Serum Lipids in Suckling and Post-Weanling Iron-Deficient Rats A D R I A R O T H M A N SHERMAN, Department of Foods and Nutrition, University of Illinois, Urbana, IL 61801 ABSTRACT
Serum lipids were studied in iron-deficient and control rats during suckling and after weaning at 21, 30, and 60 days of age. Diets providing 5 or 307 ppm iron were fed to dams and their offspring during gestation, lactation, and after weaning. Rats on the deficient diet throughout the experimental period developed a hyperlipidemia characterized by elevated triglycerides, cholesterol, and phospholipids which was present at 21, 30, and 60 days. Control pups weaned to the deficient diet developed anemia at 30 days of age and hypertriglyceridemia at 60 days of age. Repletion of deficient rats with iron after weaning caused a rapid decline in serum lipid levels after only 9 days on the control diet. The hyperlipidemia of iron deficiency thus appears to be reversible with iron supplementation. The time required to develop hypertriglyceridemia in iron deficiency is longer postweaning than during suckling. INTRODUCTION
Elevated concentrations of serum lipids have been associated with dietary iron deficiency in rats and chicks (1-4). Previous reports from this investigator have f o u n d abnormal lipid metabolism in 18-day-old offspring of rats fed irondeficient diets during pregnancy and lactation (1,2). Hyperlipidemia characterized by significant increases in the triglyceride, cholesterol, and phospholipid fractions of the serum was r e p o r t e d in pups of dams fed the deficient diet containing 5 ppm iron t h r o u g h o u t gestation and lactation (1). Sera from pups of dams fed the control diet (307 p p m iron) during either gestation a n d / o r lactation and dams fed both diets had normal lipid concentrations. The elevated serum lipids were observed only in pups whose dams were fed the lowest concentration of iron (5 ppm). A l t h o u g h feeding 29 ppm iron resulted in anemia, it was n o t associated with hyperlipidemia (2). Subsequent e x p e r i m e n t a t i o n has investigated the mechanisms which may be involved in the etiology of this hyperlipidemia. Since increases in serum lipids occurred in the offspring and n o t in the maternal organism, lipids were measured in milk of control and deficient dams to determine if iron-deficient pups had greater dietary fat levels. No differences were found in this exogenous source of lipids. Lipoprotein lipase was determined in the post-heptarin plasma of pups, and no significant differences were f o u n d b e t w e e n control and deficient pups. This indicated that decreased clearance of dietary lipids from the blood was not responsible for the hyperlipidemia. Production of triglycerides in vitro from [U-14C]glucose was found to be significantly higher in liver slices of iron-deficient pups than in controls. Apparently, during iron deficiency, this increased endogenous lipid pool may contribute to the
increased serum triglyceride concentrations (2). Lipogenesis has also been studied in irondeficient adult rats which were not hyperlipidemic (5). Triglyceride synthesis from [3H201 and [ U - 1 4 C l g l u c o s e in adipose tissue was t w o to three times greater in iron-deficient rats than in controls. Amine et al. found that iron-deficient rats had increased i n c o r p o r a t i o n of 14C-glucose into intestinal lipids than did controls and reported that the lipid synthesis increased as the level of dietary fats increased (6). The present study was initiated to investigate the effects of iron restriction during the prenatal and suckling a n d / o r postweanling periods on serum lipid levels later in life. Pups of iron-deficient dams were repleted with iron after weaning and control pups were depleted in iron after weaning to compare the effects of iron deficiency on serum lipid levels during these t w o stages in the life cycle.
888
MATERIALS AND METHODS Experimental Design
Nulliparous Sprague-Dawley CD rats (Charles River, Wilmington, MA) were obtained at a weight of 180 g and maintained on a cereal-based stock diet (Purina Rat Chow) until they weighed ca. 270 g. They were then bred and fed ad libitum either the iron-deficient diet (5 p p m i r o n ) or the control diet (307 ppm iron) (Table I) (n=16 per group) and glassdistilled water f r o m the first day of pregnancy until the pups were weaned. On the day following parturition, litters were adjusted to contain six female pups. F o o d was placed in the cage to limit access to the dams and the iron-free bedding in the solid b o t t o m "matern i t y " cages was changed frequently. On day 21 the pups were weaned to either the same or opposite diet as that fed to their dams and
SERUM LIPIDS IN IRON-DEFICIENT RATS Lipids
TABLE I Composition of Diets
Casein 2 Sucrose Cornstarch Iron-free salt mix 3 Vitamin mix 4 Corn oil 5 Cellulose 6
889
307 ppm iron 1 %
5 ppm iron %
22.00 29.70 29.70 5.48 1.00 10.00 2.00
22.00 29.76 29.76 5.48 1.00 10.00 2.00
l Iron levels determined by atomic absorption. 2Vitamin-free casein, Teklad, Chagrin Falls, OH. 3The levels of minerals used is at least 12.5% more than NRC recommendations for lactation (7). Composition of salt mixture (mg/lO0 g diet): CaCO 3, 1,680.000; COC12.6 H20 , 0.100; CUSO4"5 H20, 9.800; MgSO4.7 H20 , 278.500; MnSO4"H20, 19.000; KI, 0.022; NaCI, 1,000.000; K2HPO 4, 2,483.600; ZnCI2, 3.000. FeSO4.7 H20 was added to the supplemented diet in place of sucrose. 4Teklad, Chagrin Falls, OH, supplies in g/kg of diet; p-aminobenzoic acid, 0.110132; ascorbic acid, 0.9912; biotin, 0.000441; vitamin B12, 0.0000297; calcium pantothenate, 0.066079; choline, 1.4337; folic acid, 0.001982; inositol, 0.110132; menadione (vitamin K3) , 0.049559 ; niacin, 0.099119 ; pyridoxine HCI, 0.022026; riboflavin, 0.022026; thiamin HC1, 0.022026; supplies in units per kg of diet: dry retinyl palmitate, 19,824; dry ergocalcifrol 2.202.5; dry tocopheryl acetate, 121.15; corn starch, 4.666878 g. 5Mazola corn oil, Best Foods, Englewood Cliffs, NJ. 6Alphacel nonnutritive cellulose, Teklad, with iron extracted by the method of Houk et al. (8). placed in individual stainless steel screen b o t t o m cages. T h e r e were f o u r d i f f e r e n t experim e n t a l t r e a t m e n t s d e p e n d i n g o n the d a m ' s diet during p r e g n a n c y a n d l a c t a t i o n a n d t h e diet fed to weanlings a f t e r day 21. C o n t r o l C o n t r o l (CC) rats were b o r n to c o n t r o l dams a n d w e a n e d t o t h e c o n t r o l diet, CD ( C o n t r o l Deficient) rats were b o r n t o c o n t r o l d a m s a n d w e a n e d to t h e i r o n - d e f i c i e n t diet, DC (Defic i e n t - C o n t r o l ) rats were r e p t e t e d w i t h i r o n a f t e r weaning, DD ( D e f i c i e n t - D e f i c i e n t ) rats were b o r n to i r o n - d e f i c i e n t dams a n d w e a n e d t o i r o n - d e f i c i e n t diets ( n = 8 - 1 4 litters p e r treatm e n t ) . Diets a n d glass-distilled w a t e r were offered ad l i b i t u m a n d weekly f o o d i n t a k e s a n d b o d y weight r e c o r d s were k e p t . L i t t e r m a t e s were fasted for 4 h r o n days 21, 30 a n d 6 0 a n d taft b l o o d samples were t a k e n for h e m o g l o b i n a n d h e m a t o c r i t d e t e r m i n a t i o n s (9). A f t e r brief e x p o s u r e to c h l o r o f o r m anesthesia, b l o o d was collected b y cardiac p u n c t u r e , s e r u m r e i n o v e d a n d f r o z e n for analysis o f lipids.
S e r u m triglycerides, cholesterol, c h o l e s t e r y l ester a n d p h o s p h o l i p i d s were d e t e r m i n e d . Lipids were e x t r a c t e d f r o m s e r u m samples b y t h e m e t h o d of F o l c h et al. ( 1 0 ) a n d s e p a r a t e d o n t h i n l a y e r c h r o m a t o g r a p h y plates d e v e l o p e d in a solvent s y s t e m of p e t r o l e u m e t h e r / e t h y l e t h e r / a c e t i c acid ( 7 0 : 3 0 : 1 , v/v/v). F o l l o w i n g e x p o s u r e t o i o d i n e vapors, lipid f r a c t i o n s were i d e n t i f i e d b y c o m p a r i s o n w i t h triolein, cholesterol, cholesteryl linoleate, and lecithin s t a n d a r d s w h i c h were r u n s i m u l t a n e o u s l y . T h e spots were r e c o v e r e d a n d t h e f o l l o w i n g m e t h o d s were used to q u a n t i t a t e t h e lipid f r a c t i o n s : triglyceride c o n c e n t r a t i o n was determ i n e d b y t h e m e t h o d o f s t e r n a n d Shapiro ( 1 1 ) n o n e s t e r i f i e d c h o l e s t e r o l a n d c h o l e s t e r y l esters were m e a s u r e d b y t h e m e t h o d of Searcy a n d Bergquist (12) a n d p h o s p h o l i p i d c o n c e n t r a t i o n was d e t e r m i n e d b y m e a s u r i n g p h o s p h o r u s in t h e lipids r e m a i n i n g at t h e origin using t h e m e t h o d of C h e n et al. (13). Statistical Methods
Differences b e t w e e n t h e t w o g r o u p s at 21 days o f age were d e t e r m i n e d w i t h t h e S t u d e n t ' s t test. Analysis of v a r i a n c e was used t o determine differences among group means when four e x p e r i m e n t a l g r o u p s were c o m p a r e d (days 3 0 a n d 60). D u n c a n ' s m u l t i p l e range test was used t o find t h e m e a n s w h i c h differed significantly (14) R ESU LTS
T w e n t y - o n e - d a y - o l d p u p s of i r o n - d e f i c i e n t d a m s h a d l o w e r b o d y weights, h e m o g l o b i n a n d h e m a t o c r i t levels (Table II) a n d h i g h e r levels of s e r u m c h o l e s t e r y l ester, c h o l e s t e r o l , triglycerides, a n d p h o s p h o l i p i d s ( T a b l e III) t h a n did p u p s o f c o n t r o l dams. T h e e l e v a t i o n s in cholest e r y l ester a n d c h o l e s t e r o l were a p p r o x i m a t e l y 2-fold, triglycerides were 4-fold a n d p h o s p h o lipids were elevated 3-fold. T h e i r o n c o n t e n t of t h e w e a n i n g diet h a d d r a m a t i c effects o n b o d y weight, h e m a t o l o g i c a l values, a n d s e r u m lipid levels as early as 30 days of age. Weaning d e f i c i e n t p u p s t o t h e c o n t r o l diet (DC) increased b o d y weight, h e m o g l o b i n , a n d h e m a t o crit t o c o n t r o l levels a f t e r o n l y 9 days o n t h e diet. S e r u m c h o l e s t e r y l ester, triglycerides, a n d p h o s p h o l i p i d s r e t u r n e d to c o n t r o l levels at this t i m e also. At 3 0 days old, s e r u m c h o l e s t e r o l r e m a i n e d s o m e w h a t h i g h e r in DC rats t h a n in CC rats. Pups w e a n e d t o t h e d e f i c i e n t diet f r o m t h e c o n t r o l diet (CD) h a d l o w e r levels of h e m o g l o b i n a n d h e m a t o c r i t s t h a n CC rats b u t n o t as l o w as DD rats w h i c h h a d b e e n e x p o s e d t o t h e i r o n - d e f i c i e n t diet t h r o u g h o u t t h e LIPIDS, VOL. 14, NO. 11
890
A.R.
SHERMAN
~RR v v v
@.
t.:.Rq
§
+L
+1
.,
+1
+1
o
+,
+,.,
r
§
c
0
e~ ..
N
~
g
=
g
vvv o
....
_
:
+,
r
+,
~.
'~
g 7
+'
+' +' *1
~
=
tmq
Z
E
E
.3 _e
2 *,
+,
§
~
h3
m
o m
'9
~
'q
== +, +, +~
v
v
v
~ : ~
~176
5 ~
vvv
-~
..~
.E 7
_
~
A g .~
.1= T. ,1=
LIPIDS,
VOL.
14,
NO.
11
"
*
SERUM LIPIDS IN IRON-DEFICIENT RATS experiment. No significant elevations in serum lipids were seen in the CD rats at 30 days of age, although cholesterol concentrations were slightly elevated relative to the CC rats. The DD rats continued to have elevated serum cholesterol, triglycerides, and phospholipids after weaning. At 60 days of age, body weights were similar in CC, CD, and DC rats, and significantly lower in DD rats (Table II). Hematological values (Table II) in CC and DC rats were the same, CD rats had hemoglobin levels and hematocrits approximately one-half of the control rats. Hemoglobin levels and hematocrits in DD rats were significanlty lower than those of all of the other groups. Serum cholesterol, triglycerides and phospholipids continued to be highest in the DD group at 60 days (Table ill). Sixty-dayold DC and CC rats had serum lipid concentrations similar to those found in 30-day-old littermates. Rats restricted in iron after weaning (CD) developed a hypertriglyceridemia at 60 days of age. However, serum cholesterol and phospholipids were not significantly elevated in the CD rats at that time. Initial plans to continue the study longer than 60 days were changed when DD rats had high mortality rates after 60 days of age. DISCUSSION
The anemia and stunted growth of the suckling iron-deficient rat observed here is similar to and confirms previously reported findings (1,2). This results from iron restriction in the maternal animal during gestation and lactation and from the low concentrations of iron in milk secreted by the iron-deficient dam (2). The hyperlipidemia in the suckling irondeficient pups may be related to increased endogenous synthesis of lipid in liver (2) and/or to the decreased zinc/copper ratio in the livers of the pups (15). Rats weaned to the control diet (DC) were repleted with iron rapidly. This was evident after only 9 days on the diet when hemoglobin levels and hematocrit values increased to control values. This was probably due to the increased efficiency of iron absorption in iron-deficient rats (16) and the high level of iron found in the control diet. As the animals were repleted with iron, serum lipids decreased to normal concentrations and remained at normal levels for the duration of the study. Cholesteryl ester, cholesterol, and phospholipid in the serum decreased to approximately one-half of the preweaning levels. At 30 days of age, serum triglyceride concentrations were one-tenth of the levels found in 21-day-old
891
littermates. Apparently the hyperlipidemia of iron-deficiency is reversible when dietary iron is increased and body stores of iron are repleted. No indications of prolonged ill effects of the hyperlipidemia during suckling were observed in DC rats during this experiment. Histological studies of heart, lung, and kidney using light microscopy did not reveal any unusual pathologies in the DC rats. Although at 30 days of age the rats weaned to the deficient diet from the control diet (CD) were anemic, they were not sufficiently depleted in iron for an elevation in serum lipids to be manifested or for a depression in growth to occur. The hyperlipidemia seen in the 30-dayold DD rats was somewhat different in character than the hyperlipidemia observed during the suckling period. Cholesteryl ester was not elevated, and the levels of cholesterol and phospholipid were lower than in the sera of suckling pups. Serum triglycerides continued to be the lipid fraction most affected in the postweaning DD rats, with concentrations approximately five times greater than those found in control sera. The extent to which serum triglyceride concentrations remained elevated in the DD rats was fairly constant; concentrations at 21, 30, and 60 days did not differ significantly from each other. After 39 days on the iron-deficient diet, the 60-day-old CD rats were anemic and hypertriglyceridemic. Thus, the depression in iron status as reflected by the hematological values precedes the elevation in serum triglycerides by one month. The results of this study suggest that elevations in serum lipids during iron deficiency occur primarily in the more severely deficient rats. The age of the animal during the deficient period influences both the extent of anemia and the characteristics of the lipemia. The anemia in pups exposed to iron deficiency in utero and during suckling is quite severe as indicated by the blood hemoglobin concentrations and hematocrit values less than half of control levels and is associated with elevations in triglycerides, cholesterol, and phospholipids. When these rats are weaned to the deficient diet (DD), hemoglobin and hematocrit levels remain low and serum lipids remain high. However, serum phospholipid and cholesterol levels are not as high after weaning as during suckling. The lipemia of rats fed the deficient diet after weaning (CD) is limited to the triglyceride fraction and is not as severe as in the DD rats, and is related to a milder anemia than that found in the rats deprived of iron for a longer time period (DD). Amine and Hegsted (3) reported elevated triglycerides and phospholipids in Charles River rats fed low-iron diets for LIPIDS, VOL. 14, NO. 11
892
A.R. SHERMAN
8 weeks after weaning. The lipemia they r e p o r t e d was observed when the diet contained 15% c o c o n u t oil and 1% safflower oil. In the present "study, a hypertriglyceridemia was p r o d u c e d in the control-deficient animals fed 10% corn oil and no saturated fat for 5 89 weeks after weaning. The relationship between saturation of dietary fat, iron, and serum lipids may be related to differences in genetic strain. In a second study, Amine et al. (6) f o u n d that serum triglycerides were affected only by fat in Charles River rats, whereas, in Buffalo rats iron, fat, and an iron-fat interaction affected triglycerides. In the present study using Sprague Dawley rats, hypertriglyceridemia was observed in rats fed iron-deficient diets after weaning. However, when the deficiency was induced in utero cholesterol, triglycerides, and phospholipids were all elevated above control levels. The hypertriglyceridemia seen in the 60-dayold iron-deficient (DD) and (CD) rats may be due to increased lipogenesis in adipose and liver tissue which has been reported previously in adult iron-deficient rats (5). In a previous study, the increased i n c o r p o r a t i o n of [ 3 H 2 0 ] and [U-14C]glucose into triglycerides in adipose tissue and increased i n c o r p o r a t i o n of glucose into polar lipids in liver was not accompanied by a hypertriglyceridemia as was found in the 60-day-old deficient rats in the present study. This may be directly related to the more severe anemia seen in the present study (hemoglobin levels of 6.1 + 0.5 g/dl in CD rats and 3.0 -+ 0.7 g/dl in DD rats vs. 7.l +- 0.4 in deficient rats of the previous study [5]). The postulated sequence of events may be such that anemia is associated with increased lipogenesis in adipose and liver which precedes the appearance of increased concentrations of triglycerides in serum. When the iron deficiency anemia becomes more severe and increased lipogenesis continues, the resultant serum has a higher c o n c e n t r a t i o n of triglycerides. The hypertriglyceridemia in iron-deficient adult rats may also be related to a decreased activity in tissue lipoprotein lipase activity as reported by Lewis and l a m m a r i n o (4). The exact mechanism by which iron regulates or functions in lipid metabolism has not yet been established. Conflicting reports of serum lipid levels in anemic human subjects appear in the literature since early in this century. Bloor and MacPherson (17) and Erickson et al. (18) reported elevated lipids in anemic subjects. Others have found hypolipidemia in patients with anemias of various origins (19-22) or no significant relationships between anemia and serum lipids (23). Since hyperlipidemia is recognized as a risk factor in LIPIDS, VOL. 14, NO. 1 1
the development of atherosclerotic heart disease, all nutritional influences on serum lipid concentrations assume considerable importance and warrant further study to enable us to more clearly understand the etiology of this disease. ACKNOWLEDGMENTS Portions of this w ork were done at The Pennsylvania State University where the a ut hor was a Research Associate. This investigation was supported in part by grant HLB 18712 from the National Institutes of Health, and funds were provided by the School of Human Resources and Family Studies, University of Illinois. Animal care was provided by Nancy Tschiember, and technical assistance by Ihor M. Zulak and Karen Stratz was appreciated. REFERENCES 1.
Guthrie, H.A., M. Froozani, A.R. Sherman, and G.P. Barron, J. Nutr. 104:1273 (1974). 2. Sherman, A.R., H.A. Guthrie, I. Wolinsky, and I.M. Zulak, J. Nutr. 108:152 (1978). 3. Amine, E.K., and D.M. Hegsted, J. Nutr, 101:1575 (1971). 4. Lewis, M., and R.M. l a mma ri no, J. Lab. Clin. Med. 78:547 (1971). 5. Sherman, A.R., Lipids 13:473 (1978). 6. Amine, E.K., E.J. Desilets, and D.M. Hegsted, J. Nutr. 106:404 (1976). 7. National Research Council, "Nutritional Requirements of Laboratory A ni ma l s ," National Research Council, Washington, DC, 1972. 8. Houk, A.E.H., A.W. Thomas, and H.C. Sherman, J. Nutr. 31: 609 (1946). 9. Richterich, R., "Clinical C he mi s t ry," Academic Press, New York, 1969. 10. Folch, J., M. Lees, and G.H. Sloane-Stanley, J. Biol. Chem. 2 2 6 : 4 9 7 (1957). 11. Stern, I., and B. Shapiro, J. Clin. Pathol. 6: 158
(1953). 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Searcy, R.L., and L.M. Bergquist, Clin. Chim. Acta 5:192 (1960). Chen, P.S., T.Y. Toribara, and H. Warner, Anal. Chem. 28: 1756 (1956). Steel, R.G., and J.H. Torrie, "Principles and Procedures of Statistics," McGraw-Hill, New York, 1960. Sherman, A.R., H.A. Guthrie, and I. Wolinsky, Proc. Soc. Exp. Biol. Med. 156:396 (1977). Kinnamen, K., J. Nutr. 90:315 (1966). Bloor, W.R., and D.J. MacPherson, J. Biol. Chem. 31:79 (1917). Erickson, B.N., H.H. Williams, F.C. Hummel, P. Lee, and I.G. Macy, J. Biol. Chem. 18:569 (1937), Rifkind, B.M., and M. Gale, Am. Heart J. 76:849 (1968). Hashmi, J.A., and N. Afroz, Lancet 2:530 (1969). Elwood, P.C., P. Sweetham, R. Mahler, F. Moore, and E. Welsby, Lancet 1:589 (1970). Bottiger, L.E., and L.A. Carlson, Br. Med. J. 3:731 (1972). Fujii, R., and H. Shimizer, J. Nutr, Sci. Vitaminol. 19:23 (1973).
[Received June 11, 1979]
Serum lipids were studied in iron-deficient and control rats during suckling and after weaning at 21, 30, and 60 days of age. Diets providing 5 or 307 ppm iron were fed to dams and their offspring during gestation, lactation, and after weaning. Rats on the deficient diet throughout the experimental period developed a hyperlipidemia characterized by elevated triglycerides, cholesterol, and phospholipids which was present at 21, 30, and 60 days. Control pups weaned to the deficient diet developed anemia at 30 days of age and hypertriglyceridemia at 60 days of age. Repletion of deficient rats with iron after weaning caused a rapid decline in serum lipid levels after only 9 days on the control diet. The hyperlipidemia of iron deficiency thus appears to be reversible with iron supplementation. The time required to develop hypertriglyceridemia in iron deficiency is longer postweaning than during suckling. INTRODUCTION
Elevated concentrations of serum lipids have been associated with dietary iron deficiency in rats and chicks (1-4). Previous reports from this investigator have f o u n d abnormal lipid metabolism in 18-day-old offspring of rats fed irondeficient diets during pregnancy and lactation (1,2). Hyperlipidemia characterized by significant increases in the triglyceride, cholesterol, and phospholipid fractions of the serum was r e p o r t e d in pups of dams fed the deficient diet containing 5 ppm iron t h r o u g h o u t gestation and lactation (1). Sera from pups of dams fed the control diet (307 p p m iron) during either gestation a n d / o r lactation and dams fed both diets had normal lipid concentrations. The elevated serum lipids were observed only in pups whose dams were fed the lowest concentration of iron (5 ppm). A l t h o u g h feeding 29 ppm iron resulted in anemia, it was n o t associated with hyperlipidemia (2). Subsequent e x p e r i m e n t a t i o n has investigated the mechanisms which may be involved in the etiology of this hyperlipidemia. Since increases in serum lipids occurred in the offspring and n o t in the maternal organism, lipids were measured in milk of control and deficient dams to determine if iron-deficient pups had greater dietary fat levels. No differences were found in this exogenous source of lipids. Lipoprotein lipase was determined in the post-heptarin plasma of pups, and no significant differences were f o u n d b e t w e e n control and deficient pups. This indicated that decreased clearance of dietary lipids from the blood was not responsible for the hyperlipidemia. Production of triglycerides in vitro from [U-14C]glucose was found to be significantly higher in liver slices of iron-deficient pups than in controls. Apparently, during iron deficiency, this increased endogenous lipid pool may contribute to the
increased serum triglyceride concentrations (2). Lipogenesis has also been studied in irondeficient adult rats which were not hyperlipidemic (5). Triglyceride synthesis from [3H201 and [ U - 1 4 C l g l u c o s e in adipose tissue was t w o to three times greater in iron-deficient rats than in controls. Amine et al. found that iron-deficient rats had increased i n c o r p o r a t i o n of 14C-glucose into intestinal lipids than did controls and reported that the lipid synthesis increased as the level of dietary fats increased (6). The present study was initiated to investigate the effects of iron restriction during the prenatal and suckling a n d / o r postweanling periods on serum lipid levels later in life. Pups of iron-deficient dams were repleted with iron after weaning and control pups were depleted in iron after weaning to compare the effects of iron deficiency on serum lipid levels during these t w o stages in the life cycle.
888
MATERIALS AND METHODS Experimental Design
Nulliparous Sprague-Dawley CD rats (Charles River, Wilmington, MA) were obtained at a weight of 180 g and maintained on a cereal-based stock diet (Purina Rat Chow) until they weighed ca. 270 g. They were then bred and fed ad libitum either the iron-deficient diet (5 p p m i r o n ) or the control diet (307 ppm iron) (Table I) (n=16 per group) and glassdistilled water f r o m the first day of pregnancy until the pups were weaned. On the day following parturition, litters were adjusted to contain six female pups. F o o d was placed in the cage to limit access to the dams and the iron-free bedding in the solid b o t t o m "matern i t y " cages was changed frequently. On day 21 the pups were weaned to either the same or opposite diet as that fed to their dams and
SERUM LIPIDS IN IRON-DEFICIENT RATS Lipids
TABLE I Composition of Diets
Casein 2 Sucrose Cornstarch Iron-free salt mix 3 Vitamin mix 4 Corn oil 5 Cellulose 6
889
307 ppm iron 1 %
5 ppm iron %
22.00 29.70 29.70 5.48 1.00 10.00 2.00
22.00 29.76 29.76 5.48 1.00 10.00 2.00
l Iron levels determined by atomic absorption. 2Vitamin-free casein, Teklad, Chagrin Falls, OH. 3The levels of minerals used is at least 12.5% more than NRC recommendations for lactation (7). Composition of salt mixture (mg/lO0 g diet): CaCO 3, 1,680.000; COC12.6 H20 , 0.100; CUSO4"5 H20, 9.800; MgSO4.7 H20 , 278.500; MnSO4"H20, 19.000; KI, 0.022; NaCI, 1,000.000; K2HPO 4, 2,483.600; ZnCI2, 3.000. FeSO4.7 H20 was added to the supplemented diet in place of sucrose. 4Teklad, Chagrin Falls, OH, supplies in g/kg of diet; p-aminobenzoic acid, 0.110132; ascorbic acid, 0.9912; biotin, 0.000441; vitamin B12, 0.0000297; calcium pantothenate, 0.066079; choline, 1.4337; folic acid, 0.001982; inositol, 0.110132; menadione (vitamin K3) , 0.049559 ; niacin, 0.099119 ; pyridoxine HCI, 0.022026; riboflavin, 0.022026; thiamin HC1, 0.022026; supplies in units per kg of diet: dry retinyl palmitate, 19,824; dry ergocalcifrol 2.202.5; dry tocopheryl acetate, 121.15; corn starch, 4.666878 g. 5Mazola corn oil, Best Foods, Englewood Cliffs, NJ. 6Alphacel nonnutritive cellulose, Teklad, with iron extracted by the method of Houk et al. (8). placed in individual stainless steel screen b o t t o m cages. T h e r e were f o u r d i f f e r e n t experim e n t a l t r e a t m e n t s d e p e n d i n g o n the d a m ' s diet during p r e g n a n c y a n d l a c t a t i o n a n d t h e diet fed to weanlings a f t e r day 21. C o n t r o l C o n t r o l (CC) rats were b o r n to c o n t r o l dams a n d w e a n e d t o t h e c o n t r o l diet, CD ( C o n t r o l Deficient) rats were b o r n t o c o n t r o l d a m s a n d w e a n e d to t h e i r o n - d e f i c i e n t diet, DC (Defic i e n t - C o n t r o l ) rats were r e p t e t e d w i t h i r o n a f t e r weaning, DD ( D e f i c i e n t - D e f i c i e n t ) rats were b o r n to i r o n - d e f i c i e n t dams a n d w e a n e d t o i r o n - d e f i c i e n t diets ( n = 8 - 1 4 litters p e r treatm e n t ) . Diets a n d glass-distilled w a t e r were offered ad l i b i t u m a n d weekly f o o d i n t a k e s a n d b o d y weight r e c o r d s were k e p t . L i t t e r m a t e s were fasted for 4 h r o n days 21, 30 a n d 6 0 a n d taft b l o o d samples were t a k e n for h e m o g l o b i n a n d h e m a t o c r i t d e t e r m i n a t i o n s (9). A f t e r brief e x p o s u r e to c h l o r o f o r m anesthesia, b l o o d was collected b y cardiac p u n c t u r e , s e r u m r e i n o v e d a n d f r o z e n for analysis o f lipids.
S e r u m triglycerides, cholesterol, c h o l e s t e r y l ester a n d p h o s p h o l i p i d s were d e t e r m i n e d . Lipids were e x t r a c t e d f r o m s e r u m samples b y t h e m e t h o d of F o l c h et al. ( 1 0 ) a n d s e p a r a t e d o n t h i n l a y e r c h r o m a t o g r a p h y plates d e v e l o p e d in a solvent s y s t e m of p e t r o l e u m e t h e r / e t h y l e t h e r / a c e t i c acid ( 7 0 : 3 0 : 1 , v/v/v). F o l l o w i n g e x p o s u r e t o i o d i n e vapors, lipid f r a c t i o n s were i d e n t i f i e d b y c o m p a r i s o n w i t h triolein, cholesterol, cholesteryl linoleate, and lecithin s t a n d a r d s w h i c h were r u n s i m u l t a n e o u s l y . T h e spots were r e c o v e r e d a n d t h e f o l l o w i n g m e t h o d s were used to q u a n t i t a t e t h e lipid f r a c t i o n s : triglyceride c o n c e n t r a t i o n was determ i n e d b y t h e m e t h o d o f s t e r n a n d Shapiro ( 1 1 ) n o n e s t e r i f i e d c h o l e s t e r o l a n d c h o l e s t e r y l esters were m e a s u r e d b y t h e m e t h o d of Searcy a n d Bergquist (12) a n d p h o s p h o l i p i d c o n c e n t r a t i o n was d e t e r m i n e d b y m e a s u r i n g p h o s p h o r u s in t h e lipids r e m a i n i n g at t h e origin using t h e m e t h o d of C h e n et al. (13). Statistical Methods
Differences b e t w e e n t h e t w o g r o u p s at 21 days o f age were d e t e r m i n e d w i t h t h e S t u d e n t ' s t test. Analysis of v a r i a n c e was used t o determine differences among group means when four e x p e r i m e n t a l g r o u p s were c o m p a r e d (days 3 0 a n d 60). D u n c a n ' s m u l t i p l e range test was used t o find t h e m e a n s w h i c h differed significantly (14) R ESU LTS
T w e n t y - o n e - d a y - o l d p u p s of i r o n - d e f i c i e n t d a m s h a d l o w e r b o d y weights, h e m o g l o b i n a n d h e m a t o c r i t levels (Table II) a n d h i g h e r levels of s e r u m c h o l e s t e r y l ester, c h o l e s t e r o l , triglycerides, a n d p h o s p h o l i p i d s ( T a b l e III) t h a n did p u p s o f c o n t r o l dams. T h e e l e v a t i o n s in cholest e r y l ester a n d c h o l e s t e r o l were a p p r o x i m a t e l y 2-fold, triglycerides were 4-fold a n d p h o s p h o lipids were elevated 3-fold. T h e i r o n c o n t e n t of t h e w e a n i n g diet h a d d r a m a t i c effects o n b o d y weight, h e m a t o l o g i c a l values, a n d s e r u m lipid levels as early as 30 days of age. Weaning d e f i c i e n t p u p s t o t h e c o n t r o l diet (DC) increased b o d y weight, h e m o g l o b i n , a n d h e m a t o crit t o c o n t r o l levels a f t e r o n l y 9 days o n t h e diet. S e r u m c h o l e s t e r y l ester, triglycerides, a n d p h o s p h o l i p i d s r e t u r n e d to c o n t r o l levels at this t i m e also. At 3 0 days old, s e r u m c h o l e s t e r o l r e m a i n e d s o m e w h a t h i g h e r in DC rats t h a n in CC rats. Pups w e a n e d t o t h e d e f i c i e n t diet f r o m t h e c o n t r o l diet (CD) h a d l o w e r levels of h e m o g l o b i n a n d h e m a t o c r i t s t h a n CC rats b u t n o t as l o w as DD rats w h i c h h a d b e e n e x p o s e d t o t h e i r o n - d e f i c i e n t diet t h r o u g h o u t t h e LIPIDS, VOL. 14, NO. 11
890
A.R.
SHERMAN
~RR v v v
@.
t.:.Rq
§
+L
+1
.,
+1
+1
o
+,
+,.,
r
§
c
0
e~ ..
N
~
g
=
g
vvv o
....
_
:
+,
r
+,
~.
'~
g 7
+'
+' +' *1
~
=
tmq
Z
E
E
.3 _e
2 *,
+,
§
~
h3
m
o m
'9
~
'q
== +, +, +~
v
v
v
~ : ~
~176
5 ~
vvv
-~
..~
.E 7
_
~
A g .~
.1= T. ,1=
LIPIDS,
VOL.
14,
NO.
11
"
*
SERUM LIPIDS IN IRON-DEFICIENT RATS experiment. No significant elevations in serum lipids were seen in the CD rats at 30 days of age, although cholesterol concentrations were slightly elevated relative to the CC rats. The DD rats continued to have elevated serum cholesterol, triglycerides, and phospholipids after weaning. At 60 days of age, body weights were similar in CC, CD, and DC rats, and significantly lower in DD rats (Table II). Hematological values (Table II) in CC and DC rats were the same, CD rats had hemoglobin levels and hematocrits approximately one-half of the control rats. Hemoglobin levels and hematocrits in DD rats were significanlty lower than those of all of the other groups. Serum cholesterol, triglycerides and phospholipids continued to be highest in the DD group at 60 days (Table ill). Sixty-dayold DC and CC rats had serum lipid concentrations similar to those found in 30-day-old littermates. Rats restricted in iron after weaning (CD) developed a hypertriglyceridemia at 60 days of age. However, serum cholesterol and phospholipids were not significantly elevated in the CD rats at that time. Initial plans to continue the study longer than 60 days were changed when DD rats had high mortality rates after 60 days of age. DISCUSSION
The anemia and stunted growth of the suckling iron-deficient rat observed here is similar to and confirms previously reported findings (1,2). This results from iron restriction in the maternal animal during gestation and lactation and from the low concentrations of iron in milk secreted by the iron-deficient dam (2). The hyperlipidemia in the suckling irondeficient pups may be related to increased endogenous synthesis of lipid in liver (2) and/or to the decreased zinc/copper ratio in the livers of the pups (15). Rats weaned to the control diet (DC) were repleted with iron rapidly. This was evident after only 9 days on the diet when hemoglobin levels and hematocrit values increased to control values. This was probably due to the increased efficiency of iron absorption in iron-deficient rats (16) and the high level of iron found in the control diet. As the animals were repleted with iron, serum lipids decreased to normal concentrations and remained at normal levels for the duration of the study. Cholesteryl ester, cholesterol, and phospholipid in the serum decreased to approximately one-half of the preweaning levels. At 30 days of age, serum triglyceride concentrations were one-tenth of the levels found in 21-day-old
891
littermates. Apparently the hyperlipidemia of iron-deficiency is reversible when dietary iron is increased and body stores of iron are repleted. No indications of prolonged ill effects of the hyperlipidemia during suckling were observed in DC rats during this experiment. Histological studies of heart, lung, and kidney using light microscopy did not reveal any unusual pathologies in the DC rats. Although at 30 days of age the rats weaned to the deficient diet from the control diet (CD) were anemic, they were not sufficiently depleted in iron for an elevation in serum lipids to be manifested or for a depression in growth to occur. The hyperlipidemia seen in the 30-dayold DD rats was somewhat different in character than the hyperlipidemia observed during the suckling period. Cholesteryl ester was not elevated, and the levels of cholesterol and phospholipid were lower than in the sera of suckling pups. Serum triglycerides continued to be the lipid fraction most affected in the postweaning DD rats, with concentrations approximately five times greater than those found in control sera. The extent to which serum triglyceride concentrations remained elevated in the DD rats was fairly constant; concentrations at 21, 30, and 60 days did not differ significantly from each other. After 39 days on the iron-deficient diet, the 60-day-old CD rats were anemic and hypertriglyceridemic. Thus, the depression in iron status as reflected by the hematological values precedes the elevation in serum triglycerides by one month. The results of this study suggest that elevations in serum lipids during iron deficiency occur primarily in the more severely deficient rats. The age of the animal during the deficient period influences both the extent of anemia and the characteristics of the lipemia. The anemia in pups exposed to iron deficiency in utero and during suckling is quite severe as indicated by the blood hemoglobin concentrations and hematocrit values less than half of control levels and is associated with elevations in triglycerides, cholesterol, and phospholipids. When these rats are weaned to the deficient diet (DD), hemoglobin and hematocrit levels remain low and serum lipids remain high. However, serum phospholipid and cholesterol levels are not as high after weaning as during suckling. The lipemia of rats fed the deficient diet after weaning (CD) is limited to the triglyceride fraction and is not as severe as in the DD rats, and is related to a milder anemia than that found in the rats deprived of iron for a longer time period (DD). Amine and Hegsted (3) reported elevated triglycerides and phospholipids in Charles River rats fed low-iron diets for LIPIDS, VOL. 14, NO. 11
892
A.R. SHERMAN
8 weeks after weaning. The lipemia they r e p o r t e d was observed when the diet contained 15% c o c o n u t oil and 1% safflower oil. In the present "study, a hypertriglyceridemia was p r o d u c e d in the control-deficient animals fed 10% corn oil and no saturated fat for 5 89 weeks after weaning. The relationship between saturation of dietary fat, iron, and serum lipids may be related to differences in genetic strain. In a second study, Amine et al. (6) f o u n d that serum triglycerides were affected only by fat in Charles River rats, whereas, in Buffalo rats iron, fat, and an iron-fat interaction affected triglycerides. In the present study using Sprague Dawley rats, hypertriglyceridemia was observed in rats fed iron-deficient diets after weaning. However, when the deficiency was induced in utero cholesterol, triglycerides, and phospholipids were all elevated above control levels. The hypertriglyceridemia seen in the 60-dayold iron-deficient (DD) and (CD) rats may be due to increased lipogenesis in adipose and liver tissue which has been reported previously in adult iron-deficient rats (5). In a previous study, the increased i n c o r p o r a t i o n of [ 3 H 2 0 ] and [U-14C]glucose into triglycerides in adipose tissue and increased i n c o r p o r a t i o n of glucose into polar lipids in liver was not accompanied by a hypertriglyceridemia as was found in the 60-day-old deficient rats in the present study. This may be directly related to the more severe anemia seen in the present study (hemoglobin levels of 6.1 + 0.5 g/dl in CD rats and 3.0 -+ 0.7 g/dl in DD rats vs. 7.l +- 0.4 in deficient rats of the previous study [5]). The postulated sequence of events may be such that anemia is associated with increased lipogenesis in adipose and liver which precedes the appearance of increased concentrations of triglycerides in serum. When the iron deficiency anemia becomes more severe and increased lipogenesis continues, the resultant serum has a higher c o n c e n t r a t i o n of triglycerides. The hypertriglyceridemia in iron-deficient adult rats may also be related to a decreased activity in tissue lipoprotein lipase activity as reported by Lewis and l a m m a r i n o (4). The exact mechanism by which iron regulates or functions in lipid metabolism has not yet been established. Conflicting reports of serum lipid levels in anemic human subjects appear in the literature since early in this century. Bloor and MacPherson (17) and Erickson et al. (18) reported elevated lipids in anemic subjects. Others have found hypolipidemia in patients with anemias of various origins (19-22) or no significant relationships between anemia and serum lipids (23). Since hyperlipidemia is recognized as a risk factor in LIPIDS, VOL. 14, NO. 1 1
the development of atherosclerotic heart disease, all nutritional influences on serum lipid concentrations assume considerable importance and warrant further study to enable us to more clearly understand the etiology of this disease. ACKNOWLEDGMENTS Portions of this w ork were done at The Pennsylvania State University where the a ut hor was a Research Associate. This investigation was supported in part by grant HLB 18712 from the National Institutes of Health, and funds were provided by the School of Human Resources and Family Studies, University of Illinois. Animal care was provided by Nancy Tschiember, and technical assistance by Ihor M. Zulak and Karen Stratz was appreciated. REFERENCES 1.
Guthrie, H.A., M. Froozani, A.R. Sherman, and G.P. Barron, J. Nutr. 104:1273 (1974). 2. Sherman, A.R., H.A. Guthrie, I. Wolinsky, and I.M. Zulak, J. Nutr. 108:152 (1978). 3. Amine, E.K., and D.M. Hegsted, J. Nutr, 101:1575 (1971). 4. Lewis, M., and R.M. l a mma ri no, J. Lab. Clin. Med. 78:547 (1971). 5. Sherman, A.R., Lipids 13:473 (1978). 6. Amine, E.K., E.J. Desilets, and D.M. Hegsted, J. Nutr. 106:404 (1976). 7. National Research Council, "Nutritional Requirements of Laboratory A ni ma l s ," National Research Council, Washington, DC, 1972. 8. Houk, A.E.H., A.W. Thomas, and H.C. Sherman, J. Nutr. 31: 609 (1946). 9. Richterich, R., "Clinical C he mi s t ry," Academic Press, New York, 1969. 10. Folch, J., M. Lees, and G.H. Sloane-Stanley, J. Biol. Chem. 2 2 6 : 4 9 7 (1957). 11. Stern, I., and B. Shapiro, J. Clin. Pathol. 6: 158
(1953). 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Searcy, R.L., and L.M. Bergquist, Clin. Chim. Acta 5:192 (1960). Chen, P.S., T.Y. Toribara, and H. Warner, Anal. Chem. 28: 1756 (1956). Steel, R.G., and J.H. Torrie, "Principles and Procedures of Statistics," McGraw-Hill, New York, 1960. Sherman, A.R., H.A. Guthrie, and I. Wolinsky, Proc. Soc. Exp. Biol. Med. 156:396 (1977). Kinnamen, K., J. Nutr. 90:315 (1966). Bloor, W.R., and D.J. MacPherson, J. Biol. Chem. 31:79 (1917). Erickson, B.N., H.H. Williams, F.C. Hummel, P. Lee, and I.G. Macy, J. Biol. Chem. 18:569 (1937), Rifkind, B.M., and M. Gale, Am. Heart J. 76:849 (1968). Hashmi, J.A., and N. Afroz, Lancet 2:530 (1969). Elwood, P.C., P. Sweetham, R. Mahler, F. Moore, and E. Welsby, Lancet 1:589 (1970). Bottiger, L.E., and L.A. Carlson, Br. Med. J. 3:731 (1972). Fujii, R., and H. Shimizer, J. Nutr, Sci. Vitaminol. 19:23 (1973).
[Received June 11, 1979]
