Trisomy 21 (Down's syndrome) glutathione peroxidase, hexose monophosphate shunt and I.Q.

Pierre-Marie SINET , Jérôme LEJEUNE and Henri JÉRÔME

Life Sciences, Vol. 24, pp. 29-34

Résumé :

SUMMARY. Glutathione peroxidase activity is increased in erythrocytes and fibroblasts from trisomy 21 patients. Hexose monophosphate shunt activity in erythrocytes is also increased. The enzymatic changes may be secondary to accelerated oxidative processes within cells, which would be in accord with some of the clinical features of Down's Syndrome. A highly positive correlation exists between erythrocyte glutathione peroxidase and I. Q. This latter finding may indicate an important role for glutathione peroxidase in the cerebral status of these patients.


An increase of about 50% in the copper-zinc superoxide dismutase activity (SOD-1) has been observed in red cells (1, 2, 3), platelets (4), leucocytes and fibroblasts (5) from trisomy 21 patients (Down's Syndrome). The increase is likely the result of a gene dosage effect, the SOD-1 gene having been assigned to chromosome 21 by experiments based on cell hybridization (6). We have localized the SOD-1 gene to a segment of chromosome 21 (representing a length between 1/5 and 1/10 of the whole chromosome) (7) which appears to he responsible for the clinical features of trisomy 21. Therefore, the possible role of the SOD-1 excess in the pathogeny of this disease should be considered.

SOD catalyzes the dismutation of the superoxide radical O2- (8) according to the reaction:

O2- + O2- + 2H+ ? H2O2 + O2

Increased SOD activity should lead to a decrease in the steady state level of superoxide within cells. It seems likely that H2O2 production might also be increased, although this might depend upon other cellular reactions. However, as described in the text, we have observed increased hexose monophosphate shunt activity, which might reflect increased peroxide production. Hydrogen peroxide and organic peroxides (e. g., lipid peroxides) may exert toxic actions by oxidizing sensitive sulhydryl groups and initiating the peroxidation of unsaturated lipids. Two enzymes that protect cells by removing peroxides are catalase and glutathione peroxidase (GSHPx). Erythrocyte catalase levels are normal in trisomy 21 (9). We have studied GSHPx and the metabolic pathway with which this enzyme is connected: glutathione reductase and hexose monophosphate shunt (HMPS).


Materials and methods

For the studies in red cells, trisomy 21 patients free of congenital cardiopathy and controls were of similar age, all older than two years. Blood samples were collected with heparin and centrifuged at 1000g for 15 min at 4°C. The plasma and buffy coat were aspirated and the cells washed twice with 0.154 M NaCl.

Glutathione peroxidase activity was measured as described in (l0) by a coupled enzyme procedure (11) with glutathione reductase and NADPH using t-butyl hydroperoxide as substrate (12).

For the measure of HMPS activity one vol. of the red cell pellet was mixed with 5 vol. of a solution containing 0.1 M HEPES/NaOH pH 7.4, 4 mM KCL, 5 mM MgCl2, 55 mM NaCl, 12mM Na2HPO4, 12 mM D-glucose and 0.4 µCi per ml of D-glucose 114C (Amersham). 14CO2 production was measured by placing a sample of 1.5 ml of this suspension in the main compartment of a Warburg vessel the center well of which contained 0.2 ml of 2N NaOH on filter paper and the side arm 0.2 ml of 10.6 N perchloric acid. The flasks were gently shaken for 2 hours at 37° C in a Dubnoff shaker at 80 oscillations per min. At the end of the incubation, perchloric acid was tipped and shaking continued for 30 min. The radioactivity of the filter paper was measured using Tricarb liquid. scintillation counting equipment. Glycolysis was measured by incubating, a sample of 3.5 ml of the buffered cell suspension in erlenmeyers set besides the Warburg vessels. At the beginning and at the end of the incubation, 0.5 ml of this suspension was transferred to 1 ml of 1.2 N perchloric acid. The precipitate was withdrawn by centrifugation and the supernatant neutralized by KHCO3. Glucose was then measured with hexokinase and glucose 6 phosphate dehydrogenase (13). The total radioactivity of the buffered cell suspension was also quantitated for determination of the specific activity of D-glucose 114C.

The different fibroblast cell-lines of known karyotypes were obtained from skin biopsies or abortion material. The cells were grown in Eagle's medium with 2 p. 100 calf serum. All assays were done on confluent cells at passage numbers between 3 and 16. Before the assays, the fibroblasts were frozen, thawed once, and lysis completed as follows: one volume of 1 p. 100 (v/v) Triton x-100 was added to ten volumes of cell suspension and gently shaken at 4°C for 2 hours. The supernatant obtained by centrifugation (15,000g) was used. The assay procedure for GSHPx activity was the same as for red cells (10) except that tissue extracts were not mixed with potassium ferricyanide and KCN but 1 mM KCN was added to assay medium. The rate of oxidation of NADPH without tissue extract was subtracted from the overall rates.

I. Q. evaluation. The patients studied here come regularly (every six months) to the hospital for a visit. At each visit, their I. Q. is evaluated by psychological tests adapted to their age: the Brunet-Lezine's test was used before 3-4 years of age, the Borel-Maisonny's test between 3-4 years and 8-10 years, and the Binet-Simon's test after 8-10 years. For each patient, the evaluation of the I. Q. used in the correlation was the average of three tests: a test passed before the blood puncture for the assay of red cell GSHPx, and the two tests from the two last visits. Among these patients, 17 were less than 10 years old, 21 between 10 and 15, 12 more than 15.


Results and discussion

Table 1 shows that GSHPx activity is increased in erythrocytes of trisomy 21 patients (10)* as well as in fibroblasts in culture. No modification of glutathione reductase activity was found in erythrocytes of these patients nor any modification of the enzymes of the HMPS, viz., glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (data not shown). These last results are in agreement with other authors (9, 14, 15). Yet the cellular production of CO2 via the HMPS is significantly higher in trisomy 21 erythrocytes compared to normal controls incubated in physiological conditions (Table 2). According to Layser and Epstein (25), there is no evidence of a younger red cell population in trisomy 21. The slight increase that we observe in the number of reticulocytes in trisomy 21 blood (Legend Table 2) cannot account for a 15% increase in HMPS. This increase in red cell HMPS suggests that an increased amount of peroxide is formed and then catabolized via GSHPx in trisomy 21.

As yet the gene of GSHPx has not been firmly assigned to any human chromosome. The increase of GSHPx activity in trisomy 21 tissues may be tree result of a gene dosage effect if the GSHPx gene is on the chromosome 21**. It seems more likely that this increase of GSHPx activity is the result of a regulatory mechanism that could be secondary to an acceleration of peroxidative processes within the cells. Many features of trisomy 21 are consistent with increased oxidative damages: 1) rapid ageing with decreasing intelligence quotient (I. Q.) (16); 2) histological changes in brain similar to that seen ire neuronal degeneration (17, 18) with accumulation of lipofuscine (19) such as is observed in Alzheimer disease; and 3) shortened life span of cells in culture (21, 22).

It should be noted that GSHPx can reduce not only H2O2 but also organic hydroperoxides (22). Therefore GSHPx constitutes a defense mechanism against lipid peroxidative damage which may contribute significantly to cell ageing (23). This may be particularly important in brain which is rich in unsaturated fatty acids. Table 3 shows a highly significant positive correlation between erythrocyte GSHPx activity and I. Q. in trisomy 21 (line 1). None of the other studied enzymes (SOD-1, glutathione reductase, HMPS enzymes) correlate with I. Q. (data not shown). The correlation between GSHPx and I. Q. is not due to a variation of GSHPx and of I. Q. as a function of age (line, 2 and 3); the partial correlation coefficient GSHPx - I. Q./age (that is the correlation at constant age) remains significant (line 4).

We have no direct evidence that the observations concerning GSHPx in erythrocytes reflect the GSHPx activity in the brain. However it has been shown in growing rats that GSHPx activities vary in a constant ratio in the brain and in the erythrocytes (24). Therefore our observations concerning GSHPx and I. Q. may indicate that GSHPx plays an important role in the cerebral status of trisomy 21 patients.

TABLE 1. - Glutathione Peroxidase Activities in Red Cells and Fibroblasts: Comparison Between Controls and Trisomy 21 Patients.
Red Cells (umole NADPH oxidized/ min/ g Hb)Fib rob lasts (umole NADPH oxidized/ min/109 cells)
CONTROLS7.87 ± 2.00 (48)3.03 ± .73 (8)
TRISOMY 2110.94 ± 2.75 (50)4.43 ± .95 (5)
STATISTICAL SIG.* p < .001* p < .02
*Results as mean + S.D.: number of determinations in parentheses. Statistical significance was determined by using Student's t-test.
TABLE 2. - Glucose Catabolism Via Hexose Monophosphate Shunt (HMPS) Pathway in Red Cells: Comparison Between Controls and Trisomy 21 Patients.
Total Glucose Consumed (nmole/hr./1012 cells)Glucose via HMPS (nmole CO2/hr./ 1012 cells)% of Glucose via HMPS
CONTROLS2745 ± 365 (14)77.5 ± 10 (14)2.85 ± .38 (14)
TRISOMY 212786 ± 396 (24)90.8 ± 19 (24)3.27 ± .62 (24)
STATISTICAL SIG.* N.S.** p < .02** p < .02
Results as mean ± S. D.; number of determinations in parentheses. Statistical significance was determined by using Student's t-test (*) or Cochran's test (**) in case of significant difference in variances between controls and trisomy 21 data. N. S.: no significant difference. For each blood sample a reticulocyte count was performed; the results were (mean ± S.D.): Controls: 5.3 ± 3.9 reticulocytes per 1000 erythrocytes; Trisomy 21 patients: 8.1 ± 3.9.
TABLE 3. - Correlation Coefficients Between Glutathione Peroxidase (GSHPx) Activity in Red Cells, Intelligence Quotient (I.Q.) and Age in 50 Trisomy 21 Patients.
Correlation CoefficientStatistical Significance
GSHPx - I.Q. = + .58p < .001
GSHPx - age = - .27N.S.
I. Q. - age = - .23N.S.
GSHPx - I. Q./age = + .55p < .001
* We previously reported an increase in red cell GSHPx activity in a small number of patients (10). Table 1 provides data with a very much larger number of patients. ** At a recent meeting, International Workshop on Gene Mapping, Winnipeg, August, 1977, it was suggested that GSHPx gene is associated with chromosome 3.



1. P. M. SINET, D. ALLARD, J. LEJEUNE and H. JEROME, C. R. Acad. Sci., Paris, 278, 3267-3270 (1974).

2. S. SICHITIU, P. M. SINET, J. LEJEUNE and J. FREZAL, Humangenetik 23, 65-72 (1974).

3. R. R. FRANTS, A. N. ERIKSSON, P. H. JONGBLOET and A. J. HAMERS, Lancet ii, 42-43 (1975).

4. P. M. SINET, F. LAVELLE, A. M. MICHELSON and H. JEROME, Biochem. Biophys. Res. Commun. 67, 904-909 (1975).

5. W. W. FEASTER, L. W. KWOK and C. J. EPSTEIN, Am. J. Hum. Genet. 29, 563-570 (1977).

6. Y. H. TAN, J. TISHFIELD and F. H. RUDDLE, J Med. 137, 317-330 (1973).


8. J. M. McCORD and I. FRIDOVICH, J. Biol. Chem. 244, 6049-6055 (1969).

9. S. N. PANTEKALIS,. A. G. KARAKLIS, D. ALEXIOU, E. VARDAS and T. VALAES, Amer. J. Hum. Genet. 22, 184-193 (1970).

10. P. M. SINET, A. M. MICHELSON, A. BAZIN, J. LEJEUNE and H. JEROME, Biochem. Biophys. Res. Commun. 67, 910-915 (1975).

11. D. E. PAGLIA and W. N. VALENTINE, J. Lab. Clin. Med. 70, 158-169 (1967).

12. W. A. GUNZLER, Glutathione, p. 180, Georg Thieme Publishers, Stuttgart (1974).

13. H. U. BERGMEYER, E. BERNT, F. SCHMIDT and H. STORK, Methods of Enzymatic Anal, p. 1196, Academic Press, N. Y. and London, (1974).

14. A. G. BAIKIE, P. BRONWEN LODER, G. C. DE GRUCHY and D. B. PITT, Lancet i, 412-414 (1965).

15. P. F. BENSON, B. LINACRE and A. I. TAYLOR, Nature 220, 1235-1236 (1968).

16. G. F. SMITH and J. M. BERG, Down's Anomaly pp. 61-75, J. Churchill et A. London, (1976).

17. F. STRUEW, Z. Ges. Neurol. Psychiat. 122, 291-307 (1929).

18. P. T. OHARA, Brain 95, 681-684 (1972)

19. S. S. SCHOCHET, P. W. LAMPERT and W. J. McCORMICK, Acta Neuro ath. (Berl.) 23, 342-346 (1973) .

20. E. L. SCHNEIDER and C. J. EPSTEIN, Proc. Soc. Exp. Biol. Med. 141, 1092-1094 (1972).

21. D. J. SEGAL and E. E. McCOY, J. Cell. Physiol. 83, 85-90 (1974).

22. C. LITTLE and P. J. O'BRIEN, Biochem. Biophys. Res. Commun. 31, 145-150 (1968).

23. D. HARMAN, J. Geront. 11, 298-300 (1956).

24. J. R. PROHASKA and H. E. GANTHER, _J. Neurochem. 27, 1379-1387 (1976).

25. R. B. LAYSER and C. J. EPSTEIN, Amer. J. Hum. Genet. 24, 533-543 (1972).