Plasma lysosomal glycohydrolases in a general population

ELSEVIER

Cliniea Chimiea Acta 247 (1996) 39-49

Plasma lysosomal glycohydrolases in a general population Adriana Lombardo *a, Chiara Bairati a, Giancarlo Goi a, Carla Roggi b, Laura Maccarini b, Donatella Bollini a, Alberto Burlina c aDepartment of Medical Chemistry and Biochemistry, Medical School, University of Milan, V. Saldini 50, Milan 20133, Italy bDepartment of Preventive Occupational and Community Medicine - - Hygiene Section, University of Pavia, Pavia, Italy eDepartment of Pediatrics, Medical School, University of Padua, Padua, Italy Received 6 June 1995; revision received 20 October 1995; accepted 4 November 1995

Abstract 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-/3-~giucosaminidase, /~-D-glucuronidase, a-D-galactosidase, /~-D-galactosidase, aL-fucosidase and ~t-~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-giycosides 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./~-Glucuronidase levels were the most affected by sex and body mass. fl-I>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.

Keywords: Plasma; Lysosomal glycohydrolases; General population * Corresponding author, Tel: +39-2-70645249; Fax: +39-2-2363584. 0009-8981/96/$15.00 © 1996 Elsevier Science B.V. All fights reserved SSDI 0009-8981 (95)06218-3

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A. Lombardo et al. / Clinica Chimica Acta 247 (1996) 39-49

1. Introduction

There has been increasing interest in measuring enzymes of lysosomal origin in plasma and serum since it was found that some lysosomal glycohydrolases are of diagnostic significance, not only for some rare syndromes caused by congenital lysosomal pathology [1,2], but also for some acquired diseases, one of which is diabetes [3-7]. Before determination of these enzymes can be introduced into routine clinical chemistry laboratories to profit from their diagnostic potential, several technical problems of their analysis needed to be considered. Some of these problems have been solved [8-11]; however, we still lack reliable epidemiological data about the distribution of the enzyme levels in a general population. In this study we set out to evaluate the difference in the level of some of these lysosomal enzymes that seemed to have the most interesting diagnostic potential: N-acetyl-fl-D-glucosaminidase (EC 3.2.1.30) (NAG), /~-Dglucuronidase (EC 3.2.1.31) (GCR), fl-D-galactosidase (EC 3.2.1.23) (flGAL), t~-D-galactosidase (EC 3.2.1.22) (t~GAL), o~-t-fucosidase (EC 3.2. 1.51) (FUC) and ct-D-mannosidase (EC 3.2. 1.24) (MAN), in a general population of 417 subjects according to sex, age and to the anthropometric characteristics of body mass and to some habits, such as smoking and alcohol consumption.

2. Materials and methods

2.1. Materials

Commercial chemicals were of analytical grade or of the highest purity available. The water routinely used was freshly redistilled in a glass apparatus; 4-methylumbelliferone (Flucka GmbH, Buchs, Switzerland) was crystallized three times from ethanol; 4-methylumbelliferyl-glycosides were purchased from Melford (Suffolk, UK). 2.2. Sample collection and storage

Plasma was prepared immediately after blood sampling by centrifuging, for 15 min at 3000 x g, blood (4.5 ml) treated with sodium citrate (0.5 ml of 0.11 mol/1 sodium citrate solution). Ethylene glycol was added (30%, v/v, final concentration) to the plasma samples immediately after collection [9]. The samples were then stored at -20°C until assayed.

A. Lombardo et al. / Clinica Chimica Acta 247 (1996) 39-49

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2.3. Subjects The population studied was a stratified sample for sex and decades of age (from 20 to 79 years) corresponding to the entire population living in the town of Rovescala in the province of Pavia. Of the 767 subjects invited, 417 (187 men and 230 women) took part in the study and gave informed consent for blood collection. The mean age was 52.0 ± 16.5 years. Subjects with diabetes mellitus or obvious liver pathology [3-7] and pregnant women [12] were excluded because in these situations the levels of some lysosomal enzymes are known to be greatly increased. Venous blood samples were drawn at 09:00 h during the months of May and June, from fasting subjects. Demographic, socio-economic and anamnestic data and information about alcohol consumption and smoking were acquired by interviews and from a questionnaire filled out by specialized personnel [13]. The body mass index (BMI) (kg/m 2) for the male population was 26.00 4- 0.04 and for the females 26.00 4- 0.05. One hundred and seventy of the men (91%) declared that they drank alcoholic beverages (54.2 .4- 42.6 g/day) and 17 said they did not drink; 135 women (59%) declared an alcoholic consumption of 13.6 4- 19.0 g/day and 95 said they did not drink. Sixty-seven men (36%) and 31 women (13.5%) were smokers with declared consumption of 15 4- 8 g of tobacco per day for the men and 12 -4- 9 g for the women. The data above reported are mean .4- S.D. In addition to the determinations of the enzymes of lysosomal origin, the major blood chemistry parameters were also measured for all the subjects.

2.4. Enzyme assays All the enzymes were assayed by previously described fluorimetric procedures [8], using the corresponding 4-methylumbelliferyl-glycosides as substrates. A stable liquid material was employed for calibration purposes [91.

2.5. Statistical analysis Test of skewness and kurtosis showed significant differences from normal distribution and non-parametric techniques of analysis were used. The data were transformed into ranges, means were compared by the Mann-Whitney and Wilcoxon tests. Multiple comparisons of means were done by the Kruskal-Wallis test and, when necessary, adjusted with the Bonferroni correction; correlations were tested by the method of Spearman. For all analyses the SPSS/PC package was utilized [14].

42

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3. Results The plasma levels were higher in men than in the women (Table 1). Fig. 1 compares the empirical distributions of the data with the Gaussian distribution for the entire population. Statistical analysis after transformation of the data into ranges gave the following results: there were significant differences between the two sexes for GCR (P < 0.0001), ctGAL (P < 0.0001), MAN (P < 0.01) and FUC (P < 0.02). The levels of t~GAL,/~GAL and NAG were higher in the women than in the men in the first age class (20-29 years) (Fig. 2). In every age class, and especially for the 40-49 year class, the sex differences for GCR were strikingly greater than for any other enzyme. For this enzyme the pattern of change with age was the same for both sexes, while the other enzymes had parallel courses only during the intermediate ages (Fig. 2). Significant differences (P < 0.01) between age classes were verified only for N A G and MAN in the total population and for/~GAL in the male population. The highest and significant correlation with BMI was found for GCR in the female population (Table 2). lao ao

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Table 2 Coefficients of correlation of activities of lysosomal enzymes studied in the Rovescala population with age and body mass index Enzyme

N-acetyl-/~-D-glucosaminidasc /$-D-Glucuronidase /3-D-Galactosidase (~-D-Galactosidase ct-L-Fucosidase (~-D-Mannosidase *P < 0.01; **P < 0.001.

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0.243** n.s. n.s. n.s. n.s. 0.219

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0.234** 0.187* n.s. n.s. 0.262** 0.262**

0.222** 0.427"* n.s. 0.187" 0.206** 0.276**

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A. Lombardo et al. / Clinica Chiraica Acta 247 (1996) 39-49

In the study of correlations of the activity of each enzyme with the activity of all the others (Table 3), the higher coefficient was found between a G A L and ~GAL, NAG and MAN, NAG and GCR. In both the men and women who drank, we calculated the correlation of the enzyme levels with the declared consumption of alcohol. The only positive correlation was ~GAL in the women (r = 0.216; P < 0.01). There were no correlations at all between the enzymes and smoking. 4. Discussion

The diagnostic usefulness of some enzymes of lysosomal origin in biological fluids, especially plasma and serum, is constantly growing [3-7]. In addition to the already known correlations of plasma levels of some of these enzymes with carbohydrate dysmetabolic conditions [4-7], recent data indicate that some of these enzymes may be useful for evaluating function of some organs, e.g. liver and kidneys and to monitor the recuperation of function during the delicate post-transplantation phases [15-18]. In the past, assay of these plasma enzymes has been impeded by technical problems associated primarily with the poor stability of these enzymes in extracellular fluids [8] and by their circadian and circannual rhythms [11]. In addition, there is a tremendous variability in the levels of these enzymes even in groups homogeneous for age, sex and state of health which would indicate that there are some factors, as yet not investigated, that are able to affect the levels of these enzymes. From all of this, we deduce that a definitive confirmation of the diagnostic usefulness of plasma lysosomal enzymes will not be obtained Without having first studied the epidemiological characteristics, including variables that can influence these levels. In our study, we paid particular attention to some basic technical aspects: all blood samples were drawn at the same time of day and within a short time period (2 months) and to preserve all the enzyme activity ethylene glycol was added to the plasma sample immediately after it was obtained [9]. By excluding pregnant women and subjects with diabetes mellitus or obvious liver pathology, all conditions known to cause conspicuous changes in the distribution of these enzymes between the intra- and extra-cellular compartments [3-7,12], we obtained a general population suitable for the epidemiological study that we wished to do. The only important and highly significant difference between the two sexes was for GCR. This enzyme may be sensitive to the sex hormones, which like all steroid hormones, interact at the plasma membrane with the mechanism of release from the cell of the lysosomal enzymes [19-20]. The levels of the same enzyme were also influenced by body mass, especially in women. This is of interest since this parameter in our population was within the limits of

A. Lombardo et al. / Clinica Chimica Acta 247 (1996) 39-49

47

normal; and it is not surprising since lysosomal enzymes are altered in obese subjects [21]. In general, we observed a trend of plasma levels to increase with age even if significant differences have been found only for N A G and MAN. The lysosomal enzymes we studied do not seem to behave independently, one from the other. In fact, the good correlations of N A G with GCR and MAN and of/3GAL with c~GAL, allow coordinated behavior to be expected in patients suffering a specific pathology. This has been already verified in part [71. Neither smoking nor alcohol consumption affected enzyme levels in our population. The only positive indication was the correlation between alcohol consumption and plasma levels of/3GAL for the women. Since the levels of that enzyme are not substantially influenced by any other of the parameters we considered and the consumption of alcohol by our population was within the limits of normal, this was an interesting finding, because until now only consumption of large quantities of alcohol has been found to alter the levels of some lysosomal enzymes [22,23]. The mean levels of lysosomal enzymes measured in the population from Rovescala significantly differ from those obtained in other populations from different cities (unpublished data). The differences are particularly important for/3GAL which differs from population to population by as much as five times. Since these differences cannot be explained on the basis of the variables that we studied, we can only think that there are other variables, presumably environmental, that can have important effects on the enzyme levels and must be considered. 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.

Acknowledgements The samples analyzed were obtained in the course of a prospective CNR study of the effects of alcohol on human health (project FATMA, subproject alimentation). The work was supported by a grant to Professor Lombardo from the Italian Ministry of Education (40% contribution for 1993-1994). References

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A. Lombardo et al. /Clinica Chimica Acta 247 (1996) 39-49

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to di enzimi di origine lisosomiale durante il ciclo mestruale. Prog Med Lab 1988;2:331-343. [21] Corral J, Miralles JM, Garcia-Pascual I J, Corrales I J, Garcia-Sastre A, Villar E. Increased serum N-acetyl-/3-D-glucosaminidase and a-I>mannosidase activities in obese subjeets. Clin Invest 1992;70:880-884. [22] Karkkainen P, Poikolainen K, Salaspuro M. Serum/3-hexosaminidase as a marker of heavy drinking. Alcohol Clin Exp Res 1990;14:187-190. [23] Isaksson A, Blanche C, Hultberg B, Joelsson B. Influence of ethanol on the human serum level of B-bexosaminidase. Enzyme 1985;33:162-166.