this disease is scanty and incomplete. To elucidate the behavior of these lysosomal enzymes in the metabolic alterations occurring in the glomerular basal membrane during the initial stages of diabetic nephropathy, we conducted a prospective study to examine the urinary activities of N-acetyl-beta-D-glucosaminidase (NAG), alpha-D-mannosidase, alpha- and beta-D-glucosidase, alpha-L- and beta-D-fucosidase, and beta-D-galactosidase in patients with type I insulin-dependent diabetes mellitus, surveyed over 18 months, whose early diabetic nephropathy was detected by the presence of microalbuminuria. The simultaneous determination of beta 2-microglobulin in urine confirmed the glomerular origin of the albuminuria. No statistically significant correlation was found between the levels of albuminuria and the activities of any of the glycosidases analyzed. In the diabetic patients, a significant decrease was observed in the activities of all the enzymes (p < 0.05), except NAG and alpha-D-mannosidase, although the decrease in the latter was very close to statistical significance (p = 0.028, unilateral; p = 0.057 bilateral). Similarly, in the patients, there was a significant negative correlation (p < 0.05) with the serum levels of fructosamine, except with beta-D-galactosidase, which showed a positive correlation (p < 0.05) with fructosamine and blood HbA1c.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between urinary Tamm-Horsfall glycoprotein excretion and urinary activity of glycosidases in normal pregnancy and pre-eclampsia.

Electrophoretic analyses of the urinary proteins of pre-eclamptic patients revealed a decrease in the staining intensity of the protein band representing the Tamm-Horsfall glycoprotein (THP). In the present study the quantitative analysis of the THP excretion rate and the urinary activity of THP oligosaccharide metabolizing glycosidases were investigated. The median THP excretion rate of non-pregnant women (n = 24) was 20 mg/g creatinine (crea.). An increase in the THP excretion rate was seen in pregnancy to a level between 43 mg/g crea. (II. trimester) and 32 mg/g crea. (III. trimester) (n = 29). Hypertension in pregnancy was associated with a decrease in the THP excretion rate to 9 mg/g crea. (n = 85). Post partum, a transient elevated THP excretion rate up to 109 mg/g crea was recorded in the group of hypertensive patients. The urinary activities of the lysosomal beta-mannosidase, alpha-fucosidase, alpha-mannosidase (pH 4.5) and beta-galactosidase increased in normal pregnancy. This effect was most pronounced in the beta-galactosidase activity which increased from 50 U/mg crea. before pregnancy to 280 U/mg crea. at term. Hypertension in pregnancy was associated with a further increase in the activities of the lysosomal glycosidases. In the case of the beta-galactosidase a significant rise from 68 to 310 U/mg crea. was found. The urinary activity of the alpha-mannosidase (pH 5.5) originating from the Golgi apparatus was only elevated in patients with severe pre-eclampsia. Casuistic post partum recordings demonstrated that an elevation of the lysosomal glycosidases activities was followed by a transient increase in the THP excretion rate.

Age-related excretion of six glycosidases in rat urine.

The activity of beta-N-acetylglucosaminidase (NAG), beta-galactosidase, alpha-L-fucosidase, beta-glucuronidase, beta-glucosidase and alpha-mannosidase was determined in the urine of rats at progressive ages from newborn to old animals. The age-dependence of urinary creatinine, protein and pH values was also studied. Enzyme activity, related to urinary creatinine, was significantly higher in the newborn group than other ages. The excretion of NAG increased significantly in adult rats (3-6 months old) compared to young rats (1 month old). Most of the enzyme activities were diminished in old rats (25 months old). Increased proteinuria and creatinine excretion were observed in rats since 3 months of age. Age-related differences among enzyme activities therefore should be considered when these urinary glycosidases are to be studied in rats.

Low-dose dopamine during cardiopulmonary bypass in patients with renal dysfunction.

Thirty-six patients with preoperative renal dysfunction were studied to evaluate the effects of dopamine (D) and dopamine-nitroprusside (DN) on renal function during cardiopulmonary bypass (CPB). No differences from the control group (C) were found in creatinine clearance, fractional sodium excretion, osmolarity and free-water clearance. Sodium output/intake ratio during CPB was higher in group D than in groups C and DN (P less than 0.05); water output/intake ratio was higher in group D than in group C (P less than 0.05). Urine lysozime levels and alpha-glycosidase/creatinine ratios increased similarly in the three groups, suggesting ischemic tubular cell damage. No patients showed acute postoperative renal failure or a worsening of their renal dysfunction. The data suggest an increased water and sodium excretion during CPB with a dopamine infusion, possibly resulting from a renal vasodilator effect that was abolished by simultaneous nitroprusside administration.

Effect of acid-base changes on urinary hydrolases in Fabry's disease after renal transplantation.

Fabry's disease, which is characterized by alpha-galactosidase A (AG) deficiency, causes early renal failure. Kidney transplants do not reliably supply the deficient enzyme. To assess both urinary excretion of AG by the transplant and the relationship between urine and serum hydrolase activity, acute and chronic acid-base studies were performed in normal control subjects and in the patient with Fabry's disease who had undergone renal transplantation. For the acute studies, alkalosis was induced by intravenous infusion of sodium bicarbonate and acidosis was induced by ingestion of ammonium chloride. The chronic study involved long-term ingestion of NH4Cl by only the patient with Fabry's disease. The results show that AG is secreted by the renal graft. Urinary hydrolase excretion was increased by acute alkalinization and decreased by acute acidification. Acute, but not chronic, acidification increased the patient's serum AG activity, indicating that long-term acidification is not useful for treating Fabry's disease after transplantation. The large changes in hydrolyase excretion induced by acute and chronic acid-base changes show the difficulty of using lysosomal enzymuria as a diagnostic marker for renal disorders without knowledge of acid-base conditions.

Total amylase and pancreatic isoamylase in serum and urine: considerations from data on biological variation.

The analytical, within-subject and between-subject components of variation were estimated for amylase activity and pancreatic isoamylase activity in serum measured using newer analytical methods. Desirable analytical imprecisions based on within-subject variation were CV less than or equal to 4.4% and CV less than or equal to 7.0%, respectively. Conventional population-based reference intervals were not useful because of marked individuality; clinical decision-making points should be derived from the desired sensitivity and specificity. Serial results must change more than about 30% and 40% respectively before significance (P less than or equal to 0.05) can be claimed. Similar data on total amylase and pancreatic isoamylase activities in random and first morning urines showed that the use of conventional reference intervals was appropriate. Very large changes (greater than 100%) were required before a difference in serial results was significant. Calculation of the urine amylase/creatinine ratio appeared to confer no advantage. Derivation of the ratio of pancreatic isoamylase to total amylase activity in serum or urine was unlikely to provide additional information of value in either diagnosis or monitoring.

Role of urinary beta-glucuronidase in human bladder cancer.

It has been suggested that high levels of urinary beta-glucuronidase may increase an individual's risk of bladder cancer by releasing free carcinogens from their inactive glucuronide conjugates in the bladder. The hypothesis derives in part from the high levels of urinary beta-glucuronidase observed in bladder cancer patients. Because most of the individual variation in levels of urinary beta-glucuronidase and other lysosomal enzymes in the normal population is genetically determined, we would expect that, if high glucuronidase levels were a predisposing factor in the disease, bladder cancer patients would transmit this trait to their progeny. We have tested this hypothesis and find that levels of urinary beta-glucuronidase and three other lysosomal enzymes, alpha-galactosidase, beta-galactosidase, and beta-hexosaminidase, are not significantly elevated in 34 progeny of bladder cancer patients compared to 34 matched controls. Additionally, 15 bladder cancer patients judged to be disease free for a median time of 5 years did not have elevated levels of urinary beta-glucuronidase when compared to a normal population of 125 individuals. Thus, the high levels of glucuronidase observed in bladder cancer patients are most likely a consequence of disease rather than a cause.

Effect of tobramycin and gentamicin on the activity of some glycosidases in rat serum and urine.

beta-Galactosidase, alpha-D-mannosidase, alpha-L-fucosidase and N-acetyl-beta-D-glucosaminidase activities were assayed in serum and urine from rats treated with three different doses of the nephrotoxic antibiotic tobramycin (100 mg/kg/day for 5 days, 10 mg/kg/day for 10 days and 5 mg/kg/day for 20 days) and gentamicin (100 mg/kg/day for 5 days). A significant increase of beta-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-L-fucosidase activities occurred in urine following the administration of high doses of antibiotic. The enzyme activity was dependent on the dose level used. The excretion of alpha-D-mannosidase was atypical and elevated activities were observed on some days but no pattern of excretion of this enzyme was established. No change in any of the four glycosidase activities was found in serum of treated rats. The results obtained when high doses of gentamicin were employed are similar to those obtained with a similar dose of tobramycin. These results indicate that the assay of urinary glycosidase activities provides a useful method for monitoring the nephrotoxicity of antibiotics.

Effect of the degree of hyperglycaemia on the catalytic activities of glycosidases in kidney and urine of diabetic rats.

The catalytic activities of N-acetyl-beta-D-glucosaminidase, beta-galactosidase and alpha-glucosidase in kidney and urine of diabetic rats were investigated in relation to the duration of diabetes, to the degree of constant hyperglycaemia and to the therapeutic control in the early stage of disease. The results suggest that the degree of constant hyperglycaemia and the duration of untreated diabetes are significant determining factors for the course of morphological changes. These changes are manifested as a decrease of the glycosidases in kidney (0.5 to 0.6 time the age-matched controls) and as moderate to severe enzymurias. Daily variation of blood glucose with inadequate insulin Lente therapy caused decreased N-acetyl-beta-D-glucosaminidase and beta-galactosidase activities in kidney as well as enzymuria. Since such changes can be correlated with histologically visible changes in the kidney, the measurement of these enzymes in urine is a simple way of monitoring the development of kidney damage in poorly controlled diabetes. When constant normoglycaemia was maintained for three weeks with insulin Ultralente in diabetic rats with a confirmed decrease of kidney glycosidases, the persisting morphological alteration of the organ was reflected by a urinary output of N-acetyl-beta-D-glucosaminidase.

Increased serum N-acetyl-beta-D-glucosaminidase activity in human hypertension.

Compared to values obtained in healthy normotensive control subjects, serum activity of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (NAG) was found to be elevated in patients with untreated or treated essential hypertension and in patients with renovascular hypertension. Increased NAG activity in hypertension could not be ascribed to tissue necrosis because the serum levels of several commonly measured cytosolic enzymes were within normal limits. The kidney is a likely but unproven source of the enzyme elevation in hypertension.

Are measurements of urine enzymes useful during aminoglycoside therapy?

We prospectively evaluated concentrations of beta-D-galactosidase, alpha-L-fucosidase, beta-D-N-acetylglucosaminidase, and lysozyme in urine from normal subjects, ambulatory patients with cystic fibrosis (CF), and CF patients with previously normal renal function who were receiving intravenous aminoglycoside (AG) therapy. Enzyme activities were generally low or negligible in subjects not receiving AG. Enzymuria was documented during 12 of 13 AG treatment courses and most frequently involved beta-D-N-acetylglucosaminidase excretion. In nine courses, enzymuria occurred in the absence of proteinuria or elevations of blood urea nitrogen and serum creatinine. In three courses attended by enzymuria and evidence of nephrotoxicity, neither the time of appearance nor the magnitude of enzymuria was different from that of nonnephrotoxic patients. In two of these three treatment courses, enzymuria preceded clinical evidence of nephrotoxicity of 16 and 5 days, and in the third course enzymuria and elevation of blood urea nitrogen and serum creatinine occurred simultaneously. We conclude that enzymuria is not a reliable predictor of nephrotoxicity due to AG in CF patients and is not an indication of discontinue AG therapy.

[Renal tubular damage after rapid intravenous contrast study with iopamidol]. / Il danno tubulare renale dopo contrastografia rapida endovenosa con iopamidolo.

Reference is made to results obtained in 53 nephropathics in the study of urinary elimination of alphaglycosidase and lysozyme before and after rapid contrastography with sodium and meglumine diatrizoate and with iopamidol. A statistically significant difference in alphaglycosidase was evident with both media, whereas no change was noted for lysozyme. As far as the evaluation of alphaglycosidase was concerned, the tubular alteration induced by each medium was the same in terms of statistical comparison of the mean differences before and after urography.