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.

Iron overload and urinary lysosomal enzyme levels in beta-thalassaemia major.

UNLABELLED: The urinary levels of the lysosomal enzymes N-acetyl-beta-D-glucosaminidase (NAG) (EC 3.2.1.52) and alpha-mannosidase (EC 3.2.1.24) were evaluated in patients with beta-thalassaemia major and normal control subjects. Two groups of patients with different degrees of iron overload, as judged by their serum ferritin levels, were investigated. Renal disease was not present in any of the patients. A statistically significant increase in the levels of NAG was observed in the high ferritin (> 3,000 mg/dl) group compared to the low ferritin (< 3,000 mg/dl) and the control groups. No difference was observed in the urinary alpha-mannosidase levels between the groups examined. The finding of increased NAG levels in the patients with the increased iron load suggests that kidney lysosomes are a target of iron toxicity. The different behaviour of the two lysosomal enzymes may reflect the intra- and inter-lysosomal heterogeneity in kidney. CONCLUSION: Iron overload resulted in increased urinary levels of the lysosomal enzyme NAG which has been proposed as an early marker of kidney damage. Reduction of iron load, achieved by regular desferrioxamine infusion, resulted in normalisation of the urinary enzyme levels. Thus kidney lysosomes appear to be a target and possibly a mediator of iron toxicity in this tissue.

Lysosomal enzymuria in preeclampsia.

We hypothesize that the preeclamptic patient has proximal tubule epithelial injury, which leads to the release of lysosomal enzymes, and that the excretion of these enzymes might serve as a diagnostic or predictive marker in preeclamptic women. The study group consisted of 14 women with preeclampsia (10 severe and 4 mild, as defined by The American College of Obstetricians and Gynecologists criteria) and 28 normotensive controls with singleton pregnancies at 27 to 41 weeks. There were no significant differences between the two groups for gestational age, maternal age, or race. Maternal serum and urine specimens were prospectively obtained and analyzed for beta-glucuronidase, beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, and alpha-mannosidase using fluorometric assays. Median serum and urine activities and fractional excretions of each of the five hydrolases were compared between the two study groups using the Mann-Whitney two-sample rank test. The serum enzyme activities of beta-hexosaminidase (P = 0.002), alpha-galactosidase (P = 0.0001), and alpha-mannosidase (P = 0.02) were significantly lower in preeclamptic patients than in controls. The urine enzyme activities of beta-glucuronidase (P = 0.001), alpha-galactosidase (P = 0.002), beta-galactosidase (P 0.0003), and alpha-mannosidase (P = 0.003) were significantly higher in the preeclamptic patients. The fractional enzyme excretions of all five lysosomal hydrolases were higher in preeclamptic patients than in controls with P < or = 0.0003 for each enzyme. Preeclampsia is associated with a significant decrease in serum activities of three of the five hydrolases studied, a significant increase in urine enzyme activities in four of the five hydrolases studied, and a significant increase in the fractional excretion of all five lysosomal hydrolases.

Purification and properties of human urinary beta-D-mannosidase.

Two forms of the lysosomal enzyme beta-mannosidase were identified and purified from human urine. The purification strategy employed allowed sufficient quantities of both forms to be obtained for subunit analysis and for further characterizations. The two beta-mannosidases were identified as beta-mannosidase B and A, in order of their elution from an ion-exchange column. In all samples examined, the A form was predominant, and the B/A ratio was consistently 0.14. The two forms displayed the same optimum pH (i.e., 4.3) and both were retained by a Concanavalin-A Sepharose column, but showed different isoelectric points, molecular masses and subunit compositions. Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of pure beta-mannosidases B and A suggest that active protein B (160 kDa) consists of three subunits, one 75 kDa and two 49 kDa subunits. Protein A is smaller and appears to be composed of three subunits of 75 kDa, 49 kDa and 37 kDa. Two forms of beta-mannosidase, exhibiting a chromatographic behaviour comparable to the urinary forms, were also detected in human kidney. Nevertheless, in this tissue their relative distribution was different, the B/A ratio being 19.

Tissue and serum swainsonine concentrations in sheep ingesting Astragalus lentiginosus (locoweed).

Locoweed intoxication or locoism results when animals continuously graze certain plants of the general Astragalus or Oxytropis. The locoweed toxin, swainsonine, is water soluble and is rapidly absorbed and eliminated. The purpose of this study was to determine the distribution of swainsonine in tissues of sheep eating locoweed and to determine if the tissue swainsonine concentrations change with continued locoweed ingestion. Fifteen cross-breed whethers were divided into 3 groups of 5 each and fed alfalfa pellets (Group 1) or alfalfa pellets with 10% Astragalus lentiginosus for 13 d (Group 2) or for 21 d (Group 3). After the feeding periods, the animals were slaughtered and tissues were collected, frozen and later analyzed for swainsonine using an in vitro, alpha-mannosidase inhibition assay. Significant alpha-mannosidase inhibitory activity (interpreted as ng/ml of swainsonine) was detected in whole blood, skeletal muscle, brain, kidney, liver, thyroid and urine. The swainsonine concentrations in tissues were significantly correlated with daily swainsonine intake (r = 0.58 to 0.96). With the exception of kidney, longer exposure did not result in significant increases in the swainsonine concentrations in blood, muscle, brain, liver or thyroid. Liver had the highest swainsonine concentrations with 3049 +/- 1952 and 3947 +/- 457 ng/ml (mean +/- SD) in Groups 2 and 3 respectively. Swainsonine concentrations varied widely within the groups suggesting individual animal variability in swainsonine absorption, metabolism and excretion. These findings suggest that swainsonine is present in tissues of animals eating locoweed and that in most tissues the amount was directly correlated to the swainsonine dose ingested, but not to the length of exposure.

Altered urinary excretion of lysosomal hydrolases in pregnancy.

Creatinine concentrations and the activities of five lysosomal hydrolases were measured in the serum and urine of 14 healthy nonpregnant control women and 19 healthy pregnant women. Fractional enzyme excretion (FEE) values for beta-glucuronidase, beta-hexosaminidase, alpha-galactosidase, beta-galactosidase, and alpha-mannosidase were calculated and compared between the two groups of subjects. Fractional enzyme excretion was calculated as the ratio of enzyme clearance to creatinine clearance. The FEE values for beta-galactosidase and alpha-mannosidase between the nonpregnant and pregnant populations were not statistically different; however, relative to the nonpregnant control group, the median FEE values for beta-glucuronidase (P < 0.03), beta-hexosaminidase (P < 0.06), and alpha-galactosidase (P < 0.02) were decreased approximately 1.5-, 1.8-, and 2.7-fold, respectively, in the pregnant population. The median urinary beta-galactosidase activity for the pregnant population, when expressed on the basis of creatinine, was twofold higher than that of the control group (P < 0.0005). These data indicate that with pregnancy there are marked changes in the urinary excretion of selected lysosomal enzymes, particularly alpha-galactosidase and beta-glucuronidase. When the molecular weights of these five hydrolases were compared between kidney homogenate and control urine, a correlation of 0.96 was observed, while the correlation between control serum and control urine was 0.69. This suggests that the FEE value differences between the pregnant and control groups are most likely due to changes in tubule cell metabolism, either decreased secretion or increased reabsorption. These biochemical changes may provide a means of assessing changes in renal function during pregnancy.

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)

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.

A human lysosomal alpha-mannosidase specific for the core of complex glycans.

A novel lysosomal alpha-mannosidase, with unique substrate specificity, has been partially purified from human spleen by chromatography through concanavalin A-Sepharose, DEAE-Sephadex, and Sephacryl S-300. This enzyme can catalyze the hydrolysis of only 1 mannose residue, that which is alpha(1----6)-linked to the beta-linked mannose in the core of N-linked glycans, as found in the oligosaccharides Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc and Man alpha(1----6)Man beta(1----4) GlcNAc. The newly described alpha-mannosidase does not catalyze the hydrolysis of mannose residues outside of the core, even if they are alpha(1----6)-linked, and is not active on the other alpha-linked mannose in the core, which is (1----3)-linked. The narrow specificity of the novel mannosidase contrasts sharply with that of the major lysosomal alpha-mannosidase, which is able to catalyze the degradation of oligosaccharides containing diverse linkage and branching patterns of the mannose residues. Importantly, although the major mannosidase readily catalyzes the hydrolysis of the core alpha(1----3)-linked mannose, it is poorly active towards the alpha(1----6)-linked mannose, i.e. the very same mannose residue for which the newly characterized mannosidase is specific. The novel enzyme is further differentiated from the major lysosomal alpha-mannosidase by its inability to catalyze the efficient hydrolysis of the synthetic substrate p-nitrophenyl alpha-mannoside, and by the strong stimulation of its activity by Co2+ and Zn2+. Similarly to the major mannosidase, it is strongly inhibited by swainsonine and 1,4-dideoxy-1,4-imino-D-mannitol, but not by deoxymannojirimycin. The presence of this novel alpha-mannosidase activity in human tissues provides the best explanation, to date, for the structures of the oligosaccharides stored in human alpha-mannosidosis. In this condition the major lysosomal alpha-mannosidase activity is severely deficient, but apparently the alpha(1----6)-mannosidase is unaffected, so that the oligosaccharide structures reflect the unique specificity of this enzyme.

Quantitative analysis of disaccharides in the urine of beta-mannosidosis patients.

Until recently, there have not been any confirmed reports of beta-mannosidase deficiency in man. We have now analysed urine from two patients with confirmed beta-mannosidase deficiency and have found Man-beta(1-4)GlcNAc concentrations of 65 and 73 mg/mmol creatinine. These levels are at least 12 times higher than those found in patients with other lysosomal storage diseases. The method we report for this analysis requires only 0.1 ml of urine and uses a gas chromatograph, area ratio, calibration curve for the quantitative analysis.

beta-Mannosidase in human serum and urine. A comparative study.

All serum and urine beta-mannosidase activities are adsorbed on a DEAE-Trisacryl column at pH 6. Only one form is eluted with a NaCl linear gradient. The two enzymes, isolated from either serum or urine exhibit similar properties. Slight differences are only observed in thermostability and molecular weight.

[Alpha-mannosidase activity of different human biological fluids]. / Activité alpha-mannosidasique de quelques liquides biologiques chez l'Hommone

Two distinct alpha-mannosidases have been found in biological fluids by investigating the effect of pH on activity, the effect of Co2+ and Zn2+ and by determining the Michaelis constant. The two enzymes are designated as S, predominating in serum (optimum pH about 5.6) and U, predominating in urine (optimum pH about 4.2). In cerebro-spinal fluid, the amount of alpha-mannosidase S is almost the same as that of alpha-mannosidase U. Alpha-mannosidase S activity in sera is significantly higher in children than in adults.