@Copyright 1985 by The Humana Press Inc. All rights of any nature whatsoever reserved, 0163-4984/85/0809-0105502.00

Iron, Zinc, and Copper Content in the Tissues of the Rat During Pregnancy A. MAS, A.

R O M E U , Mo ALEMANY, A N D LL. A R O L A *

Bioquknica, Facultat de Ci6ncies Qukniques de Tarragona, Universitat de Barcelona, 43005 Tarragona, Spain Received July 23, 1984; Accepted March 19, t 9 8 5

ABSTRACT The levels of iron, zinc, and copper in the tissues of the pregnant rat, on d 12, 19, and 21 after impregnation have been determined and compared with controls. Iron levels decreased considerably in late pregnancy as a result of increased fetal requirements, thus diminishing iron stores in rat tissues, but maintaining the circulating plasma levels. Copper levels increased slightly at midpregnancy, but returned to control levels at the end of gestation. Zinc stores also increased slightly during early pregnancy, yet were decreased at the end of pregnancy, but to a lesser extent than those of iron. The data are explained on the basis of equilibrium between assimilation and fetal needs for copper, a slightly higher demand for zinc with altered equilibrium, and a much altered equilibrium for iron that provokes a dwindling of iron maternal reserves that is not compensated by dietary iron. Index Entries: Iron distribution, in the pregnant rat;zinc distribution, in the pregnant rat; copper distribution, in the pregnant rat; rat gestation, and Fe, Zn, and Cu distribution; pregnancy, Fe, Zn, and Cu distribution in rat.

INTRODUCTION The g r o w t h of the fetus i m p o s e s a severe drain on the essential m e t a l h o m e o s t a s i s of the m a t e r n a l organism. The d e m a n d s of the c o n c e p t u s for nutrients g r o w e n o r m o u s l y with the c o m p l e t i o n of its de-

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v e l o p m e n t and the end of gestation (1). There are a considerable n u m b e r of well d o c u m e n t e d descriptions of the effects of essential metal deficiency u p o n the outcome of gestation (2,5), but these deficiencies diversely affect the maternal and fetal organisms. Thus, zinc deficiency is teratogenic (6), but iron deficiency causes severe anemia in the m o t h e r (7). The different behavior of these essential metals, together with the close relationship observed between copper, zinc, and iron in deficient situations (8,9) m o v e d us to study the effects of normal pregnancy on the stores and tissue availability of these three metals. The majority of studies on this subject deal only with specific (in time or tissue) variations during pregnancy and were usually directed to altered homeostatic situations. For this reason we have studied the contents of iron, zinc, and copper in the maternal tissues during rat pregnancy.

MATERIALS AND METHODS Female virgin Wistar rats weighing initially 180-200 g were kept until impregnation (determined by the detection of spermatozoa in daily vaginal smears) with adult males, and then transferred to individual plastic cages. They were housed under standard temperature (21 -+ 1~ and light cycle (lights on from 08.00 to 20.00 hours) conditions. The rats were fed rat chow pellets (Panlab, Barcelona) that contained a m e a n 21.5 p p m of Cu, 357 p p m of Fe, and 83 p p m of Zn, and had free access to drinking water that contained less than 10 ppb of any of the metals studied. Four groups of animals were used: 12-, 19-, and 21-d pregnant animals as well as virgin controls. The rats were sacrificed by b e h e a d i n g at the beginning of a light cycle and their blood was collected in dry heparinized plastic beakers. Carcasses were dissected and pieces of liver, heart, small intestine (jejunum), lung, kidney, femur, hind-leg striated muscle, brain, placenta, whole conceptus, and fetuses were obtained, blotted, cleaned (intestine), and used, as well as blood and plasma samples for metal estimation. Samples of about 1 g were digested with 5 mL of concentrated nitric acid (Merck, low metal reagent quality) in Teflon-lined reactors at 100~ for 12 h. Clear digests were then diluted to 10 mL with pure deionized water (specific resistivity higher than 1.5 x 10 7 ohm/cm, from a Milli-Q Millipore apparatus), and were used for the estimation of copper, zinc, and iron through flame absorption spectrop h o t o m e t r y with a IL 551 apparatus. The instrument was calibrated in each series of measurements with ultrapure reagent standards (Normex, Carlo Erba). The effect of organic matrix interference was corrected by the use of standards added to randomly selected samples. This interference was in all cases lower than 2.5% of the final measures. The results were statistically compared using the Student-Neumann t-test.

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RESULTS Table 1 presents the concentrations or iron, copper, and zinc in the tissues of the pregnant rat and its conceptus. During the period from 0 to 21 d of pregnancy, a decrease occurred in the iron content of maternal brain, heart, kidney, bone, skeletal muscle, and especially in that of the liver. These changes were not reflected by corresponding changes in either plasma or blood iron concentration, and were m u c h more m a r k e d in the last stages of pregnancy. In contrast, hepatic iron increased markedly during days 0-12; however, the later d e m a n d s of fetal growth i m p o s e d a b u r d e n on iron homeostasis with a diminution of reserves in this maternal tissue. The placental and fetal iron concentrations increased up to d 19, stabilizing thereafter, despite a steady increase in fetal weight. Zinc levels remained virtually u n c h a n g e d during pregnancy, with significant decreases in the blood and small intestine noted only at the end of pregnancy. In most other tissues, no significant changes were observed, except for a transient increase in bone zinc. The placental zinc content decreased from d 12 to the end of pregnancy, whereas the concentration of zinc in the whole fetus increased considerably even from d 19 to 21, thus differing again from the iron pattern. Whole blood copper increased with pregnancy, with a m a x i m u m occurring on d 19. Plasma copper increased between 0 and 12 d but did not change thereafter. The general pattern of variation of copper tissue stores obsei'ved s h o w e d a transient increase in the concentrations by m i d p r e g n a n c y which slowly receded towards its end. The maximal values were found on both d 12 and 19, the differences versus virgin controls being significant for brain, liver, heart, lung, kidney, and skin. The values on d 21 were lower than those of midpregnancy, but w e r e statistically different from controls only in the case of whole blood.

DISCUSSION The presence of occluded blood in most tissues can contribute appreciably to their iron content. However, the concentrations found in m a n y tissues did not correlate clearly with their blood content (10) since a significant a m o u n t of iron is present in the tissues in forms other than blood (11). The pattern of change of iron concentrations in b o n e f o u n d here agreed with that of Br/itter et al. (12), but our plasma iron changes did not coincide fully with the pattern described by Finch et al. (7) for 20 d rats. The considerable d e m a n d s of the conceptus impose a severe b u r d e n on maternal iron homeostasis, with blood or plasma iron concentrations maintained at the expense of tissue iron, with practically all tissues

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~ a s et at TABLE 1 I r o n , Zi n c , a n d C o p p e r C o n t e n t of t h e O r g a n s of P r e g n a n t R a t s ~

Organ

Metal

Day 0

D a y 12

D a y 19

D a y 21

B rai n

Fe Zn Cu

31.6 -+ 1.8 13.4 -+ 1.4 2.3 • 0.2

22.6 • 1.4 b 16.2 + 0.6 3.5 + 0.2 b

24.4 • 2.1 15.4 -+- 0.2 2.8 + 0.1

18,8 - 2.2 ~ 14.8 -+ 0.5 2.5 -+ 0,1

Small intestine

Fe Zn Cu

22.7 --- 1.8 37.8 -+ 3.5 2.1 --- 0.3

22.3 • 2.0 3.5 • 2.7 2.6 • 0.3

21.9 -+ 2.6 27.6 -+ 1.3 2.8 • 0,2

10.6 -+ 1.0 b 25.9 +- 0.8 b 1.8 • 0.2

Liver

Fe Zn Cu

133 • 19 28.7 • 1.0 5.0 -+ 0.2

205 + 28 32.9 -+ 1.8 6.4 • 0.4 b

98.1 -+ 7.8 30.8 • 1.5 6.3 • 0.3 b

48.3 -+ 6.3 b 26.1 + 2.5 5.2 • 0.1 ~

Heart

Fe Zn Cu

98.2 -+ 5.5 18.6 -+ 0.5 4.8 • 0.5

89.3 + 8.8 21.1 + 1.9 7.0 + 0.2 b

78.2 + 7.4 19.4 • 0.5 7.1 -+ 0,4 b

60.3 -+ 3,9 ~ 19.3 - 0,5 5,3 -+ 0.3

Lung

Fe Zn Cu

110 + 9 19,5 • 0.5 1,7-+0,2

103 • 13 22.5 • 1.4 2.8+0,1 ~

104 -+ 11 19.4 • 0,3 3,0-~ 0.3 b

124 -+ 11 18,0 - 0,7 2,1-+0.3

Kidney

Fe Zn Cu

58.7+-4,0 23,7 -+ 0.7 14,1 -+ 1.7

53.8 • 3.8 28.5 + 1.3 22.5 • 2.7 ~

56,0 -+ 4,3 26.9 + 1,4 25,0-+3.2 b

39.9 • 2.6 ~ 26,6 --- 1,8 18.1 ~ 3 . 5

Bone

Fe Zn Cu

45.0 -+ 3.0 119 -+ 13 2.6 + 0.4

38.4 + 2.5 170 + 9 b 3.5 • 0.1

29.1 + 2.1 b 140 -+ 6 3.1 -4- 0.2

25,8 -+ 1,5 b 134 -+ 7 2.6 + 0.2

Skin

Fe Zn Cu

18.0 -+ 1.8 30.2 -+ 1.7 1.9 • 0.4

19.7 • 4.4 29.6 • 2.1 3.4 • 0.2 b

22.5 -+ 1.7 28.6 • 2.8 5.5 -+ 1.0 b

17.3 • 2,7 32.7 -+ 1,8 3.7 -+ 1,0

H i n d leg m u s cl e

Fe Zn Cu

19.5 • 1.2 13.6 + 0.7 1.2 + 0.1

19.1 • 1.2 18.8 • 1.2 1.4 • 0.1

12.6 -+ 1.0 b 15.0 -+ 1.2 1.4 -+ 0.2

14.5 -+ 1.0 b 18.5 + 1.2 1.4 -+ 0.1

Whole blood

Fe Zn Cu

386 -+ 16 6.4 • 0.1 1.4 +_ 0.1

405 • 7 5.9 • 0 . P 1.7 • 0.1

372 -+ 17 6.3 -+ 0.0 2.4 + 0.1 b

336 -+ 18 5.2 -+ 0,1 b 2.0 -+ 0,1 b

Blood plasma

Fe Zn Cu

6.2 -+ 0.4 1.3 • 0.1 1.9 -+ 0.1

5.5 • 0.3 1.4 + 0.1 2.8 • 0.1

4.6 + 0.7 1.3 -+ 0.1 2.6 -+ 0.2

5.6 • 0.7 1.5 - 0.2 2.3 +- 0,1

Placenta

Fe Zn Cu

----

54.9 _+ 5.5 16.8 • 1.8 3.4 _+ 0.3

109 -+ 2 12,7 -+ 0,3 4.9 -+ 0.2

116 --- 1 12,1 • 0.1 4.4 • 0.1

Whole fetus

Fe Zn Cu

----

17.2 -+ 1.6 11.5 • 2.0 2.1 • 0.1

42.2 -+ 3,2 16,t • 0,6 2.7 - 0,4

38.4 -+ 1. 18.6 -+ 0.9 2,8 -+ 0.1

'All values are expressed in mg/kg of tissue except for blood and plasma (rag/L), and are the m e a n -+ SEM of 5--7 different animals. bSignificance of the differences versus virgin controls (day 0): p < 0,05,

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contributing to this maintenance. This is necessary because the n e e d s of iron for fetal d e v e l o p m e n t are high (13) and cannot be met either by the observed hyperphagia associated with pregnancy (t4), or by an adaptative increase in the efficiency of iron absorption i n d u c e d by a decreasing tissue iron status. In this way, most of the fetal iron m u s t come from the maternal tissues, which thus remain grossly depleted at the end of pregnancy. Changes in the copper concentration of the plasma of p r e g n a n t rats resemble those reported for serum (15) and are similar to those described during pregnancy in h u m a n subjects (16). The d e m a n d for copper during fetal growth is not so marked as for iron, and seems to be relatively well covered by the increase in food consumption, as well by an increase in the ability to absorb copper in the intestine (17). Copper required for dev e l o p m e n t of the concepta seems to come mainly from ingested food (9), since we find no evidence of a significant withdrawal of this element from storage tissues such as the liver during pregnancy, despite the important accretion of this metal in fetal organs coming partly from maternal stores (15,19). The pattern for copper thus differs from that observed with iron. The transient increase observed by m i d p r e g n a n c y can be caused by the increased food consumption [already observable from d 12 onwards (14)], the increase in intestinal copper absorption (17), and the low needs of fetuses at this age (unpublished results), which resulted in relatively increased copper deposition in the maternal tissues that is later used for fetal and postnatal development (18). Zinc is necessary for fetal development, for maternal maintenance of the extra weight resulting from pregnancy (20), and for events leading to the initiation of parturition (21). The high increase in bone zinc at m i d p r e g n a n c y coincides with that described by Bratter et al. (12). Tissue zinc content at the end of pregnancy resembles that reported by Masters et aI. (9), and the pattern of plasma changes was similar to that described for h u m a n s (16), but more extensive than those described for h u m a n s by Tyrala et al. (22). The fact that decreases in maternal tissue zinc during p r e g n a n c y were only evident in blood and small intestine at the e n d of gestation m u s t be explicable either by the relative resistance of the maternal organism to part with its own well-fixed tissue zinc (23)--despite reports of sparing of fetal needs at the expense of maternal reserves in severe zinc deficiency (24)--or by a high efficiency of zinc absorption from the diet, containing 80 mg Zn/kg, as has been described in rats d u r i n g the last stages of pregnancy (25). It seems that dietary zinc (25) plus a small c o m p o n e n t derived from maternal tissue catabolism (9) was sufficient to cover the needs of the conceptus for this metal. It is interesting to compare the considerable difference in the patterns of change of these three metals during pregnancy. Despite a close interrelationship between them in cases of deficiency (8,9) they are stored a n d used from the maternal tissue stores with differing degrees of ease and extent. The relationship of the size of tissue stores to the de-

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m a n d s d u r i n g d e v e l o p m e n t of the concepta s e e m s to be very n a r r o w for iron (since the tissue iron includes blood tissue iron), possibly a d e q u a t e for zinc, a n d certainly adequate for copper. T h u s the expected mobility of these metals m u s t be in c o n s o n a n c e with this behavior. The extent to w h i c h food can p r o v i d e the extra n e e d s again agrees with the c h a n g e s observed a n d e n h a n c e d intestinal absorption of c o p p e r a n d zinc (19,26). From the data p r e s e n t e d , it can be concluded that adaptive m e c h a n i s m s regulating the efficiency of iron absorption were n o t entirely a d e q u a t e to p r e v e n t w i t h d r a w a l of maternal tissue iron despite a relatively h i g h dietary c o n t e n t of iron. There was no c o r r e s p o n d i n g evidence of a significant redistribution of maternal zinc or copper.

SUMMARY Iron, zinc, a n d c o p p e r were d e t e r m i n e d in the tissues of p r e g n a n t rats. The data s h o w e d that iron levels decreased considerably in late p r e g n a n c y , w h e r e a s copper a n d zinc stores increased in m i d p r e g n a n c y a n d d e c r e a s e d to control levels by the end of gestation. These results s u g g e s t an equilibrium b e t w e e n maternal u p t a k e a n d fetal n e e d s for copper a n d zinc in a lesser degree, a n d a d i m i n u t i o n of m a t e r n a l stores of iron to m e e t r e q u i r e m e n t s for fetal d e v e l o p m e n t .

ACKNOWLEDGMENT This w o r k has been s u p p o r t e d by a grant from the " C o m i s i 6 n Asesora de Investigaci6n Cientifica y T6cnica" from the G o v e r n m e n t of Spain.

REFERENCES 1. J. C. Stevenson, in Current Topics in Experimental Endocrinology, Academic Press, New York, vol. 5, 1983, pp. 177-196. 2. G. A. Hall and J. Howell, Br. J. Nutr. 23, 41 (1969). 3. G. J. Fosmire, S. Greeley, and H. H. Sandstead, [. Nutr. 107, 1543 (1977). 4. N. S. Webster, J. Nutr. 109, 1640 (1979). 5. F. H. Morris and R. M. Caprioli, in Animal Models in Fetal Medicine, P. W. Nathaniellsz, ed., Elsevier Biomedical Press, 1982, pp. 149-194. 6. I. E. Dreosti, in Clinical Applications of Recent Advances in Zinc Metabolism, Liss, New York, 1982, pp. 19--38. 7. C. A. Finch, H. A. Huebers, L. R. Miller, B. M. Josephson, T. H. Shepard, and B. Mackler, Amer. ]. Clin. Nutr. 37, 910 (1983). 8. L. S. Hurley, C. L. Keen, and B. L6nnerdal, Fed. Proc. 42, 1735 (1983). 9. D. G. Masters, C. L. Keen, B. L6nnerdal, and L. S. Hurley, J. Nutr. 113, 1448 (1983). 10. M. Robert and M. Alemany, IRCS Med. Sci. 9, 236 (1981). 11. P. Aisen and I. Listowsky, Ann. Rev. Biochem. 49, 357 (1980). 12. P. Bratter, D. Behne, D. Gawlik, T. H6fer, and U. R6sick, in Trace Element Biological Trace Element Research

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Analytical Chemistry in Medicine and Biology, P. Br~tter and P. Schnamel, eds., de Gruyter, Berlin, 1980, pp. 47-56. M. J. Murray and N. Stein, J. Nutr. 100, 1023 (1970). G. H. Wang, Amer. J. Physiol. 71, 736 (1925). W. Y. Chan, T. Z. Ramadan, M. Perlman, M. A. McCaffree, and O. M. Rennert, Nutr. Rep. Int. 22, 939 (1980). I. E. Dreosti, Ao J. McMichael, G. T. Gibson, R. A. Buckley, J~ M. Harstone, and D. P. Colley, Nutr. Res. 2~ 591 (1982). N. T. Davies and R. B. Williams, Proc. Nutr. Sci. 35, 4A (1976). R. B. Williams, N. T. Davies and I. McDonald, Br. ]. Nutr. 38, 407 (1977). C. M. Spray, Br. J. Nutr. 4, 354 (1950). S. Jameson, in Zinc Deficiency in Human Subjects, Liss, New York, 1983, pp. 53-69. G. E. Bunce, G. R. Wilson, C. F. Mills, and A. Klopper, Biochem. J. 210, 761 (1983). E. E. Tyrala, J. I. Manser, N.. Brodsky, and N. Tran, Acta Paediat. Scand., 72, 695 (1983). D. G. Masters, C. L. Keen, B. LOnnerdal, and L. Hurley, J. Nutr. 113, 905 (1983). Do J. Fosmire, S. Greeley, and H. H. Sandstead, J. Nutr. 107, 1543 (1977). N. T~ Davies and R. B. Williams, Br. J. Nutr. 38, 417 (1977).

Biological Trace Element Research

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Iron, zinc, and copper content in the tissues of the rat during pregnancy.

The levels of iron, zinc, and copper in the tissues of the pregnant rat, on d 12, 19, and 21 after impregnation have been determined and compared with...
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