ABSTRACT
Individuals with type 1 diabetes (T1D) often have higher than normal blood glucose levels, causing advanced glycation end product formation and inflammation and increasing the risk of vascular complications years or decades later. To examine the urinary proteome in juveniles with T1D for signatures indicative of inflammatory consequences of hyperglycemia, we profiled the proteome of 40 T1D patients with an average of 6.3 years after disease onset and normal or elevated HbA1C levels, in comparison with a cohort of 41 healthy siblings. Using shotgun proteomics, 1036 proteins were identified, on average, per experiment, and 50 proteins showed significant abundance differences using a Wilcoxon signed-rank test (FDR q-value ≤ 0.05). Thirteen lysosomal proteins were increased in abundance in the T1D versus control cohort. Fifteen proteins with functional roles in vascular permeability and adhesion were quantitatively changed, including CD166 antigen and angiotensin-converting enzyme 2. α-N-Acetyl-galactosaminidase and α-fucosidase 2, two differentially abundant lysosomal enzymes, were detected in western blots with often elevated quantities in the T1D versus control cohort. Increased release of proteins derived from lysosomes and vascular epithelium into urine may result from hyperglycemia-associated inflammation in the kidney vasculature.
Subject(s)
Diabetes Mellitus, Type 1/urine , Enzymes/urine , Proteome/metabolism , Proteomics/methods , Siblings , Activated-Leukocyte Cell Adhesion Molecule/metabolism , Activated-Leukocyte Cell Adhesion Molecule/urine , Adolescent , Angiotensin-Converting Enzyme 2 , Blotting, Western , Child , Chromatography, Liquid , Cohort Studies , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/metabolism , Enzymes/metabolism , Female , Humans , Lysosomes/enzymology , Lysosomes/metabolism , Male , Peptidyl-Dipeptidase A/metabolism , Peptidyl-Dipeptidase A/urine , Tandem Mass Spectrometry , alpha-L-Fucosidase/metabolism , alpha-L-Fucosidase/urine , alpha-N-Acetylgalactosaminidase/metabolism , alpha-N-Acetylgalactosaminidase/urineABSTRACT
BACKGROUND: The present study aimed to assess whether the urinary profiles of the lysosomal exoglycosidases Nacetylßhexosaminidase (HEX) and its isoenzymes A (HEX A) and B (HEX B), α-fucosidase (FUC), ß-galactosidase (GAL), α-mannosidase (MAN), and ß- glucuronidase (GLU) are useful biomarkers of tubular dysfunction in children with a solitary functioning kidney (SFK). METHODS: We measured the urinary activity of HEX, its isoenzymes HEX A, HEX B, and FUC, GAL, MAN, and GLU in 52 patients with SFK. Patients were subdivided into two groups: congenital SFK (cSFK)-unilateral renal agenesis and acquired SFK (aSFK)-unilateral nephrectomy. The reference group (RG) contained 60 healthy sex- and age-matched children. RESULTS: Urinary activity of all exoglycosidases in SFK was significantly higher than in RG (p < 0.05). There were no differences in exoglycosidase activity between cSFK and aSFK (p > 0.05). HEX and its isoenzymes HEX A and HEX B correlated negatively with estimated glomerular filtration rate (eGFR), and all estimated parameters correlated positively with albumin/creatinine ratio (p < 0.001). CONCLUSION: Urinary activity of HEX, its isoenzymes HEX A and HEX B, and FUC, GAL, MAN, and GLU is elevated in children with SFK. Long-term follow-up studies in larger groups of children with SFK may help us to better understand their clinical significance.
Subject(s)
Kidney Tubules, Proximal/injuries , Kidney/abnormalities , Urogenital Abnormalities/urine , alpha-L-Fucosidase/urine , alpha-Mannosidase/urine , beta-Galactosidase/urine , beta-N-Acetylhexosaminidases/urine , Adolescent , Biomarkers/urine , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Male , NephrectomyABSTRACT
BACKGROUND: In hospital patients suffering from adverse clinical and biochemical symptoms of malnutrition, it is often necessary to employ parenteral nutrition to avoid the body's tissue becoming broken down by being metabolised. Thus, the patient's welfare and survival can be supported throughout any periods of medical crisis. Two of the enzymes responsible for metabolising glycoconjugates are alpha-fucosidase (FUC) and beta-glucuronidase (GLU), present in lysosomes. They release fucose or glucuronic acid from the non-reducing end of oligosaccharide chains. OBJECTIVE: To determine the effect of parenteral nutrition administered to ill patients, on glycoconjugate metabolism, by measuring serum and urinary activities of FUC and GLU. Material and methods. Blood samples and the daily urine collection were taken from 23 patients' who had been undergoing parenteral nutrition for either 5 or 10 days, as well as from a baseline sample. Enzyme activities in serum and urine were determined by the method of Zwierz et al. RESULTS: Serum FUC activities were significantly lower after 10 days compared to 5, (p< 0.0172), whereas GLU activities were significantly lower after both 5 and 10 days, (p< 0.0007 and p< 0.0208 respectively), compared to levels before starting parenteral nutrition. GLU activities were however higher after 10 days than those after 5 days, (p< 0.0023). In urine, FUC activities were significantly decreased after 10 days compared to 5 days after starting parenteral nutrition, (p< 0.0245). Urine GLU activities were unaffected by parenteral nutrition nor was any effect seen on FUC or GLU activities when calculated per 1mg creatinine. CONCLUSIONS: Serum FUC and GLU activities can be used for assessing the effect of parenteral nutrition on glycoconjugate metabolism. The significant decreases of serum GLU activity observed after 5 and 10 days, may serve to indicate that the components of parental nutrition are appropriate and that the body has become suitably adapted to this form of nutrition.
Subject(s)
Glucuronidase/blood , Glucuronidase/urine , Malnutrition/metabolism , Malnutrition/therapy , Parenteral Nutrition/statistics & numerical data , alpha-L-Fucosidase/blood , alpha-L-Fucosidase/urine , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young AdultABSTRACT
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.
Subject(s)
Adenocarcinoma/enzymology , Glycoside Hydrolases/blood , Glycoside Hydrolases/urine , Lysosomes/enzymology , Pancreatic Neoplasms/enzymology , Adenocarcinoma/blood , Adenocarcinoma/urine , Adult , Aged , Case-Control Studies , Female , Glycoconjugates/metabolism , Glycoconjugates/urine , Humans , Lysosomes/metabolism , Male , Middle Aged , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/urine , Sensitivity and Specificity , Serum/enzymology , Serum/metabolism , alpha-L-Fucosidase/metabolism , alpha-L-Fucosidase/urine , alpha-Mannosidase/metabolism , beta-Galactosidase/metabolism , beta-Galactosidase/urine , beta-N-Acetylhexosaminidases/blood , beta-N-Acetylhexosaminidases/metabolism , beta-N-Acetylhexosaminidases/urineABSTRACT
The main isozyme patterns of desialylated blood plasma or serum alpha-L-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA1*1 and FUCA1*2 alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+/- SD) of the three phenotypes were 318.8 +/- 116.7 nmol/ml per h for type 1, 268.0 +/- 108.3 nmol/ml per h for type 2-1, and 233.2 +/- 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA1*1 gene product in plasma has about 1.4 times the activity of FUCA1*2.
Subject(s)
Isoenzymes/genetics , Polymorphism, Genetic , alpha-L-Fucosidase/genetics , Adult , Alleles , Child , Electrophoresis, Polyacrylamide Gel , Female , Gene Frequency , Humans , Isoelectric Focusing , Isoenzymes/blood , Isoenzymes/urine , Leukocytes/enzymology , Male , Middle Aged , Phenotype , Placenta/enzymology , Pregnancy , Semen/enzymology , alpha-L-Fucosidase/blood , alpha-L-Fucosidase/urineABSTRACT
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)
Subject(s)
Diabetes Mellitus, Type 1/enzymology , Diabetic Nephropathies/enzymology , Glycoside Hydrolases/urine , Acetylglucosaminidase/urine , Adolescent , Adult , Albuminuria , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/urine , Diabetic Nephropathies/blood , Diabetic Nephropathies/urine , Fructosamine , Glycated Hemoglobin/analysis , Hexosamines/blood , Humans , Longitudinal Studies , Mannosidases/urine , Prospective Studies , alpha-Glucosidases/urine , alpha-L-Fucosidase/urine , alpha-Mannosidase , beta-Galactosidase/urine , beta-Glucosidase/urineABSTRACT
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.
Subject(s)
Aging/urine , Glycoside Hydrolases/urine , Acetylglucosaminidase/urine , Aging/physiology , Animals , Animals, Newborn , Creatinine/urine , Glucuronidase/urine , Hydrogen-Ion Concentration , Kidney/physiology , Male , Mannosidases/urine , Proteinuria/urine , Rats , Rats, Wistar , alpha-L-Fucosidase/urine , alpha-Mannosidase , beta-Galactosidase/urine , beta-Glucosidase/urineABSTRACT
1. Enzymatic forms of alpha-L-fucosidase from human renal tissue and urine were investigated. 2. In renal tissue two different isoenzymatic patterns were obtained by chromatofocusing of either directly soluble or detergent solubilized extracts. 3. On the other hand the urinary isoenzymatic pattern is similar to that obtained for the renal soluble extract.
Subject(s)
Kidney/enzymology , alpha-L-Fucosidase/analysis , Adult , Chromatography, Ion Exchange , Detergents , Humans , Isoelectric Focusing , Isoenzymes/analysis , Isoenzymes/urine , Solubility , alpha-L-Fucosidase/urineABSTRACT
The binding of the urinary lysosomal enzyme alpha-L-fucosidase to free- and Sepharose 4B-bound concanavalin A has been compared in cystic fibrosis (CF) patients and normal controls. The concentration of methyl-alpha-D-mannoside necessary to prevent 50% of total alpha-L-fucosidase activity to bind to free and bound concanavalin A (Ki, 50%) was similar for CF (0.68 +/- 0.20 and 1.3 +/- 0.3 mmol/l, respectively) and normal controls (0.53 +/- 0.18 and 1.9 +/- 0.5 mmol/l, respectively). The CF and normal urinary alpha-L-fucosidase also showed similar pH optima (4.8), Km, app (0.071 and 0.074 mmol/l, respectively) and thermodenaturation curves at 44 degrees C (t1/2 = 108 min). We report that the kinetic and the concanavalin A-binding affinity of alpha-L-fucosidase are similar from urine of cystic fibrosis patients and controls.
Subject(s)
Cystic Fibrosis/enzymology , alpha-L-Fucosidase/urine , Concanavalin A/metabolism , Hot Temperature , Humans , Hydrogen-Ion Concentration , Kinetics , Mannosides/pharmacology , Methylmannosides , Protein Denaturation , Sepharose , alpha-L-Fucosidase/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Aminoglycosides/adverse effects , Cystic Fibrosis/enzymology , Glycoside Hydrolases/urine , Kidney/drug effects , Acetylglucosaminidase/urine , Adolescent , Adult , Child , Child, Preschool , Female , Gentamicins/therapeutic use , Humans , Lung Diseases/drug therapy , Male , Middle Aged , Muramidase/urine , Pseudomonas Infections/drug therapy , Tobramycin/therapeutic use , alpha-L-Fucosidase/urine , beta-Galactosidase/urineABSTRACT
Conditions necessary for the precise measurement of free L-fucose in urine by an enzymic method using L-fucose dehydrogenase have been studied. The normal urinary levels of L-fucose were 16.4 /+- 9.1 micrograms/ml in children (22.2 /+- 6.5 micrograms/mg of creatinine) and 17.7 /+- 8.5 micrograms/ml in adults (16.6 /+- 5.7 micrograms/mg of creatinine). There was a close correlation between the concentration of free L-fucose and that of creatinine. A close correlation was also found between the concentration of free L-fucose and alpha-L-fucosidase activity. Thus, it was suggested that the free L-fucose in urine reflected the metabolism of L-fucose or L-fucose-containing glycoconjugates. In a preliminary screening test, several urine samples which showed high concentrations of free L-fucose, namely cases of "fucosuria" were found.
Subject(s)
Carbohydrate Dehydrogenases , Fucose/urine , Adult , Child , Creatinine/urine , Female , Humans , Male , Methods , Reference Values , alpha-L-Fucosidase/urineABSTRACT
Acidic hydrolases were assayed in urines of 19 normal children, 33 children with idiopathic nephrotic syndrome of childhood (INS), 21 children with glomerulonephritides (GN) and 7 children with persistent proteinuria/hematuria, and in plasma of 10 children each with INS or GN. Both plasma and urinary acidic hydrolases were studied in intermittent orthostatic proteinuria. Cbeta-galactosidase and Cbeta-N-hexosaminidase were done in normals and children with active renal disease. Significantly (P less than 0.01) elevated urinary acidic hydrolases excretion in active renal diseases, both in INS and GN, returned to a normal range with regression of the diseases. Increased postural proteinuria was associated with normal urinary acidic hydrolases. Both beta-galactosidase and beta-N-hexosaminidase excretion was higher than similar mol wt proteins in normals and increased further in active renal diseases. The data suggests that increased urinary acidic hydrolases is related to the activity of the renal disease, and not to urinary WBC, hematuria or proteinuria. The likely source of urinary acidic hydrolases thus appears to be the injured renal parenchyma itself.
Subject(s)
Glomerulonephritis/enzymology , Hematuria/enzymology , Hydrolases/urine , Nephrotic Syndrome/enzymology , Proteinuria/enzymology , Arylsulfatases/urine , Child , Hexosaminidases/blood , Hexosaminidases/urine , Humans , Hydrolases/blood , Mannosidases/blood , alpha-L-Fucosidase/blood , alpha-L-Fucosidase/urine , beta-Galactosidase/blood , beta-Galactosidase/urineABSTRACT
1. Human alpha-L-fucosidase (EC 3.2.1.51) was studied from leucocytes, urine and serum. 2. The leucocyte and urine enzymes are similar in many properties (KM, pH optimum, electrophoretic pattern, heat stability). 3. The serum alpha-L-fucosidase differs from the leucocyte and 4rine enzyme wit,respect to: electrophoretic pattern, pH optimum and heat stapility. 4. The molecular weight of leucocyte alpha-L-fucosidase was determined to be 80 000 +/- 5000. 5. Cu2+, Hg2+ and PCMB are strong inhibitors of leucocyte alpha-L-fucosidase. This inhibition could be completely reversed by beta-mercaptoethanol, indicating that thiol groups are essential for catalytic activity.
Subject(s)
Disaccharidases/blood , Leukocytes/enzymology , alpha-L-Fucosidase/blood , Anions , Cations, Divalent , Cations, Monovalent , Chloromercuribenzoates/pharmacology , Drug Stability , Humans , Hydrogen-Ion Concentration , Iron/pharmacology , Kinetics , Mercaptoethanol/pharmacology , alpha-L-Fucosidase/urineSubject(s)
Diabetes Mellitus/urine , Hydrolases/urine , Acetylglucosaminidase/urine , Acid Phosphatase/urine , Alkaline Phosphatase/urine , Aspartate Aminotransferases/urine , Diabetes Mellitus/drug therapy , Diabetes Mellitus/physiopathology , Diuresis , Galactosidases/urine , Insulin/therapeutic use , Mannosidases/urine , Streptozocin , alpha-L-Fucosidase/urineABSTRACT
The nephrotoxicities of gentamicin and three other experimental aminoglycosides were compared at a single 60 mg. per kilogram per day dose in rats. Renal function, lysosomal enzymuria, and antibiotic concentrations in plasma, urine, and renal tissue were measured at regular intervals throughout the course of treatment. Kidney tissue was examined by light and electron microscopy in animals killed at intervals throughout the period of antibiotic administration. Proteinuria and enzymuria were early indicators of nephron dysfunction, whereas endogenous creatinine clearance declined later in the course of treatment. All animals were killed 24 hours after a previous antibiotic injection and displayed sustained renal tissue antibiotic concentrations which were 5 to 10 times higher than those in serum or urine. When assayed separately, renal cortical tissue had a fivefold greater antibiotic concentration than renal medulla. Light microscopy displayed necrosis of the pars convoluta of the proximal tubule. Electron microscopy revealed appearance of cytosegrosomes with myeloid bodies. It is possible that impaired cytoplasmic degradation of sequestered organelle membranes, resulting from aminoglycoside accumulation, is responsible for the myeloid body formation and subsequent tubular necrosis.