Lysosomal enzymuria in protein energy malnutrition.

Protein energy malnutrition (PEM) is common in underprivileged populations in many parts of the world and results from diets deficient in protein (kwashiorkor) or protein and calories (marasmus). The literature documents renal tubular abnormalities in children with PEM. In PEM the reabsorption of amino acids and phosphate is defective. In many kidney disorders in which renal tubular function is impaired (e.g., diabetes, preeclampsia, nephrotic syndrome, sickle cell anemia), lysosomal enzymuria ensues. We compared the urinary excretion of the following five lysosomal enzymes in 31 Nigerian children with marasmus, kwashiorkor, or marasmic-kwashiorkor: beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, and alpha-mannosidase. All of the protein energy malnourished children and the 18 age- and gender-matched controls were from the city of Jos, located in central Nigeria. In the severely malnourished children, the urine levels of all five lysosomal enzymes (expressed as units of enzyme activity per mg creatinine) were markedly increased. The greatest increases were seen with beta-hexosaminidase (16-fold) and beta-glucuronidase (14-fold). Routine clinical analyses also revealed that, relative to the control population, the sera of the 14 most severely malnourished patients contained 2- to 5-fold more vitamin B12 and markedly reduced levels (15%, p < 0.00001) of calcium. These data are significant in that they document lysosomal enzymuria in Nigerian children with severe PEM and point to the potential diagnostic utility of the urinary beta-galactosidase determination for assessing renal function in children with this disorder.

Some urinary enzymes in bilharziasis.

Urinary alkaline phosphatase (ALP), acid phosphatase (ACP), aryl sulphatase (Ar. sulph.), beta-glucuronidase (beta-gluc.) and galactosidase were assayed in a group of Bilharzia haematobium patients and another group of healthy subjects (control group). The results for most of the determined enzymes revealed high activities as compared to the controls. The activity of acid phosphatase in male urine samples increased also, though not significantly. These elevated enzyme activities could be used to establish the diagnosis of schistosomiasis in patients whose urine contains no ova or when it is difficult to detect them. The results are discussed in the light of localization of each enzyme in the urinary tract as well as in other organs like the liver.

Selective elevation of urinary enzyme levels after extracorporeal shock wave lithotripsy.

Urinary enzyme testing has been used by many investigators to diagnose and monitor various types of renal injury. Three urinary enzymes, N-acetyl-beta-glucosaminidase, beta-galactosidase and gamma-glutamyl transferase were monitored in 17 patients before and after a single, unilateral extracorporeal shock wave lithotripsy treatment. Stones were in the renal pelvis or calices except for 1 treated in situ in the proximal ureter. Urine specimens were collected before extracorporeal shock wave lithotripsy and at 1, 3, 5, 7, 10, 14, 21 and 28 days after treatment. N-acetyl-beta-glucosaminidase and beta-galactosidase levels increased significantly after treatment (p less than 0.05). Gamma-glutamyl transferase levels increased after treatment but this was not statistically significant. All enzyme levels were highest on days 1 and 3 after lithotripsy and returned to baseline by day 28. Factors associated with post-treatment enzyme elevation included female sex, a lower pre-treatment creatinine clearance and stone size greater than 1 cm. These findings indicate that there is a transient selective increase in urinary enzyme excretion after extracorporeal shock wave lithotripsy.

Coordinately increased lysozymuria and lysosomal enzymuria induced by maleic acid.

During the acute renal tubular dysfunction of Fanconi syndrome and type 2 renal tubular acidosis (FS/RTA2) induced by maleic acid in the unanesthetized dog, we observed: 30 minutes after the onset of FS/RTA2, the urinary excretion of lysosomal enzymes, N-acetyl-beta-glucosaminidase (NAG), beta-glucuronidase (beta-gluc) and beta-galactosidase (beta-galac), increased simultaneously with the anticipated increase in renal clearance of lysozyme; the severities of all these hyperenzymurias increased rapidly, progressively, and in parallel, all reaching a peak some 60 to 80 minutes after their onset; thereafter, while the FS/RTA2 continued undiminished in severity, the severity of the hyperenzymurias decreased rapidly, greatly, progressively, and in parallel; and sodium phosphate loading strikingly attenuated the FS/RTA2 and the hyperenzymurias. Thus, the maleic acid-induced FS/RTA2 is attended by an acute reversible-complex derangement in the renal tubular processing of proteins that: affects not only lysozyme which is normally filtered, but also NAG and other lysosomal enzymes, which are not; and is to some extent functionally separable from that of FS/RTA2. The findings suggest that the derangements in renal processing of lysozyme and lysosomal enzymes are linked, and that a phosphate-dependent metabolic abnormality in the proximal tubule can participate in the pathogenesis of both these derangements and the FS/RTA2.

Urinary endo-beta-galactosidase capable of depolymerizing polylactosaminoglycans.

Human urine was found to contain an endo-beta-galactosidase capable of depolymerizing sulfated and non-sulfated polylactosaminoglycans. Using 0.05 M sodium phosphate buffer, pH 7.0, this enzyme was not retained by DEAE-Sephadex A-50 or concanavalin A-Sepharose. The urinary endo-beta-galactosidase liberated a disaccharide with chromatographic mobility identical to 6-O-sulfo-GlcNAc beta 1----3Gal as one of the major products from keratan sulfates isolated from whale nasal cartilage, bovine cornea, and human costal cartilage. It also liberated GlcNAc beta 1----3 Gal as one of the major oligosaccharides from erythroglycan. The oligosaccharide profiles produced from various keratan sulfates and erythroglycan by the action of urinary endo-beta-galactosidase are quite similar to those produced by Escherichia freundii endo-beta-galactosidase (Nakagawa, H., Yamada, T., Chien, J.-L., Gardas, A., Kitamikado, M., Li, S.-C., and Li, Y.-T. (1980) J. Biol. Chem. 255, 5955-5959). The presence of urinary endo-beta-galactosidase indicates the existence of a new catabolic pathway for polylactosaminoglycans. This pathway involves the cleavage of internal beta-galactosyl linkages of the glycan chain.

[Normal values of the urinary activity of 4 enzymes]. / I valori normali dell'attività urinaria di 4 enzimi.

The urinary activity of 4 enzymes (NAG, GLU, GAL, GRS) was investigated in 105 healthy subjects in order to evaluate the variability of standard levels and establish the degree of such variations in relation to sex, age, weight and height. The results obtained demonstrate that these enzymurias do vary in relation to the parameters examined. Age and sex produced the most pronounced variations though height and body weight also appeared to have some influence. The study of variations in standard levels is of value in the interpretation of pathological enzymurias.

A genetic component in human lysosomal enzyme excretion.

Acid hydrolases are present in normal human urine in appreciable amounts. Their source appears to be lysosomes released by kidney proximal tubule epithelial cells. For a given lysosomal enzyme the total amount excreted is the product of two parameters, a general one describing the rate of lysosome secretion and a specific one describing the relative concentration of that enzyme in lysosomes. There is considerable population variation in both parameters. Studies of beta-glucuronidase, beta-galactosidase, beta-hexosaminidase, and alpha-galactosidase in monozygotic and dizygotic twins show that an appreciable part of this variation is genetic in origin. This appears to be true for both total enzyme excretion and lysosome composition. Although it was not possible to test directly whether this is also true for the rate of lysosome secretion, the fact that the two former parameters are both heritable strongly suggests that the rate of lysosome excretion is also a heritable trait. Taken together with previous data, the results suggest polygenic control of these biochemical traits. It is particularly significant that beta-glucuronidase excretion in normal individuals is a heritable trait since the excretion of this enzyme has frequently been used as a measure of normal and pathological physiological changes.

The diagnostic value of urinary enzyme measurements in hypertension.

N-Acetyl-beta-D-glucosaminidase (NAG), beta-D-galactosidase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) were assayed in the urine of 100 normal and 112 hypertensive subjects. Age-related urinary activities for these enzymes in the normotensive control subjects are presented. A new procedure for the assay of urinary ALP using 2-methoxy-4-(2'-nitrovinyl)phenyl (MNP) phosphate is described. Thirty-five of the hypertensive patients were considered to have primary renal disease. The urinary activity of NAG was increased in 27 (77%) of these patients and the detection of primary renal disease was not enhanced by measurements of the other urinary enzymes. Testing the urine both for NAG activity and protein, led to the detection of 91% of these patients. The assay procedures described are simple to perform and can be carried out in outpatient clinics. The measurement of urinary NAG activity is a cheap and reliable method for detecting renal disease in hypertensive patients but maximum diagnostic yield is achieved when proteinuria is determined as well.

Colorimetric assays for N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase in human urine using newly-developed omega-nitrostyryl substrates.

(1) The synthesis of 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (MNP-GlcNAc) and 2-methoxy-4-(2'nitrovinyl)-phenyl beta-D-galactopyranoside (MNP-Gal) as substrates for the assay of NAG and beta-d-galactosidase are described. (2) beta-Glycosidase activities were determined in random urine samples from normal males and females aged between 12 and 87 years and patients with renal disease. (3) Both the MNP N-acetylglucosaminide and MNP galactoside were stable indefinitely, if stored in the solid state at 4 degree C in the dark. (4) The effect of urinary inhibitors was minimized by diluting the urine in the assay procedure. A simple assay procedure has been developed using MNP substrates. A good correlation was found with established assays using 4-methylumbelliferyl and p-nitrophenyl glycosides. (5) The assay was readily automated and a good correlation was found between the automated and manual methods. (6) The assay of urinary glycosidase activity with MNP substrates is simple to perform and has been used successfully in the clinical chemistry laboratory.

Aggregation properties of beta-galactosidase of human urine and degradation of its natural substrates by a purified preparation of the enzyme.

Acid beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was purified to near homogeneity from normal human urine by two affinity chromatography steps. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the major protein band had an apparent molecular weight of 59000, thus being 5000 daltons smaller than the protein purified from human liver. Upon gel filtration on Sephadex G-150 column the purified enzyme had an apparent molecular weight of 70000 of pH 7.0. At pH 4.0 partial aggregation to a dimer of an apparent molecular weight of 150000 was found. Addition of 0.1 M galactose caused at pH 3.5, but not at pH 4.0 and 7.0, an increased formation of multimeric beta-galactosidase which eluted with the void volume of the column. Crude beta-galactosidase from human urine showed a higher aggregation tendency than the purified enzyme. None of the conditions produced an enzyme species of an apparent molecular weight of less than 40000. pH-activity profiles were measured against p-nitrophenyl-beta-D-galactoside, 3H-labelled GM1-ganglioside, [3H]keratan sulfate and the pentasaccharide O-beta-(1 leads to 4)-[6-3H]galactopyranosyl-O-beta-(1 leads to 2)-2-deoxy-2-acetamidoglycopyranosyl-O-alpha-(1 leads to 6)-mannopyranosyl-O-beta-(1 leads to 4)-mannopyranosyl-2-deoxy-2-acetamidoglucopyranoside. While p-nitrophenyl-beta-D-galactopyranoside and GM1-ganglioside were optimally hydrolyzed at pH 4.0, keratan sulfate and the pentasaccharide were optimally degraded at pH 4.3 and pH 5.0, respectively. With the chromogenic substrate and with GM1-ganglioside Km values of 0.33 mM were calculated. At pH 3.5 the hydrolysis of the synthetic substrate did not follow Michaelis-Menten kinetics. Two enzyme species appeared with Km values of 0.006 mM and 3.2 mM, respectively. The affinity of beta-galactosidase for [3H]keratan sulfate and the 3H-labelled pentasaccharide was at least one order of magnitude lower than for the amphiphilic substrates. Keratan sulfate and GM1-ganglioside did not act as competitive inhibitors of p-nitrophenyl-beta-galactosidase at the concentration tested. These findings could be explained by the existence of different binding sites for the substrates used.

Fabry's disease and cornea verticillata. A report of 3 cases.

Fabry's disease is a rare familial disorder of glycolipid metabolism which is caused by a deficiency of a lysosomal enzyme alpha-galactosidase. A Finnish family is described in which cornea verticillata was found in the father and 2 daughters. In all cases, there were symptoms suggesting Fabry's disease: febrile episodes the origin of which was not clear, limb pains and, in the case of the father, 20 years of proteinuria with elevated ESR, and hemiplegia and aphasia following a cerebral thrombosis at the age of 43. The diagnosis was confirmed by demonstration of an alpha-galactosidase deficit in the serum and urine of all patients. Deficiency of this enzyme leads to abnormally high urinary tri- and dihexosyl ceramide levels, and this was observed in the father and the elder daughter. At the age of 12, the daughter had loss of vision in her right eye as a result of occlusion of the central retinal artery. Electron microscopic (EM) examination of the father's dermal angioma suggested Fabry's disease. Computerized cranial tomography of the father revealed not only the cerebrovascular condition but also a disease affecting the white matter of the brain.

Urinary acidic glycohydrolases as an index of kidney damage in juvenile diabetes mellitus.

Two lysosomal glycohydrolases, beta-galactosidase and beta-N-hexosaminidase which have been associated with kidney disease were measured in the urine of 110 youngsters with juvenile diabetes mellitus. The mean enzyme excretions in the diabetic group were intermediate between those of normal youngsters and those with active renal disease. Three youngsters with known kidney disease had elevations comparable to others in the diabetic group but no direct correlation could be shown between enzyme elevations and proteinuria or Addis count abnormalities. Positive correlations were seen between enzyme levels and indices of metabolic balance including blood sugar, cholesterol and triglycerides but not with urine sugar or ketones. Duration and estimated stage and control of diabetes also correlated with the urinary enzymes. These preliminary studies are consistent with the possibility that the excretion of these enzymes reflects the ongoing renal damage which occurs in most juvenile diabetics.

Renal excretion of proteins and enzymes in workers exposed to cadmium.

The proteinuria rate and the relative clearances of beta 2-microglobulin, orosomucoid, albumin, transferrin and IgG were measured in forty-two workers exposed to cadmium and in seventy-seven control workers. A tubular type proteinuria with an increased excretion of beta 2-microglobulin and often also a glomerular type proteinuria with an increased excretion of orosomucoid, albumin, transferrin and IgG were observed mainly in workers exposed to cadmium for more than 25 years and whose cadmium concentration in blood exceeded 1 microgram Cd/100 ml and that in urine 10 microgram Cd/g creatinine. The glomerular dysfunction was also suggested by an increased plasma level of beta 2-microglobulin and creatinine. Both tubular and glomerular impairments occurred with the same prevalence and were not necessarily associated. The increased release of beta-galactosidase by the kidney suggested that cadmium can damage some epithelial cells.