Giancarlo Goi

Behaviour of some lysosomal enzymes in the plasma of insulin dependent diabetic patients during artificial pancreas treatment

The plasma levels of three lysosomal enzymes, beta-D-N-acetylglucosaminidase, beta-D-glucuronidase, and alpha-L-fucosidase, were fluorimetrically determined in seven insulin-dependent diabetic patients one day before, one day after, and during a two-day treatment with the artificial pancreas, at 4 to 5 h intervals. A statistically significant decrease of the plasma level of each enzyme was observed during artificial pancreas treatment. The extent of decrease was 30 to 35% for beta-N-acetylglucosaminidase, 35 to 40% for beta-D-glucuronidase, and 20 to 25% for alpha-L-fucosidase. The decrease occurred earlier (at the first day of treatment) for beta-D-N-acetylglucosaminidase, and later (at the second day of treatment, and lasting to the first day after treatment) for the other two enzymes. These results suggest a direct connection between the lysosomal apparatus and insulin-controlled metabolic pathways, and a potential role for lysosomal enzymes as indicators of the metabolic compensation in diabetes.

Serum enzymes of lysosomal origin as indicators of the metabolic control in diabetes: comparison with glycated hemoglobin and albumin.

SummarySeveral lysosomal enzymes (β-N-D-acetylglucosaminidase, β-D-glucoronidase, α-D-galactosidase, β-D-galactosidase, α-L-fucosidase, α-D-glucosidase, α-D-mannosidase, β-D-glucosidase), glycated albumin and glycated hemoglobin (HbA1c) were determined in the serum of 81 insulin-dependent diabetics with different degrees of metabolic control (optimal, 21 patients; good, 39 patients; poor, 21 patients) and without signs of complications, and in 42 control subjects. All parameters examined increased in serum in inverse proportion to the degree of metabolic control. A highly significant correlation (p<0.01) was found between lysosomal enzymes and both glycated albumin and HbA1c. All parameters correlated with hyperglycemia, glycated albumin having the highest γ-value (0.586) and lysosomal enzymes the lowest one. Unlike glycated albumin and HbA1c, serum levels of lysosomal enzymes in patients with optimal metabolic control were undistinguishable or even lower than those of controls. A 2-month longitudinal monitoring of a patient who was hospitalized in conditions of poor metabolic control and adequately treated, proved that lysosomal enzymes diminished in serum parallel to glycated albumin and HbA1c in relation to improvement of the metabolic situation. The conclusion is drawn that serum lysosomal enzymes are good indicators of the metabolic control of diabetic patients probably reflecting the overall metabolic state connected with insulin action rather than hyperglycemia.

Behaviour of several enzymes of lysosomal origin in human plasma during pregnancy

The following enzymes of lysosomal origin were fluorimetrically determined in maternal plasma from the second to the ninth month of pregnancy at 1-mth intervals: beta-D-N-acetylglucosaminidase (EC 3.2.1.30), beta-D-glucuronidase (EC 3.2.1.31), beta-D-glucosidase (EC 3.2.1.21), beta-D-galactosidase (EC 3.2.1.22), alpha-D-galactosidase (EC 3.2.1.23), alpha-L-fucosidase (EC 3.2.1.51) and alpha-D-mannosidase (EC 3.2.1.24) (pH 4.0). As reference microsomal alpha-D-mannosidase (pH 5.7) was also studied. Thirty-eight healthy women, aged 18-37 yr, who had a normal pregnancy followed by normal parturition, were studied. All enzymes, with the only exception of beta-D-galactosidase, showed a progressive and statistically significant increase of activity throughout pregnancy. At the end of pregnancy, the increase ranged from a maximum of 5.6-fold for beta-D-N-acetylglucosaminidase to a minimum of 0.55-fold for alpha-D-mannosidase, pH 5.7. In the case of beta-D-N-acetylglucosaminidase, the level at the fifth month of pregnancy was significantly higher than that at the third month, and from the sixth to the ninth month each level significantly differed from that of the month immediately preceding.

Membrane anchoring and surface distribution of glycohydrolases of human erythrocyte membranes.

The membrane anchoring of the following glycohydrolases of human erythrocyte plasma membranes was investigated: α‐ and β‐D‐glucosidase, α‐ and β‐D‐galactosidase, β‐D‐glucuronidase, N‐acetyl‐β‐D‐glucosaminidase, α‐D‐mannosidase, and α‐L‐fucosidase. Optimized fluorimetric methods for the assay of these enzymes were set up. Treatment of the ghost preparation with 1.0 mol/l (optimal concentration) NaCl caused release ranging from 4.2% of α‐D‐glucosidase to 70% of β‐D‐galactosidase; treatment with 0.4% (optimal concentration) Triton X‐100 liberated 5.1% of β‐D‐galactosidase to 89% of α‐D‐glucosidase; treatment with 1.75% (optimal concentration) octylglucoside yielded solubilization from 6.3% of β‐D‐galactosidase to 85% of α‐D‐glucosidase. Treatment with phosphoinositide‐specific phospholipase C caused no liberation of any of the studied glycohydrolases. These results are consistent with the notion that the above glycohydrolases are differently anchored or associated with the erythrocyte plasma membrane, and provide the methodological basis for inspecting the occurrence of these enzymes in different membrane microdomains.

Preparation of a Stable Liquid Material for Calibration and Quality Control for Lysosomal Enzymes in Plasma. Assay of Enzymes of Lysosomal Origin in Plasma, I.

Several lysosomal enzymes present in human plasma (N-acetyl-beta-glucosaminidase, beta-glucuronidase, beta-galactosidase, alpha-galactosidase, alpha-L-fucosidase, alpha-mannosidase, beta-glucosidase) were maintained in a fully active state for at least 8 months by the addition of ethylene glycol (300 milligrams final concentration) to freshly prepared plasma and storage at -20 degrees C. Pools of human plasma from healthy humans, stabilized and stored as above, and containing a low, medium or high content of the above enzymes, were used to establish the analytical imprecision (within-run, day-to-day and total imprecision) of the fluorimetric assay. Ten replicates in ten different analytical series, covering a period of two months, were performed. The total imprecision (expressed as coefficient of variation) was in general lower than 10%; in a few cases, particularly plasma samples with a low enzyme content, the total imprecision was 18%. The isozymes A, B, I1, and I2 of N-acetyl-beta-glucosaminidase displayed the same stability upon storage as the unfractionated enzyme. It is concluded that pools of human plasma containing known amounts of lysosomal enzymes, stabilized by the addition of 300 micrograms ethylene glycol and stored at -20 degrees C, are suitable liquid materials for calibration and quality control for the assay of the same enzymes.

Isoflavone supplementation reduces DNA oxidative damage and increases O-β-N-acetyl-d-glucosaminidase activity in healthy women

Phenolic compounds are believed to boost the human antioxidant defense system and health; therefore, the aim of this research was to investigate the hypothesis that soy isoflavones (IFs) provide antioxidant protection in healthy women by evaluating DNA resistance to oxidative damage and O-β-N-acetyl-D-glucosaminidase (OGA) activity. An IF supplement (80 mg/d) was given to 9 postmenopausal women and 13 young women for 6 months and then stopped up to the 14th month. The women were allowed to consume their normal diet. Blood samples were collected at the beginning of the study after 2, 4, and 6 months and then at the 8th and 14th months. Plasma concentrations of genistein and daidzein, total antioxidant capacity, plasma vitamin status, markers of oxidative stress (red blood cell membrane fluidity, activity of the red blood cell cytosolic enzyme OGA and lymphocyte DNA susceptibility to oxidative stress), and serum lipid profile were analyzed. Analysis of variance for repeated measures was used for statistical analysis. Plasma concentrations of IFs rose significantly during the supplementation period, and plasma total antioxidant capacity increased in young women; membrane fluidity and OGA activity increased, and DNA oxidative damage decreased (P < .05) at 4 months, then returned to the basal level. There was a significant inverse correlation between DNA damage and plasma IF concentrations (P < .01). The results indicated a positive effect of IF supplementation on oxidative stress in women, thus suggesting that the healthful action ascribed to soy consumption may be partially related to the antioxidant potential of IFs.

Erythrocyte glycohydrolases in subjects with trisomy 21: could Down's syndrome be a model of accelerated ageing?

We studied some erythrocyte glycohydrolases, erythrocyte membrane fluidity, plasma hydroperoxides and total antioxidant defences in 23 Down syndrome (DS) individuals in comparison with healthy age-matched and elderly controls. With regard to erythrocyte plasma membrane fluidity, plasma hydroperoxides and total plasma oxidative defences, DS subjects resembled the age-matched controls more than the elderly ones. Membrane glycohydrolases in DS, however, presented a pattern partly similar to age-matched controls and partly to elderly controls. Concerning cytosol glycohydrolases, DS subjects had lower levels of hexosaminidase and N-acetyl-beta-D-glucosaminidase, the latter specific for the hydrolysis of GlcNAc residues O-linked to proteins. In general, erythrocyte membrane and cytosol glycohydrolases decreased during erythrocyte ageing in DS subjects and in all controls. The increased levels of the same enzymes in DS plasma might be attributed to an alteration of their release-uptake mechanisms between the two different compartments, on account of the higher plasma hydroperoxide levels. These findings indicate that erythrocyte ageing in DS differs partially from that of age-matched and elderly controls. In any case, the accelerated ageing seen in DS is no fully comparable to physiological ageing.

Plasma lysosomal glycohydrolases in a general population

In this study we evaluated the differences in plasma levels of some glycohydrolases of lysosomal origin that appear to be the most interesting for possible usefulness for diagnosis (N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase, alpha-D-galactosidase, beta-D-galactosidase, alpha-L-fucosidase and alpha-D-mannosidase) in a general population of 417 subjects, as related to age and sex and also to body mass and to some habits, such as smoking and consumption of alcohol. The enzymatic activities were assayed by fluorimetric techniques with 4-methylumbelliferyl-glycosides as substrates. Particular attention was given to some technical aspects. Enzymatic activity was preserved by addition of ethylene glycol and stable liquid material was employed for calibration purposes. Blood was sampled rigorously at the same time of day and all the samples were obtained within a short period of time to exclude effects of the circadian and circannual rhythms. beta-Glucuronidase levels were the most affected by sex and body mass. beta-D-Galactosidase was not affected by differences in age, sex, body mass or by smoking, but appeared to be the most sensitive to modification by alcohol consumption. The data in this report emphasize that, whenever changes or differences in the levels of lysosomal enzymes in body fluids are studied, it is essential to have a reference population rigorously correlated with the study population. When possible, repetitive measurements in the same subject could better indicate a clinical trend.

Stability of enzymes of lysosomal origin in human cerebrospinal fluid

The optimal assay conditions and the stability of the following enzymes of lysosomal origin in human cerebrospinal fluid (CSF) were studied: acid phosphatase, beta-D-N-acetylglucosaminidase, alpha-D-galactosidase, beta-D-galactosidase, alpha-D-glucosidase, beta-D-glucosidase, alpha-L-fucosidase, alpha-D-mannosidase, beta-D-glucuronidase. The microsomal alpha-D-mannosidase, pH 5.7, was used as a reference non-lysosomal glycohydrolase. All the examined enzymes, with the only exception of beta-D-glucuronidase, underwent a more or less rapid loss of activity upon CSF storage in the temperature range from 37 degrees C to -80 degrees C. Storage in liquid nitrogen (-196 degrees C) was the only condition in which full activity for all tested enzymes was maintained for at least 15 days. Addition of human serum albumin to CSF, immediately after withdrawal, had a double effect in favouring enzyme stabilization and causing enzyme activation in some cases, and enzyme inhibition in others. Using conditions warranting enzyme stability the fluorimetric methods for lysosomal enzymes determination in cerebrospinal fluid appear to be highly reproducible (CV less than 5%) and simple enough for routine use.

The esters of p-hydroxy-benzoate (parabens) inhibit the release of lysosomal enzymes by mitogen-stimulated peripheral human lymphocytes in culture

An in vitro test was set up to assess the release of lysosomal enzymes from cells and the effect on this process of the commonly used preservatives, parabens. Human peripheral lymphocytes, cultivated in vitro for 24 h in the presence or absence of phytohaemagglutinin (PHA; 5 mg/l), were used. After 1 day of incubation, PHA treatment caused an increased release (from 220 to 500%) of the lysosomal enzymes N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase, alpha-L-fucosidase and alpha-D-galactosidase. This enhancement was analytically reliable, and detectable with 1-5 micrograms of cell protein. Leakage of lactate dehydrogenase (LDH) underwent only a 20% increase on PHA treatment, indicating that the increased release of lysosomal enzymes was presumably due to secretion, not to cell damage. In PHA-stimulated lymphocytes, methyl-, ethyl-, propyl- and butyl-parabens caused a concentration-dependent diminution of the secretion of lysosomal enzymes. Butyl-paraben appeared to be the most potent inhibitor, causing a 45-50% inhibition at 0.06 mmol/l. With the other parabens, the inhibitory effect became statistically significant at about 0.25 mmol/l for alpha-L-fucosidase and alpha-D-galactosidase, and at 0.5 mmol/l for N-acetyl-beta-D-glucosaminidase and beta-D-glucuronidase. At 1 mmol/l inhibition was greater than 50% for all the enzymes and was more marked with the propyl derivative. Parabens did not influence the release of LDH, suggesting that they affected particularly the secretion of lysosomal enzymes. This supports the hypothesis that parabens are capable of affecting cellular function at concentrations which are likely to be reached in blood or tissues under conditions of common use.