We have earlier described the increase of erythrocuprein (superoxide
dismutase, E. C. 1. 15. 1. 1. ) for which the gene is located on chromosome 21
(1, 2) in erythrocytes (3-6) and platelets (7) from trisomy 21 patients
compared with controls. As demonstrated in the pioneer work of McCord and
Fridovich (8) this enzyme catalyses the dismutation of superoxide radicals to
oxygen and H202.
As part of a programme of study of oxygen metabolism in trisomy 21
cases we have also examined the activity of erythrocyte glutathione
In the red blood cell, H202 produced biochemically or otherwise, can
be eliminated by the action of various enzymes such as catalase and glutathione
peroxidase (GPX). It is generally considered (9) that in erythrocytes, at low
concentrations of H202 the second enzyme catalyses reduction of H202
preferentially by the reaction :
2 GSH + H202 ---GPX---> GSSG + 2 H20
Previous studies have shown that catalase activity in erythrocytes
from cases of trisomy 21 is completely normal (5,10). It was thus of interest
to examine levels of glutathione peroxidase in such patients.
Material and methods
Twelve trisomy 21 subjects free of congenital cardiopathy were
compared with eighteen normal individuals of similar age, all older than three
Dosage of glutathione peroxidase (11) was by the modified method
described by Gunzler (12) using t-butyl hydroperoxide as substrate. Oxidised
glutathione (GSSG) produced by action of glutathione peroxidase and peroxide
was reconverted to reduced glutathione (GSH) by glutathione reductase (GR) and
nicotinamide adenine dinucleotide phosphate (NADPH). Decrease in concentration
of NADPH was recorded at 340 nm in a Gilford 2400 spectrophotometer.
Blood was drawn by veinous puncture using heparin as anticoagulant.
The red cells were separated by low speed centrifugation and washed three times
in 0.154 M NaCI, then lysed by freezing (at -70°C) and thawing. The lysates
were adjusted by dilution with distilled water to a uniform concentration of 5
g hemoglobin per 100 ml (using the method of Drabkin (13) for estimation of
hemoglobin) then mixed with an equal volume of a solution of 4 x
10-3 M potassium ferricyanide and 2 x 10-2 M KCN in 0.1 M
phosphate buffer pH 7.0.
The incubation mixture (3 ml) maintained at 37° C contained 5 x
10-2 M phosphate pH 7.0, 2 x 10-4 M NADPH,
10-3 M reduced glutathione, 2 units of yeast glutathione reductase
(Sigma type III) and 0.1 ml of the above erythrocyte lysate mixture. After 10
min pre-incubation, the reaction was initiated by addition of t-butyl
hydroperoxide (to a final concentration of 10-3 M). Kinetics of
oxidation of NADPH were calculated using a molar extinction coefficient for
NADPH of 6.22 x 103 at 340 nm. The slow oxidation of NADPH observed
before addition of t-butyl hydroperoxide was substracted from the values
obtained in presence of the peroxide.
As control, the same technique was used throughout except that the
erythrocyte lysate mixture was replaced by an equivalent amount of hemoglobin
free of glutathione peroxidase, obtained by treatment of a red blood cell
lysate in 3 x 10-3 M phosphate buffer pH 7.0 with DEAF-Sephadex A-50
to absorb the peroxidase. This blank was then substracted from the rates
observed in presence of the various samples.
Results and discussion
The results are shown in figure 1, expressed as µMoles of NADPH
consumed per min per mg of hemoglobin. Athough activities are relative, the
technique described above, with a molar ratio of K3FeCN6 : KCN : heme of 1. 2 :
6 : 1 and a corresponding heme control (see methods) gives a relatively
reliable estimate of glutathione peroxidase activity in erythrocytes (12).
The difference between average levels of glutathione peroxidase in the
two populations is highly significant (table 1) and the ratio of the averages
for trisomy 21 compared with normal subjects is 1. 55, closely similar to that
observed (3, 5) in the case of determinations of erythrocuprein (SOD - 1).
In contrast with the clear cut difference in distribution for
erythrocuprein, the levels of glutathione peroxidase activity for trisomy 21
cases and normal subjects partially overlap. It is not excluded that the
distribution of glutathione peroxidase values observed with trisomy 21 is
bimodal (see fig. 1).
Two hypotheses may be presented at the moment to explain the
concurrent increase of activity to the same extent for glutathione peroxidase
and erythrocuprein. The first implies that the genes for expression of both
proteins are located on chromosome 21 (as is the case for erythrocuprein) while
the second invokes participation of intracellular O2- or H2O2, or of
superoxide dismutase in the regulation of erythrocyte glutathione peroxidase
activity. With respect to the effect of superoxide dismutase, it is to be noted
that this enzyme can in fact increase intracellular levels of H2O2 by catalysed
dismutation of O2- as opposed, not to spontaneous dismutation (which
gives the same quantity of H2O2), but to elimination of O2- by
oxidative processes or by diffusion (given the relatively long life time of
O2- and the kinetics of dismutation by superoxide dismutase).
Apart from direct roles in the metabolism of activated oxygen
derivatives played by these two enzymes, glutathione peroxidase forms a link
between destruction of H2O2 and the hexose monophosphate metabolic pathway via
glutathione reductase and NADPH. Thus the often contradictory observations of
glucose metabolism (14,15) in cases of trisomy 21 could well be reconsidered in
the light of the results presented in this communication.
Figure 1. - Glutathione peroxidase activity in normal and trisomy 21
Table I. - Comparison of lutathione peroxidase activity in
erythrocytes from normal subjects and trisomy 21 patients.
|Number of subjects||µM NADPH/min/mg Hb||P
|Average||Standard deviation|| Standard
deviation of the mean
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