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*
Department of Human Nutrition, Kansas State University, Manhattan, KS 66505 and
Department of Nutritional Sciences, Oklahoma State University, Stillwater, OK 74078
1To whom correspondence should be addressed. E-mail: Medeiros{at}ksu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • bone • calcium restriction • iron deficiency • dual-energy X-ray absorptiometry
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–4
). Other nutrients that may have an impact on bone physiology include protein, vitamin C, copper and iron. Scurvy and associated bone anomalies are due to impairment of the enzymes prolyl and lysyl hydroxylases that catalyze an ascorbate-dependent hydroxylation of select prolyl and lysyl residues and are essential steps before crosslinking by the copper-dependent enzyme lysyl oxidase (5
). These enzymes apparently need iron and vitamin C to function normally. Previously our laboratory reported decreased mechanical strength in femurs from rats fed an iron-deficient diet compared with controls. Even though these animals were only mildly deficient there were significant changes in cortical area and bone physiology, which may be attributable to impaired function of prolyl and lysyl hydroxylase (6
).
Iron deficiency anemia continues to be the number two public health nutrition problem in the United States after obesity. Teenage girls are likely to be afflicted with iron deficiency at a time when peak bone mass and bone formation are critical. It is not clear why the role of iron in bone formation and disease has not received much attention given the known biochemical role that iron has upon collagen maturation and the prevalence of iron deficiency anemia in this country. The Center for Disease Control refers to iron deficiency as the most common known form of nutritional deficiency, with its prevalence being highest among young children and women of childbearing age (7
). Beard (8
) concludes that the increased iron requirements in adolescent women are unlikely to be met based on available data on iron intakes in adolescents. Collectively, these studies indicate that there is a need for more information on the health consequences of iron deficiency.
This study presents results on the impact of iron deficiency in rats on femur and tibia density and morphology as compared with rats fed either a calcium-restricted diet or a control diet. Furthermore, we report here the combined impact upon bone when both dietary iron and calcium are limited simultaneously.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The basal diets used were formulated after the recommendations of the American Institute of Nutrition (9
), with casein contributing 20%, corn oil 5%, sucrose 50% and corn starch 15% of the energy yielding macronutrients. Normally cellulose is used as a source of fiber, but because it has rather high levels of contaminating iron we used 5% Avicel as a source of fiber, because it is much lower in iron. The remainder of the diet consisted of a vitamin and mineral mix that meets the requirements of growing rats.
The following four diets were composed: 1) a control diet that follows the American Institute of Nutrition (AIN) 1980 recommendations, 2) an iron-deficient diet, 3) a calcium-restricted diet and 4) an iron-deficient and calcium-restricted diet. The modifications of the diets for each treatment are primarily in the mineral mix, as described below.
The normal iron diet formulated after recommendations is 
40 mg/kg Fe (716 µmol/kg). To produce iron deficiency in rats, it is necessary to have a level of 5–8 mg/kg Fe (89–143 µmol/kg). To accomplish this, in addition to using Avicel as a source of fiber, we used chemically pure calcium phosphate dibasic and magnesium carbonate. Standard chemicals have background iron that often makes obtaining iron-deficient rats problematic. With respect to the calcium-restricted diet, 0.1% calcium (1 g/kg Ca or 0.025 mol/kg) by weight was used, as opposed to the standard AIN diet of 0.52% (5.2 g/kg Ca or 0.130 mol/kg). The phosphorus content of the diet remained at the recommended levels of 0.56% (5.6 g/kg P or 0.186 mol/kg) by weight. This approach was chosen so that any changes in the calcium-restricted diet could be attributed to calcium restriction directly and not to a decrease in both calcium and phosphorus. We were aware that this may have led to an imbalance of these two minerals; however, we used the calcium-restricted diet primarily as a basis of comparison for what occurs in the iron-deficient group. In preparing the diet, we assumed that, for phosphorus, 0.16% (1.6 g/kg diet or 0.052 mol/kg) came from casein and 0.4% (4.0 g/kg P diet or 0.129 mol/kg) came from calcium phosphate. The diets were prepared by Research Diets (New Brunswick, NJ). Calcium and iron contents of all diets were verified by atomic absorption spectrophotometry (AAS) analysis and were well within the mean targets.
Experimental design
Thirty-two weanling Long-Evans male rats were assigned to one of the four dietary groups described above, with a sample size of eight in each group. This number was used in our previous studies and proved to be of sufficient power to determine significant differences in bone mass, breakage and radiometry parameters. The rats were allowed free access to the diets for a period of 5 wk. Rats were housed individually in stainless steel cages and were provided with deionized distilled water. At the end of the 5-wk period, rats were weighed, then anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) as approved by Kansas State University’s Animal Care and Use Committee. The thoracic cavity was opened after the rats had achieved a plane of anesthesia, then 1 mL of blood was removed and placed in nonheparinized tubes and hematocrit was determined. Hearts were removed and weighed because heart enlargement is one sign of iron deficiency anemia. Femurs and tibias were dissected from the left side of the animals, trimmed of muscle tissue, weighed, defatted by shaking in hexane for an 18-h period, dried and reweighed for a dry weight. Bones had densitometry measurements, radiometry, mechanical testing and mineral analysis as described below.
Densitometric and morphometry measurements
The femur and tibia of each rat were scanned using a dual-energy X-ray absorptiometry (DEXA) small animal high-resolution scan module (Hologic QDR-4500A, Bedford, MA) at Oklahoma State University. Bone mineral content and density were recorded from the DEXA. X-rays of the left femurs and tibias were developed and cortical bone area, medullary area and total bone length and width were measured as described earlier (10
).
Mechanical testing
A three-point breakage test using an Instron Universal Testing System (model 4466; Instron, Dayton, OH) interfaced with their Series IX software for Windows was used to measure bone strength (11
). The midpoints of the femurs and tibias were determined by measuring the length of each bone and dividing that length by two using a vernier caliper. The length of the femur was determined as the highest point of the head of the femur to the lowest point of the inner condyle. Similarly, the length of the tibia was measured as the highest point of the external tuberosity to the lowest point of the external malleolus. The maximum breakage point was measured for each bone.
Mineral analysis of bone and diet
After completion of the mechanical testing of the bones, the fractured bones were digested in trace element grade nitric acid and diluted up to 10 mL with deionized distilled water. For calcium analysis, the samples were serial diluted with 1% lanthanum as lanthanum chloride to break any calcium phosphate bonds, followed by calcium analysis via flame AAS (model 5000; Perkin-Elmer, Norwalk, CT). Iron levels were determined from the original 1/10 diluted samples via flame AAS. Calcium and iron standards were prepared from Fisher Scientific (Fairlawn, NJ) certified standards. Phosphorus levels were determined using the acid-molybdate method with a commercial kit purchased from Sigma-Aldrich (St. Louis, MO). Diets were analyzed in a similar fashion using <1 g of sample.
Statistical analysis
Data were analyzed by the following separate approaches: 1) as a one-way ANOVA for the four groups and 2) by a two-way ANOVA with iron level and calcium level as the main treatment effects. When significant F-values were obtained for the one-way ANOVA or for the interaction with the two-way ANOVA, the least significant difference method was used to separate means at the 0.05 level. Both analysis methods revealed identical results and results of the two-way analysis suggested that when iron and calcium were lacking in the diets the effect was additive. We report the results from the one-way ANOVA to simplify interpretation. The Statistical Analysis System (SAS, Cary, NC) was used for these analyses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The calcium-restricted + iron-deficient (Ca-Fe-) rats were the lightest (201 ± 8.3 g), followed by the iron-deficient (Fe-) group (229 ± 8.9 g), the calcium-restricted (Ca-) group (268 ± 3.2 g) and finally the controls (295 ± 7.2 g) (P 
0.05). As expected, heart weight in the Fe- group was greater than in the other three groups (P 0.05) and the heart:body weight was greater in both the Fe- and the Ca- Fe- (P 0.05) groups. Anemia as reflected by significantly lower hematocrit readings (P 0.05) was present in the Fe- and Ca-Fe- groups compared with the other two groups (P 0.05). Femur and tibia wet weights were significantly lower (P 0.05) in the Ca-Fe- rats compared with the other three groups and the femurs from the Fe- rats were lighter or weighed less than the controls (P 0.05). However, when adjusted for final body weight, femur:body weight was greater in the Fe- rats (P 0.05) than in the other three groups and the tibia:body weight in the Fe- groups was greater than the control (P 0.05).
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). Rats fed the Ca-, Fe- and Ca-Fe- diets had decreased total and cortical femur and tibia widths compared with controls (Fig. 2A, B
) (P 0.05). Ca- and Ca-Fe- animals had the smallest cortical widths, followed by the iron-deficient group (P 0.05). The medullary width was significantly decreased in the iron-deficient rats compared with the other three groups (P 0.05). Total cortical bone areas were markedly decreased in all three mineral-restricted groups compared with the control (Fig. 2
C, D) (P 0.05). The only exception was the tibia cortical area in the Fe- rats, which was greater than that of either the Ca- or Ca-Fe- rats (Fig. 2
D). DEXA analysis of bone tended to reflect a similar pattern (Fig. 3A, B
). Bone mineral content and density were markedly reduced in the Ca- and Ca-Fe- groups compared with the control and Fe- groups (P 0.05). However, the Fe- group did have significantly lower mineral content and bone density than the controls (P 0.05). Collectively these data revealed that calcium restriction either singly or with iron deficiency tended to affect bone morphometry and bone mineral density the most. However, iron-deficient rats also had compromised bones compared with controls.
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0.05) (Fig. 4A
). However, the femur iron content of rats fed the Ca- diet was significantly greater than in the other three groups (P 0.05). Calcium content of both femurs and tibias was dramatically reduced in rats fed either the Ca- or Ca-Fe- diets compared with the controls and Fe- rats (P 0.05) (Fig. 4
B). Also, the Fe- rats had lower calcium levels in the tibias compared with the controls (P 0.05), and there was a nonsignificant trend in the femurs. Femur phosphorus content of the Ca- and Ca-Fe- groups were approximately one-half the value of the controls and Fe- rats (Fig. 4
C) (P 0.05) and the phosphorus level in the Fe- group was greater than controls (P 0.05). For the tibias, the phosphorus level was greater in the control than in the Ca- and Ca-Fe- rats (P 0.05).
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0.05; Fig. 5
). The Ca- rats required less force to fracture the femurs than the Fe- or control rats (P 0.05). However, the femur of Fe- rats also had significantly lower force necessary for fracturing compared with the control group (P 0.05). With respect to the tibias, Ca- and Ca-Fe- had similar decreases in the force needed for fracturing, but both required less fracture force than the other two groups (P 0.05). The necessary fracture force for tibias of the Fe- group was intermediary between the Ca- groups and the controls, similar to what was observed for femurs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and Marra and de Alaniz (13
) both used 0.5 g/kg Ca diet to assess calcium restriction upon bone parameters. Peterson et al. (14
) used 1.5 g/kg Ca diet in rats, and Kochanowski (15
) used 3 g/kg Ca diet. In most cases rats were fed for a period of several weeks (up to 12 wk in these studies) and beginning at young ages. Thomas et al. (16
) used 1 g/kg Ca diet to examine calcium metabolism in growing rats and thus we chose that level as a good midpoint range for a calcium-restricted diet.
Tibia weights by treatments were similar for all groups except for the Ca-Fe- groups, which were lighter. When corrected for by body weight, the Fe- group tibia weights were greater than those of the other groups. Similar observations were made for the femurs, with Fe- and Ca-Fe- femur weights being lighter than controls, but when corrected for body weight there was an increase in the Fe- group. Collectively, these data are significant in that any potential decrease in food intake, particularly protein, is not likely to have played a role in the results of bone measurements for animals fed the modified diets. A study using mice and protein restriction resulted in no differences in bone mineral content (17
). Reduced bone mineral content is not always associated with protein depletion or food intake (18
). Studies in rats and humans have shown that moderate protein depletion reduces calcium excretion, indicating a protective mechanism (18
,19
). Differences in body weight could explain some difference between groups; however, Kipp et al. (20
) recently reported that, when iron-deficient rats were compared with a weight-matched control group, cortical and cancellous bone were significantly decreased, supporting an independent effect of iron upon bone morphometry. A previous study on magnesium deficiency and bone calcium and phosphorus content suggested that pair-feeding had no effect (21
). Three-week-old rats were fed moderately or severely magnesium-restricted diets for 3 wk and included pair-fed groups. There was a 36% decline in body weight in the severely magnesium-deficient group compared with the adequate groups. Both restricted groups as well as the respective pair-fed groups did not differ from controls in bone calcium and phosphorus. In our study, the Fe- group had a 22% reduction in body weight, and the Ca-Fe- group had a 32% reduction. Phosphorus levels in the Fe- group were not lower than controls, but, for femurs, were greater than controls. Calcium levels between controls and Fe- groups did not differ in femurs. Thus, it is highly unlikely that the observed changes reported are a result of food intake.
The many changes we report for the calcium-restricted groups (Ca- and Ca-Fe-) were not unexpected. However, the most salient results came from the apparent role iron deficiency had upon several bone parameters we measured and the consistency of our findings in terms of pathology, mechanical strength and bone mineral density and content in iron-deficient groups compared with the control group.
It is of interest to note that the changes in bone morphology were mirrored by changes in bone mineral density, with calcium restriction having a greater impact than iron deficiency. Calcium restriction plus iron deficiency resulted in an additive decrease in femur bone mineral density and bone breaking strength. It was also notable that in iron deficiency there was lower tibia calcium content. These two minerals are both often limiting in terms of diets among westerners. Results obtained from this study are the results of severe calcium and/or restriction. Although some portions of the globe may have this severe level of iron deficiency, this level of severity in the U.S. population is not likely. However, these results set the stage for future studies on graded levels of both iron and calcium over protracted time periods because they may negatively impact bone mechanics and physiology.
Within nutrition, calcium is regarded as the most important nutrient concerning bone health (22
–25
), and a lifelong low calcium intake seems to be another risk factor for osteoporosis (2
). This is because calcium and phosphorus together build hydroxyapatite, the main mineral of the bone matrix (26
) that comprises 90% of the mineral content of bone (23
) and functions as a reservoir for these minerals (1
). It is widely accepted that extra calcium shows a positive effect upon bone health (1
,22
, 26
–28
). Comparing bone density of adults with low calcium intake to high calcium intake groups, high dietary calcium seems to increase bone density between 5 and 6% (1
).
A significant part of bone is collagen, primarily type I. Copper and iron are two nutritive trace elements known to influence collagen maturation. Lysyl oxidase is a copper-containing enzyme that catalyzes the crosslinking of the 
amino groups lysine and hydroxyproline between adjacent collagen fibrils, thereby increasing the mechanical strength of the protein. Iron is known to be a cofactor for prolyl and lysyl hydroxylases, which catalyze an ascorbate-dependent hydroxylation of both select prolyl and lysyl residues and are essential steps before crosslinking by lysyl oxidase (5
). The role of copper in maintaining good bone health is not new. Jonas et al. (29
) reported that femurs from copper-deficient rats had decreased maximal torque, angular distortion and toughness compared with pair-fed controls. Ash weight and calcium content did not differ between the two groups, suggesting that decreased mechanical strength could be due to decreased lysyl oxidase activity leading to lower crosslinking of the collagen. Rucker et al. (30
) made similar observations with chick bones that had copper deficiency. With respect to iron, our laboratory previously reported that iron deficiency in young rats leads to decreased mechanical strength of femurs (6
). In that study both copper-deficient and iron-deficient rats had smaller cortical and larger medullary areas in a portion of the femurs.
Hyroxylase enzymes apparently require both ascorbate and iron to function properly. Some of the results from our present study may be also confounded by decreased hydroxylation of 1,25-dihydroxylcholcalciferol, the active form of vitamin D, in which there would be an anticipated decline in calcium absorption. It is known that the 25-hydroxycholcalciferol hydroxylase is located within the renal mitochondria and the enzyme is a three-component system: a flavoprotein, an iron-sulfur protein and a cytochrome P-450 (31
). A lack of iron could have an impact on this enzyme system, leading to decreased renal reabsorption of calcium as well as decreased intestinal calcium absorption when iron is limiting. Although vitamin D was not the focus of the present study, it is important that, in future studies, this possibility be considered.
In summary, iron deficiency has significant impact upon bone mineral density, content and fragility. Although the degree of pathology observed among just the Fe- group is not as severe as that observed with calcium restriction, this study for the first time suggests that simultaneous restriction of both calcium and iron, two minerals likely to be low in western diets, exacerbate the variables studied. Human data on iron status and evidence of bone fractures may help give some insight into this area. Additional studies on marginal intakes of iron as well as more definitive studies on the mechanism(s) by which iron restriction leads to these findings are warranted.
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FOOTNOTES |
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Manuscript received 23 May 2002. Initial review completed 25 June 2002. Revision accepted 31 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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20. Kipp, D. E., Beard, J. L. & Lees, C. J. (2002) Mild iron deficiency results in altered bone mass and histomorphometry in growing female rats. FASEB J 16:A273(abs.).
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