- Email: [email protected]
Iron and copper absorption in young and elderly men
NUTRITION RESEARCH, Vol. 8, pp. 333-343, 1988 0271-5317/88 $3.00 + .00 Printed in the USA. Pergamon Press plc. All rights reserved.
IRON AND COPPERABSORPTIONIN YOUNGAND ELDERLYMEN[I2 Judith R. Turnlund, Ph.D.3, Ross D. Reager, B.S. and Francoise Costa, B.S. USDA, Western Human Nutrition Research Center, Albany, CA 94710, General Electric Company, Box 460, Pleasanton, CA 94566, and Department of Nutritional Sciences, University of California, Berkeley, 94720.
ABSTRACT Six healthy young men were confined to a metabolic unit to study iron and copper absorption from constant diets containing 10 mg of iron and 3 mg of copper daily. Iron status of all the men was normal. The men were confined to a metabolic unit for 78 days. Absorption was determined twice during the stud~using iron,and copper enriched with the stable isotopes ~ F e and Q~Cu. Stable isotopes were analyzed by magnetic sector, thermal ionization mass spectrometry. Iron and copper balances were determined for two 21 day periods and blood parameters of iron and copper status were measured at the beginnlng and end of the study. Data of the young men were combined with data of elderly men studied earlier. Blood indicators reflected adequate iron status in all subjects at the end of the study and serum copper was in the normal range for all subjects. The following indicators of iron status differed significantly among subjects: hemoglobin, hematocrit, TIBC, f e r r i t i n , and serum transferrin. Urinary and serum copper also differed significantly among subjects. Neither iron nor copper absorption differed between age groups or study periods. Iron absorption differed s l g n i f i c a n t l y among individuals, but copper absorption did not. Key words: stable isotopes, iron, copper, absorption, elderly, young men
INTRODUCTION Metabolism of iron, in contrast to some other minerals, is not regulated through excretion, but through absorption (1). It is poorly absorbed compared to most essential minerals, but absorption is known to increase in blood donors, phlebotomized individuals, and individuals with reduced iron stores ( I ) . Isupported in part by USDA Competitive Grants 82-CRCR-I-I072 and 85-CRCR-I21581 presented in part at American Society for Clinical Nutrition Meeting, Washington, D.C., 1984. Am. J. Clin. Nutr. 39, 664. To whom correspondence should be addressed.
333
334
J.R. TURNLUND et al.
Despite the regulation of iron absorption, iron deficiency anemia is prevalent throughout the world (2, 3). This is due in part to dietary or extrinsic factors and in part to individual or i n t r i n s i c factors. The dietary factors contributing to iron deficiency anemia include inadequate intake of iron and consumption of diets in which the iron is poorly available. Many studies have been conducted on the dietary factors affecting iron b i o a v a i l a b i l i t y and these factors are r e l a t i v e l y well understood (4,5). The i n t r i n s i c factors affecting iron absorption are not well understood (5,6). The turnover rate of iron in the body, as estimated by whole body counting using radioactive iron, is variable (7), suggesting that dietary requirements may vary considerably among individuals. L i t t l e is known about the effect of age on iron absorption. Radioisotopes have been used for most studies of iron absorption, but stable isotopes have recently been used to determine iron absorption. Either neutron activation analysis (8) or mass spectrometry (9) have been used to measure the stable isotopes. Much less is known about copper absorption and the regulation of copper absorption than is known about iron. A number of studies of copper metabolism were conducted in the 1950's by Cartwright, Wintrobe, and their colleagues using radioactive copper (10). However, radioisotopes of copper have short half l i v e s , which has limited their use in human subjects. A stable isotope of copper has been used e f f e c t i v e l y to determine copper absorption in humans (11,12). However, copper absorption in the young and elderly has not been compared. The absorption study described herein was conducted in non-anemic healthy young men consuming formula diets containing the recommended intake of dietary iron and dietary copper levels of approximately the recommended safe and adequate range. Absorption data from the young men were combined with data from elderly men studied e a r l i e r under similar conditions (8, 12, 13). The objectives were to compare inter- and intra-individual v a r i a b i l i t y in absorption of iron and copper in healthy men and to compare iron absorption in young and elderly men. METHODS This study of iron and copper absorption was conducted in conjunction with a larger study on protein and energy requirements of young men (14). Six healthy young men between the ages of 22 and 30 were confined to a metabolic unit for 78 days, The procedures followed were approved by the Committee for the Protection of Human Subjects, University of California, Berkeley. Their heights averaged 182 + 2 cm (mean + SEM) and their weights averaged 74 + 6 kg. Zinc absorption and stat~s of these ~ n have been reportea (15). A formula diet, supplemented with a few food items, was fed to the men. The formula contained egg albumen, cornstarch, maltodextrins, sucrose, cottonseed o i l , shortening, methylcellulose, raffinose, minerals, and deionized water. Other items fed were peaches, low protein rusks, margarine, tea, decaffeinated coffe, supplements of vitamns and minerals, and flavorlngs for the formula. The diet was constant throughout the study and has been described in detail (15). Diets contained the recommended intake of a l l nutrients except protein and contained 10.4 mg iron (as FeCl3-6HpO, reduced with dilute HCI) and 2.70 mg copper (as CuS04) d a i l y . 9 No smoking, alcohol, or medications were allowed during the study. Three-week iron and copper balances were determined twice during the study beginning on days 15 and 57. Balances were dietary less fecal iron and dietary copper less fecal and urinary copper. Procedures for fecal collections and iron and copper determinations by atomic absorption spectrophotometry (AAS) have been described (13). Urinary iron was also determined by heated graphite atomizer AAS. The analytical values were highly variable but very low (from 0.02 to 0.05 mg/day) and did not affect balances. Blood was drawn at the beginning and end of the study for determination of parameters of iron status. The following parameters were determined by a
IRON AND COPPER ABSORPTION
335
commercial laboratory: hemoglobin, hematocrit, serum iron, serum f e r r i t i n , mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and t o t a l iron binding capacity (TIBC). Serum copper was determined by AAS at the beginning, midpoint, and end of the study. 5~
Iron a~# copper absorption were determined twice during the study, using re and CU stable isotopes ~f~ iron and copper. On days 24 and 63, i0 ~Q of iron containing 97.23% ~ and 3 mg of copper containing 99.69% O~Cu were added to the diet in ~Qlace of I0 mg of unenriched iron and 2.~mg of unenriched copper. The ~ was purchased as Fe~OR and the O~ as CuO (Oak Ridge National Laboratory, Oak Ridge, Tennessee).L Each was dissolved in HCL, then diluted to provide I0 mg Fe and 3 mg Cu in 20 ml of solution. The iron was reduced with ascorbic acid (15 mg per subject per day). The isotope solution was added to the formula d i e t consumed in four equal servings on each of the two days. Polyethyleneglycol (PEG), a fecal marker, was added to the diet formulas containing the isotope solution to determine i n t e s t i n a l t r a n s i t time and to assure complete c o l l e c t i o n of a l l unabsorbed iron isotope. PEG was determined using a t u r b i d i m e t r i c method (16). Subsamples of homogenized 3-day fecal p ~ I s were combined for 12 d a y s following isotope feedings for ~Fe determinations. The 12-day composites w e r e then prepared for 54Fe and 65CU determinations. Organic material in the samples was destroyed in a muffle furnace, as previously described (17). The minerals remaining in the samples were dissolved in I0 mls of 6 N Ultrex (Baker Company, P h i l l i p s b u r g , NJ) HCI. Iron and copper were separated from the other minerals and p u r i f i e d using ion exchange chromatography. Anion exchange resin, chloride form, quaternary ammonium styrene type (Bio-Rad Laboratorles, Richmond, C a l i f o r n i a ) was washed thoroughly with deionized water and U l t r e x HCI, and loaded into a 7-mm inner diameter column to a height of i i cm resin. The column was again washed with deionized water and Ultrex HCI. The dissolved sample was applied to the column and washed with 6N H C I . Copper was eluted with 2.5 N Ultrex HCI. Iron was eluted with I N Ultrex HCI in about 6 drops of eluento P u r i f i c a t i o n of copper analysis by thermal i o n i z a t i o n mass spectrometry (TIMS), and calculations have been described ( i i ) . We previously reported a procedure for p u r i f i c a t i o n of iron and isotopic measurements (9). D i f f e r e n t separation procedures and improved a n a l y t i c a l methods were used in t h i s study and are described below. The iron eluent was a c i d i f i e d to 2.5 N and applied to a 3-mm column containing I i cm of the same type of resin. Fe was eluted with 1 N HCI. ~ h e ~ - m m column procedure was repeated for each Fe sample. The r a t i o of ~176176 was then determined in the p u r i f i e d sample by thermal i o n i z a t i o n mass spectrometry using an automated magnetic sector mass spectrometer with a 13 sample t u r r e t and controlled with a desktop computer system (Finnigan MAT 261, Bremen, Germany). A double filament design was used with an ionizing and a sample filament. Filaments were made from zone refined rhenium, 0.00157" thick and 0.0275" wide. A 4ul sample containing approximately I00 + 20 ug Fe was applied to a rhenium f i l a m e n t , using a m i c r o l i t e r pipet (Eppendor-T, Brinkman I n s t . , Westbury, N.Y.) The l i q u i d in the sample was evaporated by applying a 0.7 amp current u n t i l the sample was dry. The current was then ra~ed 0.5 amps/min to a current of 1.25 amps. A faraday detector with a 9 X I0T M ohm feedback r e s i s t o r was used for measurement. The ionizing filament was set to a temperature at which 0.6 v o l t s at mass 187 was measured on the faraday detector. The sample filament was heated at 0 . i amp/min u n t i l a current of 0.5 v o l t s was achieved at mass 56, a f t e r 12 min, the current was raised to obtain a I v o l t output for mass 56. Peak i n t e n s i t y (V) for iron masses 54, 56, 57, and 58 and a background peak at 52.5 were s e q u e n t i a l l y measured for 9 scans. An i n t e g r a t i o n time of 8 seconds was used f o r a l l peaks except mass 56, the most abundant peak, which was integrated for 4 seconds. The 54Fe:56Fe r a t i o and the total iron con~t#nt of the 12-day composite were used to determine the amount of the ~Fe from the isotope feeding elimintated in the feces, The equations used have been described in d e t a i l previously (9). The amount of isotope absorbed was calculated by
336
J.R. TURNLUND et al.
subtracting the amount appearing in the feces from the amount fed. The percentage of the isotope which was absorbed was then calculated by d i v i d i n g by the amount fed and multiplying by 100. Prior to the above analyse~s~, we validated the method of 54Fe measurement as follows. Known amounts of ~Fe, were added to 5 replicate fecal samples to evaluate reproducibility of the method. Iron content of the samples determined by isotope dilution was compared to iron content determined by atomic ~sorption spectrophotometry. Correlations v~e~e c#~culated between the amount of b~Fe added to the samples and the Fe:uuFe r a t i o . The measured ratios were compared with theoretical ratios. Iron contamination was evaluated by taking a sample of the enriched isotopic diluent through the sample preparation procedures and determining the isotopic ratios before and following the procedures. Data from the young men were combined with data acquired previously in elderly men (9, 12). Similar measurements were made in the elderly and youn~ men, e x c e p t serum f e r r i t i n was determined only in the young men ano transferrin was determined only in the elderly men. The combined data were subjected to s t a t i s t i c a l analysis. Repeated measures analysis of variance was used to compare iron and copper absorption, balance, and blood parameters in young and elderly men (18). Inter- and intra-individual v a r i a b i l i t y were also compared. Kendall correlations were calculated between iron absorption, balance, and the blood parameters of iron status and between copper absorption, balance, and serum copper (19). Differences in repeated measures ANOVA and in correlations were considered to be significant i f P < 0.05. RESULTS
The iron concentration of the fecal samp~, determined by isotope dilution following the addition of several levels of ~Fe, was 1.110 + 0.0059 mg Fe/g dry fecal sample, as shown in Table 1. The coefficient of v~riation of 0.6% reflects the combination of several sources of error including sample homogeneity, errors in weighing the fecal sample and isotope solution, and the isotope ratio determinations. This value compared with 1.13 + 0.017 mg/g determined by atomic absorption spectrpphotometry. The correlatioff between the amount of Fe enri~edrwith 97.23% ~ which was added to replicate fecal samples and the ~176 ratio was 0.99999, as shown in Figure 1. All ~asu~d ratios were within_O 5% of the calculated ratios. The comparison of the "Fe:~uFe ratio of the 54Fe enriched iron before and after i t was put through the sample preparation procedure demonstrated that there was no detectable contamination during sample preparation. The apparent iron absorption and status of the young men is shown in Table 2. Their copper absorption and status is shown in Table 3. All blood values were within normal ranges, except at the end of the study serum f e r r i t i n was elevated in three men, serum iron was elevated in three men, and TIBC was elevated in one man. Serum copper, MCV, MCH, and MCHC were within the normal range for a l l subjects. A sample collected from one young man for serum iron and TIBC at the beginning of the study was hemolized and was not used. Individual data for the elderly men have been reported (9, 13). Iron balance and absorption data for the young men and for the elderly men studied previously are summarized in Table 4. There were no significant differences between age groups or metabolic periods in fecal iron, iron balance, or iron absorption. However, iron absorption differed s i g n i f i c a n t l y (P < 0.05) among subjects within age group. The variance estimate between subjects was 37.12, while the within subject variance estimate was 9.36, demonstrating much greater v a r i a b i l i t y among subjects than within subjects. A summary comparing the blood parameters of iron status of the young and elderly is given in Table 5. Hemoglobin, hematocrit, TIBC, f e r r i t i n and transferrin levels differed s i g n i f i c a n t l y among subjects. There was a
IRON AND COPPERABSORPTION
337
significant interaction between age groups and periods for hemoglobin and hematocrit. These levels for the elderTy men at the beginning of the study were lower than at the end of the study, or than levels of the young men at either the beginning or end of the study. MCV, MCH, and MCHCwere within the normal range for all subjects. There were significant positive correlations (P <0.05) between iron absorption and the following: hemoglobin (r = 0.387), hematocrit (r = 0.305), and serum iron (r = 0.349). TABLE I 54 Fe Isotope Dilution Determination of Iron Concentration by TIMS Sample No.
Sample Weight
54Fea
(g) 2.480
(mg) 0.1436
2.480
0.1436
1.510
0.02752
1.527
0.04942
1.512
0.21040
54Fe:56Feb
Fe Contentc (mglg) 1.103 1.111 1.106 1.123 1.117 1.101 1.101 1.115 1.111 1.112 1.115
0.1215 0.1210 0.1213 0.1204 0.1207 0.08198 0.08198 0.09568 0.09579 0.2010 0.2006
1.11 0.0059 0.6%
Mean SD CV
54Fe added to ~97.23% Determined by TIMS
fecal sample
CBased on isotope dilution calculations
0.200-
0m ,< N 0.100-
jloJ ~ r = 0.99999
0.000
I
I
0.050
0.100
M G FE-54 A D D E D / M G
F E I N FECAL SAMPLE
FIG. I X~otopic ratio of 54Fe:56Fe as a function of amount of o~Fe added to a fecal sample containing 1.1mg Fe/g.
0.150
338
O.R. TURNLUND e t a ] . TABLE 2 Iron Absorption and Status of Young Men 1
Subject 3 4
2
5
"6
Mean
SEM
Absorption %) MP 1~ 17.9 2.9 3.4 3.5 15.7 4.5 8.0 2.8 6.0 2.2 -0.5 15.2 9.1 8.2 MP 2 17.2 2.6 Iron Balance (mg/day) MP 1 -0.4 -1.1 -3.9 -0.2 -1.0 -1.4 -1.3 0.5 MP 2 0.3 0.1 1.3 -0.9 0.8 0.3 0.3 0.5 Hemoglobin (g/dl) Prestudy 16.9 16.0 1 5 . 2 1 6 . 2 16.5 16.2 16.2 0.2 End study 16.0 14.6 1 5 . 1 1 5 . 2 15.7 15.7 15.4 0.2 Hematocrit (%) Prestudy 47.3 46.8 4 4 . 7 4 5 . 9 46.9 47.2 46.5 0.4 End study 42.7 43.4 4 4 . 1 4 4 . 1 45.7 46.0 44.3 0.5 Serum iron (ug/dl) Prestudy 143 76 88 147. 158b 122c 15 129 142 169b 173D 166 155 End study 123 9 Serum f e r r i t i n (ng/ml) Prestudy 157 85 95 178. 36 269 137 34 End study 381b 82 193 305D 46 344D 255 57 TIBC (ug/dl) 341 283 367 251 307c Prestudy 292 19 402b 333 273 354 300 304 End study 262 16 aMetabolic Period 1 ~Above normal range ~ of 5 subjects
TABLE 3 Copper Absorption, Balance, and Serum Levels of Young Men .... 1 Absorption (%) MP 1 40.8 MP 2 27.6 Balance (mg/day) MP 1 9.35 MP 2 0.23 Serum Copper (ug/dl) Prestudy 69 End MP 1 66 End MP 2 77
2
Subject 3
4 ....
5
6
Mean
SEM
24.2 24.3
26.7 27.1
22.5 22.0
27.3 23.0
33.5 29.2 30.6 25.8
2.8 1.3
0.17 0.30
-0.53 0.49
0.23 -0.07
0.21 0.06
0.24 0.11 0.25 0.21
0.13 O.08
93 88 85
87 83 77
4 6 5
100 199 110
88 76 88
79 62 75
86 79 85
Table 6 summarizes the copper data for the young and elderly men. Fecal copper differed s i g n i f i c a n t l y between age groups, but this reflected higher dietary copper intake by the elderly. Serum copper was s i g n i f i c a n t l y higher in elderly men than in young men. Urinary copper was lower in both groups in MP2 than in M P I . Serum and urinary copper differed s i g n i f i c a n t l y among subjects. Neither copper absorption nor balance differed between age groups or among subjects. DISCUSSION Evaluation of the measurement of 54Fe in the fecal samples demonstrated that the analysis was done with a high degree of precision and accuracy. This is essential to reliable absorption determinations when fecal monitoring is used.
IRON AND COPPER ABSORPTION
339
TABLE 4 Summary of Iron Balance and Absorption Data for Young and Elderly Men
MP 1 Dietary Iron (mg/day) Young men 10.4 Elderly men 9.8 Fecal Iron (mg/day) Young men 11.7
MP 2
Elderly men
6.6 b
ANUVA between among MO subjects (p)
10.4 10.0 10.0
Elderly men Urinary Iron (mg/day) Youn9 and elderly <0.1 <0.1 Iron Balance (mg/day) Young men -1.3 0.3 Elderly men -0.2 b Apparent absorption (%) Young men 8.0
~a
between age groups
-0.4 8.2 8.7
0.5 10.0b
NS 10.4
NS
NS
(I0.6 b)
O. b)
NS
NS
NS
1.2 (I.8 b)
NS
NS
0.0024
(0.
Standard error of pooled mean ~Mean of 4, other means are of 6 men Since the amount of 54Fe excreted is measured and iron is poorly absorbed, errors are compounded in calculations. The CV we observed, 0.6%, in the determination of excreted isotope would result in a 7% CV on the average when absorption averages 8% of the amount fed, as i t did in these men. An individual absorption measurement of 8% would have a SD of 0.6. The precision of analyses was improved considerably compared to e a r l i e r work (9) by the new purification procedures and improved analytical instrumentation described in the Methods Section. A number of studies have demonstrated that labeled iron added e x t r i n s i c a l l y , as was done in this study, equilibrates with the miscible~Qon-heme iron pool (5,6). Therefore, the absorption of the stable isotope, ~Fe should reflect absorption of the iron in the diet, which was non-heme iron. The data on iron absorption obtained in this work demonstrates that iron absorption can be determined with s u f f i c i e n t precision to clearly detect differences in absorption. Iron absorption and status data of these young men suggests that the currently recommended intake of 10 mg Fe/day is adequate to maintain iron status in normal healthy young men. Blood indicators of iron status remained in the normal range except for some individuals with elevated values as discussed in the Results Section. Iron balance was negative in the young men in the f i r s t period, but became positive in a l l but one man in the second balance period. We observed negative iron balance previously when other indicators of iron status were normal and when iron status improved during the course of the study (13). Therefore, we consider iron balance r e l a t i v e l y unreliable for assessing iron requirements and status. Copper balance was s l i g h t l y positive, on the average, in the young men. Serum copper levels were within the normal range for a l l the young men. These data suggest that a copper intake of 2.7 mg/day was adequate. When iron and copper absorption data from the two groups of men were compared, absorption was similar in both groups. Iron absorption averaged 6.6 and 8.7 % in the elderly men and 8.0 and 8.2 % in t h e young men. Copper absorption averaged 25.3 and 27.7 % in the elderly men and 29.2 and 25.8 % in the young men. Z i n c absorption was compared in these subjects previously and a
340
3.R. TURNLUND et al.
TABLE 5
Summary of Blood Iron Data for Young and Elderly Men
Day 1 Hemoglobin (mg/dl) Young men
Day 91
5.4
13.4
14.6
46.5
44.3
Elderly men MCV (u ~) Young men
40.0
43.4
84.5
86.2
Elderly men MCH (pg) Young men
91.2
83.8
30.3
29.3
Elderly men MCHC (%) Young men
30.6
28.1
33.9
34.0
Elderly men Serum iron (ug/dl) Young men
33.5
33.7
118c
150
94c
146
323c
321
Elderly men TIBC Young men
among subjects
0.2
b
NS
0.0012
0.6
b
NS
0.0085
3.6
NS
NS
NS
I.I
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.048
0.025
14 (17c)
15 NS Elderly men 292c 261 (13c ) Ferritin (mg/dl) Young men 137 225 24 Saturation (%) Elderly men 36c 56 6 Transferrin (mg/dl) Elderly men 264 281 Standard error of pooled m e a n . . . Significant interaction. Lower for elderly men, Day 1. CMean of 5 due to sample hemolysis.
~
ANUVA between MP (P)
16.2
Elderly men Hematocrit (%) Young men
S~a
6etween age grouPs
NS .
NS .
NS 0.014
marked difference was observed between the young and elderly (15). Zinc absorption in elderly men averaged 17.3 and 18.1% in MP I and MP 2 respectively, and 29.9 and 33.0 % in young men. I t appears that while zinc absorption changes markedly with age, iron and copper absorption may not be affected. While copper absorption did not d i f f e r between age groups, serum co~per was s i g n i f i c a n t l y higher in the elderly men. In contrast, serum zlnc was s i g n i f i c a n t l y lower in these elderly men than in young men. The higher serum copper levels we observed in elderly men agrees with the results of a population survey of serum copper (20). The investigators divided men in six population ~roups by age and found serum copper was higher in men over 60 years of age than in men in five younger population groups. There were large differences among individuals in iron absorption. Zinc absorption also differed among subjects Within age groups, but the differences were small compared to the differences between age groups. Iron absorption is known to d i f f e r among individuals, but the difference has been attributed primarily to enhanced absorption when stores are low or with irondeficiency (21). Our subjects were not iron depleted, based on serum f e r r i t i n in the young men and transferrin in the elderly men, and these large differences in absorption were unexpected.
IRON AND COPPERABSORPTION
341
TABLE 6 Summary of Copper Data for Young and Elderly
MP 1
MP 2
Dietary copper (mg/day) Young men 2.70 Elderly men 3.24 Urinary copper (mg/day) Young men 0.0114 Elderly men 0.0113b Fecal copper (mg/day) Young men 2.58 Elderly men 3.22 D
S'~a
25.3 b
Serum copper (ug/dl) Pre Study Young men 86 Elderly men
98
0.0068 0.0087 2.48 3.22
27.7d
0.00006 (0.00076)
NS
0.0004
0.01~ (0.14 u)
0.0007c
NS
NS
0.12L (0.14 ~)
NS
NS
NS
1.3 (1.6 b)
NS
NS
NS
0.0005
NS
0.0001
Mid End S t u d y Study 79 85 103b
among subjects
(P)
2.70 3.28
Copper balance (mg/day) Young men 0.11 0.21 Elderly men 0.01D 0.05 Apparent absorption (%) Young men 29.2 25.8 Elderly men
ANOVA ~e~-6etween age MP groups
105
(b)
0.0064
Standard error of pooled mean ~Mean of 4, other means are of 6 Due to level ~Mean of 5
of dietary copper
The hemoglobins and hematocrits of the elderly men improved during the course of the study, while those of the young men did not. At the end o f the study hemoglobin and hematocrit levels were similar in b o t h age groups. The comparisons of hemoglobin and hematocrit levels in the two groups of men suggests that the prestudy dletary iron intake of the elderly men may have been lower than the recommended intake they were fed during the study. Or i t may be that the prestudy diets of the elderly contained iron that was poorly available, as has been suggested by Lynch, et al (21). The significant positive correlations between iron absorption and three indicators of iron status were also unexpected. However, our subjects were not iron depleted. When absorption from the same diet was consistently higher in some individuals than others for an extended period of time (78 and 83 days), higher hemoglobin and hematocrit levels might be expected in those absorbing more iron. Serum f e r r i t i n and transferrin were not significantly correlated with iron absorption in our subjects, but these were each done In only one population group; therefore, the number of observations was small. I t is well established that the efficiency of absorption increases with iron deficiency anemia and when iron stores are low. This would result in a negative correlation between indicators of iron status and absorption. In those studies, the negative correlations were attributed to individuals whose iron stores were depleted (6). The data from these studies suggests that iron and copper absorption are similar in healthy young and elderly men. The data suggest that dietary iron and copper requirements are not affected specifically by age alone. T h i s conclusion would be valid only for healthy individuals with no chronic diseases and not taking medication. The incidence of chronic disease and use of medications is substantially higher in the elderly and b o t h could affect iron and copper
342
J.R. TURNLUNDet al.
requirements. The marked differences in homeostatic control of iron absorption, appears to be adequate. Therefore, individuals consuming similar diets could
iron absorption among subjects suggests even amon~ subjects whose iron status iron requirements of normal, healthy d i f f e r considerably.
ACKNOWLEDGEMENTS The authors thank Drs. Sheldon Margen, Ned Durkin, and Yves Schutz who made i t possible to conduct these studies of mineral absorption in conjunction with t h e i r studies of protein and energy requirements, Bonnie Gong who carried out the mineral separations, and William Keyes who carried out TIMS analysis of copper isotopes. REFERENCES 1.
RecommendedDietary Allowances, 9th edition. Wash. D.C., 1980; 137-144.
National Academy of Sciences,
2.
Baker SJ, DeMaeyer EM. Nutritional anemia: its understanding and control with special reference to the work of the World Health Organization. Am. J. Clin. Nutr. 1979; 32:368-417.
3.
Dallman PR, Siimes MA, Stekel A. Iron deficiency in infancy and childhood. Am. J. Clin. Nutr. 1980; 33:86-118.
4.
Morck TA, Cook JD. Factors affecting the bioavailability of dietary iron. Cereal Foods World 1981; 26:667-72.
5.
Hallber~ L. Bioavailability of dietary iron in man. ~n Annual Review of Nutrltion (Darby, W.J,, Broquist, H.P., and Olson, .E., eds.) 1981; p 123-47, Annual Reviews, Inc., Palo Alto, CA.
6.
Kuhn IN, Monsen ER, Cook, JD, and Finch CA. Clin. Med. 1968; 71:715-721.
7.
Bowering J, Sanchez AM, Irwin MI. A conspectus of research on iron requirements of man. J. Nutr. 1976; 106:985-1074.
8.
King JC, Raynolds WL, Margen S. Absorption of stable isotopes of iron, copper, and zinc during oral contraceptive use. Am. J. Clin. Nutr. 1981;31:1198-1203
9.
Turnlund JR, Michel MC, Keyes WR, King JC andMargen S. Use of enriched stable isotopes to determine zinc and iron absorption in elderly men. Am. J. Clin. Nutr. 1982;35:1033-40.
Iron absorption in man.
10. Cartwright GE, Wintrobe MM. Copper metabolism in normal subjects. Clin. Nutr. 1964;14:224-32.
J. Lab.
Am. J.
11. Turnlund JR, King JC, Gong B, Keyes WR, Michel MC. A stable isotope study of copper absorption in young men: effect of phytate and cellulose. Am. J. Clin. Nutr. 1985;42:18-23. 12. Turnlund JR, Michel MC, Keyes WR- Schutz Y, Margen S. Copper absorption in elderly men determined by using 65Cu. Am. J. Clin. Nutr. 1982;36:587-91, 13. Turnlund JR, Costa F, Margen S. Zinc, copper and iron balance in elderly men. Am. J. Clin. Nutr. 1981; 34:2641-7. 14. Durkin N, Margen S, Ogar DA, Tilve SG. Human protein requirements: autocorrelation and adaptation to a low protein diet. In: Protein-Energy Requirement Studies in Developing Countries: Results of International Research. (Rand, WM, Uauy, R, Schrimshaw N, eds) 1984;pp:57-62, United Nations University, Tokyo.
IRON AND COPPER ABSORPTION
343
15. Turnlund JR, Durkin N, Costa F, Margen S. Stable isotope studies of zinc absorption and retention in young and elderly men. J. Nutr. 1986;116:1239-47.
16. Malawer SJ, Powell DW. An improved turbidimetric analysis of polyethylene glycol u t i l i z i n g an emulsifier. Gastroenterology 1967;53:250-6. 17. Turnlund JR, King JC, Keyes WR, Gong B, Michel MC. A stable isotope study of zinc absorption In young men: effects of phytate and alpha-cellulose. Am. J.Clin. Nutr. 1967;40:1071-7. Milliken GA, and Johnson DE. Analysis of messy data, Vol. 1. Designed 18. Experiments. 407.
Lifetime Learning Publications, Belmont, CA. 1984. pp:322-
19. Siegel S. Nonparametric Statistics for the Behavorial Sciences. McGraw-Hill, New York. 1956;pp:213-22. 20. Schreurs WHP, Klosse JA, Muys T, Haesen JP. Serum copper levels in relation to sex and age. Internat. J. Vit. Nutr. Res. 1982;52:68-74. 21, Lynch SR, Finch CA, Monsen ER, Cook JD. Iron status of elderly Americans. Am. J. Clin. Nutr. 1982;36:1032-45.
Accepted for publication September 28, 1987.
IRON AND COPPERABSORPTIONIN YOUNGAND ELDERLYMEN[I2 Judith R. Turnlund, Ph.D.3, Ross D. Reager, B.S. and Francoise Costa, B.S. USDA, Western Human Nutrition Research Center, Albany, CA 94710, General Electric Company, Box 460, Pleasanton, CA 94566, and Department of Nutritional Sciences, University of California, Berkeley, 94720.
ABSTRACT Six healthy young men were confined to a metabolic unit to study iron and copper absorption from constant diets containing 10 mg of iron and 3 mg of copper daily. Iron status of all the men was normal. The men were confined to a metabolic unit for 78 days. Absorption was determined twice during the stud~using iron,and copper enriched with the stable isotopes ~ F e and Q~Cu. Stable isotopes were analyzed by magnetic sector, thermal ionization mass spectrometry. Iron and copper balances were determined for two 21 day periods and blood parameters of iron and copper status were measured at the beginnlng and end of the study. Data of the young men were combined with data of elderly men studied earlier. Blood indicators reflected adequate iron status in all subjects at the end of the study and serum copper was in the normal range for all subjects. The following indicators of iron status differed significantly among subjects: hemoglobin, hematocrit, TIBC, f e r r i t i n , and serum transferrin. Urinary and serum copper also differed significantly among subjects. Neither iron nor copper absorption differed between age groups or study periods. Iron absorption differed s l g n i f i c a n t l y among individuals, but copper absorption did not. Key words: stable isotopes, iron, copper, absorption, elderly, young men
INTRODUCTION Metabolism of iron, in contrast to some other minerals, is not regulated through excretion, but through absorption (1). It is poorly absorbed compared to most essential minerals, but absorption is known to increase in blood donors, phlebotomized individuals, and individuals with reduced iron stores ( I ) . Isupported in part by USDA Competitive Grants 82-CRCR-I-I072 and 85-CRCR-I21581 presented in part at American Society for Clinical Nutrition Meeting, Washington, D.C., 1984. Am. J. Clin. Nutr. 39, 664. To whom correspondence should be addressed.
333
334
J.R. TURNLUND et al.
Despite the regulation of iron absorption, iron deficiency anemia is prevalent throughout the world (2, 3). This is due in part to dietary or extrinsic factors and in part to individual or i n t r i n s i c factors. The dietary factors contributing to iron deficiency anemia include inadequate intake of iron and consumption of diets in which the iron is poorly available. Many studies have been conducted on the dietary factors affecting iron b i o a v a i l a b i l i t y and these factors are r e l a t i v e l y well understood (4,5). The i n t r i n s i c factors affecting iron absorption are not well understood (5,6). The turnover rate of iron in the body, as estimated by whole body counting using radioactive iron, is variable (7), suggesting that dietary requirements may vary considerably among individuals. L i t t l e is known about the effect of age on iron absorption. Radioisotopes have been used for most studies of iron absorption, but stable isotopes have recently been used to determine iron absorption. Either neutron activation analysis (8) or mass spectrometry (9) have been used to measure the stable isotopes. Much less is known about copper absorption and the regulation of copper absorption than is known about iron. A number of studies of copper metabolism were conducted in the 1950's by Cartwright, Wintrobe, and their colleagues using radioactive copper (10). However, radioisotopes of copper have short half l i v e s , which has limited their use in human subjects. A stable isotope of copper has been used e f f e c t i v e l y to determine copper absorption in humans (11,12). However, copper absorption in the young and elderly has not been compared. The absorption study described herein was conducted in non-anemic healthy young men consuming formula diets containing the recommended intake of dietary iron and dietary copper levels of approximately the recommended safe and adequate range. Absorption data from the young men were combined with data from elderly men studied e a r l i e r under similar conditions (8, 12, 13). The objectives were to compare inter- and intra-individual v a r i a b i l i t y in absorption of iron and copper in healthy men and to compare iron absorption in young and elderly men. METHODS This study of iron and copper absorption was conducted in conjunction with a larger study on protein and energy requirements of young men (14). Six healthy young men between the ages of 22 and 30 were confined to a metabolic unit for 78 days, The procedures followed were approved by the Committee for the Protection of Human Subjects, University of California, Berkeley. Their heights averaged 182 + 2 cm (mean + SEM) and their weights averaged 74 + 6 kg. Zinc absorption and stat~s of these ~ n have been reportea (15). A formula diet, supplemented with a few food items, was fed to the men. The formula contained egg albumen, cornstarch, maltodextrins, sucrose, cottonseed o i l , shortening, methylcellulose, raffinose, minerals, and deionized water. Other items fed were peaches, low protein rusks, margarine, tea, decaffeinated coffe, supplements of vitamns and minerals, and flavorlngs for the formula. The diet was constant throughout the study and has been described in detail (15). Diets contained the recommended intake of a l l nutrients except protein and contained 10.4 mg iron (as FeCl3-6HpO, reduced with dilute HCI) and 2.70 mg copper (as CuS04) d a i l y . 9 No smoking, alcohol, or medications were allowed during the study. Three-week iron and copper balances were determined twice during the study beginning on days 15 and 57. Balances were dietary less fecal iron and dietary copper less fecal and urinary copper. Procedures for fecal collections and iron and copper determinations by atomic absorption spectrophotometry (AAS) have been described (13). Urinary iron was also determined by heated graphite atomizer AAS. The analytical values were highly variable but very low (from 0.02 to 0.05 mg/day) and did not affect balances. Blood was drawn at the beginning and end of the study for determination of parameters of iron status. The following parameters were determined by a
IRON AND COPPER ABSORPTION
335
commercial laboratory: hemoglobin, hematocrit, serum iron, serum f e r r i t i n , mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and t o t a l iron binding capacity (TIBC). Serum copper was determined by AAS at the beginning, midpoint, and end of the study. 5~
Iron a~# copper absorption were determined twice during the study, using re and CU stable isotopes ~f~ iron and copper. On days 24 and 63, i0 ~Q of iron containing 97.23% ~ and 3 mg of copper containing 99.69% O~Cu were added to the diet in ~Qlace of I0 mg of unenriched iron and 2.~mg of unenriched copper. The ~ was purchased as Fe~OR and the O~ as CuO (Oak Ridge National Laboratory, Oak Ridge, Tennessee).L Each was dissolved in HCL, then diluted to provide I0 mg Fe and 3 mg Cu in 20 ml of solution. The iron was reduced with ascorbic acid (15 mg per subject per day). The isotope solution was added to the formula d i e t consumed in four equal servings on each of the two days. Polyethyleneglycol (PEG), a fecal marker, was added to the diet formulas containing the isotope solution to determine i n t e s t i n a l t r a n s i t time and to assure complete c o l l e c t i o n of a l l unabsorbed iron isotope. PEG was determined using a t u r b i d i m e t r i c method (16). Subsamples of homogenized 3-day fecal p ~ I s were combined for 12 d a y s following isotope feedings for ~Fe determinations. The 12-day composites w e r e then prepared for 54Fe and 65CU determinations. Organic material in the samples was destroyed in a muffle furnace, as previously described (17). The minerals remaining in the samples were dissolved in I0 mls of 6 N Ultrex (Baker Company, P h i l l i p s b u r g , NJ) HCI. Iron and copper were separated from the other minerals and p u r i f i e d using ion exchange chromatography. Anion exchange resin, chloride form, quaternary ammonium styrene type (Bio-Rad Laboratorles, Richmond, C a l i f o r n i a ) was washed thoroughly with deionized water and U l t r e x HCI, and loaded into a 7-mm inner diameter column to a height of i i cm resin. The column was again washed with deionized water and Ultrex HCI. The dissolved sample was applied to the column and washed with 6N H C I . Copper was eluted with 2.5 N Ultrex HCI. Iron was eluted with I N Ultrex HCI in about 6 drops of eluento P u r i f i c a t i o n of copper analysis by thermal i o n i z a t i o n mass spectrometry (TIMS), and calculations have been described ( i i ) . We previously reported a procedure for p u r i f i c a t i o n of iron and isotopic measurements (9). D i f f e r e n t separation procedures and improved a n a l y t i c a l methods were used in t h i s study and are described below. The iron eluent was a c i d i f i e d to 2.5 N and applied to a 3-mm column containing I i cm of the same type of resin. Fe was eluted with 1 N HCI. ~ h e ~ - m m column procedure was repeated for each Fe sample. The r a t i o of ~176176 was then determined in the p u r i f i e d sample by thermal i o n i z a t i o n mass spectrometry using an automated magnetic sector mass spectrometer with a 13 sample t u r r e t and controlled with a desktop computer system (Finnigan MAT 261, Bremen, Germany). A double filament design was used with an ionizing and a sample filament. Filaments were made from zone refined rhenium, 0.00157" thick and 0.0275" wide. A 4ul sample containing approximately I00 + 20 ug Fe was applied to a rhenium f i l a m e n t , using a m i c r o l i t e r pipet (Eppendor-T, Brinkman I n s t . , Westbury, N.Y.) The l i q u i d in the sample was evaporated by applying a 0.7 amp current u n t i l the sample was dry. The current was then ra~ed 0.5 amps/min to a current of 1.25 amps. A faraday detector with a 9 X I0T M ohm feedback r e s i s t o r was used for measurement. The ionizing filament was set to a temperature at which 0.6 v o l t s at mass 187 was measured on the faraday detector. The sample filament was heated at 0 . i amp/min u n t i l a current of 0.5 v o l t s was achieved at mass 56, a f t e r 12 min, the current was raised to obtain a I v o l t output for mass 56. Peak i n t e n s i t y (V) for iron masses 54, 56, 57, and 58 and a background peak at 52.5 were s e q u e n t i a l l y measured for 9 scans. An i n t e g r a t i o n time of 8 seconds was used f o r a l l peaks except mass 56, the most abundant peak, which was integrated for 4 seconds. The 54Fe:56Fe r a t i o and the total iron con~t#nt of the 12-day composite were used to determine the amount of the ~Fe from the isotope feeding elimintated in the feces, The equations used have been described in d e t a i l previously (9). The amount of isotope absorbed was calculated by
336
J.R. TURNLUND et al.
subtracting the amount appearing in the feces from the amount fed. The percentage of the isotope which was absorbed was then calculated by d i v i d i n g by the amount fed and multiplying by 100. Prior to the above analyse~s~, we validated the method of 54Fe measurement as follows. Known amounts of ~Fe, were added to 5 replicate fecal samples to evaluate reproducibility of the method. Iron content of the samples determined by isotope dilution was compared to iron content determined by atomic ~sorption spectrophotometry. Correlations v~e~e c#~culated between the amount of b~Fe added to the samples and the Fe:uuFe r a t i o . The measured ratios were compared with theoretical ratios. Iron contamination was evaluated by taking a sample of the enriched isotopic diluent through the sample preparation procedures and determining the isotopic ratios before and following the procedures. Data from the young men were combined with data acquired previously in elderly men (9, 12). Similar measurements were made in the elderly and youn~ men, e x c e p t serum f e r r i t i n was determined only in the young men ano transferrin was determined only in the elderly men. The combined data were subjected to s t a t i s t i c a l analysis. Repeated measures analysis of variance was used to compare iron and copper absorption, balance, and blood parameters in young and elderly men (18). Inter- and intra-individual v a r i a b i l i t y were also compared. Kendall correlations were calculated between iron absorption, balance, and the blood parameters of iron status and between copper absorption, balance, and serum copper (19). Differences in repeated measures ANOVA and in correlations were considered to be significant i f P < 0.05. RESULTS
The iron concentration of the fecal samp~, determined by isotope dilution following the addition of several levels of ~Fe, was 1.110 + 0.0059 mg Fe/g dry fecal sample, as shown in Table 1. The coefficient of v~riation of 0.6% reflects the combination of several sources of error including sample homogeneity, errors in weighing the fecal sample and isotope solution, and the isotope ratio determinations. This value compared with 1.13 + 0.017 mg/g determined by atomic absorption spectrpphotometry. The correlatioff between the amount of Fe enri~edrwith 97.23% ~ which was added to replicate fecal samples and the ~176 ratio was 0.99999, as shown in Figure 1. All ~asu~d ratios were within_O 5% of the calculated ratios. The comparison of the "Fe:~uFe ratio of the 54Fe enriched iron before and after i t was put through the sample preparation procedure demonstrated that there was no detectable contamination during sample preparation. The apparent iron absorption and status of the young men is shown in Table 2. Their copper absorption and status is shown in Table 3. All blood values were within normal ranges, except at the end of the study serum f e r r i t i n was elevated in three men, serum iron was elevated in three men, and TIBC was elevated in one man. Serum copper, MCV, MCH, and MCHC were within the normal range for a l l subjects. A sample collected from one young man for serum iron and TIBC at the beginning of the study was hemolized and was not used. Individual data for the elderly men have been reported (9, 13). Iron balance and absorption data for the young men and for the elderly men studied previously are summarized in Table 4. There were no significant differences between age groups or metabolic periods in fecal iron, iron balance, or iron absorption. However, iron absorption differed s i g n i f i c a n t l y (P < 0.05) among subjects within age group. The variance estimate between subjects was 37.12, while the within subject variance estimate was 9.36, demonstrating much greater v a r i a b i l i t y among subjects than within subjects. A summary comparing the blood parameters of iron status of the young and elderly is given in Table 5. Hemoglobin, hematocrit, TIBC, f e r r i t i n and transferrin levels differed s i g n i f i c a n t l y among subjects. There was a
IRON AND COPPERABSORPTION
337
significant interaction between age groups and periods for hemoglobin and hematocrit. These levels for the elderTy men at the beginning of the study were lower than at the end of the study, or than levels of the young men at either the beginning or end of the study. MCV, MCH, and MCHCwere within the normal range for all subjects. There were significant positive correlations (P <0.05) between iron absorption and the following: hemoglobin (r = 0.387), hematocrit (r = 0.305), and serum iron (r = 0.349). TABLE I 54 Fe Isotope Dilution Determination of Iron Concentration by TIMS Sample No.
Sample Weight
54Fea
(g) 2.480
(mg) 0.1436
2.480
0.1436
1.510
0.02752
1.527
0.04942
1.512
0.21040
54Fe:56Feb
Fe Contentc (mglg) 1.103 1.111 1.106 1.123 1.117 1.101 1.101 1.115 1.111 1.112 1.115
0.1215 0.1210 0.1213 0.1204 0.1207 0.08198 0.08198 0.09568 0.09579 0.2010 0.2006
1.11 0.0059 0.6%
Mean SD CV
54Fe added to ~97.23% Determined by TIMS
fecal sample
CBased on isotope dilution calculations
0.200-
0m ,< N 0.100-
jloJ ~ r = 0.99999
0.000
I
I
0.050
0.100
M G FE-54 A D D E D / M G
F E I N FECAL SAMPLE
FIG. I X~otopic ratio of 54Fe:56Fe as a function of amount of o~Fe added to a fecal sample containing 1.1mg Fe/g.
0.150
338
O.R. TURNLUND e t a ] . TABLE 2 Iron Absorption and Status of Young Men 1
Subject 3 4
2
5
"6
Mean
SEM
Absorption %) MP 1~ 17.9 2.9 3.4 3.5 15.7 4.5 8.0 2.8 6.0 2.2 -0.5 15.2 9.1 8.2 MP 2 17.2 2.6 Iron Balance (mg/day) MP 1 -0.4 -1.1 -3.9 -0.2 -1.0 -1.4 -1.3 0.5 MP 2 0.3 0.1 1.3 -0.9 0.8 0.3 0.3 0.5 Hemoglobin (g/dl) Prestudy 16.9 16.0 1 5 . 2 1 6 . 2 16.5 16.2 16.2 0.2 End study 16.0 14.6 1 5 . 1 1 5 . 2 15.7 15.7 15.4 0.2 Hematocrit (%) Prestudy 47.3 46.8 4 4 . 7 4 5 . 9 46.9 47.2 46.5 0.4 End study 42.7 43.4 4 4 . 1 4 4 . 1 45.7 46.0 44.3 0.5 Serum iron (ug/dl) Prestudy 143 76 88 147. 158b 122c 15 129 142 169b 173D 166 155 End study 123 9 Serum f e r r i t i n (ng/ml) Prestudy 157 85 95 178. 36 269 137 34 End study 381b 82 193 305D 46 344D 255 57 TIBC (ug/dl) 341 283 367 251 307c Prestudy 292 19 402b 333 273 354 300 304 End study 262 16 aMetabolic Period 1 ~Above normal range ~ of 5 subjects
TABLE 3 Copper Absorption, Balance, and Serum Levels of Young Men .... 1 Absorption (%) MP 1 40.8 MP 2 27.6 Balance (mg/day) MP 1 9.35 MP 2 0.23 Serum Copper (ug/dl) Prestudy 69 End MP 1 66 End MP 2 77
2
Subject 3
4 ....
5
6
Mean
SEM
24.2 24.3
26.7 27.1
22.5 22.0
27.3 23.0
33.5 29.2 30.6 25.8
2.8 1.3
0.17 0.30
-0.53 0.49
0.23 -0.07
0.21 0.06
0.24 0.11 0.25 0.21
0.13 O.08
93 88 85
87 83 77
4 6 5
100 199 110
88 76 88
79 62 75
86 79 85
Table 6 summarizes the copper data for the young and elderly men. Fecal copper differed s i g n i f i c a n t l y between age groups, but this reflected higher dietary copper intake by the elderly. Serum copper was s i g n i f i c a n t l y higher in elderly men than in young men. Urinary copper was lower in both groups in MP2 than in M P I . Serum and urinary copper differed s i g n i f i c a n t l y among subjects. Neither copper absorption nor balance differed between age groups or among subjects. DISCUSSION Evaluation of the measurement of 54Fe in the fecal samples demonstrated that the analysis was done with a high degree of precision and accuracy. This is essential to reliable absorption determinations when fecal monitoring is used.
IRON AND COPPER ABSORPTION
339
TABLE 4 Summary of Iron Balance and Absorption Data for Young and Elderly Men
MP 1 Dietary Iron (mg/day) Young men 10.4 Elderly men 9.8 Fecal Iron (mg/day) Young men 11.7
MP 2
Elderly men
6.6 b
ANUVA between among MO subjects (p)
10.4 10.0 10.0
Elderly men Urinary Iron (mg/day) Youn9 and elderly <0.1 <0.1 Iron Balance (mg/day) Young men -1.3 0.3 Elderly men -0.2 b Apparent absorption (%) Young men 8.0
~a
between age groups
-0.4 8.2 8.7
0.5 10.0b
NS 10.4
NS
NS
(I0.6 b)
O. b)
NS
NS
NS
1.2 (I.8 b)
NS
NS
0.0024
(0.
Standard error of pooled mean ~Mean of 4, other means are of 6 men Since the amount of 54Fe excreted is measured and iron is poorly absorbed, errors are compounded in calculations. The CV we observed, 0.6%, in the determination of excreted isotope would result in a 7% CV on the average when absorption averages 8% of the amount fed, as i t did in these men. An individual absorption measurement of 8% would have a SD of 0.6. The precision of analyses was improved considerably compared to e a r l i e r work (9) by the new purification procedures and improved analytical instrumentation described in the Methods Section. A number of studies have demonstrated that labeled iron added e x t r i n s i c a l l y , as was done in this study, equilibrates with the miscible~Qon-heme iron pool (5,6). Therefore, the absorption of the stable isotope, ~Fe should reflect absorption of the iron in the diet, which was non-heme iron. The data on iron absorption obtained in this work demonstrates that iron absorption can be determined with s u f f i c i e n t precision to clearly detect differences in absorption. Iron absorption and status data of these young men suggests that the currently recommended intake of 10 mg Fe/day is adequate to maintain iron status in normal healthy young men. Blood indicators of iron status remained in the normal range except for some individuals with elevated values as discussed in the Results Section. Iron balance was negative in the young men in the f i r s t period, but became positive in a l l but one man in the second balance period. We observed negative iron balance previously when other indicators of iron status were normal and when iron status improved during the course of the study (13). Therefore, we consider iron balance r e l a t i v e l y unreliable for assessing iron requirements and status. Copper balance was s l i g h t l y positive, on the average, in the young men. Serum copper levels were within the normal range for a l l the young men. These data suggest that a copper intake of 2.7 mg/day was adequate. When iron and copper absorption data from the two groups of men were compared, absorption was similar in both groups. Iron absorption averaged 6.6 and 8.7 % in the elderly men and 8.0 and 8.2 % in t h e young men. Copper absorption averaged 25.3 and 27.7 % in the elderly men and 29.2 and 25.8 % in the young men. Z i n c absorption was compared in these subjects previously and a
340
3.R. TURNLUND et al.
TABLE 5
Summary of Blood Iron Data for Young and Elderly Men
Day 1 Hemoglobin (mg/dl) Young men
Day 91
5.4
13.4
14.6
46.5
44.3
Elderly men MCV (u ~) Young men
40.0
43.4
84.5
86.2
Elderly men MCH (pg) Young men
91.2
83.8
30.3
29.3
Elderly men MCHC (%) Young men
30.6
28.1
33.9
34.0
Elderly men Serum iron (ug/dl) Young men
33.5
33.7
118c
150
94c
146
323c
321
Elderly men TIBC Young men
among subjects
0.2
b
NS
0.0012
0.6
b
NS
0.0085
3.6
NS
NS
NS
I.I
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.048
0.025
14 (17c)
15 NS Elderly men 292c 261 (13c ) Ferritin (mg/dl) Young men 137 225 24 Saturation (%) Elderly men 36c 56 6 Transferrin (mg/dl) Elderly men 264 281 Standard error of pooled m e a n . . . Significant interaction. Lower for elderly men, Day 1. CMean of 5 due to sample hemolysis.
~
ANUVA between MP (P)
16.2
Elderly men Hematocrit (%) Young men
S~a
6etween age grouPs
NS .
NS .
NS 0.014
marked difference was observed between the young and elderly (15). Zinc absorption in elderly men averaged 17.3 and 18.1% in MP I and MP 2 respectively, and 29.9 and 33.0 % in young men. I t appears that while zinc absorption changes markedly with age, iron and copper absorption may not be affected. While copper absorption did not d i f f e r between age groups, serum co~per was s i g n i f i c a n t l y higher in the elderly men. In contrast, serum zlnc was s i g n i f i c a n t l y lower in these elderly men than in young men. The higher serum copper levels we observed in elderly men agrees with the results of a population survey of serum copper (20). The investigators divided men in six population ~roups by age and found serum copper was higher in men over 60 years of age than in men in five younger population groups. There were large differences among individuals in iron absorption. Zinc absorption also differed among subjects Within age groups, but the differences were small compared to the differences between age groups. Iron absorption is known to d i f f e r among individuals, but the difference has been attributed primarily to enhanced absorption when stores are low or with irondeficiency (21). Our subjects were not iron depleted, based on serum f e r r i t i n in the young men and transferrin in the elderly men, and these large differences in absorption were unexpected.
IRON AND COPPERABSORPTION
341
TABLE 6 Summary of Copper Data for Young and Elderly
MP 1
MP 2
Dietary copper (mg/day) Young men 2.70 Elderly men 3.24 Urinary copper (mg/day) Young men 0.0114 Elderly men 0.0113b Fecal copper (mg/day) Young men 2.58 Elderly men 3.22 D
S'~a
25.3 b
Serum copper (ug/dl) Pre Study Young men 86 Elderly men
98
0.0068 0.0087 2.48 3.22
27.7d
0.00006 (0.00076)
NS
0.0004
0.01~ (0.14 u)
0.0007c
NS
NS
0.12L (0.14 ~)
NS
NS
NS
1.3 (1.6 b)
NS
NS
NS
0.0005
NS
0.0001
Mid End S t u d y Study 79 85 103b
among subjects
(P)
2.70 3.28
Copper balance (mg/day) Young men 0.11 0.21 Elderly men 0.01D 0.05 Apparent absorption (%) Young men 29.2 25.8 Elderly men
ANOVA ~e~-6etween age MP groups
105
(b)
0.0064
Standard error of pooled mean ~Mean of 4, other means are of 6 Due to level ~Mean of 5
of dietary copper
The hemoglobins and hematocrits of the elderly men improved during the course of the study, while those of the young men did not. At the end o f the study hemoglobin and hematocrit levels were similar in b o t h age groups. The comparisons of hemoglobin and hematocrit levels in the two groups of men suggests that the prestudy dletary iron intake of the elderly men may have been lower than the recommended intake they were fed during the study. Or i t may be that the prestudy diets of the elderly contained iron that was poorly available, as has been suggested by Lynch, et al (21). The significant positive correlations between iron absorption and three indicators of iron status were also unexpected. However, our subjects were not iron depleted. When absorption from the same diet was consistently higher in some individuals than others for an extended period of time (78 and 83 days), higher hemoglobin and hematocrit levels might be expected in those absorbing more iron. Serum f e r r i t i n and transferrin were not significantly correlated with iron absorption in our subjects, but these were each done In only one population group; therefore, the number of observations was small. I t is well established that the efficiency of absorption increases with iron deficiency anemia and when iron stores are low. This would result in a negative correlation between indicators of iron status and absorption. In those studies, the negative correlations were attributed to individuals whose iron stores were depleted (6). The data from these studies suggests that iron and copper absorption are similar in healthy young and elderly men. The data suggest that dietary iron and copper requirements are not affected specifically by age alone. T h i s conclusion would be valid only for healthy individuals with no chronic diseases and not taking medication. The incidence of chronic disease and use of medications is substantially higher in the elderly and b o t h could affect iron and copper
342
J.R. TURNLUNDet al.
requirements. The marked differences in homeostatic control of iron absorption, appears to be adequate. Therefore, individuals consuming similar diets could
iron absorption among subjects suggests even amon~ subjects whose iron status iron requirements of normal, healthy d i f f e r considerably.
ACKNOWLEDGEMENTS The authors thank Drs. Sheldon Margen, Ned Durkin, and Yves Schutz who made i t possible to conduct these studies of mineral absorption in conjunction with t h e i r studies of protein and energy requirements, Bonnie Gong who carried out the mineral separations, and William Keyes who carried out TIMS analysis of copper isotopes. REFERENCES 1.
RecommendedDietary Allowances, 9th edition. Wash. D.C., 1980; 137-144.
National Academy of Sciences,
2.
Baker SJ, DeMaeyer EM. Nutritional anemia: its understanding and control with special reference to the work of the World Health Organization. Am. J. Clin. Nutr. 1979; 32:368-417.
3.
Dallman PR, Siimes MA, Stekel A. Iron deficiency in infancy and childhood. Am. J. Clin. Nutr. 1980; 33:86-118.
4.
Morck TA, Cook JD. Factors affecting the bioavailability of dietary iron. Cereal Foods World 1981; 26:667-72.
5.
Hallber~ L. Bioavailability of dietary iron in man. ~n Annual Review of Nutrltion (Darby, W.J,, Broquist, H.P., and Olson, .E., eds.) 1981; p 123-47, Annual Reviews, Inc., Palo Alto, CA.
6.
Kuhn IN, Monsen ER, Cook, JD, and Finch CA. Clin. Med. 1968; 71:715-721.
7.
Bowering J, Sanchez AM, Irwin MI. A conspectus of research on iron requirements of man. J. Nutr. 1976; 106:985-1074.
8.
King JC, Raynolds WL, Margen S. Absorption of stable isotopes of iron, copper, and zinc during oral contraceptive use. Am. J. Clin. Nutr. 1981;31:1198-1203
9.
Turnlund JR, Michel MC, Keyes WR, King JC andMargen S. Use of enriched stable isotopes to determine zinc and iron absorption in elderly men. Am. J. Clin. Nutr. 1982;35:1033-40.
Iron absorption in man.
10. Cartwright GE, Wintrobe MM. Copper metabolism in normal subjects. Clin. Nutr. 1964;14:224-32.
J. Lab.
Am. J.
11. Turnlund JR, King JC, Gong B, Keyes WR, Michel MC. A stable isotope study of copper absorption in young men: effect of phytate and cellulose. Am. J. Clin. Nutr. 1985;42:18-23. 12. Turnlund JR, Michel MC, Keyes WR- Schutz Y, Margen S. Copper absorption in elderly men determined by using 65Cu. Am. J. Clin. Nutr. 1982;36:587-91, 13. Turnlund JR, Costa F, Margen S. Zinc, copper and iron balance in elderly men. Am. J. Clin. Nutr. 1981; 34:2641-7. 14. Durkin N, Margen S, Ogar DA, Tilve SG. Human protein requirements: autocorrelation and adaptation to a low protein diet. In: Protein-Energy Requirement Studies in Developing Countries: Results of International Research. (Rand, WM, Uauy, R, Schrimshaw N, eds) 1984;pp:57-62, United Nations University, Tokyo.
IRON AND COPPER ABSORPTION
343
15. Turnlund JR, Durkin N, Costa F, Margen S. Stable isotope studies of zinc absorption and retention in young and elderly men. J. Nutr. 1986;116:1239-47.
16. Malawer SJ, Powell DW. An improved turbidimetric analysis of polyethylene glycol u t i l i z i n g an emulsifier. Gastroenterology 1967;53:250-6. 17. Turnlund JR, King JC, Keyes WR, Gong B, Michel MC. A stable isotope study of zinc absorption In young men: effects of phytate and alpha-cellulose. Am. J.Clin. Nutr. 1967;40:1071-7. Milliken GA, and Johnson DE. Analysis of messy data, Vol. 1. Designed 18. Experiments. 407.
Lifetime Learning Publications, Belmont, CA. 1984. pp:322-
19. Siegel S. Nonparametric Statistics for the Behavorial Sciences. McGraw-Hill, New York. 1956;pp:213-22. 20. Schreurs WHP, Klosse JA, Muys T, Haesen JP. Serum copper levels in relation to sex and age. Internat. J. Vit. Nutr. Res. 1982;52:68-74. 21, Lynch SR, Finch CA, Monsen ER, Cook JD. Iron status of elderly Americans. Am. J. Clin. Nutr. 1982;36:1032-45.
Accepted for publication September 28, 1987.