Serum Exoglycosidases in Children and Adolescents With Harmful Alcohol Use.

OBJECTIVE: There is a lack of accurate alcohol-use biomarkers in children/adolescents due to a short drinking duration/rapid normalization of elevated markers. We checked if lysosomal exoglycosidases, elevated earlier in binge-drinking young adults, can be applicable in children/adolescents as markers of harmful alcohol use. METHODS: The serum activities (pKat/mL) of α-fucosidase (FUC), ß-galactosidase (GAL), ß-glucuronidase (GLU), ß-hexosaminidase (HEX; its HEX A and HEX B isoenzymes), and α-mannosidase (MAN) were determined in 20 healthy controls (C) and 25 children/adolescents with harmful alcohol use (intoxicated by alcohol at hospital admission -AI1 and on the next day -AI2). RESULTS: The serum HEX A and alanine aminotransferase (ALT) activity was significantly higher in the AI1 group than in the control. The activities of FUC, GAL, GLU, HEX B, and MAN were lower in the AI group. We found fair and poor accuracy, respectively, for increased enzymes HEX A and ALT. We found fair accuracy for decreased HEX B (AI1) and MAN (AI1), good accuracy for GLU (AI2), FUC (AI2), GAL (AI1, AI2), MAN (AI2), and excellent for FUC (AI1). Correlations were found: ALT with C-reactive protein (CRP), HEX A with white blood cell (WBC) count, blood alcohol concentration with FUC, MAN and HEX B, and WBC with FUC. CONCLUSIONS: Decreased FUC, GLU, GAL, MAN values, and especially FUC (AI1) have the potential to be markers of harmful alcohol use in children/adolescents. The raised activity of HEX A and ALT points to the need for further research to check another inflammatory agent as potential alcohol marker in children and adolescents. Samples need to be collected before intravenous fluid therapy.

Glycosidases in porcine follicular fluid and their effect on zona pellucida-AWN 1 spermadhesin interaction.

Oligosaccharide moieties on the surface of the oocyte belong to the key molecules that direct the course of fertilization and are subjected to changes during oocyte maturation in the follicle. In our study, we focused on the activities of five glycosidases in the fluids from porcine secondary and preovulatory follicles (α-l-fucosidase, α-d-galactosidase, ß-d-galactosidase, ß-D-N-acetylhexosaminidase, and α-d-mannosidase). All of them were detected active at neutral and acidic pH. However, changes in their activities associated with follicle development were observed only in the case of α-d-mannosidase, which was increased (P < 0.001), and ß-d-galactosidase, which was decreased (P < 0.001) at neutral pH, and of α-d-galactosidase and ß-N-acetylhexosaminidase, which were decreased (P < 0.0001) at the acidic pH. The comparison of glycosidases from follicular fluid and from blood plasma using red native electrophoresis revealed that most of the glycosidases are present in more than one isoenzyme form; some of them were detected mainly in the follicular fluid. Finally, we tested the effect of glycosidases on the interaction between zona pellucida and AWN 1 spermadhesin (putative sperm receptor of zona pellucida) and demonstrated that the effect of both ß-d-galactosidase and to a lesser degree α-d-mannosidase led to a decrease in this interaction. We can hypothesize that these two glycosidases modulate the amount of zona pellucida oligosaccharide moieties and/or their structures for an optimal sperm binding in pigs.

Reference intervals of α-glycosidase, ß-glycosidase, and α-galactosidase in dried blood spot in a Turkish newborn population.

Inherited lysosomal storage diseases (LSDs) are rare, and diagnosis is often delayed for 7-10 years. Since the therapies have become available for a limited number of LSDs, (Fabry, Gaucher, Pompe, and MPS-1), early diagnosis of treatable LSDs can be lifesaving or ameliorating and allows timely treatment before irreversible damage occurs. Recently, the use of dried blood spot test (DBS) for newborn screening of LSDs has been proposed for newborn screening tests. They are noninvasive, sensitive, and specific assays with the further advantage of a fast turnaround time compared to measurement in leukocyte and/or fibroblast culture. We aimed to determine the reference intervals for lysosomal enzyme activities of newborn babies in our population and to investigate the effect of gestational week on enzyme activity. One hundred thirty healthy newborn babies (70 girls, 60 boys) were included into the study. α-Glycosidase, ß-glycosidase, and α-galactosidase activities in DBS samples of newborns were determined fluorometrically. Reference intervals were calculated using Dixon's rule and percentiles of 2.5-97.5. Cutoff limits (5 %) for α-glycosidase, ß-glycosidase, and α-galactosidase activities were 0.57, 0.92, and 2.18, respectively. α-Galactosidase activity was higher in girls compared to boys (p < 0.05). Interestingly, α-glycosidase and ß-glycosidase activities of newborns who were delivered before 38 weeks were significantly lower than those who were delivered at 39-40 weeks. Conclusion It is of utmost importance to define the reference intervals for lysosomal enzyme activities as well as cutoff limits for newborn babies with regard to gestational age and sex. More studies to clarify the reason for the change in enzyme activity by gestational week will be required.

Analysis of haptoglobin N-glycome alterations in inflammatory and malignant lung diseases by capillary electrophoresis.

A CE-based method was introduced to compare the N-glycosylation profile of haptoglobin in normal and pathologic conditions. To assess the biomarker potential of glycosylation changes in various lung diseases, haptoglobin was isolated from plasma samples of healthy, pneumonia, chronic obstructive pulmonary disease, and lung cancer patients by means of two haptoglobin-specific monoclonal antibodies. Haptoglobin N-glycans were then enzymatically released, fluorescently labeled, and profiled by CE. Disease-associated changes of core and antennary fucosylation were identified by targeted exoglycosidase digestions and their levels were compared in the different patient groups. Terms such as core- and arm-fucosylation degree, as well as branching degree, were introduced for easier characterization of the changes and statistical analysis was used to examine which structures were responsible for the observed differences. Increased level of α1-6 fucosylated tri-antennary glycans was found in all disease groups compared to the control. Elevated amounts of core- and arm-fucosylation on tetra-antennary glycans were detected in the lung cancer group compared to the chronic obstructive pulmonary disease group. A larger scale study is necessary to confirm and validate these preliminary findings in the glycosylation changes of haptoglobin, so could then be used as biomarkers in the diagnosis of malignant and inflammatory lung diseases.

Some heterocyclic thione derivatives exhibit anticoccidial activity by inhibiting glycosidases.

Coccidiosis is one of the most common parasitic diseases affecting many species of domestic animals. This disease has a major economic significance and the search for new compounds having anticoccidial activity is of great importance. In this article, different levels of protection from coccidian infection by Eimeria stiedae were developed in rabbits by treatment with compounds incorporating the skeleton of thiourea. These compounds include 4,5-diphenylimidazole-2-thione (1), 4,5-Diphenyl-1,2,4-triazole-3-thiol (2) and 5-(2-Hydroxyphenyl)-4-phenyl-1,2,4-triazole-3-thiol (3) compared to the anticoccidial drug toltrazuril as a reference compound. Compounds 1-3 inhibit coccidiosis-induced activity of α-glucosidase. The protection from coccidial infection by compound 1 was higher than that shown for compounds 2 and 3. These data suggest that diazole and triazole thione derivatives have a mimetic effect for anticoccidial drugs through their inhibition of glycosidases.

Using next-generation sequencing for the diagnosis of rare disorders: a family with retinitis pigmentosa and skeletal abnormalities.

Linkage analysis with subsequent candidate gene sequencing is typically used to diagnose novel inherited syndromes. It is now possible to expedite diagnosis through the sequencing of all coding regions of the genome (the exome) or full genomes. We sequenced the exomes of four members of a family presenting with spondylo-epiphyseal dysplasia and retinitis pigmentosa and identified a six-base-pair (6-bp) deletion in GNPTG, the gene implicated in mucolipidosis type IIIγ. The diagnosis was confirmed by biochemical studies and both broadens the mucolipidosis type III phenotype and demonstrates the clinical utility of next-generation sequencing to diagnose rare genetic diseases.

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.

The activity of lysosomal exoglycosidases in serum of alcohol-dependent men supplemented with borage oil enriched with vitamin E.

The aim of this study was to determine the activity of the lysosomal exoglycosidases: alpha-mannosidase (MAN), alpha-fucosidase (FUC), and beta-glucuronidase (GLUCUR) in serum of alcohol-dependent men supplemented and not supplemented with borage oil enriched with vitamin E. Serum was collected from eight social drinkers and 16 alcohol-dependent men after a drinking period. The activity of exoglycosidases and the concentration of protein in serum were determined. The increase in specific activity of MAN and GLUCUR was significant in serum of alcohol-dependent men both not supplemented and supplemented with borage oil enriched with vitamin E, in comparison with the specific activity in serum of social drinkers. In serum of alcohol-dependent men treated with borage oil enriched with vitamin E, specific activity of MAN and GLUCUR fluctuated in comparison with alcohol-dependent men not supplemented. Specific activity of FUC in serum of alcohol-dependent men both not supplemented and supplemented with borage oil enriched with vitamin E showed a tendency to increase, in comparison with social drinkers. Specific activity of FUC had a tendency to decrease in serum of alcohol-dependent men supplemented with borage oil enriched with vitamin E, in comparison with alcohol-dependent men not supplemented. Thus, supplementation of alcohol-dependent men after a long-lasting drinking period with borage oil and vitamin E did not change the rate of catabolism of the oligosaccharide chains of glycoconjugates, as evaluated by serum activity of exoglycosidases.

Mice lacking alpha/beta subunits of GlcNAc-1-phosphotransferase exhibit growth retardation, retinal degeneration, and secretory cell lesions.

PURPOSE: Mucolipidosis II and III (ML II; ML III) are lysosomal storage diseases characterized by a deficiency in GlcNAc-1-phosphotransferase. Patients with ML III have retinal disease, but in cases of the more clinically severe ML II, human ophthalmic studies are limited. In this study, retinal function and overall disease were assessed in mice lacking GNPTAB, the gene mutated in patients with ML II. METHODS: Mice deficient in GNPTAB were generated from Omnibank, a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones as part of a large-scale effort to knock out, phenotypically screen, and thereby validate pharmaceutically tractable genes for drug development. Routine diagnostics, expression analysis, histopathology, and ERG analyses were performed on mice lacking GNPTAB. In addition, measurements of serum lysosomal enzymes were performed. RESULTS: Severe retinal degeneration was observed in mice deficient in GNPTAB. Heterozygous mice were phenotypically normal and in situ hybridization showed expression across the neural retina. Compared to wild-type mice, the GNPTAB homozygous mice were smaller, had elevated levels of serum lysosomal enzymes, exhibited cartilage defects, and had cytoplasmic alterations in secretory cells of several exocrine glands. CONCLUSIONS: Mice deficient in GNPTAB exhibited severe retinal degeneration. Additional features observed in patients with ML II, a lysosomal storage disease, are also present in these mice. Understanding underlying mechanisms of this gene in the eye will increase its therapeutic potential for the treatment of retinal diseases.

Lysosomal leukocyte beta-D-glucuronidase during enzyme replacement therapy in Fabry disease.

OBJECTIVE: Fabry disease results from a deficiency in the activity of alpha-d-galactosidase A and subsequent accumulation of neutral glycosphingolipids in lysosomes. This study investigated whether lysosomal enzymes can indicate biochemical changes in the lysosomal apparatus induced by enzyme replacement therapy (ERT). DESIGN AND METHODS: Eight patients were monitored by clinical and biochemical tests and several lysosomal glycohydrolases were measured in plasma and leucocytes. RESULTS: Before starting ERT, beta-d-glucuronidase in leukocytes was markedly increased. After 1 month of therapy, enzyme levels dropped in all patients. In the patients who regularly followed the therapy, the enzyme levels remained stable for the next 20 months. In one patient who interrupted therapy for 2 months, the enzyme levels rose again. CONCLUSIONS: Lysosomal enzymes can be useful for monitoring biochemical changes in patients with Fabry disease receiving ERT. Though these findings refer to only a small number of patients, the correlation between beta-d-glucuronidase levels and ERT is interesting and might serve as a basis for further studies to define the potential of this enzyme in monitoring the effects of ERT in lysosomal storage disorders.

Low levels of occupational exposure to arsenic and antimony: effects on lysosomal glycohydrolase levels in plasma of exposed workers and in lymphocyte cultures.

BACKGROUND: Heavy metals have been shown to alter the mechanism and release of lysosomal enzymes. In the present study, the activities of lysosomal glycohydrolases were determined in order to evaluate the asymptomatic toxic effects of low levels of exposure to arsenic (As) and antimony (Sb) in art glass workers. METHODS: N-acetyl-beta-D-glucosaminidase (NAG), beta-D-glucuronidase (GCR), alpha- and beta-D-galactosidase, alpha-D-glucosidase, and alpha-D-mannosidase were determined by a fluorimetric assay in the plasma of 26 art glass workers. Lymphocytes cultured in the presence of different species of As and Sb served as an in vitro model for the study of the protective action of selenium and zinc. RESULTS: No significant difference in the plasma levels of the various enzymes was detected in art glass workers or control subjects. The in vitro experiments demonstrated that secretion of lysosomal glycohydrolases was increased by Sb (225%) and decreased by As (57%) at the same concentration of elements (200 microg/L). The addition of bivalent selenium to the culture neutralized the effects of both metals, while zinc chloride did not show any protective effect. CONCLUSIONS: As for the plasma glycohydrolases, no praecox signs of toxicity related to a low concentration of As and Sb was evident in art glass workers. This may be due to the antagonistic effects demonstrated by these two metals in vitro. Their different mechanism of action on release of glycohydrolases is being discussed.

Transglycosidase activity of chitotriosidase: improved enzymatic assay for the human macrophage chitinase.

Chitotriosidase is a chitinase that is massively expressed by lipid-laden tissue macrophages in man. Its enzymatic activity is markedly elevated in serum of patients suffering from lysosomal lipid storage disorders, sarcoidosis, thalassemia, and visceral Leishmaniasis. Monitoring of serum chitotriosidase activity in Gaucher disease patients during progression and therapeutic correction of their disease is useful to obtain insight in changes in body burden on pathological macrophages. However, accurate quantification of chitotriosidase levels by enzyme assay is complicated by apparent substrate inhibition, which prohibits the use of saturating substrate concentrations. We have therefore studied the catalytic features of chitotriosidase in more detail. It is demonstrated that the inhibition of enzyme activity at excess substrate concentration can be fully explained by transglycosylation of substrate molecules. The potential physiological consequences of the ability of chitotriosidase to hydrolyze as well as transglycosylate are discussed. The novel insight in transglycosidase activity of chitotriosidase has led to the design of a new substrate molecule, 4-methylumbelliferyl-(4-deoxy)chitobiose. With this substrate, which is no acceptor for transglycosylation, chitotriosidase shows normal Michaelis-Menten kinetics, resulting in major improvements in sensitivity and reproducibility of enzymatic activity measurements. The novel convenient chitotriosidase enzyme assay should facilitate the accurate monitoring of Gaucher disease patients receiving costly enzyme replacement therapy.

Poly(ADPR) polymerase-1 and poly(ADPR) glycohydrolase level and distribution in differentiating rat germinal cells.

Poly(ADP-ribose)polymerase (PARP-1) and poly(ADP-ribose)glycohydrolase (PARG) are responsible for the transient poly(ADP-ribosyl)ation of proteins in eukaryotic cells. This biochemical reaction plays an active role in DNA replication and repair, transcription, cell differentiation and death. The aim of this study was to investigate the levels and the sub-cellular distribution of such enzymes in rat germinal cells at different stages of differentiation, i.e. in primary spermatocytes and round spermatids, representing meiotic and post-meiotic cells, respectively. The determination of the level of PARP-1 mRNA and protein revealed its higher expression in primary spermatocytes, thus implying that PARP-1 is one of the meiotic genes whose expression is requested at the pachytene phase of the meiosis. We also demonstrated that rat germinal cells contain both the forms of PARG (i.e. of 110 and 60 kDa) so far described in somatic cells. In our experimental system, the large PARG was present and active mainly in the nuclear fraction of primary spermatocytes, whereas round spermatids showed a higher level of the 60 kDa PARG in the post-nuclear fraction. Collectively, our data show a different expression level of PARP-1 and a different endocellular distribution of PARG and suggest a role for the poly(ADP-ribose) turnover in distinct pathways in meiotic and post-meiotic germinal cells.

Graves' disease-associated changes in the serum lysosomal glycosidases activity and the glycosaminoglycan content.

BACKGROUND: This study was undertaken to elucidate the influence of Graves' hyperthyroidism upon the metabolism of proteoglycans (PGs), the extracellular matrix (ECM) components. We determined the serum activity of lysosomal hydrolases contributing to GAGs degradation (N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase, beta-D-galactosidase, alpha-D-mannosidase, beta-D-xylosidase and alpha-L-fucosidase). An effect of Graves' hyperthyroidism on total serum GAGs content was also analysed. METHODS: Blood samples were taken from 30 patients with newly diagnosed Graves' disease, prior to antithyroid treatment and after attainment of euthyroid state, as well as from 30 healthy individuals. RESULTS: The activity of all investigated enzymes involved in GAGs degradation was found markedly increased in blood serum of patients with hyperthyroidism, except for alpha-D-mannosidase, which was not significantly modified. Antithyroid treatment with thiamazole resulted in normalization of the lysosomal glycosidases activity, so they no longer differed from the healthy subjects. The total glycosaminoglycans content in blood serum of patients with newly diagnosed untreated Graves' disease significantly increased compared to control group. Following thiamazole therapy total serum amount of GAGs decreased significantly, but was still markedly increased as compared to serum of healthy individuals. CONCLUSIONS: The obtained results indicate that Graves' hyperthyroidism is associated with extracellular matrix components' alterations. Furthermore, we suggest that general increase of the serum lysosomal glycosidases activity and serum GAG concentration may both result from the same reason, i.e. excessive reactive oxygen species formation in the course of hyperthyroidism due to Graves' disease.

YKL-40 is elevated in cerebrospinal fluid from patients with purulent meningitis.

YKL-40, a member of the family 18 glycosyl hydrolases, is secreted by activated neutrophils and macrophages. It is a growth factor for connective tissue cells and a potent migration factor for endothelial cells and may function in inflammation and tissue remodeling. YKL-40 was determined in 134 cerebrospinal fluid (CSF) samples taken on admission from patients suspected of having meningitis (48 with purulent meningitis, 49 with lymphocytic meningitis, 5 with encephalitis, and 32 without evidence of meningitis). YKL-40 levels in CSF were significantly higher in patients with purulent meningitis (median, 663 microg/liter [range, 20 to 8,960]) and encephalitis (5,430 microg/liter [620 to 11,600]) than in patients with lymphocytic meningitis (137 microg/liter [41 to 1,865]) or patients without meningitis (167 microg/liter [24 to 630]) (Kruskal-Wallis and Dunn multiple comparison tests, P < 0.001). CSF YKL-40 levels were also determined for 26 patients with purulent meningitis having a repuncture, and patients who died (n = 5) had significantly higher YKL-40 levels than patients who survived (n = 21) (2,100 microg/liter [1,160 to 7,050] versus 885 microg/liter [192 to 15,400], respectively; Mann-Whitney test, P = 0.018). YKL-40 was most likely locally produced, since patients with infections of the central nervous system had CSF YKL-40 levels that were at least 10-fold higher than the corresponding levels in serum (2,033 microg/liter [470 to 11,600] versus 80 microg/liter [19 to 195]). The CSF neopterin level was the biochemical parameter in CSF and blood that correlated best with CSF YKL-40 levels, indicating that YKL-40 may be produced by activated macrophages within the central nervous system. In conclusion, high levels of YKL-40 in CSF are found in patients with purulent meningitis.

Plasma glycohydrolase levels in patients with type 1 diabetes at onset and in subjects undergoing an intravenous glucose tolerance test.

The effect of hyperglycemia and insulin deficiency on the plasma level of lysosomal glycohydrolases, namely N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase, alpha-D-galactosidase, and alpha-D-glucosidase, was investigated. Two patient groups were assessed: (1) 28 children with type 1 diabetes at onset (fasting blood glucose, 444+/-154 mg/100 mL; hemoglobin A1c, 11.9%+/-2.4%; symptom duration, 15.9+/-8 days; and absence of complications), (2) 14 adult subjects undergoing an intravenous glucose tolerance test (IVGTT), consisting of 8 non-obese subjects (body mass index, 26+/-0.04 kg/m2; fasting blood glucose, 82+/-13 mg/100 mL; blood insulin, 6+/-0.04 mU/L) and 6 obese subjects (fasting blood glucose, 97+/-3.5 mg/100 mL; blood insulin, 27+/-6 mU/L, with normal oral glucose tolerance test). Enzyme activity was determined with the fluorimetric method. The mean level of all evaluated enzymes was significantly increased in patients with type 1 diabetes at diagnosis compared with normal controls. Increased enzyme levels were also detected in the group of adults undergoing an IVGTT in whom hyperglycemia was accompanied by insulin resistance (ie, obese subjects). Glycohydrolase abnormalities appear to be related to insulin deficiency rather than hyperglycemia. Lysosomal apparatus abnormalities seem to be an inherent feature of diabetes that is present at disease onset. The possible role of insulin in the regulation of plasma glycohydrolase levels is discussed.

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes.

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ss-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ss-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ss-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ss-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ss-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes Beta-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and Beta-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10 percent heparin, 5 percent heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of Beta-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and Beta-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that Beta-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice

Alterations in the activity of several glycohydrolases in red blood cell membrane from type 2 diabetes mellitus patients.

The erythrocyte membrane in 71 patients with type 2 diabetes mellitus was assessed for glycohydrolase activity: N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase, alpha- and beta-D-galactosidase, alpha- and beta-D-glucosidase, alpha-D-mannosidase, and alpha-L-fucosidase. Only beta-D-glucuronidase, alpha-D-glucosidase, and beta-D-glucosidase showed markedly elevated levels with respect to the controls regardless of the presence of complications. Among the examined patients, those with good metabolic control (not yet submitted to any therapy) showed the same enzyme levels as the reference subjects, while the levels in patients with unsatisfactory metabolic control (treated with oral hypoglycemic and/or insulin) significantly differed from the control levels. For alpha-D-glucosidase and beta-glucosidase, a correlation with glycemia and the parameters of metabolic control was also evidenced. Alterations of beta-D-glucuronidase, alpha-D-glucosidase, and beta-D-glucosidase were also ascertained in the plasma of the same diabetic patients according to the literature; each enzyme correlated with the other, either in plasma or in the erythrocyte membrane. This study shows a correlation between plasma and erythrocyte membrane levels for these three enzymes. The strict parallelism of the glycohydrolases in the two different compartments provides a profile of these enzymes in the pathology of diabetes.