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Reticulocyte 59Fe uptake and total protein synthesis in riboflavin deficient rats
NUTRITION RESEARCH, Vol. 9, pp. 645-652, 1989 0271-5317/89 $3.00 + .00 Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.
RETICULOCYTE 59Fe UPTAKE AND TOTAL PROTEIN SYNTHESIS IN RIBOFLAVIN DEFICIENT RATS
Delana A. ADELEKAN, Ph.D Department of Human Nutrition London School of Hygiene & Tropical Medicine Keppel ~t. (Gower St.) London WCIE 7HT. U.K.
ABSTRACT Reticulocytosis was induced in rats depleted of riboflavin for 8 weeks and in their weight-matched (WM) controls by removal of 600ul of blood from the tall vein representing approximately 10% of total blood volume in each rat. Weight-matched rats responded to the bleeding with almost 10% reticulocytosis while the response in RD rats was significantly less (6%). Furthermore, reticulocytes from RD rats took up less iron from an incubation medium containing 59FeCIs, than retlculocytes from WM rats but the difference was not significant. Incorporation of a tritiated amino acid (3H-Leucine) into total proteins in reticulocytes was significantly less in those from RD than WM rats. The results of the present study suggest that the ineffective response of RD rats to bleeding and the decreased protein synthesis may be secondary to impairment of iron metabolism by riboflavin defiency. Key words: Riboflavin Deficiency, Reticulocytosis, Iron Uptake, Total Protein Synthesis. INTRODUCTION Studies in both humans and laboratory animals have shown an association between riboflavin and iron metabolism but the mechanism is not well understood. There is evidence however, that riboflavin plays a role in the mobilisation of iron from storage compartments via the action of the flavin-dependent enzyme, NADH-FMN Oxidoreductase (Ferriductase, EC 1.6.8.1.) (i) and hence in haemoglobin synthesis (2-4).
Present Address: Department of Paediatrics & Child Health Faculty of Health Sciences Obafemi Awolowo University Ile-Ife, Nigeria. 645
646
D.A. ADELEKAN
Experimental riboflavin deficiency has been associated with bone marrow hypoplasia and a reduced rate of iron utilisation for haemoglobin synthesis (5-7). Riboflavin deficiency has also been associated with varying degrees of anaemia in a variety of animal species and man (7-10). Recently we have shown that both absorption and liver storage of iron are impaired in riboflavin deficienbt rats
(11/. The present study was undertaken to investigate more closely haemopoietic activity in immature red cells from riboflavin deficient rats.
MATERIALS
AND METHODS
(a)
Animals - Male, weanling specific pathogen-free (SPF) albino rats (mean weight 46g; range 43.5 48g) of Wister strain were used. Rats were housed individually in white plastic wire bottommed cages in a well ventilated room at a temperature of about 26oC. One group of rats was fed a riboflavin-deficient diet ad llb for a period of 8 weeks. A second group of rats was fed the basal diet plus added riboflavin (l.lg/kg diet) in sufficient quantity only to maintain the same rate of growth as rats fed the riboflavin deficient diet. These are the weight-matched (WM) controls. The composition of the basal diet has been described else where (121.
(b)
Induction of reticulocytosis - At the end of the feeding period, riboflavin deficient (RD) rats or their weight-matched controls were bled from the tail vein on 3 consecutive days. 200ul of blood was removed on each day, making a total of 600ul removed from each rat; this volume represented approximately 10% of the total blood volume of each rat (5). Rats were killed 2 days after the last bleeding by drawing blood from the heart under light ether anaesthesia. Reticulocytes were counted by the method of Dacie and Lewis (13).
(c)
Radio-Iron (59Fe) Uptake by Reticulocytes - This was measured by the method of Edwards et al (14) with modifications. Briefly, normal rat plasma from fasted ad llb-control rats was tagged with radioactive iron by adding 2.3 ml of 59FeClz solution (specific activity 11.gmCi/mgFe; Amersham International plc, Amersham, Bucks, UK) to 7.7mi of normal rat plasma (final concentration 1.932ug Fe/ml plasma). This was incubated at 37oC for 80 minutes. Next, heparinised blood
RIBOFLAVIN DEFICIENCY from RD or WM rats was centrifuged at 2500xg for i0 minutes at room temperature. Plasma and bully coat layer were removed. The sendimented red cells were washed twice with 5 volumes of a solution containing 154mM - sodium chloride, 5mM potassium chloride and 5mM - magnesium chloride. Then 200ul of washed red cells were mixed with 800ul of 59Fe-labelled normal rat plasma and the mixture incubated at 37oC for 60 minutes. After incubation, the red cells were washed 3 times in 5 volumes of 154mM sodium chloride solution and then lysed in 4 volumes of cold distilled water. The haemolysate was centrifuged at 30,000xg for 30 minutes at room temperature to remove red cell "ghosts". The extent of haemolysis in the red cells was not further determined. 59Fe radioactivity in the red cell lysate was counted in a well-type gamma counter (model LKB 1280 Ultrogamma). Reticulocyte iron uptake was expressed as 59Fe cpm/107 reticulocytes. (d)
Total Protein Synthesis in Retlculocytes this was measured by the method of KonlJn, Hershko and Izak (15) with modifications. 200ul of saline-washed red cells from RD and WM rats were incubated with 10mCi of a tritiated amino acid (L (4,5,-3H) Leucine, 46Ci/mmol; Amersham International plc) at 37oC for 60 minutes. After incubation, the red cells were washed 3 times with cold normal saline, lysed in 4 volumes of cold distilled water and then centrifuged as described earlier. Then 50ul of lysate together with 0.Smg albumin (Sigma Chemical Company) was added to 3 ml of 5% (W/V) cold trichloroacetic acid (TCA), and the mixture centrifuged at l,O00xg for 5 minutes. The precipitate was washed once in cold 5% TCA, heated for 20 minutes at 85oC in 2ml of 5%c TCA and then sequentially washed with cold 5% TCA (2 times}, a I:I (V/V) mixture of ethanol and ether ( 2 times) and ether (once}. -
(e)
Riboflavin status of the rats was determined as previously described (12). Haematocrit was measured by the microhaematocrit method and total red blood cells were counted in a Coulter counter.
(f)
Statistical Analysis - Significance of differences between mean values was tested using the Student's "t" test with level of significance fixed at 5%.
647
648
D.A. ADELEKAN RESULTS
Table 1 shows the final body weights and haematological parameters of the lid and WM control rats. Although there was no significant difference in the body weights of rats in the 2 groups at the termination of the experiment, there were significant differences in the haematological parameters examined. Both the haematocrit (41.0 +/- 0.63%) and total red blood cell counts (6.88 +/- 0.43 x 106/ui) of RD rats were significantly lower than those of WM rats. However, the percentage of reticulocytes in circulation in RD rats (2.25%) prior bleeding was significantly higher than that in WM rats (1.2%; p < 0.01).
Table I: Final Body Weights and Haematological Parameters in Riboflavin Deficient and Control Rats Body Wt
(g) Riboflavin Deficient
RBC (xl06/ul)
Retic (%)
HCT (%)
103.0 +/-1.48
6.8 +/-0.43
2.25 +/-0.34
41.0 +/-0.63
105.0 +/-2.22
8.02 +/-0.33
1.2 +/-0.30
43.0 +/-1.09
0.01
0.01
(5) WM Controls (5) P <
ns
0.025
* Figures in parenthesis indicate number of rats in each group.
Table 2 shows the riboflavin status (EGRAC) and degree of reticulocytosis in RD or WM rats fed appropriate diets for 8 weeks. The mean +/- sem AC of RD rats, 3.13 +/- 0.45 was significantly higher than AC of WM controls, (1.64 +/0.22), indicating a poorer riboflavin status. RD rats responded to the repeated bleeding by producing significantly fewer reticulocytes than their weight-matched controls. Following withdrawal of approximately 10% of total blood volume, reticulocyte production in liD rats was only about 6% of total red cell in circulation; this represented approximately a 2 fold increase over the value before bleeding (2.25%). By contrast WM rats responded with reticulocyte production which was nearly 10% of total red cell in circulation, an increase of more than 7 fold over the mean value before bleeding (1.2%). The difference in mean values between WM controls and RD rats in both cases was statistically significant (p <
0.001).
RIBOFLAVIN DEFICIENCY
649
Table 2: Radio-iron Uptake and Total Protein Reticulocytes from RD and WM Rats. RD
Synthesis
WM
p <
59Fe Radioactivity (cpm/107 Reticulocytes)
302 +/-4.12
SH Radioactivity (DPM/106 Reticulocytes)
1744 +/-9.41
Reticulocytes
6.58 +/-0.4
9,78 +/-0.45
3.13
1.64
(%)*
EGRAC
in
350 +/-4.69
2346 +/-Ii.i
ns
0.05
0.001
0.01
Values are mean +/- sem for 5 rats in each group. *Rats bled from the tail vein on 3 consecutive days after 8 weeks of feeding a RD or control diet. Reticulocytes in blood were counted 2 days after the last bleeding.
The pattern of radio-iron uptake and total protein synthesis in RD and WM rats is shown in Table 2. When reitculocytes from RD and WM rats were incubated with radlo-iron (59Fe), RD reticulocytes incorporated less radio-iron than reticulocytes from WM rats but the difference was not statistically significant. However, incorporation of tritiated leucine into the cellular proteins was significantly less in reticulocytes from RD rats than in those from NM controls. Because of the it was not possible correlation between 59Fe or amino acid
small number of rats used in this study, to establish whether there was any riboflavin status (EGRAC) of rats and incorporation by reticulocytes.
DISCUSSION It has been reported that the rate of red blood cell production following repeated blood removal (phlebotomy) depends chiefly on availability of iron from body stores (16). In the present study, although the percent reticulocytes in circulation in RD rats was significantly higher than that in WM controls before bleeding, the value following repeated blood removal was significantly less in them. This indicated an impairment in the ability of the RD
650
D.A. ADELEKAN
rats to respond adequately to the further stress of bleeding. Iron for red cell synthesis reaches the bone marrow directly from transferrin in the plasma (17,18). Most of the iron in transferrin comes from the reticuloendothelial system where the iron is derived chiefly from haemoglobin catabolism of effete red cells (19,20). We have recently shown that iron concentration in the livers of riboflavin deficient rats was significantly less than that in weight matched controls from 3 weeks of deficiency (Ii). This partly explains the significantly lower reticulocytosis in RD rats in the present study by comparison with their WM controls. However, in another study (12) we also reported that there was no significant difference in total iron concentration in the bones of riboflavin deficient or weight-matched rats; this suggests that there was probably sufficient iron available for normal red cell synthesis. This result could be interpreted to suggest that there is a direct influence of the iron mobilising enzyme, Ferriductase (I). It is possible therefore that Ferriductase activity in the erythroid marrow is reduced as in the liver of riboflavin deficient rats reported by other workers (1,2), leading to a failure to fully mobilise iron from ferritin in the marrow. Even though there was no impairment of iron uptake by reticulocytes from RD rats in the present study, total protein synthesis as estimated from incorporation of SH Leucine into total proteins in vitro was significantly depressed in RD rats (Table 2); whether this represents the specific inhibition of a particular protein or proteins, is however, not known. No attempt was made in this study to measure synthesis of haem or ferritin separately in reticulocytes. It has been shown though that both DNA and protein syntheses as well as haem synthesis are considerably less in iron deficient bone marrow cells (21). The effect of riboflavin deficiency therefore in depressing total protein synthesis may be secondary to its impairment of iron metabolism.
REFERENCES I. OSAKI, S and SIRIVECH, S. Identification and partial purification of ferritin reducing enzyme in liver. Fed. Proe. 30: 1292, 1971. 2.
3.
SIRIVECH, S, DRISKELL, J AND FRIEDEN, E. NADH-FMN Oxidoreductase activity and iron content of organs riboflavin and iron-deficient rats. J. Nutr 107: 739-745, 1977. ZAMAN,
Z and VERWILGHEN,
R.L.
Effect
of riboflavin
from
RIBOFLAVIN DEFICIENCY
651
deficiency on activity of NADH-FMN Oxidoreductase and iron content of rat liver. Biochem. SOc. Trans. ~: 306-308, 1977. 4.
CHRICHTON, R.R. WAUTERS, M and ROMANS, F.: Ferric iron uptake and release. In: Proteins of Iron Stora=e and Transport in Biochemistry and Medicine. R.R. Chrlchton (ed), North-Holland Publishing Co., Amsterdam. 1975, pp. 287-294.
5.
ALFREY, C.P., JNR., and LANE, M. The effect of riboflavin deficiency on erythropoiesis. ~@minars in Hematol 7: 49-54, 1970.
6.
JAMDAR, S.C., UDUPA, K.B. and CHATTERJI, A. Study of hematopoiesis in riboflavin deficient rats with 59Fe as tracer. ~ _ Y ~ ~: 219-222, 1968.
7.
MOOKERJEA, S. and HAWKINS, W.W. Haematopoiesis in the rat in riboflavin deficiency. Brit, J. Nutr. 14: 239-246, 1960.
B.
WAISMAN, B.A. Production of riboflavin deficiency in the monkey. Proc. SoQ, ExDtl. Biol. Med. 5_~: 69-71, 1944.
9.
WINTROBE, M.M., BUSCHKE, W., FOLLIS, R.H. and HUMPHREYS, S. Riboflavin deficiency in swine. ~ i i . John Hopkins Bosp. 75: 102-114, 144.
I0. LANE, M., ALFREY, C.P.Jnr., MENGEL, D.E., DOHERTY, M.A. and DOHERTY, J. The rapid induction of human riboflavin deficiency with galactoflavin. J. Clin. Invest. 43: 357-373, 1964. 11. ADELEKAN, D.A. and THURNHAM, D.I. The influence of riboflavin deficiency on absorption and liver storage of iron in the growing rat. Brit. J. N u t ~ 56: 171-179, 1986. 12. ADELEKAN, D.A. and THURNHAM, D.I. Effects of combined riboflavin and iron deficiency on the hematological status and tissue iron concentration of the rat. J. Nutr. 116: 1257-1265, 1986. 13. DACIE, J.V. and LEWIS, S.M. Practical haematolo~y, A. Churchill Ltd., London, 1963.
J &
14. EDWARDS, J.A., GARRICK, L.M. and HOKE, J.E. Defective iron uptake and globin synthesis by erythroid cells in the anaemia of Belgrade laboratory rat. BLOOD 5_i: 347-357, 1978. 15. KONIJN, A.M., HERSHKO, C. and IZAK, G. Ferritin synthesis and iron uptake in developing erythroid cells. Amer. J. Hematol. ~: 373-379, 1979.
652
D.A. ADELEKAN
16. FINCH, S., HASKINS, D. and FINCH, S.A. Iron Metabolism: Hematopoiesis following phlebotomy. Iron as a limiting factor. J. Clin. Invest 29: 1078-1086, 1950. 17. WALSH, R.J., THOMAS, E.D., CHOW, S.K., FLUHARTY, R.G. and FINCH, C.A. Iron Metabolism. Heme synthesis in vitro by immature erythrocytes. Science (Wash. D.C.) Ii0: 396-398, 1949. 18. JANDL, J.H., INMAN, J.K. SIMMONS, R.L. and ALLEN, D.W. Transfer of iron from serum iron binding protein to human reticulocytes. J. Olin. Invest. 38: 161-185, 1959 19. JACOBS, A. Erythropoiesis and iron deficiency anaemia. In: Iron in Biochemistry and Medicine A. Jacobs and M. Worwood (eds). Academic Press, London and New York, 1974, pp. 405-432. 20. HERSHKO, C. In: Pro~res in Hematology. E.B. Brown (ed). Grune and Stratton, 1977, pp. 105-148. 21. HERSHKO, C. KARSAI, A., EYLON, L. and IZAK, G. The effect of chronic iron deficiency on some biochemical functions of the human hemopoletic system. Blood 3~: 321-329, 1970.
Accepted for publication February 13, 1989.
RETICULOCYTE 59Fe UPTAKE AND TOTAL PROTEIN SYNTHESIS IN RIBOFLAVIN DEFICIENT RATS
Delana A. ADELEKAN, Ph.D Department of Human Nutrition London School of Hygiene & Tropical Medicine Keppel ~t. (Gower St.) London WCIE 7HT. U.K.
ABSTRACT Reticulocytosis was induced in rats depleted of riboflavin for 8 weeks and in their weight-matched (WM) controls by removal of 600ul of blood from the tall vein representing approximately 10% of total blood volume in each rat. Weight-matched rats responded to the bleeding with almost 10% reticulocytosis while the response in RD rats was significantly less (6%). Furthermore, reticulocytes from RD rats took up less iron from an incubation medium containing 59FeCIs, than retlculocytes from WM rats but the difference was not significant. Incorporation of a tritiated amino acid (3H-Leucine) into total proteins in reticulocytes was significantly less in those from RD than WM rats. The results of the present study suggest that the ineffective response of RD rats to bleeding and the decreased protein synthesis may be secondary to impairment of iron metabolism by riboflavin defiency. Key words: Riboflavin Deficiency, Reticulocytosis, Iron Uptake, Total Protein Synthesis. INTRODUCTION Studies in both humans and laboratory animals have shown an association between riboflavin and iron metabolism but the mechanism is not well understood. There is evidence however, that riboflavin plays a role in the mobilisation of iron from storage compartments via the action of the flavin-dependent enzyme, NADH-FMN Oxidoreductase (Ferriductase, EC 1.6.8.1.) (i) and hence in haemoglobin synthesis (2-4).
Present Address: Department of Paediatrics & Child Health Faculty of Health Sciences Obafemi Awolowo University Ile-Ife, Nigeria. 645
646
D.A. ADELEKAN
Experimental riboflavin deficiency has been associated with bone marrow hypoplasia and a reduced rate of iron utilisation for haemoglobin synthesis (5-7). Riboflavin deficiency has also been associated with varying degrees of anaemia in a variety of animal species and man (7-10). Recently we have shown that both absorption and liver storage of iron are impaired in riboflavin deficienbt rats
(11/. The present study was undertaken to investigate more closely haemopoietic activity in immature red cells from riboflavin deficient rats.
MATERIALS
AND METHODS
(a)
Animals - Male, weanling specific pathogen-free (SPF) albino rats (mean weight 46g; range 43.5 48g) of Wister strain were used. Rats were housed individually in white plastic wire bottommed cages in a well ventilated room at a temperature of about 26oC. One group of rats was fed a riboflavin-deficient diet ad llb for a period of 8 weeks. A second group of rats was fed the basal diet plus added riboflavin (l.lg/kg diet) in sufficient quantity only to maintain the same rate of growth as rats fed the riboflavin deficient diet. These are the weight-matched (WM) controls. The composition of the basal diet has been described else where (121.
(b)
Induction of reticulocytosis - At the end of the feeding period, riboflavin deficient (RD) rats or their weight-matched controls were bled from the tail vein on 3 consecutive days. 200ul of blood was removed on each day, making a total of 600ul removed from each rat; this volume represented approximately 10% of the total blood volume of each rat (5). Rats were killed 2 days after the last bleeding by drawing blood from the heart under light ether anaesthesia. Reticulocytes were counted by the method of Dacie and Lewis (13).
(c)
Radio-Iron (59Fe) Uptake by Reticulocytes - This was measured by the method of Edwards et al (14) with modifications. Briefly, normal rat plasma from fasted ad llb-control rats was tagged with radioactive iron by adding 2.3 ml of 59FeClz solution (specific activity 11.gmCi/mgFe; Amersham International plc, Amersham, Bucks, UK) to 7.7mi of normal rat plasma (final concentration 1.932ug Fe/ml plasma). This was incubated at 37oC for 80 minutes. Next, heparinised blood
RIBOFLAVIN DEFICIENCY from RD or WM rats was centrifuged at 2500xg for i0 minutes at room temperature. Plasma and bully coat layer were removed. The sendimented red cells were washed twice with 5 volumes of a solution containing 154mM - sodium chloride, 5mM potassium chloride and 5mM - magnesium chloride. Then 200ul of washed red cells were mixed with 800ul of 59Fe-labelled normal rat plasma and the mixture incubated at 37oC for 60 minutes. After incubation, the red cells were washed 3 times in 5 volumes of 154mM sodium chloride solution and then lysed in 4 volumes of cold distilled water. The haemolysate was centrifuged at 30,000xg for 30 minutes at room temperature to remove red cell "ghosts". The extent of haemolysis in the red cells was not further determined. 59Fe radioactivity in the red cell lysate was counted in a well-type gamma counter (model LKB 1280 Ultrogamma). Reticulocyte iron uptake was expressed as 59Fe cpm/107 reticulocytes. (d)
Total Protein Synthesis in Retlculocytes this was measured by the method of KonlJn, Hershko and Izak (15) with modifications. 200ul of saline-washed red cells from RD and WM rats were incubated with 10mCi of a tritiated amino acid (L (4,5,-3H) Leucine, 46Ci/mmol; Amersham International plc) at 37oC for 60 minutes. After incubation, the red cells were washed 3 times with cold normal saline, lysed in 4 volumes of cold distilled water and then centrifuged as described earlier. Then 50ul of lysate together with 0.Smg albumin (Sigma Chemical Company) was added to 3 ml of 5% (W/V) cold trichloroacetic acid (TCA), and the mixture centrifuged at l,O00xg for 5 minutes. The precipitate was washed once in cold 5% TCA, heated for 20 minutes at 85oC in 2ml of 5%c TCA and then sequentially washed with cold 5% TCA (2 times}, a I:I (V/V) mixture of ethanol and ether ( 2 times) and ether (once}. -
(e)
Riboflavin status of the rats was determined as previously described (12). Haematocrit was measured by the microhaematocrit method and total red blood cells were counted in a Coulter counter.
(f)
Statistical Analysis - Significance of differences between mean values was tested using the Student's "t" test with level of significance fixed at 5%.
647
648
D.A. ADELEKAN RESULTS
Table 1 shows the final body weights and haematological parameters of the lid and WM control rats. Although there was no significant difference in the body weights of rats in the 2 groups at the termination of the experiment, there were significant differences in the haematological parameters examined. Both the haematocrit (41.0 +/- 0.63%) and total red blood cell counts (6.88 +/- 0.43 x 106/ui) of RD rats were significantly lower than those of WM rats. However, the percentage of reticulocytes in circulation in RD rats (2.25%) prior bleeding was significantly higher than that in WM rats (1.2%; p < 0.01).
Table I: Final Body Weights and Haematological Parameters in Riboflavin Deficient and Control Rats Body Wt
(g) Riboflavin Deficient
RBC (xl06/ul)
Retic (%)
HCT (%)
103.0 +/-1.48
6.8 +/-0.43
2.25 +/-0.34
41.0 +/-0.63
105.0 +/-2.22
8.02 +/-0.33
1.2 +/-0.30
43.0 +/-1.09
0.01
0.01
(5) WM Controls (5) P <
ns
0.025
* Figures in parenthesis indicate number of rats in each group.
Table 2 shows the riboflavin status (EGRAC) and degree of reticulocytosis in RD or WM rats fed appropriate diets for 8 weeks. The mean +/- sem AC of RD rats, 3.13 +/- 0.45 was significantly higher than AC of WM controls, (1.64 +/0.22), indicating a poorer riboflavin status. RD rats responded to the repeated bleeding by producing significantly fewer reticulocytes than their weight-matched controls. Following withdrawal of approximately 10% of total blood volume, reticulocyte production in liD rats was only about 6% of total red cell in circulation; this represented approximately a 2 fold increase over the value before bleeding (2.25%). By contrast WM rats responded with reticulocyte production which was nearly 10% of total red cell in circulation, an increase of more than 7 fold over the mean value before bleeding (1.2%). The difference in mean values between WM controls and RD rats in both cases was statistically significant (p <
0.001).
RIBOFLAVIN DEFICIENCY
649
Table 2: Radio-iron Uptake and Total Protein Reticulocytes from RD and WM Rats. RD
Synthesis
WM
p <
59Fe Radioactivity (cpm/107 Reticulocytes)
302 +/-4.12
SH Radioactivity (DPM/106 Reticulocytes)
1744 +/-9.41
Reticulocytes
6.58 +/-0.4
9,78 +/-0.45
3.13
1.64
(%)*
EGRAC
in
350 +/-4.69
2346 +/-Ii.i
ns
0.05
0.001
0.01
Values are mean +/- sem for 5 rats in each group. *Rats bled from the tail vein on 3 consecutive days after 8 weeks of feeding a RD or control diet. Reticulocytes in blood were counted 2 days after the last bleeding.
The pattern of radio-iron uptake and total protein synthesis in RD and WM rats is shown in Table 2. When reitculocytes from RD and WM rats were incubated with radlo-iron (59Fe), RD reticulocytes incorporated less radio-iron than reticulocytes from WM rats but the difference was not statistically significant. However, incorporation of tritiated leucine into the cellular proteins was significantly less in reticulocytes from RD rats than in those from NM controls. Because of the it was not possible correlation between 59Fe or amino acid
small number of rats used in this study, to establish whether there was any riboflavin status (EGRAC) of rats and incorporation by reticulocytes.
DISCUSSION It has been reported that the rate of red blood cell production following repeated blood removal (phlebotomy) depends chiefly on availability of iron from body stores (16). In the present study, although the percent reticulocytes in circulation in RD rats was significantly higher than that in WM controls before bleeding, the value following repeated blood removal was significantly less in them. This indicated an impairment in the ability of the RD
650
D.A. ADELEKAN
rats to respond adequately to the further stress of bleeding. Iron for red cell synthesis reaches the bone marrow directly from transferrin in the plasma (17,18). Most of the iron in transferrin comes from the reticuloendothelial system where the iron is derived chiefly from haemoglobin catabolism of effete red cells (19,20). We have recently shown that iron concentration in the livers of riboflavin deficient rats was significantly less than that in weight matched controls from 3 weeks of deficiency (Ii). This partly explains the significantly lower reticulocytosis in RD rats in the present study by comparison with their WM controls. However, in another study (12) we also reported that there was no significant difference in total iron concentration in the bones of riboflavin deficient or weight-matched rats; this suggests that there was probably sufficient iron available for normal red cell synthesis. This result could be interpreted to suggest that there is a direct influence of the iron mobilising enzyme, Ferriductase (I). It is possible therefore that Ferriductase activity in the erythroid marrow is reduced as in the liver of riboflavin deficient rats reported by other workers (1,2), leading to a failure to fully mobilise iron from ferritin in the marrow. Even though there was no impairment of iron uptake by reticulocytes from RD rats in the present study, total protein synthesis as estimated from incorporation of SH Leucine into total proteins in vitro was significantly depressed in RD rats (Table 2); whether this represents the specific inhibition of a particular protein or proteins, is however, not known. No attempt was made in this study to measure synthesis of haem or ferritin separately in reticulocytes. It has been shown though that both DNA and protein syntheses as well as haem synthesis are considerably less in iron deficient bone marrow cells (21). The effect of riboflavin deficiency therefore in depressing total protein synthesis may be secondary to its impairment of iron metabolism.
REFERENCES I. OSAKI, S and SIRIVECH, S. Identification and partial purification of ferritin reducing enzyme in liver. Fed. Proe. 30: 1292, 1971. 2.
3.
SIRIVECH, S, DRISKELL, J AND FRIEDEN, E. NADH-FMN Oxidoreductase activity and iron content of organs riboflavin and iron-deficient rats. J. Nutr 107: 739-745, 1977. ZAMAN,
Z and VERWILGHEN,
R.L.
Effect
of riboflavin
from
RIBOFLAVIN DEFICIENCY
651
deficiency on activity of NADH-FMN Oxidoreductase and iron content of rat liver. Biochem. SOc. Trans. ~: 306-308, 1977. 4.
CHRICHTON, R.R. WAUTERS, M and ROMANS, F.: Ferric iron uptake and release. In: Proteins of Iron Stora=e and Transport in Biochemistry and Medicine. R.R. Chrlchton (ed), North-Holland Publishing Co., Amsterdam. 1975, pp. 287-294.
5.
ALFREY, C.P., JNR., and LANE, M. The effect of riboflavin deficiency on erythropoiesis. ~@minars in Hematol 7: 49-54, 1970.
6.
JAMDAR, S.C., UDUPA, K.B. and CHATTERJI, A. Study of hematopoiesis in riboflavin deficient rats with 59Fe as tracer. ~ _ Y ~ ~: 219-222, 1968.
7.
MOOKERJEA, S. and HAWKINS, W.W. Haematopoiesis in the rat in riboflavin deficiency. Brit, J. Nutr. 14: 239-246, 1960.
B.
WAISMAN, B.A. Production of riboflavin deficiency in the monkey. Proc. SoQ, ExDtl. Biol. Med. 5_~: 69-71, 1944.
9.
WINTROBE, M.M., BUSCHKE, W., FOLLIS, R.H. and HUMPHREYS, S. Riboflavin deficiency in swine. ~ i i . John Hopkins Bosp. 75: 102-114, 144.
I0. LANE, M., ALFREY, C.P.Jnr., MENGEL, D.E., DOHERTY, M.A. and DOHERTY, J. The rapid induction of human riboflavin deficiency with galactoflavin. J. Clin. Invest. 43: 357-373, 1964. 11. ADELEKAN, D.A. and THURNHAM, D.I. The influence of riboflavin deficiency on absorption and liver storage of iron in the growing rat. Brit. J. N u t ~ 56: 171-179, 1986. 12. ADELEKAN, D.A. and THURNHAM, D.I. Effects of combined riboflavin and iron deficiency on the hematological status and tissue iron concentration of the rat. J. Nutr. 116: 1257-1265, 1986. 13. DACIE, J.V. and LEWIS, S.M. Practical haematolo~y, A. Churchill Ltd., London, 1963.
J &
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Accepted for publication February 13, 1989.