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).
Haut
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.
Haut
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) |
|---|
| CONTROLS | 7.87 ± 2.00 (48) | 3.03 ± .73
(8) |
| TRISOMY 21 | 10.94 ± 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 |
|---|
| CONTROLS | 2745 ± 365 (14) | 77.5 ± 10
(14) | 2.85 ± .38 (14) |
| TRISOMY 21 | 2786 ± 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 Coefficient | Statistical Significance |
|---|
| GSHPx - I.Q. = + .58 | p < .001 |
| GSHPx - age = - .27 | N.S. |
| I. Q. - age = - .23 | N.S. |
| GSHPx - I. Q./age = + .55 | p < .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. |
Haut
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