BIOCHEMICAL
20, 336-343
MEDICINE
( 1978)
Racial and Age-Related Differences a-N-AcetybGlucosaminidase
in the Activity of in Man
JACAT SINGH AND FERENC GYORKEY Department of Pathology, Veterans’ Administration Received
May
Hospital, Houston, Texas 772/l
16, 1978
The interaction between plasma lipoproteins and glycosaminoglycans (GAG) has attracted a great deal of attention since it has been suggested that it might play a role in the intimal accumulation of lipoproteins in the early stages of atherosclerosis (1). In fact, in species susceptible to spontaneous or experimentally induced atherosclerosis, one of the initial abnormalities described is the appearance of intimal accumulation of amorphous material which stains metachromatically (2,3) and may be identified as GAG. It is at this level that lipid deposition starts to take place (4) as demonstrated by the isolation of GAG-lipoprotein complexes from fatty streaks of human aortas (1,5). Although the mechanisms responsible for GAG accumulation in early atherosclerotic lesions are not clear, it is known that young children with lysosomal storage diseases affecting the degradation of GAG (such as Hurler disease, MPS I) develop sclerotic, proliferative, and finally occlusive vascular lesions in absence of hypertension and of abnormalities of serum lipid levels (6). Since each of these diseases is caused by an inherited deficiency of a specific lysosomal hydrolase, it seems relevant to explore whether acquired dysfunctions of these hydrolases might be a factor contributing to the vascular accumulation of GAG. Heparin sulfate (HS) is the GAG which has been demonstrated to have the highest affinity for low density and very low density lipoproteins (7); it is also the one which, with dermatan sulfate, accumulates in Hurler disease (MPS I) because of L-iduronidase deficiency (8). Since in individuals without hyperlipoproteinemia, the severity of atherosclerosis is age related (9), we have decided to measure the serum levels of an enzyme involved in HS degradation (cY-N-acetyl-D336 OOO6-2944/78/0203-0336$02.00/O Copyright All rights
@ 1978 by Academic Press, Inc. of reproduction in any form raewed.
RACIAL
DIFFERENCES
IN wACETYLGLUCOSAMINIDASE
337
glucosaminidase) in individuals of increasing age, belonging to races (white and black) known to have a different incidence of atherosclerosis (9). Our results show that the serum enzyme levels indeed decrease with age and are lowest in those groups (whites) with higher incidence of atherosclerosis. MATERIALS
AND METHODS
The substratep-nitrophenyl-2-acetamido-2-deoxy-a-D-glucopyranoside was obtained from Research Products International Corporation, Elk Grove, Illinois: para-nitrophenol was from Fisher Scientific, Houston. Texas. Serum was obtained in the fasting state from blood of voluntary healthy individuals with no history or evidence of heart or metabolic diseases. An aliquot of the serum was used for the measurement of triglycerides and total cholesterol, while the other aliquot was stored frozen until assayed for a-N-acetyl-D-glycosaminidase. The 200 individuals included in this study were divided as follows: 64 (16 white males, 16 black males, 16 white females, 16 black females) were in group I (age, 20-29 years); 48 (12 white males, 12 black males, 12 white females, 12 black females) were in group II (age, 30-39 years): 46 (13 white males, 13 black males, 10 white females, 10 black females) were in group III (age, 40-49 years); and 42 (13 white males, 13 black males, 8 white females, 8 black females) were in group IV (age, 50-59 years). Triglycerides were measured with an enzymatic method (IO) and total serum cholesterol with a modified Liebermann-Burchard method (11) using a Technicon high speed computerized biochemical analyzer, SMAC.’ a-N-acetyl-D-glucosaminidase was measured as follows: 0.3 ml of a solution containing 2.92 pmole of substrate/ml of 0.8 M citrate-phosphate buffer, pH 4.5, 0.2 ml of serum, and 0.5 ml of 0.9% NaCl were incubated at 37°C for 3 hr. Thereafter, 0.3 ml of 40% trichloroacetic acid was added to each tube to precipitate the protein and the supernatants obtained by centrifugation at 2000g for 45 min at 5°C were collected. To 0.75 ml of each supernatant, 1.5 ml of 1.6 M glycine-NaOH buffer, was added, pH 10.6, and the yellow color produced by the release of p-nitrophenol was measured at 420 nm against water. Appropriate blanks were made by incubating serum and substrate separately and combining them immediately after adding trichloroacetic acid to the serum tube. The results in Fig. 1 are based on the net absorbancies obtained by subtracting the values of the blanks from those of the respective reaction mixtures. The ’ The mention of a trade name does not imply an endorsement instrument may be equally effective for these analyses.
as another suitable
338
SINGH AND GYORKEY 6
F
OVERALL
4; 2
iY22
0
I MALES:
POPULATION
I
I BLACK
I O---o
WHITE
M
FEMALES:
BLACK
O--O
WHITE
O-O
t
I 8 AGE
GROUP
I
I
1
I
I
I
I
I
I
II
Ill
IV
I
II
Ill
IV
FIG. I. a-N-Acetyl-o-glucosaminidase activity (1 unit = 1 pmole of p-nitrophenol released per hour per deciliter of serum at 37°C) in healthy individuals of various age, race, and sex. Overall population means white and black males and females combined. Values found to be significantly different at a 5% level have been marked by arrows connected by a solid horizontal line (when differences were found between age groups within a given race) or by a vertical dotted line (when differences were found between corresponding age groups of different race). For example, in black males while age group I has significantly higher activity than group IV, no significant differences occur in any other age group; in females, while blacks have significantly higher activity than the whites in each of age groups I, II, and III, no significant racial difference exists in group IV. For groups I. II, III, and IV, the age in years is. respectively. 20 to 29. 30 to 39. 40 to 49. and 50 to 59.
enzyme activity is expressed in units, pmole of p-nitrophenol released at 37°C The statistical significance of differences groups was determined by the t test at
where 1 unit corresponds to 1 per hour per deciliter of serum. in the enzyme activity in various 5% level of significance.
RESULTS Since all the individuals in the study had normal levels of triglycerides and total cholesterol for their age and sex, the single values have not been reported. The triglycerides and total cholesterol levels were considered normal only if the values were within the normal range for these constituents as listed by Fredrickson and Levy [see Table 28-2 of Ref. (12)].
RACIAL
DIFFERENCES
339
IN a-ACETYLGLUCOSAMINIDASE
The results of the enzyme assay and their statistical analyses for the various groups are reported in Fig. 1 and in Table 1. Blacks had two- to threefold higher activity of this enzyme for the corresponding age groups than the whites. These differences were statistically significant for all age groups of males but only for age groups I, II, and III of females. For a given race, there was no significant difference between males and females of the same age group. Although, there was ample variation in the enzyme activity within each age group, the blacks appeared more heterogeneous than the whites, as evident from their respective coefficient of variation. When the values of all the individuals (blacks and whites) belonging to each age group were considered (overall population), the enzyme activity decreased with age from 4.8 units in group I to 3.3 units in group IV. While the difference in these two age groups is statistically significant, there was no significant difference between the other groups. Although the decrease in the enzyme activity with age in the males of both races TABLE 1 RAW STATISTICAL DATA ON THE VALUES IN FIG. 1 ON HUMAN SERUM CY-N-ACETYL-D-GLUCOSAMINIDASE ACTIVITY Race and sex Overall populalion Black Males
White Males
Black Females
White females
Group and age Enzyme activity t-P (years) Mean Range” I II III IV I II III IV I II III IV I II III IV I II III IV
(20-29) (30-39) (40-49) (50-59) (20-29) (30-39) (40-49) (50-59) (20-29) (30-39) (40-49) (50-59) (20-29) (30-39) (40-49) (50-59) (20-29) (30-39) (40-49) (50-59)
4.8 4.5 3.8 3.3 7.4 6.5 5.7 4.1 3.0 2.5 2.4 2.2 6.3 6.8 4.8 4.3 2.4 2.2 2.2 2.8
1.8-13.1 1.5-19.8 1.3-14.3 1.2- 9.5 4.0-12.6 2.0-19.8 1.3-14.3 1.4- 9.5 1.8-5.6 1.8-3.1 1.5-3.9 1.2-3.3 2.0-13.1 1.6-16.4 2.6- 8.9 2.2- 8.9 1.8-4.0 1.5-4.1 1.5-3.5 1.3-5.1
64 48 46 42 I6 12 13 13 16 12 13 13 16 12 10 8 16 12 10 8
c.v.c
SEMZd
65.0899 91.7285 67.9632 61.2857 34.8850 80.3779 61.4725 61.3885 34.5211 16.7305 30.2823 29.6448 61.4769 71.2857 42.3511 57.393 1 24.3 146 32.5668 32.3444 45.4056
0.1547 0.3638 0.1510 0.1000 0.4397 2.5142 1.0318 0.5287 0.0696 0.0158 0.0458 0.0363 0.98% 2.1641 0.4651 0.8644 0.0266 0.0446 0.0555 0.2273
” Range represents the lowest and the highest activity observed. * Number of samples. c Coefficient of variation in percent. d Standard error of the mean.
340
SINGH
AND
GYORKEY
was identical to that in the overall population, there was no significant age-related decrease in the enzyme activity in the females of either race. DISCUSSION
The lysosomal enzyme cr-N-acetyl-D-glucosaminidase is essential for the degradation of HS (13) and presumably participates in the regulation of HS levels in various tissues. Our study shows that in the males of both races, there is a significant decrease in the enzyme activity with age (cf. group I versus group IV, Fig. l), the blacks maintaining significantly higher levels than the whites of the same group. While for the females there is not a significant decrease of enzyme activity with age, the blacks show significantly higher levels of activity than the whites for age groups I, II, and III. These results prompt several comments. It is not clear at this point whether such decline in the activity of the enzyme might affect the degradation of HS and favor its tissue accumulation. Nevertheless, it is tempting to correlate our findings with the known incidence of severe atherosclerosis (fatty plaques and complicated lesions) which is higher in aged males than females and higher in white males than black males (9). An argument in support of the view of a possible inverse relationship between the severity of atherosclerosis and serum levels of this enzyme concerns the experimentally induced atherosclerosis in several species of laboratory animals. Those resistant to the development of atherosclerosis (rat) show consistently higher levels of activity of lysosomal enzymes (14.15) known to be involved in the degradation of several GAG. To further explore if such a correlation also exists in man, apparently the measurement of other lysosomal enzymes relevant to GAG degradation in the individuals of different race, age, and sex is desirable. There are many medical as well as biochemical differences between the white and the black races. Some examples are as follow. As compared to whites, blacks are known to have less incidence of coronary heart disease (16) and myocardial infarction (17), and these have been attributed (16) to their higher levels of serum high density lipoproteins. Sickle cell disease almost exclusively occurs in the blacks (18). Glucose-6-phosphate dehydrogenase deficiency disease is found in both races; however, blacks with the defect have significantly higher mean enzyme levels than those of deficient whites (19). As compared to whites, there is a higher incidence of cancer: of stomach (20,21) and pancreas (20) in the blacks of either sex: of liver (20,21), larynx, and prostate (20) in black males; and of cervix and bladder (20) in black females. The occurrence of Hodgkin’s disease is higher in blacks than in whites in the early childhood, but in young adults and in older age groups more whites are affected than the blacks (22).
RACIAL
DIFFERENCES
IN a-ACETYLGLLJCOSAMINIDASE
341
Tumors of the central nervous system have been reported to be less prevalent in the blacks than in the whites (23). Recently, certain biochemical differences found among Chinese and Caucasians have been explained on the basis of the “Justification Theory” (24,25). The race with the low protein intake (Chinese) retain more nitrogen and have a higher level of serum nonessential amino acids than the race (Caucasian) with the high protein intake (24). These observations conform to the “Justification Theory” (24); the Chinese retain more nitrogen and have higher serum nonessential amino acids than the Caucasians as a guard against possible mutation which might affect the synthesis of nonessential amino acids and which could be of dire consequences in the Chinese than in the whites even if the mutation occurred at the same rate in the two races. The variation known to exist in the intelligence among the siblings of phenylketonuric children has been explained recently on the basis of this theory (26). It appears that this theory may provide an explanation for a large number of medical and biochemical racial variations (26). Additional research is needed to fully determine the medical and biochemical implications of the racial variability we observed in the level of cY-N-acetyl-D-glucosaminidase activity. The apparent significant difference in the activity of this enzyme between the white and the black women of childbearing age suggests that this and other disease-relevant lysosomal enzymes be measured in serum and in fibroblasts or leukocyte extracts in various racial groups separately, in order to establish possible race-related “normal“ values to be used for screening and diagnostic purposes. Moreover, since the parents of children affected by autosomal recessive mucopolysaccharidoses involving the degradation of HS have levels of specific lysosomal enzymes about 50% lower than the normal ones, a study of the age of incidence of atherosclerosis in these individuals would indicate the relevance of HS degrading enzymes in the appearance of the disease. SUMMARY
Serum cy-N-acetyl-D-glucosaminidase, essential for the degradation of heparan sulfate, was measured in the serum of 200 healthy individuals (male and female, whites and blacks) of various ages: 20 to 29 (group I); 30 to 39 (group II); 40 to 49 (group III), and 50 to 59 (group IV) years old. Blacks had two- to threefold higher enzyme activity than the whites of the corresponding age groups. These differences were statistically significant for all age groups in males, but only for groups I, II, and III in females. These observations suggest that other disease-relevant lysosomal enzymes be measured in various genetic groups individually in order to
342
SINGH AND GYORKEY
establish possible race-related “normal” values to be used for diagnostic purposes. Furthermore, we observed that in the overall population (white and black males and females combined), the enzyme activity decreased significantly with age (from 4.8 units/d1 in group I to 3.3 units/d1 in group IV). Since it has been postulated that the accumulation of GAG occurring in the vascular wall is involved in the pathogenesis of the early stages of human and experimental atherosclerosis, it is of interest to note that in the individuals 50 to 59 years old of various population groups, the levels of enzyme activity which we have observed are inversely related to the incidence of microscopically evident atherosclerotic lesions (fatty plaques and complicated lesions), as studied by McGill et al. (9). ACKNOWLEDGMENT This work was supported in part by a grant-in-aid from the American Heart Association, Texas Affiliate, Inc., Houston Chapter, and Veterans Administration Pathology Research.
REFERENCES 1. Berenson, G. S.. Srinivasan, S. R., Radhakrishnamurthy, B., and Dalferes, E. R., Advan. Exp. Med. Biol. 43, 141 (1973). 2. Mancini, M., Rossi, G. B., Oriente, P.. and Cali, A., Nature (London) 207, 1206 (1965). 3. Rossi, G. B., Mancini, M., Oriente, P., Vecchione, A., Vecchione, R., Cerqua, R., and Cuzzupoli, M., J. Atheroscler. Res. 5, 569 (1965). 4. Gresham, G. A.. and Howard. A. N. J. Atheroscler, Res. 1, 413 (1961). 5. Srinivasan, S. R., Dolan, P., Radhakrishnamurthy, B., and Berenson, G. S.. Atherosclerosis 16, 95 (1972). 6. Goldfischer, S., Coltoff-Schiller. B., Biempica, L.. and Wolinsky, H. Hum. Patho/. 6, 633 (1975). 7. Srinivasan, S. R., Lopez, S. A., Radhakrishnamurthy, B., and Berenson. G. S.. Atherosclerosis 12, 321 (1970). 8. Dorfman, A., and Matalon, R., Proc. Nat. Acad. Sci. USA 73, 630 (1976). 9. McGill, H. C.. Jr., Geer, J. C., and Strong, J. P., in “Atherosclerosis and Its Origin” (M. Sandler and G. H. Bourne, Eds.), p, 39. Academic Press, New York, 1963. IO. Levine, J., Morgenstern, S., and Vlastelica, D.. in “Automation in Analytical Chemistry,” p. 25. Technicon Symposia, White Plains, N.Y.. 1968. II. Bucolo, G.. and David, H., Clin. Chem. 19, 475 (1973). 12. Fredrickson, D. S., and Levy, R. I., in “The Metabolic Basis of Inherited Diseases” (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, Eds.), p. 545. McGraw-Hill, New York, 1972. 13. O’Brien, J. S., Proc. Nat. Acad. Sci. USA 69, 1720 (1972). 14. Bonner, M. J., Miller, B. F., and Kothari. H. V.. Proc. Sot. Exp. Biol. Med. 139, 1359 (1972). 15. Howard, A. N., in “Atherosclerosis III, Proceedings of Third International Symposium” (G. Schettler and A. Weizel, Eds.), p. 308. Springer-Verlag, New York/ Berlin, 1974. 16. Tyroler, H. A., Heyden, S., Bartel, A., Cassel, J., Cornoni, J.. Hames, C. G., and Kleinbaum, D., Arch. Intern. Med. 128,. 907 (1971). 17. Weisse, A. B., Abiuso, P. D., and Thind, I. S., Arch. In/era. Med. 137, 1402 (1977).
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IN a-ACETYLGLUCOSAMINIDASE
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18. Davidsohn, I.. and Nelson, D. A., in “Clinical Diagnosis by Laboratory Methods” (I. Davidsohn and J. B. Henry, Eds.), p. 100. Saunders, Philadelphia, 1974. 19. Allison, A. C., in “Genetical Variation in Human Populations” (G. A. Harrison, Ed.), p. 16. Pergamon, New York, l%l. 20. Henschke. U. H., Leffall, L. D., Mason, C. H., Reinhold, A. W., Schneider, R. L., and White, J. E., Cancer 31, 763 (1973). 21. U.S. Cancer Mortality by County, 1950-1969. DHEW Publication No. (NIH) 74-615 (1974). U.S. Department of Health, Education and Welfare, Public Health Service, NIH, National Cancer Institute, Bethesda, Md. 22. Vianna, N. J.. Thind, I.S., Louria, D. B., Polman, A., Kirmss, V., and Davies, J. N. P.. Cancer 40, 3133 (1977). 23. Heshmat, M. Y., Kovi, J., Simpson, C., Kennedy, J., and Fan. K. J.. Cancer, 38, 2135 (1976). 24. Bessman, S. P., and Huang, P. C., Fed. Proc. 37, 840 (1978). 25. Bessman, S. P., J. Pediat. 81, 834 (1972). 26. Bessman. S. P., Williamson, M. L., and Koch, R., Proc. Nat. Acad. Sci. USA 75, 1562 (1978).
20, 336-343
MEDICINE
( 1978)
Racial and Age-Related Differences a-N-AcetybGlucosaminidase
in the Activity of in Man
JACAT SINGH AND FERENC GYORKEY Department of Pathology, Veterans’ Administration Received
May
Hospital, Houston, Texas 772/l
16, 1978
The interaction between plasma lipoproteins and glycosaminoglycans (GAG) has attracted a great deal of attention since it has been suggested that it might play a role in the intimal accumulation of lipoproteins in the early stages of atherosclerosis (1). In fact, in species susceptible to spontaneous or experimentally induced atherosclerosis, one of the initial abnormalities described is the appearance of intimal accumulation of amorphous material which stains metachromatically (2,3) and may be identified as GAG. It is at this level that lipid deposition starts to take place (4) as demonstrated by the isolation of GAG-lipoprotein complexes from fatty streaks of human aortas (1,5). Although the mechanisms responsible for GAG accumulation in early atherosclerotic lesions are not clear, it is known that young children with lysosomal storage diseases affecting the degradation of GAG (such as Hurler disease, MPS I) develop sclerotic, proliferative, and finally occlusive vascular lesions in absence of hypertension and of abnormalities of serum lipid levels (6). Since each of these diseases is caused by an inherited deficiency of a specific lysosomal hydrolase, it seems relevant to explore whether acquired dysfunctions of these hydrolases might be a factor contributing to the vascular accumulation of GAG. Heparin sulfate (HS) is the GAG which has been demonstrated to have the highest affinity for low density and very low density lipoproteins (7); it is also the one which, with dermatan sulfate, accumulates in Hurler disease (MPS I) because of L-iduronidase deficiency (8). Since in individuals without hyperlipoproteinemia, the severity of atherosclerosis is age related (9), we have decided to measure the serum levels of an enzyme involved in HS degradation (cY-N-acetyl-D336 OOO6-2944/78/0203-0336$02.00/O Copyright All rights
@ 1978 by Academic Press, Inc. of reproduction in any form raewed.
RACIAL
DIFFERENCES
IN wACETYLGLUCOSAMINIDASE
337
glucosaminidase) in individuals of increasing age, belonging to races (white and black) known to have a different incidence of atherosclerosis (9). Our results show that the serum enzyme levels indeed decrease with age and are lowest in those groups (whites) with higher incidence of atherosclerosis. MATERIALS
AND METHODS
The substratep-nitrophenyl-2-acetamido-2-deoxy-a-D-glucopyranoside was obtained from Research Products International Corporation, Elk Grove, Illinois: para-nitrophenol was from Fisher Scientific, Houston. Texas. Serum was obtained in the fasting state from blood of voluntary healthy individuals with no history or evidence of heart or metabolic diseases. An aliquot of the serum was used for the measurement of triglycerides and total cholesterol, while the other aliquot was stored frozen until assayed for a-N-acetyl-D-glycosaminidase. The 200 individuals included in this study were divided as follows: 64 (16 white males, 16 black males, 16 white females, 16 black females) were in group I (age, 20-29 years); 48 (12 white males, 12 black males, 12 white females, 12 black females) were in group II (age, 30-39 years): 46 (13 white males, 13 black males, 10 white females, 10 black females) were in group III (age, 40-49 years); and 42 (13 white males, 13 black males, 8 white females, 8 black females) were in group IV (age, 50-59 years). Triglycerides were measured with an enzymatic method (IO) and total serum cholesterol with a modified Liebermann-Burchard method (11) using a Technicon high speed computerized biochemical analyzer, SMAC.’ a-N-acetyl-D-glucosaminidase was measured as follows: 0.3 ml of a solution containing 2.92 pmole of substrate/ml of 0.8 M citrate-phosphate buffer, pH 4.5, 0.2 ml of serum, and 0.5 ml of 0.9% NaCl were incubated at 37°C for 3 hr. Thereafter, 0.3 ml of 40% trichloroacetic acid was added to each tube to precipitate the protein and the supernatants obtained by centrifugation at 2000g for 45 min at 5°C were collected. To 0.75 ml of each supernatant, 1.5 ml of 1.6 M glycine-NaOH buffer, was added, pH 10.6, and the yellow color produced by the release of p-nitrophenol was measured at 420 nm against water. Appropriate blanks were made by incubating serum and substrate separately and combining them immediately after adding trichloroacetic acid to the serum tube. The results in Fig. 1 are based on the net absorbancies obtained by subtracting the values of the blanks from those of the respective reaction mixtures. The ’ The mention of a trade name does not imply an endorsement instrument may be equally effective for these analyses.
as another suitable
338
SINGH AND GYORKEY 6
F
OVERALL
4; 2
iY22
0
I MALES:
POPULATION
I
I BLACK
I O---o
WHITE
M
FEMALES:
BLACK
O--O
WHITE
O-O
t
I 8 AGE
GROUP
I
I
1
I
I
I
I
I
I
II
Ill
IV
I
II
Ill
IV
FIG. I. a-N-Acetyl-o-glucosaminidase activity (1 unit = 1 pmole of p-nitrophenol released per hour per deciliter of serum at 37°C) in healthy individuals of various age, race, and sex. Overall population means white and black males and females combined. Values found to be significantly different at a 5% level have been marked by arrows connected by a solid horizontal line (when differences were found between age groups within a given race) or by a vertical dotted line (when differences were found between corresponding age groups of different race). For example, in black males while age group I has significantly higher activity than group IV, no significant differences occur in any other age group; in females, while blacks have significantly higher activity than the whites in each of age groups I, II, and III, no significant racial difference exists in group IV. For groups I. II, III, and IV, the age in years is. respectively. 20 to 29. 30 to 39. 40 to 49. and 50 to 59.
enzyme activity is expressed in units, pmole of p-nitrophenol released at 37°C The statistical significance of differences groups was determined by the t test at
where 1 unit corresponds to 1 per hour per deciliter of serum. in the enzyme activity in various 5% level of significance.
RESULTS Since all the individuals in the study had normal levels of triglycerides and total cholesterol for their age and sex, the single values have not been reported. The triglycerides and total cholesterol levels were considered normal only if the values were within the normal range for these constituents as listed by Fredrickson and Levy [see Table 28-2 of Ref. (12)].
RACIAL
DIFFERENCES
339
IN a-ACETYLGLUCOSAMINIDASE
The results of the enzyme assay and their statistical analyses for the various groups are reported in Fig. 1 and in Table 1. Blacks had two- to threefold higher activity of this enzyme for the corresponding age groups than the whites. These differences were statistically significant for all age groups of males but only for age groups I, II, and III of females. For a given race, there was no significant difference between males and females of the same age group. Although, there was ample variation in the enzyme activity within each age group, the blacks appeared more heterogeneous than the whites, as evident from their respective coefficient of variation. When the values of all the individuals (blacks and whites) belonging to each age group were considered (overall population), the enzyme activity decreased with age from 4.8 units in group I to 3.3 units in group IV. While the difference in these two age groups is statistically significant, there was no significant difference between the other groups. Although the decrease in the enzyme activity with age in the males of both races TABLE 1 RAW STATISTICAL DATA ON THE VALUES IN FIG. 1 ON HUMAN SERUM CY-N-ACETYL-D-GLUCOSAMINIDASE ACTIVITY Race and sex Overall populalion Black Males
White Males
Black Females
White females
Group and age Enzyme activity t-P (years) Mean Range” I II III IV I II III IV I II III IV I II III IV I II III IV
(20-29) (30-39) (40-49) (50-59) (20-29) (30-39) (40-49) (50-59) (20-29) (30-39) (40-49) (50-59) (20-29) (30-39) (40-49) (50-59) (20-29) (30-39) (40-49) (50-59)
4.8 4.5 3.8 3.3 7.4 6.5 5.7 4.1 3.0 2.5 2.4 2.2 6.3 6.8 4.8 4.3 2.4 2.2 2.2 2.8
1.8-13.1 1.5-19.8 1.3-14.3 1.2- 9.5 4.0-12.6 2.0-19.8 1.3-14.3 1.4- 9.5 1.8-5.6 1.8-3.1 1.5-3.9 1.2-3.3 2.0-13.1 1.6-16.4 2.6- 8.9 2.2- 8.9 1.8-4.0 1.5-4.1 1.5-3.5 1.3-5.1
64 48 46 42 I6 12 13 13 16 12 13 13 16 12 10 8 16 12 10 8
c.v.c
SEMZd
65.0899 91.7285 67.9632 61.2857 34.8850 80.3779 61.4725 61.3885 34.5211 16.7305 30.2823 29.6448 61.4769 71.2857 42.3511 57.393 1 24.3 146 32.5668 32.3444 45.4056
0.1547 0.3638 0.1510 0.1000 0.4397 2.5142 1.0318 0.5287 0.0696 0.0158 0.0458 0.0363 0.98% 2.1641 0.4651 0.8644 0.0266 0.0446 0.0555 0.2273
” Range represents the lowest and the highest activity observed. * Number of samples. c Coefficient of variation in percent. d Standard error of the mean.
340
SINGH
AND
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was identical to that in the overall population, there was no significant age-related decrease in the enzyme activity in the females of either race. DISCUSSION
The lysosomal enzyme cr-N-acetyl-D-glucosaminidase is essential for the degradation of HS (13) and presumably participates in the regulation of HS levels in various tissues. Our study shows that in the males of both races, there is a significant decrease in the enzyme activity with age (cf. group I versus group IV, Fig. l), the blacks maintaining significantly higher levels than the whites of the same group. While for the females there is not a significant decrease of enzyme activity with age, the blacks show significantly higher levels of activity than the whites for age groups I, II, and III. These results prompt several comments. It is not clear at this point whether such decline in the activity of the enzyme might affect the degradation of HS and favor its tissue accumulation. Nevertheless, it is tempting to correlate our findings with the known incidence of severe atherosclerosis (fatty plaques and complicated lesions) which is higher in aged males than females and higher in white males than black males (9). An argument in support of the view of a possible inverse relationship between the severity of atherosclerosis and serum levels of this enzyme concerns the experimentally induced atherosclerosis in several species of laboratory animals. Those resistant to the development of atherosclerosis (rat) show consistently higher levels of activity of lysosomal enzymes (14.15) known to be involved in the degradation of several GAG. To further explore if such a correlation also exists in man, apparently the measurement of other lysosomal enzymes relevant to GAG degradation in the individuals of different race, age, and sex is desirable. There are many medical as well as biochemical differences between the white and the black races. Some examples are as follow. As compared to whites, blacks are known to have less incidence of coronary heart disease (16) and myocardial infarction (17), and these have been attributed (16) to their higher levels of serum high density lipoproteins. Sickle cell disease almost exclusively occurs in the blacks (18). Glucose-6-phosphate dehydrogenase deficiency disease is found in both races; however, blacks with the defect have significantly higher mean enzyme levels than those of deficient whites (19). As compared to whites, there is a higher incidence of cancer: of stomach (20,21) and pancreas (20) in the blacks of either sex: of liver (20,21), larynx, and prostate (20) in black males; and of cervix and bladder (20) in black females. The occurrence of Hodgkin’s disease is higher in blacks than in whites in the early childhood, but in young adults and in older age groups more whites are affected than the blacks (22).
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Tumors of the central nervous system have been reported to be less prevalent in the blacks than in the whites (23). Recently, certain biochemical differences found among Chinese and Caucasians have been explained on the basis of the “Justification Theory” (24,25). The race with the low protein intake (Chinese) retain more nitrogen and have a higher level of serum nonessential amino acids than the race (Caucasian) with the high protein intake (24). These observations conform to the “Justification Theory” (24); the Chinese retain more nitrogen and have higher serum nonessential amino acids than the Caucasians as a guard against possible mutation which might affect the synthesis of nonessential amino acids and which could be of dire consequences in the Chinese than in the whites even if the mutation occurred at the same rate in the two races. The variation known to exist in the intelligence among the siblings of phenylketonuric children has been explained recently on the basis of this theory (26). It appears that this theory may provide an explanation for a large number of medical and biochemical racial variations (26). Additional research is needed to fully determine the medical and biochemical implications of the racial variability we observed in the level of cY-N-acetyl-D-glucosaminidase activity. The apparent significant difference in the activity of this enzyme between the white and the black women of childbearing age suggests that this and other disease-relevant lysosomal enzymes be measured in serum and in fibroblasts or leukocyte extracts in various racial groups separately, in order to establish possible race-related “normal“ values to be used for screening and diagnostic purposes. Moreover, since the parents of children affected by autosomal recessive mucopolysaccharidoses involving the degradation of HS have levels of specific lysosomal enzymes about 50% lower than the normal ones, a study of the age of incidence of atherosclerosis in these individuals would indicate the relevance of HS degrading enzymes in the appearance of the disease. SUMMARY
Serum cy-N-acetyl-D-glucosaminidase, essential for the degradation of heparan sulfate, was measured in the serum of 200 healthy individuals (male and female, whites and blacks) of various ages: 20 to 29 (group I); 30 to 39 (group II); 40 to 49 (group III), and 50 to 59 (group IV) years old. Blacks had two- to threefold higher enzyme activity than the whites of the corresponding age groups. These differences were statistically significant for all age groups in males, but only for groups I, II, and III in females. These observations suggest that other disease-relevant lysosomal enzymes be measured in various genetic groups individually in order to
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establish possible race-related “normal” values to be used for diagnostic purposes. Furthermore, we observed that in the overall population (white and black males and females combined), the enzyme activity decreased significantly with age (from 4.8 units/d1 in group I to 3.3 units/d1 in group IV). Since it has been postulated that the accumulation of GAG occurring in the vascular wall is involved in the pathogenesis of the early stages of human and experimental atherosclerosis, it is of interest to note that in the individuals 50 to 59 years old of various population groups, the levels of enzyme activity which we have observed are inversely related to the incidence of microscopically evident atherosclerotic lesions (fatty plaques and complicated lesions), as studied by McGill et al. (9). ACKNOWLEDGMENT This work was supported in part by a grant-in-aid from the American Heart Association, Texas Affiliate, Inc., Houston Chapter, and Veterans Administration Pathology Research.
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18. Davidsohn, I.. and Nelson, D. A., in “Clinical Diagnosis by Laboratory Methods” (I. Davidsohn and J. B. Henry, Eds.), p. 100. Saunders, Philadelphia, 1974. 19. Allison, A. C., in “Genetical Variation in Human Populations” (G. A. Harrison, Ed.), p. 16. Pergamon, New York, l%l. 20. Henschke. U. H., Leffall, L. D., Mason, C. H., Reinhold, A. W., Schneider, R. L., and White, J. E., Cancer 31, 763 (1973). 21. U.S. Cancer Mortality by County, 1950-1969. DHEW Publication No. (NIH) 74-615 (1974). U.S. Department of Health, Education and Welfare, Public Health Service, NIH, National Cancer Institute, Bethesda, Md. 22. Vianna, N. J.. Thind, I.S., Louria, D. B., Polman, A., Kirmss, V., and Davies, J. N. P.. Cancer 40, 3133 (1977). 23. Heshmat, M. Y., Kovi, J., Simpson, C., Kennedy, J., and Fan. K. J.. Cancer, 38, 2135 (1976). 24. Bessman, S. P., and Huang, P. C., Fed. Proc. 37, 840 (1978). 25. Bessman, S. P., J. Pediat. 81, 834 (1972). 26. Bessman. S. P., Williamson, M. L., and Koch, R., Proc. Nat. Acad. Sci. USA 75, 1562 (1978).
