C. Tassi

Lysosomal hydrolases in cerebrospinal fluid from subjects with Parkinson's disease

Recent studies have shown a genetic association between glucocerebrosidase deficiencies and Parkinsons disease (PD). To further explore this issue the activity of β‐glucocerebrosidase and the activities of other lysosomal enzymes, α‐mannosidase, β‐mannosidase, β‐hexosaminidase, and β‐galactosidase have been evaluated in the cerebrospinal fluid (CSF) of PD patients. The activities of α‐mannosidase, β‐mannosidase, β‐glucocerebrosidase, and β‐hexosaminidase were substantially decreased in the CSF of PD patients, while levels of β‐galactosidase were essentially identical to controls. This study indicates that in PD several lysosomal hydrolases have decreased activities, further supporting a possible link between pathophysiological mechanisms underlying PD and lysosomal hydrolases.

Activity and isoenzyme profile of N-acetyl-β-d-glucosaminidase in urine from workers exposed to cadmium

The urinary excretion of N-acetyl-beta-D-glucosaminidase (U-NAG) and urinary Cadmium (U-Cd) concentration, a measure of the metal load in the body, were evaluated in 28 workers exposed to Cd, to determine the relation between the two parameters. In urine from 22 exposed workers with U-Cd<2 microg/g creatinine (Cr) there was no significant difference in U-NAG value (0.98+/-0.59 U/gCr) compared to non-exposed (0.73+/-0.48 U/gCr). In the six workers with 2 microg/gCr < or =U-Cd<10 microg/gCr the U-NAG (2.32+/-0.61 U/gCr) was statistically (P<0.05) higher than in other workers. In both the U-Cd intervals examined there were no altered values of beta2-microglobulin from urine of exposed workers compared to non-exposed (<0.30 mg/l). The U-NAG isoenzymes were separated by DEAE-cellulose chromatography from urine of non-exposed subjects and exposed workers. The U-NAG isoenzyme profile in urine of non-exposed subjects showed a high percentage (about 95%) of the A (acid) form, a much lower percentage (about 4.5%) of B (basic) form and a negligible percentage (about 0.5%) of I (intermediate) form. In the urine of 22 exposed workers with U-Cd<2 microg/gCr, the percentages of U-NAG isoenzymes were not different from non-exposed. In the urine of six workers with 2 microg/gCr< or =U-Cd<10 microg/gCr the percentage (8.34+/-0.91) of isoenzyme B (U-NAG-B), a marker of lesional enzymuria, was statistically increased (P<0.05) compared to non-exposed (4.42+/-0.56). These results suggest that adopting a biological limit for U-Cd equal to 10 microg/gCr might not be sufficiently protective. The increase in U-NAG-B content at 2 microg/gCr

β-Hexosaminidase, α-d-mannosidase, and β-mannosidase expression in serum from patients with carbohydrate-deficient glycoprotein syndrome type I

The activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, and of beta-D-mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrate, and the activity of alpha-D-mannosidase. Using DEAE-cellulose chromatography, a greater amount of hexosaminidase B than hexosaminidase A was detected in CDG serum. In CDG serum, hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of alpha-D-mannosidase and beta-D-mannosidase was identified in control and CDG sera. alpha-D-Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas beta-D-mannosidase isoenzyme was eluted in the same position.

Post operative surveillance of human hydatidosis: evaluation of immunodiagnostic tests

Summary We investigated the kinetics of antibodies detected by indirect hemagglutination (IHA), IgE Elisa and immunoelectrophoresis (IEP) in patients with hydatid disease operated on and continuously followed in the pre‐operative and post‐operative periods. In the pre‐operative phase the IgE Elisa test was found to be adequately sensitive (68.4%) compared with IHA (79%), with a ratio of IgE Elisa/IHA positivity of 87%, while IEP was positive in 55.3% of cases (IEP/IHA ratio = 70%). During post‐operative follow‐up IHA became negative late in patients who were cured (7 out of 11 were still positive after 4 yrs), whereas IEP and IgE Elisa became negative within 2 yrs of operation (apart from 1 patient with a persisting positive IgE Elisa 3 yrs later). However, IgE Elisa appeared clearly more sensitive in revealing postoperative recurrences (13 out of 13 patients had positive IgE Elisa, vs. 6 out of 13 IEP).

Lysosomal hydrolases in serum from human immunodeficiency virus-infected patients.

beta-Hexosaminidase isoenzymes were separated by DEAE-cellulose chromatography in the serum of 23 patients infected with human immunodeficiency virus at different stage of the disease. Forms corresponding to hexosaminidase B, I and A were present in pathological sera. There is an increase in the percentage of hexosaminidase I in pathological sera, that could be used as an additional marker to monitor the clinical stage of the disease. Furthermore, total activities of some lysosomal enzymes were determined in these sera. Activities of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, alpha-mannosidase and beta-mannosidase were significantly higher in the serum of patients at the C3 stage of disease than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate substrate, beta-glucuronidase and beta-galactosidase.

β-N-Acetylhexosaminidase in the urine, kidney and serum of bromobenzene-intoxicated mice

beta-N-Acetylhexosaminidase isoenzymes were separated from the kidney, serum and urine of normal mice and mice intoxicated with bromobenzene, using DEAE-cellulose chromatography. Both mouse serum and urine showed hexosaminidase profiles similar to the human counterparts with the presence of B (basic), I (intermediate) and A (acidic) isoenzymes. A notable feature was the presence of a high proportion of an intermediate form in mouse urine which is not always present in human urine. Hexosaminidase activity increased significantly in urine of mice intoxicated with bromobenzene. Its increase was time-dependent and due to kidney damage with a release in the urine of hexosaminidase A, I and, in higher proportion, B. No significant differences were observed in mouse kidney and serum profiles following intoxication with bromobenzene. The total activity of hexosaminidase, using 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside as substrate, did not increase in the serum of mice intoxicated with bromobenzene. Both hexosaminidase activity and the isoenzyme pattern in urine can be used as indicators of kidney damage by bromobenzene intoxication.

beta-hexosaminidase, alpha-D-mannosidase, and beta-mannosidase expression in serum from patients with carbohydrate-deficient glycoprotein syndrome type I.

The activity of beta-hexosaminidase, determined with 4-methylumbelliferyl-beta-N-acetylglucopyranoside substrate, and of beta-D-mannosidase was significantly higher in the serum of patients with carbohydrate-deficient glycoprotein (CDG) syndrome type IA (phosphomannomutase deficiency) than in controls. No significant differences were observed in the activity of beta-hexosaminidase, determined using 4-methylumbelliferyl-beta-N-acetylglucopyranoside-6-sulphate as substrate, and the activity of alpha-D-mannosidase. Using DEAE-cellulose chromatography, a greater amount of hexosaminidase B than hexosaminidase A was detected in CDG serum. In CDG serum, hexosaminidase A was eluted in a more basic position in the salt gradient. An isoenzyme of alpha-D-mannosidase and beta-D-mannosidase was identified in control and CDG sera. alpha-D-Mannosidase isoenzyme was eluted in a slightly more basic position in CDG serum than in control serum, whereas beta-D-mannosidase isoenzyme was eluted in the same position.

The behaviour of specific antibody classes in human hydatid disease

&NA; The behaviour of IgE antibodies was investigated by the micro‐ELISA method (IgE‐ELISA) in patients affected by hydatidosis or in patients who had already been operated on. The results were correlated with those obtained by IgG‐ELISA, IgM‐ELISA, IgA‐ELISA and IHA tests. In the preoperative phase the IgG‐ELISA method proved to be as sensitive (80%) as the IHA test (78.5%); the IgE‐ELISA method showed a good sensitivity (72.8%) with a positive rate for the IgE‐ELISA/IHA of 92.7%. The IgM‐ and IgA‐ELISA methods were of moderate sensitivity. The IgE‐ELISA proved to be much more suitable than the other methods for postoperative control, since the persistence of positivity 4 years after surgery suggests the involvement of a relapse.

Isoenzyme A and urinary N-acetyl-β-D-glucosaminidase activity in normal pregnancy.

Objectives: Urinary N-acetyl-β-d-glucosaminidase (NAG) activity has been found to increase during normal uncomplicated pregnancy and such behavior could limit the diagnostic value of this enzyme for detection of subclinical tubular injury. The aim of this study was to evaluate urinary NAG activity and isoenzyme A in normal pregnant women at 30th week of pregnancy and in healthy women, to discriminate between physiological and lesional enzymuria. Design and methods: Enzyme activities in first morning fasting urine samples from 20 nonpregnant control and 20 normal pregnant women at 30th gestational week were evaluated by fluorometric methods. Results: Both total and isoenzyme A activity was significantly higher ( p < 0.01) in urines of normal pregnant women compared with control urines, whereas ratio between these two parameters was significantly lower ( p < 0.001). Conclusions: The increase of urinary NAG activity during normal uncomplicated pregnancy appears to be characterized by a prevalent increase in isoenzyme A form, a finding associated with functional (not lesional) enzymuria. The fluorometric assays may represent a simple and rapid method to evaluate whether increase in urinary NAG activity represents a renal physiological adaptation during pregnancy.

Fluorimetric determination of activity and isoenzyme composition of N-acetyl-β-D-hexosaminidase in seminal plasma of fertile men and infertile patients with secretory azoospermia

Abstract Background: The activity and isoenzyme composition of N-acetyl-β-D-hexosaminidase (EC.3.2.1.52) in seminal plasma of fertile and infertile men have been evaluated. However, no data are available on the isoenzyme content in seminal plasma from patients with secretory azoospermia. Methods: The activity and isoenzyme composition of seminal plasma from 15 normozoospermic controls and 18 patients with secretory azoospermia were determined by fluorimetric methods. 4-Methylumbelliferil-2-acetamido-2-deoxy-β-D-glucopyranoside and 4-methylumbelliferil-2-acetamido-2-deoxy-β-D-glucopyranoside-6-sulfate were used as fluorigenic substrates. Receiver-operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic efficiency of the assays. Results: No significant difference was found in total enzyme activity between the two groups, while isoenzyme A activity was significantly lower (p=0.004) and the ratio between total enzyme activity and isoenzyme A activity was significantly higher (p=0.04) in azoospermic patients compared to controls. The diagnostic efficiency of these evaluations was low (≤75.7%). Conclusions: Our findings show that the isoenzyme composition of N-acetyl-β-D-hexosaminidase in seminal plasma from patients with secretory azoospermia is significantly different from controls, but this difference does not represent a useful marker of secretory azoospermia. The fluorimetric assays are simple and rapid methods for evaluating the isoenzyme composition. Clin Chem Lab Med 2006;44:843–7.