HELEN K. ONDERKA ANDAVANELLE KIRKSEY Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907 ABSTRACT Interrelationships between oral contraceptives and dietary lipids on iron and copper levels in plasma and tissues were investigated in rats. Diets containing either 20% (by weight) safflower oil or hydrogenated coconut oil with and without cholesterol (0.5%) were fed to weanling, female, Wistar-strain rats for a period of 19 weeks. Three types of oral contraceptive agents differing in estrogen/progesterone ratios were administered during weeks 16 through 19 of the experiment. Control rats received the dietary treatments without oral contraceptives. Hemoglobin concentration, hematocrit, reel blood cell counts, mean cell hemoglobin and hemoglobin concentration, and mean cell volume values were similar among the various dietary and drug treatment groups. Elevated levels of copper were found in livers of drug-treated animals fed diets containing cholesterol and safflower oil, whereas levels of copper or iron in spleen and kidney were not influenced by oral contraceptives. Dietary safflower or coconut oil had no influence on levels of iron or copper in plasma. However, iron levels were higher in liver, spleen, and kidneys of rats fed coconut oil compared with those fed safflower oil. Cholesterol-fed rats had reduced levels of iron in plasma and tissues and increased levels of copper in plasma and liver. Iron deficiency in cholesterol-fed rats was indicated by low levels of iron in plasma, liver, spleen, and kidney. In experiment 2, animals were fed the 20% safflower oil diet, with and without sodium glycocholate or cholesterol, to determine whether the apparent malabsorption of iron resulted from sodium glyco cholate or cholesterol. Sodium glycocholate resulted in a marked increase in the ab sorption of iron, whereas cholesterol depressed absorption. J. Nutr. 105: 12691277, 1975. INDEXING KEY WORDS oral contraceptives •iron •copper •cholesterol safflower oil •coconut oil
Seemingly diverse metabolic changes have been reported to occur after the use of oral contraceptive agents (OC). Among these alterations are elevated levels of iron and copper in tissues and of cholesterol and/or triglycérides in serum (1-3). Whether the elevated levels of iron and copper are a direct effect of OC or an indirect effect of hyperlipemia, produced by the drugs, is not established. Some evidence suggests that dietary fats and cho-
induced similar anemia in man (7). Amine and Hegsted (8) reported that anemic female rats fed a diet containing 15% partially hydrogenated vegetable oil and coconut oil developed severe lipemia. Interrelationships of dietary lipids with iron metabolism are better documented than those with copper. However, atherogenesis in rabbits has been reported 3 to be intensified by high levels of copper in the Recelvertfor publlcatlonJanuary 28. 1OT5.
lesterol and iron metabolism are interret From Purdue University Agricultural Experiment laf-prl TTvnprprinlpsf-prnlpTni'l-i'nrliipincr rhVt« Station and School of Home Economics, Department
larea. nypercnoiesreroiemia inducing aieis have been demonstrated to induce hemolytic anemia resulting in elevated iron in tissues in the rat, rabbit, and guinea pig (4-6), and rat emulsion mhisions 2 have
nf poo(,Ban(, Nlltrltlon r,afayette,ind. 47907,Jour»aipaperno. 4965. Î ^Ä^^e^^ÄjÄ m -^ %£S8tà *Vm£SS Chicago,in., Abstr.36.
1269
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Influence of Dietary Lipids on Iron and Copper Levels of Rats Administered Oral Contraceptives1
1270
HELEN
K. ONDERKA
AND AVANELLE
g/100 g diel
Casein1 DL-Methionine1 Sucrose Starch Fat2 Salt mix' Vitamin mix1-4
Cholesterol1-1
18.0 0.3 18.7 36.0 20.0 4.0 3.0
Sodium glycocholate5
0.5
0.5
1 Nutritional Biochemicals, Inc., Cleveland, Ohio. 2 Coco nut oil or safflower oil; safflower oil (0.3%) was added to coco nut oil to prevent EFA deficiency. >Jones and Foster (33). 4 mg/100 g of diet: thiamin, 1.5; riboflavin, 1.5; niacin, 3.0; pyridoxine HC1, 0.8; DL-calcium pantothenate, 7.0; biotin, 0.2; p-aminobenzoic acid, 1.5; inositol, 100.0; choline chloride 180.0; folie acid 1.0; H-12 (triturate 1%) 3.0; menadione, 0.1; vitamin A powder, largely palmitate (400 IU), 20.0; cholecalciferol (50 IU), 0.005; DL-«-tocopherol, 12.0. s Added only to diets of rats fed cholesterol.
diet. Also in atherogenesis, copper concen trations in plasma and tissues are elevated (9). Conceivably the combined effects of OC and dietary lipids on iron and copper metabolism could result in an intensifica tion of the separate effects of drugs and diet that have been reported. The purpose of this investigation was to explore the ef fects of OC and diets high in saturated and unsaturated fat with and without added cholesterol on plasma and tissue levels of iron and copper in rats. A second study investigated the effects of choles terol and sodium glycocholate on iron ab sorption. MATERIALS
AND METHODS
Animals and diets. Wistar-strain female rats weighing approximately 40 to 50 g were randomly divided into 4 groups of 32 animals each. For a period of 15 weeks, two of the groups were fed a diet (table 1) containing (by weight) 20% safflower oil4 and two received the diets with 20% hydrogenated coconut oil.5 Cholesterol (0.5%) was included in the diets fed to one-half of the rats receiving safflower or coconut oil. Sodium glycocholate (0.5%) was added to diets containing cholesterol to aid in its absorption (10). All fats used in the experiment were refrigerated and stored in the dark, and diets were frozen immediately after mixing to prevent fatty acid oxidation. For 4 additional weeks, after the 15-week period of dietary treat ment, three OC agents *• '•8differing in
estrogen/progesterone ratios were ran domly administered to three groups of eight animals in each of the four dietary treatment groups; the remaining eight ani mals in each group received no OC and were designated as controls. The daily dosage of OC was incorporated into 15 g of diet.6'7-s Rats received a physiological drug dosage equivalent to 1.5 to 2 times the usual human dosage on a drug per body weight basis. Vaginal smears were taken daily for a period of 2 weeks. During this time, no animal receiving an OC was found to be in estrus. On this basis, the OC treatment was judged to be effective. Experiment 2 was carried out to investi gate the effects of sodium glycocholate and cholesterol on iron absorption. Female weanling Wistar rats were divided into four groups of seven animals each. One group (control) was fed the 20% safflower oil diet described in table 1 but without the addition of cholesterol or sodium glyco cholate. The remaining groups were fed the same diet as that of the control group with the addition of (1) 0.5% sodium glycocholate, (2) 0.5% cholesterol, or (3) 0.5% sodium glycocholate and 0.5% cho lesterol. All diets were fed for a 7-week period. Rats were housed individually in wirebottomed suspended cages in a room maintained at 25°and 40% relative hu midity and with alternate periods of light and dark. Diets and water were fed ad libitum. Weight gain and food intake were measured 3 times weekly. Methods. On day 28 of OC administra tion, animals were fasted for 4 hours, anesthetized with ether, and 4.5 ml of blood was drawn by heart puncture into a syringe containing 0.25 ml of 3.8% sodium citrate. To minimize diurnal variation in assays, blood was drawn at the same time each day. Blood samples were immedi ately placed on ice and were then centrifuged at 500 X g for 10 minutes at 5°.The 1 Pacific Vegetable Oil Porp.. Sun Francisco. Calif. 6 Procter and (ïamble, Cincinnati. Ohio. "Ovulen (S.G «g ethynodiol acetate and 0.8« ug mestranol/15 g diet) G. D. Searle Co.. Chicago, 111. 7 Provest (86 ug medroxyprogesterone acetate and 0.43 ug ethlnylestradiol/ln g diet) Upjohn Co.. Kalamnzoo. Mich. 8Bnovid (22 jjg norethynodrel and 0.80 «gmestranol/15 g diet) G. D. Searle Co., Chicago, 111.
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TABLE 1 Composition of diets
KIRKSEY
DIET AND ORAL CONTRACEPTIVES
IN THE RAT
1271 Downloaded from https://academic.oup.com/jn/article-abstract/105/10/1269/4768653 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
plasma was removed and stored at —20° sign. The Newman-Keuls Sequential Range until analysis. Test was used to determine significant dif Hemoglobin was measured spectrophoferences among means. The t test was tometrically as cyanomethemoglobin using used to determine differences between a commercial standard.9 Red blood cells means of certain data collected prior to were counted by use of a hemacytometer, and during OC administration. Experiment and hematocrits were measured in a micro- 2 was designed as a 2x2x2 factorial, hematocrit reader after centrifugation in and data were analyzed by analysis of a high speed centrifuge. Plasma iron and variance. copper were measured by atomic absorp RESULTS AND DISCUSSION tion spectrophotometry (11, 12). An atomic absorption spectrophotometer I0 fitted with Food consumption, iron and copper in the Intensitron series hollow cathode iron takes, and body weights. In the 28-day and copper tubes was used. The samples period prior to OC administration, food were aspirated into an air-acetylene mix consumption was not significantly affected ture. by any of the dietary treatments. However, All equipment used for iron and copper animals fed safflower oil weighed more determinations was soaked at least 3 hours than those fed coconut oil (table 2). During the period of OC treatment, a sig in 50% nitric acid and was thoroughly nificant overall reduction in food consump rinsed with distilled water. tion was observed in animals receiving Copper oxidase activity was measured drugs compared with values for animals according to the method of Houchin (13), and the activity was used to calculate not fed OC (table 2). Aftergood et al. (16) reported food consumption to be re ceruloplasmin concentration by use of the following formula: Y = a + b x, where duced by over 50% in rats after the ad Y = ceruloplasmin concentration in mg ministration of 1 mg of an oral contra ceptive,8 which was also used in this study. per 100 ml, x = absorbance of the product formed by copper oxidase activity, a — However, in this study food consumption —1.7,and b = 150. Plasma samples were was reduced by only 11%. The differences in findings may be attributed to the size of analyzed for copper oxidase activity within the OC dosage used. The amount of drug 10 days after they were collected. administered in this study was smaller Liver, kidney, and spleen samples were than that given by Aftergood et al. and digested by a modification of a wet oxida tion procedure reported by Reit/ et al. was a more physiological dosage. In the (14). The iron and copper content in the present study, body weights were not sig digested tissue samples was measured by nificantly affected by OC treatment. The effectiveness of the dosage was confirmed atomic absorption spectrophotometry. by anestrus and by a statistically signifi In experiment 2, feces were collected during the last 7 days of the study and cant (P < 0.01) increase in uterine weights were wet digested and analyzed for iron at the end of the experiment. Differences in food consumption related content in the same manner as that de scribed for the tissue samples. Fecal iron to diet were observed during the OC treat content was compared with dietary iron ment period. Animals fed diets containing coconut oil had higher food consumption intake to determine the approximate per by approximately 18% than those fed diets centage absorption of dietary iron. Plan of experiment. This experiment was containing safflower oil; however, body weights were lower. This appeared to in designed as a 4 X 2 X 2 factorial. Data by analysis 4were X 2 analyzed X 2'factorial and thenofasvariance a 3 X 2 as X 2a dicate that coconut oil was not as well uti lized as safflower oil. Animals receiving dietary cholesterol had higher food con factorial to determine if there were sta tistically significant differences among the sumption by approximately 10% than those not receiving cholesterol; however, three OC treatments (15). When no dif ferences were found, the data from the body weights were not significantly differanimals administered OC were pooled and » Ilycel. Inc., Houston, Texas. 10Perkin-Elmer Corp., Norwalk, Conn., model 303. were analyzed as a 2 X 2 X 2 factorial de
1272
HELEN K. ONDERKA AND AVANELLE KIRKSEY
tration, and mean cell volume were ob served among the various dietary and drug treatments. OC treatment did not alter the levels of iron or copper in plasma. The efiFectsof estrogen therapy and OC admin istration in increasing the levels of serum copper and ceruloplasmin are well docu mented in the literature (18-20). Some in vestigators (21, 22) have reported changes in hematological values due to estrogen treatment or OC administration. The level of OC agent administered in this study was considerably less than that used to in duce these reported changes. The type of dietary fat used in this study had no significant influence upon plasma iron, copper, ceruloplasmin, or other hematological indices. Ahlstrom and Jar-
TABLE2 Total fowl consumption and Itody weight«prior to and during llu- administration of oral contraceptive agents (OC) Food consumption Treatment1
No cholesterol Safflower No OC2 OC* OC« OC« Coconut No OC OC OC OC Cholesterol Safflower No OC OC OC OC
No.
rats
Prior to OC
During OC
Body weight Prior to OC
During OC
8 8 7 8
370±423 366±43 376±34 386±27
300±42*Significantly different from A (P < 0.01) and from a (P < 0.05). t»> Significantly different from B (P < 0.01 and from 6 (P < 0.05). Significantly different from C (P < 0.01) and from c (P < 0.05).
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ent due to cholesterol. An explanation for the poorer food efficiency in animals fed cholesterol was not apparent from the data. The intakes of iron and copper equaled or exceeded the NRG (17) requirements of the rat for growth. Intakes of iron ranged from 2.2 to 3.2 mg daily and copper from 0.05 to 0.07 mg. Hence the changes in iron and copper levels in plasma and tissues observed in this study were not re lated to an inadequacy of dietary iron and copper. Hematological measurements and plasma iron and copper levels. No significant dif ferences in hemoglobin levels (table 3), hematocrit, red blood cell counts, mean cell hemoglobin and hemoglobin concen
1273
DIET AND ORAL CONTRACEPTIVES IN THE RAT
Treatment1No
ironlig/
rats7 ml13.
ml368±56 100
copperng/100 ml156.8Ü8
ml30.9±
cholesterolSafflowerNo OC2 OC4CoconutNo OC OCCholesterol SafflowerNo OC OCCoconutNo
197
9±1.82' 371 ±55416±79 13.5±1.513.6±0.6
364 ±76306±66 1661!)6 13.8±0.613.6±0.6
13.4±0.712.4±0.4NSOC ±64294±40 281
OC
201 OCSignificant
6.732.6±10.733.9±
162.5±32157.6±26 3.8 149.0±30255.9±68 32.6± 7.963.5±
7.4 62.0±19.279.2±20.0 243.9±48304.5±94
328±98CholesterolPlasma ±92Cholesterolweeks. 254.4
57.8±14.6Cholesterol1
values(P F < 0.01)No. Diet«were fed for 19 weeks after weaning ;Hemoglobina/100 ! No oral contraceptive.Ceruloplasminmo/100 Mean ±so. *Value! were fed the last 4Plasma for animals treated with three oral contraceptives were similar and have been pooled.
vinen (23) reported that various levels of fresh and heated safflower oil ( 10, 20, and 30% of the diet) had no effect on hemo globin, packed cell volume, and mean cell volume. Rats fed cholesterol had significantly lower plasma iron levels (table 3) than those receiving no cholesterol. Other in vestigators (6, 24 ) have reported hemolytic anemia and lowered levels of hemoglobin and of serum iron in animals fed dietary cholesterol. Ostwald et al. (25) recently described the effects of dietary modifica tion on a cholesterol-induced hemolytic anemia in guinea pigs in which iron depo sition was increased in liver, spleen, and other organs. This is in contrast to this study in which hemolytic anemia was not observed in cholesterol-treated rats, and iron levels in liver and spleen were de creased compared with values for animals receiving no cholesterol. In contrast to the lowered levels of iron in plasma of cholesterol-fed rats, plasma copper and ceruloplasmin levels were sig nificantly increased (table 3). Total plasma copper and ceruloplasmin were measured in the present study, and the direct-react ing fraction of plasma copper was calcu
lated from these measurements. Cerulo plasmin contains 0.34% copper and ac counts for 90% of the copper in plasma. The total copper in plasma minus the cop per in ceruloplasmin is the direct-reacting fraction of plasma copper. From this cal culation, it appeared that the major in crease of plasma copper was due to the elevation in ceruloplasmin. Osaki et al. (26) proposed that the physiological role of ceruloplasmin is to stimulate the release of iron from tissue stores into plasma. Evans and Abraham (27) have recently demonstrated this role of ceruloplasmin in vivo. In the present study, animals with elevated ceruloplasmin levels had lower iron stores and lower plasma iron values. The depression in iron levels in plasma in conjunction with the findings of Roeser et al. (28) that plasma iron levels increased after the administration of ceruloplasmin appeared to contraindÃ-cate the possibility that the elevated ceruloplasmin levels ob served in this study resulted in the in creased iron mobilization from tissues. Weight and iron and copper content of organs. Livers of rats fed dietary choles terol were fatty in appearance, pale yellow in color, and larger in size than those of
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TABLE 3 Hemoglobin, plasma iron, copper, and ceruloplasmin
1274
HELEN
K. ONDERKA AND AVANELLE
MO22
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Seemingly diverse metabolic changes have been reported to occur after the use of oral contraceptive agents (OC). Among these alterations are elevated levels of iron and copper in tissues and of cholesterol and/or triglycérides in serum (1-3). Whether the elevated levels of iron and copper are a direct effect of OC or an indirect effect of hyperlipemia, produced by the drugs, is not established. Some evidence suggests that dietary fats and cho-
induced similar anemia in man (7). Amine and Hegsted (8) reported that anemic female rats fed a diet containing 15% partially hydrogenated vegetable oil and coconut oil developed severe lipemia. Interrelationships of dietary lipids with iron metabolism are better documented than those with copper. However, atherogenesis in rabbits has been reported 3 to be intensified by high levels of copper in the Recelvertfor publlcatlonJanuary 28. 1OT5.
lesterol and iron metabolism are interret From Purdue University Agricultural Experiment laf-prl TTvnprprinlpsf-prnlpTni'l-i'nrliipincr rhVt« Station and School of Home Economics, Department
larea. nypercnoiesreroiemia inducing aieis have been demonstrated to induce hemolytic anemia resulting in elevated iron in tissues in the rat, rabbit, and guinea pig (4-6), and rat emulsion mhisions 2 have
nf poo(,Ban(, Nlltrltlon r,afayette,ind. 47907,Jour»aipaperno. 4965. Î ^Ä^^e^^ÄjÄ m -^ %£S8tà *Vm£SS Chicago,in., Abstr.36.
1269
Downloaded from https://academic.oup.com/jn/article-abstract/105/10/1269/4768653 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
Influence of Dietary Lipids on Iron and Copper Levels of Rats Administered Oral Contraceptives1
1270
HELEN
K. ONDERKA
AND AVANELLE
g/100 g diel
Casein1 DL-Methionine1 Sucrose Starch Fat2 Salt mix' Vitamin mix1-4
Cholesterol1-1
18.0 0.3 18.7 36.0 20.0 4.0 3.0
Sodium glycocholate5
0.5
0.5
1 Nutritional Biochemicals, Inc., Cleveland, Ohio. 2 Coco nut oil or safflower oil; safflower oil (0.3%) was added to coco nut oil to prevent EFA deficiency. >Jones and Foster (33). 4 mg/100 g of diet: thiamin, 1.5; riboflavin, 1.5; niacin, 3.0; pyridoxine HC1, 0.8; DL-calcium pantothenate, 7.0; biotin, 0.2; p-aminobenzoic acid, 1.5; inositol, 100.0; choline chloride 180.0; folie acid 1.0; H-12 (triturate 1%) 3.0; menadione, 0.1; vitamin A powder, largely palmitate (400 IU), 20.0; cholecalciferol (50 IU), 0.005; DL-«-tocopherol, 12.0. s Added only to diets of rats fed cholesterol.
diet. Also in atherogenesis, copper concen trations in plasma and tissues are elevated (9). Conceivably the combined effects of OC and dietary lipids on iron and copper metabolism could result in an intensifica tion of the separate effects of drugs and diet that have been reported. The purpose of this investigation was to explore the ef fects of OC and diets high in saturated and unsaturated fat with and without added cholesterol on plasma and tissue levels of iron and copper in rats. A second study investigated the effects of choles terol and sodium glycocholate on iron ab sorption. MATERIALS
AND METHODS
Animals and diets. Wistar-strain female rats weighing approximately 40 to 50 g were randomly divided into 4 groups of 32 animals each. For a period of 15 weeks, two of the groups were fed a diet (table 1) containing (by weight) 20% safflower oil4 and two received the diets with 20% hydrogenated coconut oil.5 Cholesterol (0.5%) was included in the diets fed to one-half of the rats receiving safflower or coconut oil. Sodium glycocholate (0.5%) was added to diets containing cholesterol to aid in its absorption (10). All fats used in the experiment were refrigerated and stored in the dark, and diets were frozen immediately after mixing to prevent fatty acid oxidation. For 4 additional weeks, after the 15-week period of dietary treat ment, three OC agents *• '•8differing in
estrogen/progesterone ratios were ran domly administered to three groups of eight animals in each of the four dietary treatment groups; the remaining eight ani mals in each group received no OC and were designated as controls. The daily dosage of OC was incorporated into 15 g of diet.6'7-s Rats received a physiological drug dosage equivalent to 1.5 to 2 times the usual human dosage on a drug per body weight basis. Vaginal smears were taken daily for a period of 2 weeks. During this time, no animal receiving an OC was found to be in estrus. On this basis, the OC treatment was judged to be effective. Experiment 2 was carried out to investi gate the effects of sodium glycocholate and cholesterol on iron absorption. Female weanling Wistar rats were divided into four groups of seven animals each. One group (control) was fed the 20% safflower oil diet described in table 1 but without the addition of cholesterol or sodium glyco cholate. The remaining groups were fed the same diet as that of the control group with the addition of (1) 0.5% sodium glycocholate, (2) 0.5% cholesterol, or (3) 0.5% sodium glycocholate and 0.5% cho lesterol. All diets were fed for a 7-week period. Rats were housed individually in wirebottomed suspended cages in a room maintained at 25°and 40% relative hu midity and with alternate periods of light and dark. Diets and water were fed ad libitum. Weight gain and food intake were measured 3 times weekly. Methods. On day 28 of OC administra tion, animals were fasted for 4 hours, anesthetized with ether, and 4.5 ml of blood was drawn by heart puncture into a syringe containing 0.25 ml of 3.8% sodium citrate. To minimize diurnal variation in assays, blood was drawn at the same time each day. Blood samples were immedi ately placed on ice and were then centrifuged at 500 X g for 10 minutes at 5°.The 1 Pacific Vegetable Oil Porp.. Sun Francisco. Calif. 6 Procter and (ïamble, Cincinnati. Ohio. "Ovulen (S.G «g ethynodiol acetate and 0.8« ug mestranol/15 g diet) G. D. Searle Co.. Chicago, 111. 7 Provest (86 ug medroxyprogesterone acetate and 0.43 ug ethlnylestradiol/ln g diet) Upjohn Co.. Kalamnzoo. Mich. 8Bnovid (22 jjg norethynodrel and 0.80 «gmestranol/15 g diet) G. D. Searle Co., Chicago, 111.
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TABLE 1 Composition of diets
KIRKSEY
DIET AND ORAL CONTRACEPTIVES
IN THE RAT
1271 Downloaded from https://academic.oup.com/jn/article-abstract/105/10/1269/4768653 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
plasma was removed and stored at —20° sign. The Newman-Keuls Sequential Range until analysis. Test was used to determine significant dif Hemoglobin was measured spectrophoferences among means. The t test was tometrically as cyanomethemoglobin using used to determine differences between a commercial standard.9 Red blood cells means of certain data collected prior to were counted by use of a hemacytometer, and during OC administration. Experiment and hematocrits were measured in a micro- 2 was designed as a 2x2x2 factorial, hematocrit reader after centrifugation in and data were analyzed by analysis of a high speed centrifuge. Plasma iron and variance. copper were measured by atomic absorp RESULTS AND DISCUSSION tion spectrophotometry (11, 12). An atomic absorption spectrophotometer I0 fitted with Food consumption, iron and copper in the Intensitron series hollow cathode iron takes, and body weights. In the 28-day and copper tubes was used. The samples period prior to OC administration, food were aspirated into an air-acetylene mix consumption was not significantly affected ture. by any of the dietary treatments. However, All equipment used for iron and copper animals fed safflower oil weighed more determinations was soaked at least 3 hours than those fed coconut oil (table 2). During the period of OC treatment, a sig in 50% nitric acid and was thoroughly nificant overall reduction in food consump rinsed with distilled water. tion was observed in animals receiving Copper oxidase activity was measured drugs compared with values for animals according to the method of Houchin (13), and the activity was used to calculate not fed OC (table 2). Aftergood et al. (16) reported food consumption to be re ceruloplasmin concentration by use of the following formula: Y = a + b x, where duced by over 50% in rats after the ad Y = ceruloplasmin concentration in mg ministration of 1 mg of an oral contra ceptive,8 which was also used in this study. per 100 ml, x = absorbance of the product formed by copper oxidase activity, a — However, in this study food consumption —1.7,and b = 150. Plasma samples were was reduced by only 11%. The differences in findings may be attributed to the size of analyzed for copper oxidase activity within the OC dosage used. The amount of drug 10 days after they were collected. administered in this study was smaller Liver, kidney, and spleen samples were than that given by Aftergood et al. and digested by a modification of a wet oxida tion procedure reported by Reit/ et al. was a more physiological dosage. In the (14). The iron and copper content in the present study, body weights were not sig digested tissue samples was measured by nificantly affected by OC treatment. The effectiveness of the dosage was confirmed atomic absorption spectrophotometry. by anestrus and by a statistically signifi In experiment 2, feces were collected during the last 7 days of the study and cant (P < 0.01) increase in uterine weights were wet digested and analyzed for iron at the end of the experiment. Differences in food consumption related content in the same manner as that de scribed for the tissue samples. Fecal iron to diet were observed during the OC treat content was compared with dietary iron ment period. Animals fed diets containing coconut oil had higher food consumption intake to determine the approximate per by approximately 18% than those fed diets centage absorption of dietary iron. Plan of experiment. This experiment was containing safflower oil; however, body weights were lower. This appeared to in designed as a 4 X 2 X 2 factorial. Data by analysis 4were X 2 analyzed X 2'factorial and thenofasvariance a 3 X 2 as X 2a dicate that coconut oil was not as well uti lized as safflower oil. Animals receiving dietary cholesterol had higher food con factorial to determine if there were sta tistically significant differences among the sumption by approximately 10% than those not receiving cholesterol; however, three OC treatments (15). When no dif ferences were found, the data from the body weights were not significantly differanimals administered OC were pooled and » Ilycel. Inc., Houston, Texas. 10Perkin-Elmer Corp., Norwalk, Conn., model 303. were analyzed as a 2 X 2 X 2 factorial de
1272
HELEN K. ONDERKA AND AVANELLE KIRKSEY
tration, and mean cell volume were ob served among the various dietary and drug treatments. OC treatment did not alter the levels of iron or copper in plasma. The efiFectsof estrogen therapy and OC admin istration in increasing the levels of serum copper and ceruloplasmin are well docu mented in the literature (18-20). Some in vestigators (21, 22) have reported changes in hematological values due to estrogen treatment or OC administration. The level of OC agent administered in this study was considerably less than that used to in duce these reported changes. The type of dietary fat used in this study had no significant influence upon plasma iron, copper, ceruloplasmin, or other hematological indices. Ahlstrom and Jar-
TABLE2 Total fowl consumption and Itody weight«prior to and during llu- administration of oral contraceptive agents (OC) Food consumption Treatment1
No cholesterol Safflower No OC2 OC* OC« OC« Coconut No OC OC OC OC Cholesterol Safflower No OC OC OC OC
No.
rats
Prior to OC
During OC
Body weight Prior to OC
During OC
8 8 7 8
370±423 366±43 376±34 386±27
300±42*Significantly different from A (P < 0.01) and from a (P < 0.05). t»> Significantly different from B (P < 0.01 and from 6 (P < 0.05). Significantly different from C (P < 0.01) and from c (P < 0.05).
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ent due to cholesterol. An explanation for the poorer food efficiency in animals fed cholesterol was not apparent from the data. The intakes of iron and copper equaled or exceeded the NRG (17) requirements of the rat for growth. Intakes of iron ranged from 2.2 to 3.2 mg daily and copper from 0.05 to 0.07 mg. Hence the changes in iron and copper levels in plasma and tissues observed in this study were not re lated to an inadequacy of dietary iron and copper. Hematological measurements and plasma iron and copper levels. No significant dif ferences in hemoglobin levels (table 3), hematocrit, red blood cell counts, mean cell hemoglobin and hemoglobin concen
1273
DIET AND ORAL CONTRACEPTIVES IN THE RAT
Treatment1No
ironlig/
rats7 ml13.
ml368±56 100
copperng/100 ml156.8Ü8
ml30.9±
cholesterolSafflowerNo OC2 OC4CoconutNo OC OCCholesterol SafflowerNo OC OCCoconutNo
197
9±1.82' 371 ±55416±79 13.5±1.513.6±0.6
364 ±76306±66 1661!)6 13.8±0.613.6±0.6
13.4±0.712.4±0.4NSOC ±64294±40 281
OC
201 OCSignificant
6.732.6±10.733.9±
162.5±32157.6±26 3.8 149.0±30255.9±68 32.6± 7.963.5±
7.4 62.0±19.279.2±20.0 243.9±48304.5±94
328±98CholesterolPlasma ±92Cholesterolweeks. 254.4
57.8±14.6Cholesterol1
values(P F < 0.01)No. Diet«were fed for 19 weeks after weaning ;Hemoglobina/100 ! No oral contraceptive.Ceruloplasminmo/100 Mean ±so. *Value! were fed the last 4Plasma for animals treated with three oral contraceptives were similar and have been pooled.
vinen (23) reported that various levels of fresh and heated safflower oil ( 10, 20, and 30% of the diet) had no effect on hemo globin, packed cell volume, and mean cell volume. Rats fed cholesterol had significantly lower plasma iron levels (table 3) than those receiving no cholesterol. Other in vestigators (6, 24 ) have reported hemolytic anemia and lowered levels of hemoglobin and of serum iron in animals fed dietary cholesterol. Ostwald et al. (25) recently described the effects of dietary modifica tion on a cholesterol-induced hemolytic anemia in guinea pigs in which iron depo sition was increased in liver, spleen, and other organs. This is in contrast to this study in which hemolytic anemia was not observed in cholesterol-treated rats, and iron levels in liver and spleen were de creased compared with values for animals receiving no cholesterol. In contrast to the lowered levels of iron in plasma of cholesterol-fed rats, plasma copper and ceruloplasmin levels were sig nificantly increased (table 3). Total plasma copper and ceruloplasmin were measured in the present study, and the direct-react ing fraction of plasma copper was calcu
lated from these measurements. Cerulo plasmin contains 0.34% copper and ac counts for 90% of the copper in plasma. The total copper in plasma minus the cop per in ceruloplasmin is the direct-reacting fraction of plasma copper. From this cal culation, it appeared that the major in crease of plasma copper was due to the elevation in ceruloplasmin. Osaki et al. (26) proposed that the physiological role of ceruloplasmin is to stimulate the release of iron from tissue stores into plasma. Evans and Abraham (27) have recently demonstrated this role of ceruloplasmin in vivo. In the present study, animals with elevated ceruloplasmin levels had lower iron stores and lower plasma iron values. The depression in iron levels in plasma in conjunction with the findings of Roeser et al. (28) that plasma iron levels increased after the administration of ceruloplasmin appeared to contraindÃ-cate the possibility that the elevated ceruloplasmin levels ob served in this study resulted in the in creased iron mobilization from tissues. Weight and iron and copper content of organs. Livers of rats fed dietary choles terol were fatty in appearance, pale yellow in color, and larger in size than those of
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TABLE 3 Hemoglobin, plasma iron, copper, and ceruloplasmin
1274
HELEN
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