Automated assay of N-acetyl-β-GLucosaminidase in normal and pathological human urine

An automated method for the assay of N-acetyl-β-glucosaminidase in human urine is described and a normal range of urinary N-acetyl-β-glucosaminidase activity is reported. The automated method has been used routinely over a twelve month period in two London hospitals for monitoring the excretion of N-acetyl-β-glucosaminidase in renal transplant patients. The abnormal elevation of urinary N-acetyl-β-glucosaminidase provides an early warning of rejection by indicating the presence of renal parenchymal damage. The automated assay of urinary N-acetyl-β-glucosaminidase may also be used in screening for the presence of renal disease.     

Biochemical activities of lysosomal acid hydrolases in leukemic cells

The biochemical activities of 8 lysosomal acid hydrolases in leukemic cells from 48 patients were examined. Characteristic alterations were found in α-mannosidase, β-galactosidase and N-acetyl-β-glucosaminidase activities of leukemic cells. The level of α-mannosidase activity was much higher in myelo(mono)genous leukemias (AML, AMoL, AMMoL, CML and CMMoL) than in lymphogenous ones (ALL, T-cell leukemia, hairy cell leukemia and CLL) without exception. The β-galactosidase activity also differed as a result of α-mannosidase, except in T-cell leukemia. In T-cell leukemia it was within the range of normal lymphocytes, but in the other lymphogenous leukemias it was significantly below normal. N-acetyl-β-glucosaminidase activity in myelo(mono)genous leukemic cells was above the range of normal granulocytes. The changes in these enzyme levels were consistent. The lymphocytic or myelocytic nature of three cases of acute undifferentiated leukemia could be determined by enzyme studies. In two cases it was lymphocytic and in one it was myelocytic. The enzymatic abnormalities were also found in morphologically mature neutrophils from patients with not only chronic types (CML, CMMoL) but also acute types (AMoL, AMMoL) of leukemias, and were similar to those of their respective leukemic cells. Analysis of lysosomal enzymes (at least three of those mentioned above), can elucidate one of the biochemical properties of leukemic cells and may be valuable in the differentiation of leukemias.     

Acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy

A study was made at various stages of pregnancy of five acid hydrolases which occur in amniotic fluid and chorionic villi and which are relevant to serious storage disorders.In amniotic fluid β-galactosidase and α-mannosidase decreased moderately towards term, while β-glucosidase decreased markedly. N-Acetyl-β-glucosaminidase and β-glucuronidase were relatively unchanged.In chorionic villi N-acetyl-β-glucosaminidase, β-galactosidase, and α-mannosidase were substantially decreased towards term, while β-glucosidase was unchanged and β-glucuronidase markedly increased.In both amniotic fluid and chorionic villi the enzyme pattern was approximately the same as that found in liver in a previous study.The findings suggest that these enzyme assays might be useful in the diagnosis of inborn errors prenatally by using amniotic fluid, and early postnatally by using chorionic villi.     

Detection of Fabry hemizygotes and heterozygotes by measurement of -galactosidase in urine

The determination of α-galactosidase in urine can be used as a simple method for the diagnosis of Fabry hemizygotes. The activity of this enzyme was related to that of N-acetyl-β-glucosaminidase. The ratio N-acetyl-β-glucosaminidase/α-galactosidase in urine was relatively constant in any one individual. In the control group, the mean value of this ratio was 7.4 (range 1.2–20.5). In Fabry hemizygotes (n = 6) the ratio was 50 or higher. Three types of carriers could be recognized, with high (n = 1), intermediate (n = 2) and normal (n = 3) values, so that with this procedure some of the carriers are detected.     

Isoenzymes of four acid hydrolases in human kidney and urine

The occurrence of different isoenzymes of β-glucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, and α-mannosidase in human urine and kidney tissue was studied by isoelectric focusing. Artificial substrates were used for the enzymatic assays. There was a predominance of isoenzymes with a low isoelectric point in the urine. In the kidney tissue isoenzymes with higher isoelectric point predominated. This difference may be due to a higher proportion of N-acetylneuraminic acid-containing enzymes in the urine than in the kidney tissue.     

Analysis of urinary albumin,transferrin, N-acetylβ-D-glucosaminidase and β2-microglobulin in patients with impaired glucose tolerance

We investigated the changes in urinary albumin and urinary transferrin as glomerular proteins, and in urinary N-acetyl-β-D -glucosaminidase and urinary β₂-microglobulin as tubular proteins, in patients with impaired glucose tolerance. We attempted to compare the proteins of normal subjects to those of diabetics with pre-nephropathy. Transferrin and N-acetyl-β-D -glucosaminidase levels were significantly increased in patients with impaired glucose tolerance, while albumin and β₂-microglobulin levels were only slightly increased. In addition, there was no significant difference in transferrin levels between patients with impaired glucose tolerance and type 2 diabetics with pre-nephropathy. In our observation, although albumin levels were only slightly increased in patients with impaired glucose tolerance, a sharp increase in transferrin levels was reflected in patients with glomerular disorders. In addition, since N-acetyl-β-D -glucosaminidase levels varied markedly, tubular disorders were suspected. It should be stressed that increased parameters for both glomerular and tubular disorders in group C—patients who showed abnormal levels in three proteins—had already been observed in some patients with impaired glucose tolerance. Therefore, the evaluation of the mutual relationships between various urinary protein components in patients with impaired glucose tolerance will become a more important assessment tool than that of single urinary protein components. J. Clin. Lab. Anal. 12:351–355, 1998. © 1998 Wiley-Liss, Inc.     

The effect of fosfomycin on nedaplatin-induced nephrotoxicity in rats

The effect of coadministration of fosfomycin (FOM) on nedaplatin-induced nephrotoxicity in rats was investigated for 6 days. FOM decreased nedaplatin-induced nephrotoxicity, as shown by reduced blood urea nitrogen (BUN), serum creatinine levels, and urinary excretion of N-acetyl--d-glucosaminidase (NAG). Further, there were fewer histopathological signs of nephrotoxicity in the groups treated with the combination of nedaplatin and FOM as compared with the nedaplatin-alone group. The concentration of nedaplatin was significantly lower in the renal cortex of rats treated with the combination of nedaplatin and FOM as compared with those treated with nedaplatin alone (p < 0.05). In conclusion, the concomitant administration of FOM and nedaplatin may help to achieve a chemotherapeutic strategy that reduces the nephrotoxic effects of nedaplatin.     

Urinary TGF-β1 as an indicator of antiproteinuric response to angiotensin II receptor blocker in proteinuric renal diseases

Background

Angiotensin II receptor blockers (ARBs) reduce proteinuria, however, with large inter-individual variability. The present study investigated whether urinary transforming growth factor-β1 (TGF-β1) might predict the antiproteinuric efficacy of ARB in non-diabetic chronic renal disease.

Methods

Non-diabetic patients with proteinuria (>1 g/day) received 50 mg of losartan daily followed by 100 mg in two treatment periods, each lasting 12 weeks. Clinical parameters and urinary TGF-β1 levels were measured at baseline and during the treatment period.

Results

In the whole group of patients, losartan treatment effectively decreased proteinuria. However, considerable differences existed among individual antiproteinuric responses. Good (n = 34) or low (n = 15) responders showed average proteinuria reduction of 69% or 17% from baseline, respectively. Both groups showed similar baseline biochemical and renal parameters and comparable degree of mean arterial blood pressure (MAP) reduction. However, the low responders were older and showed significantly higher baseline urinary TGF-β1 levels. On multiple regression analysis, age, baseline urinary TGF-β1 and % reduction in urinary TGF-β1 and % reduction in MAP significantly predicted antiproteinuric response to losartan therapy.

Conclusion

The present data suggest that the determination of baseline urinary TGF-β1 could be an useful indicator of short-term antiproteinuric response to ARB treatment in non-diabetic nephropathy.     

Evaluation of Urinary Elimination of N-Acetyl-β-Glucosaminidase in Healthy Volunteers Treated with Dibekacin or Gentamicin

The urinary excretion of N-acetyl-β-glucosaminidase was studied in healthy subjects during and after treatment with aminoglycosides. In terms of this parameter dibekacin appeared to be less nephrotoxic than gentamicin.     

Activity of beta-galactosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in human rectal mucosa

Activity of β-galactosidase, β-glucuronidase and N-acetl-β-glucosaminidase was determined by biopsy specimens of rectal mucosa of control subjects, cystinotic children and their parents.The studied enzymes exhibited maximal activity at pH 5.0, 4.0 and 4.5, respectively. Apparent K_m values using P-nitrophenyl-β-galactoside, p-nitrophenyl-β-glucuronide and p-nitropheny-β-glucosaminide were found to be 0.52 mM, 0.70 mM, and 0.67 mM.The activity of all three enzymes was found to be closely correlated in the 11 subjects of the control group. The values found in parents and their cystinotic children fit into these correlations, but show higher scatter of data caused by the fact that values of β-galactosidase were found to be higher and of β-glucuronidase and N-acetyl-glucosaminidase lower in the group of parents than in the other two groups.     

Mucolipidosis I: Studies of sialidase activity and a prenatal diagnosis

The characteristics of the sialidase (N-acetyl-α-neuraminidase) of human leukocytes, fibroblasts and amniotic fluid cell cultures were determined with a radioactive assay method utilizing neuramin-[³H]actitol as the enzyme substrate. Fibroblast cultures from patients with the inherited sialidase deficiency diseases including mucolipidosis I, sialidosis I and sialidosis II, juvenile type have less than 10% of normal sialidase activity using either this substrate, 2-(3′-methoxyphenyl)-N-acetyl-α-neuraminic acid, or 2′-(4-methylumbelliferyl)-Nacetyl-α-neuraminic acid. The total sialic acid content of fibroblasts and leukocytes from mucolipidosis I and sialidosis I patients is greatly elevated; this parameter is useful in establishing a diagnosis of sialidase deficiency. The sialic acid content of sialidosis II, juvenile type, with coexistent sialidase and β-galactosidase deficiencies, is only slightly elevated above normal levels. A patient with mucolipidosis I has 16% of normal neuramin-[³H]lactitol sialidase activity in his peripheral leukocytes. His parents were clearly distinguished from the normal range using leukocyte enzyme levels and a maternal aunt was identified as a possible carrier. The presence of this enzyme in amniotic fluid cell cultures, both fibroblastic and mixed cell type, makes possible the prenatal detection of these diseases. A pregnancy from a family at risk for having a child with mucolipidosis I was monitored by amniocentesis and subsequent sialidase measurement of the amniotic fluid cell culture.     

beta-Mercaptolactate cysteine disulfiduria in two normal sisters. Isolation and characterization of beta-mercaptolactate cysteine disulfide

β-Mercaptolactate cysteine disulfide (βMLCD) was detected in two mentally normal sisters, 11 and 13 years old. After isolation by ion-exchange chromatography, high-voltage electrophoresis, and adsorption chromatography on Porapak Q, βMLCD was identified by mass spectrometry of its following methyl ester derivatives: O, N-di-trifluoracetyl, O-trifluracetyl-N-dinitrophenyl, O, N-diacetyl, and O-acetyl-N-dinitrophenyl, with acetyl groups 50% perdeuterated, as well as by gas chromatography-mass spectrometry combination of the desulfuration products alanine (as N-trifluotacetyl-methy; ester) and lactic acid (as methyl ester). The isolated βMLCD contained no sulfoxide nor sulfone and exhibited an UV spectrum closely related to cystine. In urine the concentration range of βMLCD was 345–751 μmole/1 (n=5) and 54–133 mg/g creatinine; in plasma a trace of βMLCD could be detected only in one case. Oral administration of L-cysteine or L-methionine elevated the concentration of βMLCD in urine and in plasma. A further disulfide accompanying βMLCD in urine was isolated and identified as thioglycolate cysteine disulfide.     

Megalin Contributes to Kidney Accumulation and Nephrotoxicity of Colistin

Interest has recently been shown again in colistin because of the increased prevalence of infections caused by multidrug-resistant Gram-negative bacteria. Although the potential for nephrotoxicity is a major dose-limiting factor in colistin use, little is known about the mechanisms that underlie colistin-induced nephrotoxicity. In this study, we focused on an endocytosis receptor, megalin, that is expressed in renal proximal tubules, with the aim of clarifying the role of megalin in the kidney accumulation and nephrotoxicity of colistin. We examined the binding of colistin to megalin by using a vesicle assay. The kidney accumulation, urinary excretion, and concentrations in plasma of colistin in megalin-shedding rats were also evaluated. Furthermore, we examined the effect of megalin ligands and a microtubule-depolymerizing agent on colistin-induced nephrotoxicity. We found that cytochrome c, a typical megalin ligand, inhibited the binding of colistin to megalin competitively. In megalin-shedding rats, renal proximal tubule colistin accumulation was decreased (13.5 ± 1.6 and 21.3 ± 2.6 μg in megalin-shedding and control rats, respectively). Coadministration of colistin and cytochrome c or albumin fragments resulted in a significant decrease in urinary N-acetyl-β-d-glucosaminidase (NAG) excretion, a marker of renal tubular damage (717.1 ± 183.9 mU/day for colistin alone, 500.8 ± 102.4 mU/day for cytochrome c with colistin, and 406.7 ± 156.7 mU/day for albumin fragments with colistin). Moreover, coadministration of colistin and colchicine, a microtubule-depolymerizing agent, resulted in a significant decrease in urinary NAG excretion. In conclusion, our results indicate that colistin acts as a megalin ligand and that megalin plays a key role in the accumulation in the kidney and nephrotoxicity of colistin. Megalin ligands may be new targets for the prevention of colistin-induced nephrotoxicity.     

Role of transforming growth factor-β2 in, and apossible transforming growth factor-β2 gene polymorphism as a marker of, renal dysfunction in essential hypertension: A study in Turkish patients

Background:

Many studies have shown that transforming growth factor(TGF)-β has a major role in renal scarring in many renal diseases and hypertension.

Objectives:

The primary aim of this study was to investigate both the relationship between hypertension and serum and urinary levels of TGF-β₂ (a more sensitive isoform for glomeruli than TGF-β₁), and the effects of combination therapy with perindopril + indapamide on microalbuminuria, which becomes an early indicator of hypertensive benign nephropathy, and serum and urinary TGF-β₂ levels in patients with mild to moderate essential hypertension. In addition, we examined the possible relationship between TGF-β₂ gene polymorphism and essential hypertension.

Methods:

This study was conducted at the Department of Nephrology, Medical Faculty, Gazi University, Ankara, Turkey. Patients aged ≥18 years with newly diagnosed mild to moderate essential hypertension (systolic/diastolic blood pressure [SBP/DBP] >120/>80 mm Hg) who had not previously received antihypertensive treatment were included in the study. Patients with stage I hypertension received perindopril 2 mg + indapamide 0.625 mg (tablet), and patients with stage lI hypertension received perindopril 4 mg + indapamide 1.125 mg (tablet). All study drugs were given OD (morning) PO with food for 6 months. Serum and urinary TGF-β₂ and creatinine levels and serum and urinary albumin levels were measured before and after perindopril + indapamide administration. Amplified DNA fragments of the TGF-β₂ primer region were screened using amplification refractory mutation system polymerase chain reaction analysis, and the number of ACA repeats was confirmed by DNA sequencing. Genetic studies were performed using a commercial TGF-β₂ kit.

Results:

Forty patients were enrolled in the study, and 38 patients (27 women, 11 men; mean [SD] age, 46.3 [6.5] years) completed it. SBP and DBP were significantly decreased from baseline with perindopril/indapamide (both, P < 0.001). Microalbuminuria and urinary TGF-β₂ levels also decreased significantly from baseline (P = 0.04 and P < 0.001, respectively), whereas the serum TGF-β₂ level did not change significantly. Three patients, all of whom were found to have TGF-β₂ gene mutations, had increased urinary TGF-β₂ levels despite good blood pressure control.

Conclusions:

The results of this study in patients with mild to moderate hypertension suggest that, despite good clinical control of blood pressure, the persistence of microalbuminuria and high urinary TGF-β₂ levels might predict renal impairment. When treating these patients, genetic tendencies and possible polymorphisms on the TGF-β₂ locus should be kept in mind.     

Urinary beta-2 microglobulin and alpha-1 microglobulin are useful screening markers for tenofovir-induced kidney tubulopathy in patients with HIV-1 infection: a diagnostic accuracy study

Kidney tubulopathy is a well-known adverse event of antiretroviral agent tenofovir. A cross-sectional study was conducted to compare the diagnostic accuracy of five tubular markers, with a collection of abnormalities in these markers as the reference standard. The study subjects were patients with HIV-1 infection on ritonavir-boosted darunavir plus tenofovir/emtricitabine with suppressed viral load. Kidney tubular dysfunction (KTD) was predefined as the presence of at least three abnormalities in the following five parameters: β2-microglobulinuria (β2M), α1-microglobulinuria (α1M), high urinary N-acetyl-β-d-glucosaminidase (NAG), fractional excretion of phosphate (FE_IP), and fractional excretion of uric acid (FE_UA). Receiver operating characteristic curves and areas under the curves (AUC) were estimated, and the differences between the largest AUC and each of the other AUCs were tested using a nonparametric method. The cutoff value of each tubular marker was determined using raw data of 100 % sensitivity with maximal specificity. KTD was diagnosed in 19 of the 190 (10 %) patients. The AUCs (95 % CIs) of each tubular marker were β2M, 0.970 (0.947–0.992); α1M, 0.968 (0.944–0.992); NAG, 0.901 (0.828–0.974); FE_IP, 0.757 (0.607–0.907), and FE_UA, 0.762 (0.653–0.872). The AUCs of β2M and α1M were not significantly different, whereas those of the other three markers were smaller. The optimal cutoff values with 100 % sensitivity were 1,123 μg/gCr (β2M, specificity 89 %), 15.4 mg/gCr (α1M, specificity 87 %), 3.58 U/gCr (NAG, specificity 46 %), 1.02 % (FE_IP, specificity 0 %), and 3.92 % (FE_UA, specificity 12 %). Urinary β2M and α1M are potentially suitable screening tools for tenofovir-induced KTD. Monitoring either urinary β2M or α1M should be useful in early detection of tenofovir nephrotoxicity.     

Preliminary Study on the Role of Virtual Touch Tissue Quantification Combined with a Urinary β2-Microglobulin Test on the Early Diagnosis of Gouty Kidney Damage

The goal of the work described here was to evaluate the role of virtual touch tissue quantification (VTQ) combined with urinary β2-microglobulin (β2-MG) measurement in the early diagnosis of gouty kidney damage. Two hundred fifty-nine patients with gouty kidney damage and 200 healthy control subjects were tested. The shear wave velocity (SWV) of the renal parenchyma and sinus as determined with VTQ and the urinary β2-MG level of the two groups were analyzed. Although there were no significant differences in age, body mass index, creatinine level and blood urea nitrogen between the two groups (all p's > 0.05), the aforementioned parameters were higher in the group with gouty kidney damage than in the control group. Urinary β2-MG levels of the patients with kidney damage were significantly higher than those of the control subjects (t = 6.38, p < 0.01). The SWV of the renal parenchyma was higher than that of the sinus in both groups. Compared with controls, patients with kidney damage had significantly increased renal parenchyma and sinus SWVs (all p-values < 0.05). Urinary β2-MG level was positively linearly correlated with the SWV of renal parenchyma in patients with kidney damage (r = 0.442, p < 0.0001). However, there was no correlation between urinary β2-MG level and the SWV of the sinus in patients with kidney damage (r = 0). In the control group, there was no correlation between urinary β2-MG level and the SWV of the renal parenchyma or sinus. The elasticity of the kidney as determined with VTQ, combined with the urinary β2-MG level, may be helpful in the early diagnosis of gouty kidney damage.     

The effect of glutamine administration on urinary ammonium excretion in normal subjects and patients with renal disease

The effect of acute changes in the delivery rate of glutamine to the kidney on urinary ammonium excretion was studied in man. Healthy subjects and patients with intrinsic renal disease were studied under three different acid-base conditions: unaltered acid-base balance; NH₄Cl-induced acidosis; and NaHCO₃-induced alkalosis. Anhydrous L-glutamine was administered orally in a single dose of 260 mmoles during each of these three acid-base states. We found that endogenous venous plasma glutamine concentration fell during acidosis and rose during alkalosis in both healthy subjects and patients with renal disease. In healthy subjects, orally administered glutamine raised plasma glutamine concentration markedly over a 2-3 hr period. This was accompanied by an increase in urinary ammonium excretion and a rise in urine pH under normal acid-base conditions and during metabolic acidosis. No increase in ammonium excretion occurred when glutamine was administered during metabolic alkalosis in spite of an equivalent rise in plasma glutamine concentration. In patients with renal disease, endogenous venous plasma glutamine concentration was lower than in healthy subjects, perhaps as a result of mild metabolic acidosis. Acute oral glutamine loading failed to increase urinary ammonium excretion significantly during either unaltered acid-base conditions or after NH₄Cl-induced acidosis, even though plasma glutamine rose as high as in healthy subjects. We conclude from these observations that glutamine delivery to the kidney is a rate-limiting factor for ammonium excretion in healthy subjects, both before and after cellular enzyme adaptation induced by metabolic acidosis. In contrast, in patients with renal disease, glutamine delivery is not rate-limiting for ammonium excretion. Presumably other factors, such as surviving renal mass and the activity of intracellular enzymes necessary for ammonia synthesis limit ammonium excretion in these patients.     

Linear relationship between the R- and S-enantiomers of β-aminoisobutyric acid in human urine

Urine from untreated patients with various tumours and patients not having tumours has been examined for the excretion of β-aminoisobutyric acid enantiomers. It was shown that the ratio of urinary R- to S-β-aminoisobutyric acid was nearly constant indicating a correlation between the production of both forms. The results are discussed in relation to thymine and valine metabolism, and a metabolic scheme explaining the observed relationship between both forms of β-aminoisobutyric acid is presented.     

Urinary enzyme excretion by renal-transplant recipients in relation to interval after transplantation

We determined the urinary excretion of the enzymes aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) in two groups of renal-transplant recipients at different times after transplantation (1.8 months and 52 months, respectively). Both groups of patients showed a higher rate of enzyme excretion than did a reference group of healthy persons. More aminopeptidase and N-acetyl-beta-glucosaminidase were excreted during the early period after transplantation than later. The time-dependence of urinary enzyme excretion was confirmed in six renal-transplant recipients studied during the course of 15 months after transplantation. There was a general correlation between the extent of urinary enzyme excretion and both the time after transplantation and the daily dose of prednisolone. Therefore, it is necessary to take into account this influence on the extent of urinary enzyme in renal-transplant recipients if urinary enzyme excretion is used as an indicator of renal disorder and especially as an early predictor of transplant rejection.     

Impaired renal function is associated with greater urinary strong ion differences in critically ill patients with metabolic acidosis

Purpose

Urinary excretion of chloride corrects metabolic acidosis, but this may be hampered in patients with impaired renal function. We explored the effects of renal function on acid-base characteristics and urinary strong ion excretion using the Stewart approach in critically ill patients with metabolic acidosis.

Materials and Methods

We examined the plasma and urine chemistry in 65 critically ill (mixed medical and surgical) patients with metabolic acidosis. The apparent strong ion difference, effective strong ion difference, strong ion gap, and urinary simplified strong ion difference (urinary SID) were calculated. Linear regression analyses were used (1) to assess whether plasma creatinine concentrations were related to urinary SIDs values, adjusted for blood pH levels, and (2) to determine whether urinary SID values were associated with blood pH levels.

Results

Creatinine concentrations were positively and significantly (P < .001) associated with urinary SIDs values, adjusted for pH levels. Urinary simplified strong ion difference values were inversely and significantly (P < .001) related to pH levels.

Conclusions

In critically ill patients with metabolic acidosis, impaired renal function was associated with greater urinary SIDs. Subsequently, the higher urinary SIDs values were related to lower pH levels, illustrating the importance of renal chloride excretion to correct for acidosis.