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The effect of experimental anaemia on red cell glutathione reductase
Camp.
Biochem. Phvsiol. Vol.
0300-9629/84$3.00
79A. No. 3, pp. 413418, 1984
8
Printed in Great Britain
THE EFFECT OF EXPERIMENTAL ANAEMIA RED CELL GLUTATHIONE REDUCTASE
1984
fO.00
Pergamon Press Ltd
ON
T. SUZUKI*, N. S. AGAR~ and M. SUZUKI Department of Veterinary Medicine, Faculty of Agriculture, Gifu University, Gifu, Gifu 501-l 1, Japan (Received 9 March 1984)
Abstract-l. The effect of experimental anaemia on red cell glutathione reductase (GR) was investigated in rabbits, guinea-pigs and cattle. 2. The ratio of active and inactive forms of GR did not change during the experimental period in either the rabbits or the cattle. However, there was a marked rise in the ratio of inactive form in the guinea-pigs during anaemia. 3. It appears that, in addition to the most important regulator of GR activity, namely the flavin content of the red blood cells, there are other mechanisms which are operating in the red blood cells of anaemic animals and that these mechanisms vary among different species of animals.
has been observed in several animal species (see Agar and Board, 1983), none of these studies clearly report whether the rise in CR is related to the changes in the active or inactive forms. The aim of the present investigation was, therefore, to examine the changes in both active and inactive forms of GR during experimental anaemia in rabbits, guinea-pigs and cattle.
INTRODUCTION Glutathione reductase GSSG to GSH: NADPH
(or NADH)
+ GSSG 2
(GR) catalyses
the reaction
of
+ H+
NADP+
(or NAD+)
+ 2GSH.
Thus, GR is primarily responsible for the maintenance of the intercellular GSH concentration which, in turn, plays an important role in the protection of haemoglobin and several other structural proteins and enzymes of the red blood cells against the oxidative damage. It is now generally accepted that red cell GR consists mainly of two forms, an “active form”, which is saturated with FAD and an “inactive form”, which is not saturated with FAD (Beutler, 1969; Staal et al., 1969). The degree of in vitro stimulation of GR activity is dependent on the saturation with FAD, and hence this activation has been used as an indication of the riboflavin status in humans and rats (Glatzle et al., 1968; Bamji, 1969; Prentice and Bates, 1981). GR in the red blood cells of human and other animals has been the subject of extensive study. Higher activity of GR observed in the patient with severe hepatic cirrhosis, chronic uremia and G-6-PD deficiency has been suggested to be due to a marked increase in the degree of saturation of GR with FAD (Yawata and Tanaka, 1974). Brady et al. (1977) demonstrated that the red cell GR activity rapidly increases in equine red cell in response to exercise. The activity is also elevated in fasting dogs. The ratio of active forms also show marked changes in both these species (Brady et al., 1977, 1978). Although the effect of experimental anaemia on the activity of GR
*Present address: Department of Human Biology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601, Australia. tPresent address: Department of Physiology, University of New England, Armidale, N.S.W. 2351, Australia.
MATERIALS AND
METHODS
Rabbits
Eight rabbits were used, four of these (males aged four months, New Zealand White) served as a control and in the other four (males, aged nine months, Japanese White) anaemia was produced by subcutaneous injection of phenvlhvdrazine-hvdrochloride (PHH) (16 ma/kg bodv wt) on day -0 and 3 as previously‘described by &uk; and Kataoka (1983). The blood was obtained by cardiac puncture from both control and anaemic groups on days 0, 5, 9, 12 and 20 (Fig. 1) and was collected into 5 ml vials containing heparin. Guinea-pigs
Twenty guinea-pigs (males, aged three months, Hartley) were used; four of these provided control values on day 0 and the remaining sixteen animals were injected with PHH intradermally (16 mg/kg body wt) on days 0 and 3 by the method of Suzuki and Kataoka (1983). The blood samples were obtained by cardiac puncture from all the animals. Four separate animals were bled on days 5, 7, 9 and 12 and blood was collected into 5 ml vials containing heparin (Fig. 1). Cattle Three cattle (females, aged about one year, HolsteinFriesian) were used. Normal values were established in these cows over a period of about one week. The animals were then bled by jugular venepuncture at a rate of approximately 10 ml/kg body wt. After 3 or 4 days, when packed cell volume (PCV) had dropped to under 22”/, and haemoglobin (Hb) concentration- to under 7.5 g/d< the phlebotomy was discontinued. The blood samples for analysis, taken at regular intervals over the next 44 days, were obtained by jugular venepuncture and collected into 10 ml vials containing heparin (Fig. 1). 473
474 Guinea
Rabbit
pig
Colltr0l (4) Anaemia (4)
Cattle
(3)
t t
\
+
+
t
+
t
t
t
\
t0
+
2
4
t
6
6
t
+ 10
-
+
12
14
+ 16
16
. ..L.t..t..f 20
23 303744
DSYS
Fig. 1. Schematic representation
of experimental anaemia in guinea pigs, rabbits
PCV and Hb levels were determined by standard laboratory methods. Red cell GR activity was measured by the method of Beutler (1975). GR activity measured with FAD is referred to as total GR, without FAD as active form and the difference between total GR and active form as inactive form of the enzyme. RESULTS
PCV and Hb were decreased to about half of their control values in all three species of animals; in the rabbit to 20.1% and 5.5 g/d1 after day 5 (control values; 40.5% and 14.3 g/dl), in the guinea-pigs to 29.4% and 7.5 g/d1 after day 7 (control values; 44.3% and 15.1 g/dl), and in cattle 21.7% and 7.4 g/d1 after day 4 (control value: 37.7% and 12.6 g/dl). Both PCV and Hb returned to normal levels by day 20 in the rabbits, day 12 in the guinea-pigs and day 18 in cattle. Total GR activity increased in all three species of animals examined. The maximum activity was reached on day 12 in the rabbits (from the control value of 6.46 IU/gHb to 12.05 IU/gHb), on day 9 in the guinea-pigs (from 19.36 IU/gHb to 26.61 IU/ gHb) and on day 18 in the cattle (from 1.16 IU/gHb to 2.05 IU/gHb). There were marked species differences in the changes of active and inactive forms of GR and also in the relative ratio of the two forms of GR. It is evident from Figs 2 and 4 that the activities of both forms of GR increased in the rabbits and cattle, implying that there was no change in the ratio of activity of active form of the enzyme. The activity of inactive form rose sharply in the guinea-pigs, especially on days 7 and 9. Consequently, there was a marked rise in the ratio of inactive form (from 23.3% to 43.9% by day 7, Fig. 3). In the control rabbits, PCV, Hb, the activity of total GR, active form and inactive form, and the ratio of active and inactive forms did not change during the whole experimental period (Fig. 5). This indicates that repeated blood collections by cardiac puncture does not appear to affect the red cell GR activity. DISCUSSION
The present observations that the activity of total GR increases after the anaemia is induced, confirm
and cattle.
the previous investigations on sheep (Agar et al., 1975; Smith and Lee, 1976), dogs (Smith and Agar, 1975), cattle (Smith et al., 1972), rabbits (Rodovien et al., 1974; Suzuki and Kataoka, 1983) and guineapigs (Suzuki and Kataoka, 1983). No change in GR activity was, however, found by Kaneko et al. (1969) during equine infectious anaemia. It is therefore suggested that the response of red cell metabolism to anaemia induced by the phlebotomy or by the injection of PHH is different from that induced by infection. As mentioned above, previous studies on the effects of anaemia on the levels of the red cell GR in several animals did not include the relative changes in active and inactive forms of the enzyme. The present results clearly indicate that the changes in active and inactive forms of GR during the recovery period of anaemia varies in different animals; the degree of saturation of GR with FAD (i.e. active form) is lower in the guinea-pig during anaemia. No such change occurs in the saturation of GR in the rabbits and cattle (Figs 2 and 4). These results, therefore, indicate that the activity of total GR in the young red cells is higher in all the three species; however, there appears to be interspecies variation with regard to the relative ratio of active and inactive forms of GR. The young red cells produced during anaemia in the guinea-pigs contain GR which is less saturated with FAD (i.e. active form) than those produced during normal conditions. However, both young and normal red cells contain similar proportions of active and inactive forms of GR in rabbits and cattle. Studies carried out on young and old red cells from humans obtained by fractionation procedures are also conflicting. Powers and Thurnham (1977) reported that the degree of saturation of GR in the young red cells is higher than in the old red cells, while Yamauchi et al. (1978) have shown that the ratio of active and inactive forms of GR are the same in young and old red cells. Extensive investigations have also been carried out on the changes in active and inactive forms of GR in red blood cells obtained from normal humans and those from patients suffering from various metabolic disorders. Yawata and Tanaka (1971) have shown that there is a rise of GR activity in red blood cells from uremics, cirrhotics and G-6-PD deficient sub-
Red cell glutathione reductase during anaemia GUINEA
RABBI’1
PIG
Pm 1 50
i
20 t
00 0
cn.ctrv* 3
5
e
12
20
Days
term
0 0
3
5 Days
7
9
12
Fig. 2. PCV, Hb, total CR, GR active form, GR inactive form and percentage of both GR forms in rabbits during experimental anaemia.
Fig. 3. PCV, Hb, total GR, GR active form, GR inactive form and percentage of both GR forms in guinea-pigs during experimental anaemia.
jects, and that this rise in GR is due to elevation
and also from adult patients with metabolic disorders, GR in cord red cells is only partially saturated with FAD in spite of a marked increase of flavin compounds in plasma and red blood ceils (Yawata and Tanaka, 1974). Pispa and Huttenen (1966) have suggested that although both reticuiocytes and mature red cells in rat incorporate radioactive riboflavin into FAD, the reticulocytes are more effective in this
active form. the degree represent a integrity of
in They have suggested that the increase in of saturation of GR with FAD might regulatory mechanism to maintain the the red blood cells in severe metabolic
disorders. They have also shown that although total GR activity of red blood cells from neonates is considerably higher than those from adult humans
476
T.
?h~zUKl
et
al.
Fig. 4. PCV, Hb, total GR, GR active form, GR inactive form and percentage of both GR forms in cattle during experimental anaemia.
than the mature red blood cells. Intact human red cells have been shown to synthesize FAD from the riboflavin by ATP and the rate of synthesis of FAD is dependent on the concentration of riboflavin in the medium (Mandula and Beutler, 1970). These results suggest that although the most important relator of GR activity is the flavin content incorporation
of the red blood cell, other m~hanisms are also operating which regulate the GR activity in the red blood cells produced during anaemia. One such mechanism may be directly related to the capacity of the red blood cells to incorporate riboflavin from plasma. Another mechanism influencing the level of active and inactive forms of GR in the red blood cells may be blood pH. Miller et al. (1976) have shown a
Red cell glutathione
“,I
.
__
reductase
j
during
411
anaemia
or jointly regulates GR activity in the guinea-pigs during experimental anaemia. It is, however, evident that the “stress” reticulocytes in the guinea-pigs have higher activity of GR and lower saturation of GR with FAD, while this is not so in the rabbits and cattle. The results obtained in the present study tend to suggest that the biochemistry of red cell ageing in general and GR activity in relation to FAD (i.e. active and inactive forms) during experimental anaemia in particular, is more complex and that in addition to the kinetic properties of GR, additional mechanisms, yet unknown, control the inter-relationship of GR and FAD. Acknowledgements-We would like to thank Dr M. Nakagawa (National Institute of Health, Japan) for providing guinea-pigs. This work was supported by a grant from the University of New England (N.S.A.), and a Visiting Fellowship (MS.) and a Student Exchange Scholarship (T.S.) from the Ministry of Education, Japanese Government. REFERENCES
5
9
20
12
20
Fig. 5. PCV, Hb, total GR, GR active form, GR inactive form and percentage of both GR forms in rabbits as control.
rise in blood pH during anaemia in the rat. Brady et al. (1977) found that the inactive form of GR in-
creases in horses after exercise and suggested that this increase could reflect a response to insufficient absolute concentrations of NADPH or a response to apparent insufficiency brought on by declining blood pH which would increase the X, for NADPH. It is possible
that
either
one of these factors
independently
Agar N. S. and Board P. G. (1983) Red cell metabolism. In Red Blood Cells of Domestic Mammals (Edited by Agar N. S. and Board P. G.), pp. 227-251. Elsevier, Amsterdam. Agar N. S., Roberts J. and Mulley A. (1975) The effect of experimental anaemia on the levels of glutathione and glycolytic enzymes of the erythrocytes of normal and glutathione-deficient merino sheep. Aust. J. biol. Sci. Zs, 233-238. Bamji M. S. (1969) Glutathione reductase activity in red blood cells and riboflavin nutritional status in humans. Clin. chim. Acta 26, 263-269. Beutler E. (1969) Effect of flavin compounds on glutathione reductase activity: In viva and in vitro studies. J. clin. Invest. 48, 1951-1966. Beutler E. (1975) Red Cell Metabolism: A Manual of Biochemical Methods, 2nd edn. Grune & Stratton, New York. Brady P. S., Brady L. J. and Ullrey D. E. (1978) Interrelationship of erythrocyte glutathione peroxidase system enzymes in the fasted beagle. Am. J. vet. Res. 29,261-210. Brady P. S., Shelle J. E. and Ullrey D. E. (1977) Rapid changes in equine erythrocyte glutathione reductase with exercise. Am. J. vet. Res. 38, 1045-1047. Glatzle D., Weber F. and Wiss 0. (1968) Enzymatic test for the detection of riboflavin deficiency. NADPH-dependent glutathione reductase of red cells and its activation by FAD in v&-o. Experientia 24, 1122. Kaneko J. J., Tanaka J. and Nakajima H. (1969) Enzymes of equine erythrocytes: Changes during equine infectious anaemia. Am. J. vet. Res. 30, 543-549. Mandula B. and Beutler E. (1970) Synthesis of riboflavin nucleotides by mature human erythrocytes. Blood 36, 491499. Miller E. F., Rosth M. and Stohlman F. Jr. (1976) The effects of acute bleeding on acid-base balance, erythropoietin (Ep) production and in viva P50 in the rat. Br. J. Haematol. 33, 379-385. Pispa J. and Huttenen M. (1966) In vifro incorporation of 14C-riboflavin into flavin adenine dinucleotide in rat red cells. Annls med. exp. Biol. Fenn. 44, 504-506. Powers H. J. and Thurnham D. J. (1977) Influence of red cell on the measurement of riboflavin status. Nutr. Metab. 21, 155-157. Prentice A. M. and Bates C. J. (1981) A biochemical evaluation of the erythrocyte glutathione reductase (EC 1.6.4.2) test for riboflavin status. 1. Rate and specificity of response in acute deficiency. Br. J. Nutr. 45, 37-52.
478
T. SUZUKI et al.
Rodvien R., Gillum A. and Weintraub L. R. (1974) Decreased glutathione peroxidase activity secondary to severe iron deficiency: A possible mechanism responsible for the shortened life span of the iron-deficient red cell. Blood 43, 281-289. Smith J. E. and Agar N. S. (1975) The effect of phlebotomy on canine erythrocyte metabolism. Res. vet. Sci. 18, 23 l-236. Smith J. E. and Lee M. (1976) How phlebotomy affects the ervthrocvte metabolism of hiah- and low-elutathione sheep. Ru. vet. Sci. 21, 127-l%. Smith J. E., McCarto M. and Parks P. (1972) Influence of erthrocyte age on enzyme activity in the bovine. Camp. Biochem. Physiol. 41B, 551-558. Staal G. E. J., Nisser J. and Neeger C. (1969) Purifications
and properties of glutathione reductase of human erythrocytes. Biochim. biophys. Acre 185, 39-48. Suzuki M. and Kataoka T. (1983) Effect of experimental anaemia on red cell GSH and enzyme activities in guinea pig and rabbit. Comp. Biochem. Physiol. 74B, 5-8. Yamauchi K., Nakashima K. and Miwa S. (1978) Human erthrocyte glutathione reductase. II. Evidence for molecular ageing of normal erythrocytes and apoenzyme defect in aplastic anaemia. Acta haem. jap. 41, 3444. Yawata Y. and Tanaka K. R. (1971) Effect of metabolic stress on activation of glutathione reductase by FAD in human red cells. Experientio 27, 1214-1215. Yawata Y. and Tanaka K. R. (1974) Regulatory mechanism of glutathione reductase activity in human red cell. Blood 43, 99-109.
Biochem. Phvsiol. Vol.
0300-9629/84$3.00
79A. No. 3, pp. 413418, 1984
8
Printed in Great Britain
THE EFFECT OF EXPERIMENTAL ANAEMIA RED CELL GLUTATHIONE REDUCTASE
1984
fO.00
Pergamon Press Ltd
ON
T. SUZUKI*, N. S. AGAR~ and M. SUZUKI Department of Veterinary Medicine, Faculty of Agriculture, Gifu University, Gifu, Gifu 501-l 1, Japan (Received 9 March 1984)
Abstract-l. The effect of experimental anaemia on red cell glutathione reductase (GR) was investigated in rabbits, guinea-pigs and cattle. 2. The ratio of active and inactive forms of GR did not change during the experimental period in either the rabbits or the cattle. However, there was a marked rise in the ratio of inactive form in the guinea-pigs during anaemia. 3. It appears that, in addition to the most important regulator of GR activity, namely the flavin content of the red blood cells, there are other mechanisms which are operating in the red blood cells of anaemic animals and that these mechanisms vary among different species of animals.
has been observed in several animal species (see Agar and Board, 1983), none of these studies clearly report whether the rise in CR is related to the changes in the active or inactive forms. The aim of the present investigation was, therefore, to examine the changes in both active and inactive forms of GR during experimental anaemia in rabbits, guinea-pigs and cattle.
INTRODUCTION Glutathione reductase GSSG to GSH: NADPH
(or NADH)
+ GSSG 2
(GR) catalyses
the reaction
of
+ H+
NADP+
(or NAD+)
+ 2GSH.
Thus, GR is primarily responsible for the maintenance of the intercellular GSH concentration which, in turn, plays an important role in the protection of haemoglobin and several other structural proteins and enzymes of the red blood cells against the oxidative damage. It is now generally accepted that red cell GR consists mainly of two forms, an “active form”, which is saturated with FAD and an “inactive form”, which is not saturated with FAD (Beutler, 1969; Staal et al., 1969). The degree of in vitro stimulation of GR activity is dependent on the saturation with FAD, and hence this activation has been used as an indication of the riboflavin status in humans and rats (Glatzle et al., 1968; Bamji, 1969; Prentice and Bates, 1981). GR in the red blood cells of human and other animals has been the subject of extensive study. Higher activity of GR observed in the patient with severe hepatic cirrhosis, chronic uremia and G-6-PD deficiency has been suggested to be due to a marked increase in the degree of saturation of GR with FAD (Yawata and Tanaka, 1974). Brady et al. (1977) demonstrated that the red cell GR activity rapidly increases in equine red cell in response to exercise. The activity is also elevated in fasting dogs. The ratio of active forms also show marked changes in both these species (Brady et al., 1977, 1978). Although the effect of experimental anaemia on the activity of GR
*Present address: Department of Human Biology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601, Australia. tPresent address: Department of Physiology, University of New England, Armidale, N.S.W. 2351, Australia.
MATERIALS AND
METHODS
Rabbits
Eight rabbits were used, four of these (males aged four months, New Zealand White) served as a control and in the other four (males, aged nine months, Japanese White) anaemia was produced by subcutaneous injection of phenvlhvdrazine-hvdrochloride (PHH) (16 ma/kg bodv wt) on day -0 and 3 as previously‘described by &uk; and Kataoka (1983). The blood was obtained by cardiac puncture from both control and anaemic groups on days 0, 5, 9, 12 and 20 (Fig. 1) and was collected into 5 ml vials containing heparin. Guinea-pigs
Twenty guinea-pigs (males, aged three months, Hartley) were used; four of these provided control values on day 0 and the remaining sixteen animals were injected with PHH intradermally (16 mg/kg body wt) on days 0 and 3 by the method of Suzuki and Kataoka (1983). The blood samples were obtained by cardiac puncture from all the animals. Four separate animals were bled on days 5, 7, 9 and 12 and blood was collected into 5 ml vials containing heparin (Fig. 1). Cattle Three cattle (females, aged about one year, HolsteinFriesian) were used. Normal values were established in these cows over a period of about one week. The animals were then bled by jugular venepuncture at a rate of approximately 10 ml/kg body wt. After 3 or 4 days, when packed cell volume (PCV) had dropped to under 22”/, and haemoglobin (Hb) concentration- to under 7.5 g/d< the phlebotomy was discontinued. The blood samples for analysis, taken at regular intervals over the next 44 days, were obtained by jugular venepuncture and collected into 10 ml vials containing heparin (Fig. 1). 473
474 Guinea
Rabbit
pig
Colltr0l (4) Anaemia (4)
Cattle
(3)
t t
\
+
+
t
+
t
t
t
\
t0
+
2
4
t
6
6
t
+ 10
-
+
12
14
+ 16
16
. ..L.t..t..f 20
23 303744
DSYS
Fig. 1. Schematic representation
of experimental anaemia in guinea pigs, rabbits
PCV and Hb levels were determined by standard laboratory methods. Red cell GR activity was measured by the method of Beutler (1975). GR activity measured with FAD is referred to as total GR, without FAD as active form and the difference between total GR and active form as inactive form of the enzyme. RESULTS
PCV and Hb were decreased to about half of their control values in all three species of animals; in the rabbit to 20.1% and 5.5 g/d1 after day 5 (control values; 40.5% and 14.3 g/dl), in the guinea-pigs to 29.4% and 7.5 g/d1 after day 7 (control values; 44.3% and 15.1 g/dl), and in cattle 21.7% and 7.4 g/d1 after day 4 (control value: 37.7% and 12.6 g/dl). Both PCV and Hb returned to normal levels by day 20 in the rabbits, day 12 in the guinea-pigs and day 18 in cattle. Total GR activity increased in all three species of animals examined. The maximum activity was reached on day 12 in the rabbits (from the control value of 6.46 IU/gHb to 12.05 IU/gHb), on day 9 in the guinea-pigs (from 19.36 IU/gHb to 26.61 IU/ gHb) and on day 18 in the cattle (from 1.16 IU/gHb to 2.05 IU/gHb). There were marked species differences in the changes of active and inactive forms of GR and also in the relative ratio of the two forms of GR. It is evident from Figs 2 and 4 that the activities of both forms of GR increased in the rabbits and cattle, implying that there was no change in the ratio of activity of active form of the enzyme. The activity of inactive form rose sharply in the guinea-pigs, especially on days 7 and 9. Consequently, there was a marked rise in the ratio of inactive form (from 23.3% to 43.9% by day 7, Fig. 3). In the control rabbits, PCV, Hb, the activity of total GR, active form and inactive form, and the ratio of active and inactive forms did not change during the whole experimental period (Fig. 5). This indicates that repeated blood collections by cardiac puncture does not appear to affect the red cell GR activity. DISCUSSION
The present observations that the activity of total GR increases after the anaemia is induced, confirm
and cattle.
the previous investigations on sheep (Agar et al., 1975; Smith and Lee, 1976), dogs (Smith and Agar, 1975), cattle (Smith et al., 1972), rabbits (Rodovien et al., 1974; Suzuki and Kataoka, 1983) and guineapigs (Suzuki and Kataoka, 1983). No change in GR activity was, however, found by Kaneko et al. (1969) during equine infectious anaemia. It is therefore suggested that the response of red cell metabolism to anaemia induced by the phlebotomy or by the injection of PHH is different from that induced by infection. As mentioned above, previous studies on the effects of anaemia on the levels of the red cell GR in several animals did not include the relative changes in active and inactive forms of the enzyme. The present results clearly indicate that the changes in active and inactive forms of GR during the recovery period of anaemia varies in different animals; the degree of saturation of GR with FAD (i.e. active form) is lower in the guinea-pig during anaemia. No such change occurs in the saturation of GR in the rabbits and cattle (Figs 2 and 4). These results, therefore, indicate that the activity of total GR in the young red cells is higher in all the three species; however, there appears to be interspecies variation with regard to the relative ratio of active and inactive forms of GR. The young red cells produced during anaemia in the guinea-pigs contain GR which is less saturated with FAD (i.e. active form) than those produced during normal conditions. However, both young and normal red cells contain similar proportions of active and inactive forms of GR in rabbits and cattle. Studies carried out on young and old red cells from humans obtained by fractionation procedures are also conflicting. Powers and Thurnham (1977) reported that the degree of saturation of GR in the young red cells is higher than in the old red cells, while Yamauchi et al. (1978) have shown that the ratio of active and inactive forms of GR are the same in young and old red cells. Extensive investigations have also been carried out on the changes in active and inactive forms of GR in red blood cells obtained from normal humans and those from patients suffering from various metabolic disorders. Yawata and Tanaka (1971) have shown that there is a rise of GR activity in red blood cells from uremics, cirrhotics and G-6-PD deficient sub-
Red cell glutathione reductase during anaemia GUINEA
RABBI’1
PIG
Pm 1 50
i
20 t
00 0
cn.ctrv* 3
5
e
12
20
Days
term
0 0
3
5 Days
7
9
12
Fig. 2. PCV, Hb, total CR, GR active form, GR inactive form and percentage of both GR forms in rabbits during experimental anaemia.
Fig. 3. PCV, Hb, total GR, GR active form, GR inactive form and percentage of both GR forms in guinea-pigs during experimental anaemia.
jects, and that this rise in GR is due to elevation
and also from adult patients with metabolic disorders, GR in cord red cells is only partially saturated with FAD in spite of a marked increase of flavin compounds in plasma and red blood ceils (Yawata and Tanaka, 1974). Pispa and Huttenen (1966) have suggested that although both reticuiocytes and mature red cells in rat incorporate radioactive riboflavin into FAD, the reticulocytes are more effective in this
active form. the degree represent a integrity of
in They have suggested that the increase in of saturation of GR with FAD might regulatory mechanism to maintain the the red blood cells in severe metabolic
disorders. They have also shown that although total GR activity of red blood cells from neonates is considerably higher than those from adult humans
476
T.
?h~zUKl
et
al.
Fig. 4. PCV, Hb, total GR, GR active form, GR inactive form and percentage of both GR forms in cattle during experimental anaemia.
than the mature red blood cells. Intact human red cells have been shown to synthesize FAD from the riboflavin by ATP and the rate of synthesis of FAD is dependent on the concentration of riboflavin in the medium (Mandula and Beutler, 1970). These results suggest that although the most important relator of GR activity is the flavin content incorporation
of the red blood cell, other m~hanisms are also operating which regulate the GR activity in the red blood cells produced during anaemia. One such mechanism may be directly related to the capacity of the red blood cells to incorporate riboflavin from plasma. Another mechanism influencing the level of active and inactive forms of GR in the red blood cells may be blood pH. Miller et al. (1976) have shown a
Red cell glutathione
“,I
.
__
reductase
j
during
411
anaemia
or jointly regulates GR activity in the guinea-pigs during experimental anaemia. It is, however, evident that the “stress” reticulocytes in the guinea-pigs have higher activity of GR and lower saturation of GR with FAD, while this is not so in the rabbits and cattle. The results obtained in the present study tend to suggest that the biochemistry of red cell ageing in general and GR activity in relation to FAD (i.e. active and inactive forms) during experimental anaemia in particular, is more complex and that in addition to the kinetic properties of GR, additional mechanisms, yet unknown, control the inter-relationship of GR and FAD. Acknowledgements-We would like to thank Dr M. Nakagawa (National Institute of Health, Japan) for providing guinea-pigs. This work was supported by a grant from the University of New England (N.S.A.), and a Visiting Fellowship (MS.) and a Student Exchange Scholarship (T.S.) from the Ministry of Education, Japanese Government. REFERENCES
5
9
20
12
20
Fig. 5. PCV, Hb, total GR, GR active form, GR inactive form and percentage of both GR forms in rabbits as control.
rise in blood pH during anaemia in the rat. Brady et al. (1977) found that the inactive form of GR in-
creases in horses after exercise and suggested that this increase could reflect a response to insufficient absolute concentrations of NADPH or a response to apparent insufficiency brought on by declining blood pH which would increase the X, for NADPH. It is possible
that
either
one of these factors
independently
Agar N. S. and Board P. G. (1983) Red cell metabolism. In Red Blood Cells of Domestic Mammals (Edited by Agar N. S. and Board P. G.), pp. 227-251. Elsevier, Amsterdam. Agar N. S., Roberts J. and Mulley A. (1975) The effect of experimental anaemia on the levels of glutathione and glycolytic enzymes of the erythrocytes of normal and glutathione-deficient merino sheep. Aust. J. biol. Sci. Zs, 233-238. Bamji M. S. (1969) Glutathione reductase activity in red blood cells and riboflavin nutritional status in humans. Clin. chim. Acta 26, 263-269. Beutler E. (1969) Effect of flavin compounds on glutathione reductase activity: In viva and in vitro studies. J. clin. Invest. 48, 1951-1966. Beutler E. (1975) Red Cell Metabolism: A Manual of Biochemical Methods, 2nd edn. Grune & Stratton, New York. Brady P. S., Brady L. J. and Ullrey D. E. (1978) Interrelationship of erythrocyte glutathione peroxidase system enzymes in the fasted beagle. Am. J. vet. Res. 29,261-210. Brady P. S., Shelle J. E. and Ullrey D. E. (1977) Rapid changes in equine erythrocyte glutathione reductase with exercise. Am. J. vet. Res. 38, 1045-1047. Glatzle D., Weber F. and Wiss 0. (1968) Enzymatic test for the detection of riboflavin deficiency. NADPH-dependent glutathione reductase of red cells and its activation by FAD in v&-o. Experientia 24, 1122. Kaneko J. J., Tanaka J. and Nakajima H. (1969) Enzymes of equine erythrocytes: Changes during equine infectious anaemia. Am. J. vet. Res. 30, 543-549. Mandula B. and Beutler E. (1970) Synthesis of riboflavin nucleotides by mature human erythrocytes. Blood 36, 491499. Miller E. F., Rosth M. and Stohlman F. Jr. (1976) The effects of acute bleeding on acid-base balance, erythropoietin (Ep) production and in viva P50 in the rat. Br. J. Haematol. 33, 379-385. Pispa J. and Huttenen M. (1966) In vifro incorporation of 14C-riboflavin into flavin adenine dinucleotide in rat red cells. Annls med. exp. Biol. Fenn. 44, 504-506. Powers H. J. and Thurnham D. J. (1977) Influence of red cell on the measurement of riboflavin status. Nutr. Metab. 21, 155-157. Prentice A. M. and Bates C. J. (1981) A biochemical evaluation of the erythrocyte glutathione reductase (EC 1.6.4.2) test for riboflavin status. 1. Rate and specificity of response in acute deficiency. Br. J. Nutr. 45, 37-52.
478
T. SUZUKI et al.
Rodvien R., Gillum A. and Weintraub L. R. (1974) Decreased glutathione peroxidase activity secondary to severe iron deficiency: A possible mechanism responsible for the shortened life span of the iron-deficient red cell. Blood 43, 281-289. Smith J. E. and Agar N. S. (1975) The effect of phlebotomy on canine erythrocyte metabolism. Res. vet. Sci. 18, 23 l-236. Smith J. E. and Lee M. (1976) How phlebotomy affects the ervthrocvte metabolism of hiah- and low-elutathione sheep. Ru. vet. Sci. 21, 127-l%. Smith J. E., McCarto M. and Parks P. (1972) Influence of erthrocyte age on enzyme activity in the bovine. Camp. Biochem. Physiol. 41B, 551-558. Staal G. E. J., Nisser J. and Neeger C. (1969) Purifications
and properties of glutathione reductase of human erythrocytes. Biochim. biophys. Acre 185, 39-48. Suzuki M. and Kataoka T. (1983) Effect of experimental anaemia on red cell GSH and enzyme activities in guinea pig and rabbit. Comp. Biochem. Physiol. 74B, 5-8. Yamauchi K., Nakashima K. and Miwa S. (1978) Human erthrocyte glutathione reductase. II. Evidence for molecular ageing of normal erythrocytes and apoenzyme defect in aplastic anaemia. Acta haem. jap. 41, 3444. Yawata Y. and Tanaka K. R. (1971) Effect of metabolic stress on activation of glutathione reductase by FAD in human red cells. Experientio 27, 1214-1215. Yawata Y. and Tanaka K. R. (1974) Regulatory mechanism of glutathione reductase activity in human red cell. Blood 43, 99-109.