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Iron Deficiency Negatively Affects Vertebrae and Femurs of Rats Independently of Energy Intake and Body Weight1

November 27, 2004

ABSTRACT

The question of whether iron deficiency has direct adverse effects on vertebral trabecular bone and long bones was answered by this study. Four groups of female weanling rats were fed for 5 wk diets that were 1) control; 2) calcium restricted, 1.0 g Ca/kg diet; 3) iron deficient,

KEY WORDS: * iron * calcium * rat * bone density

Osteoporsosis has been linked to life-long inadequate calcium intakes in humans (1) and calcium is often considered the most important nutrient concerning bone health (2-5). Sufficient calcium has a positive effect upon bone quality (2,6-9); however, other minerals also may affect bone biology. Iron is a cofactor for prolyl and lysyl hydroxylases, enzymes that catalyze an ascorbate- dependent hydroxylation of prolyl and lysyl residues, essential steps to crosslinking by lysyl oxidase (10). Iron deficiency in young rats leads to decreased mechanical strength of femurs and reduced cortical bone (11). In severe iron deficiency, bone strength and mineral density are reduced (12). Calcium restriction, when combined with iron deficiency, enhances the pathology in several measures. However, the issue of decreased weight gain observed during iron deficiency has not been addressed in any of these studies. Also, these studies centered only on the long bones and did not include vertebrae.

Iron deficiency anemia remains a substantial public health nutrition problem in the United States. The Center for Disease Control refers to iron deficiency as the most common known nutritional deficiency, with its prevalence being highest among young children and women of childbearing age (13). Beard (14) reviewed the large increase in iron requirements of adolescent women due to growth spurt and menstrual losses and the improbability of meeting these additional recommendations. Collectively, these studies suggest that the magnitude of iron deficiency among adolescent females in this country might also affect bone health during an age when peak bone mass attainment is critical.

In this study, we evaluated rats fed calcium-restricted or iron- deficient diets and a group that was pair-fed a diet adequate in nutrients, but in amounts eaten by the iron-deficient group. Because of the links between dietary calcium restriction and bone pathogenesis, a calcium-restricted group was included as a basis of comparison for our results dealing with iron deficiency. We took advantage of imaging and simulation technologies to detect changes in the microarchitecture and potential strength of trabecular bone from vertebrae.

MATERIALS AND METHODS

Diets. The basal diets used were formulated after the recommendations of the American Institute of Nutrition as modified in 1980 (15) with casein contributing 20%, corn oil 5%, sucrose 50%, and corn starch 15% of the energy-yielding macronutrients. Because cellulose has rather high levels of contaminating iron, we used 5% Avicel as a source of fiber. The remainder of the diet consisted of vitamin and mineral mixes that meet the requirements of growing rats except as modified by the experimental protocol.

Diet groups were: 1) rats fed a control diet based on the AIN- 1980 recommendations (control); 2) rats fed an iron-deficient diet; 3) rats fed a calcium-restricted diet; and 4) a group given the control diet but pair-fed (PF)3 to the iron-deficient group. The normal diet contained about 40 nig Fe/kg (716 mol/kg) diet and 0.52% calcium (5.2 g Ca/kg diet or 0.130 mol/kg). The iron-deficient diet was formulated to contain 5-8 mg Fe/kg (89-143 mol/kg) diet. The calcium-restricted diet contained 0.1% calcium (1 g Ca/kg diet or 0.025 mol/kg) by weight. Modifications of the diets for each treatment were primarily in the mineral mix, as we described in detail previously (11). Calcium and iron concentrations of all diets were verified by atomic absorption spectrophotometry analysis. For iron, the mean was 40.1 mg/kg and for calcium, 6.2 g/kg. The iron- deficient diet contained 7.7 mg Fe/kg and the calcium-restricted diet had 1 g Ca/kg. The amount of food consumed by the iron- deficient group was determined daily and an equal amount of control diet was given to the pair-fed rats.

Experimental design. Thirty-two weanling Long-Evans female rats were randomly assigned to 1 of the 4 groups. The black fur of Long- Evans rats turns gray within 2 wk of consuming an iron-deficient diet and is used by our group as an indicator of iron-deficiency. We chose females because human females are more prone to iron deficiency. Eight rats per group were used in our previous studies and provided sufficient power to determine significant differences in bone mass, breakage, and radiometry parameters (11). The rats were allowed free access to the diets for 5 wk with the exception of the pair-fed group. Rats were housed individually in stainless- steel cages and provided with deionized-distilled water. Twenty- four-hour urine samples were collected at wk 3 and 5 by placing the rats in metabolic cages. At the end of the 5-wk period, rats were weighed and then anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body wt) 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, ~ 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 were dissected from the left legs of the rats, trimmed of muscle tissue, weighed, defatted by shaking in hexane for 18 h, dried, and reweighed for a dry weight. Bones received densitometry measurements, radiometry, mechanical testing, micro-CT scans, and mineral analysis as described below. The L-3 vertebrae were removed for analysis by micro-CT scans as described below.

Densitometric and morphometry measurements. Total body composition and bone mineral density were measured with dual-energy X-ray absorptiometry (DEXA) using small animal software (v5.6, Prodigy, General-Electric Lunar). Rats were anesthesized with sodium pentobarbitol prior to scanning. Whole-body DEXAs were conducted at 3 wk and a final 5-wk determination was recorded. Analysis of each rat was made using a customized region of interest that encapsulated the entire animal into one total body region. Subsequently, an excised femur from each rat was scanned using a DEXA small animal high-resolution scan module (Hologic QDR-4500A) at Oklahoma State University. Bone mineral content and density were recorded from the DEXA. X-rays of the left femurs were developed and total and cortical hone areas were measured as described earlier (8,11).

The structural aspects of the L-3 trabecular bone were determined using a Micro-CT 40 (Scanco Medical) capable of analyzing the 3- dimensional architecture of the bone. Information on bone volume fraction, trabecular number, thickness, and separation was obtained on L-3 vertebrae and cortical thickness also was determined. The specimens were placed in 16-mm tubes and imaged every 0.0165 m. For trabecular bone between the cranial and caudal growth plates, every 10th serial section was outlined, and intermediate sections were interpolated with the contouring alogorithm to create a volume of interest. Approximately 250 images using a 1024 1024 matrix in an isotropic resolution of 22 m^sup 3^ were analyzed to acquire 3- dimensional images and conduct the morphometric analysis. Segmentation values used for analysis were sigma 0.7, support 1, and threshold 210. A 3-dimensional analysis was conducted to determine bone volume fraction (BV/TV), trabecular number, trabecular thickness, trabecular separation,and structural model index.

The force required to compress each vertebrae was determined by simulation using the finite element software (Scanco Medical) available for use with the micro-CT scanner. The TV used for morphometric analysis of vertebral trabecular bone was the volume of interest for compression. Total force for compression, stiffness, size-independent stiffness, and von Mises stresses were obtained from the analysis and reported here.

Mechanical testing. Femur strength was measured using a 3-point breakage test at the midpoints of the bones using an Instron Universal Testing System (Model No. 4466, Instron) interfaced with their Series IX software for Windows as previously described (11).

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 to 10 mL with deionized-distilled water. For calcium analysis, the samples were serially diluted with 1% lanthanum (10 g La/L) as lanthanum chloride to break any calcium phosphate bonds and analyzed by flame atomic absorption spectrophotometry (AAS) (Perkin-Elmer Model No. 5000). Iron levels were determined from the original 1:10 diluted samples via flame AAS. Calcium and iron standards were prepared from Fisher Scientific certified standards. Phosphorus levels were determined using the acid-molybdate method with a commercial kit purchased from Sigma Chemical. Diets were analyzed in a similar fashion using

Urinary pyridinium crosslinks and serum osteocalcin and 1,25- dihydoxycholcalciferol analysis. Twenty-four-hour urine deoxypyridinoline crosslinks were determined using the Pyrilinks-D kit (Cat. No. 8030) from Metra Biosystems. This assay is a competitive enzyme immunoassay that uses a monoclonal anti- deoxypyridionoline anti-body and is standardized to creatinine determined by the Jaffe reaction. Bound crosslinks are removed from small peptides using acid hydrolysis. Serum rat osteocalcin levels were assayed using an immunoradiometric assay (Cat. No. 50-1500) from Immunotopics. This assay uses 2 antibodies; an affinity- purified goat antibody to immobilize the osteocalcin onto beads and another affinity-purified goat antibody radiolabeled with ^sup 125^I that recognizes the amino-terminal end of the molecule. Serum 1,25- dihydoxycholcalciferol levels were determined using a radioimmunoassay kit purchased from IDS. The IDS Gamma-B kit (Cat. No. AA-54F) is a complete assay system for the measurement of 1,25- dihydroxy vitamin D (1,25D) in human serum or plasma. This assay requires purification of 1,25D from the sample by monoclonal immuiioextraction followed by quantification by ^sup 125^I radioimmunoassay.

Statistical analysis. Data were analyzed by a one-way ANOVA using the Statistical Analysis System. For whole-body DEXAs and urinary deoxypyridinium crosslinks, a one-way ANOVA with repeated measures was used. When significant F values were obtained, the least significance differences method was used to separate means.

RESULTS

All rats survived the 5-wk study. The final body weights were lower (P

TABLE 1

Body weight, heart weight, heart:body weight, hematocrit, serum 1,25 dihydroxycholcalciferol and osteocalcin, and urinary excretion of deoxypyridinium crosslinks of rats fed control, calcium- restricted, iron-deficient, and PF diets for 5 wk1

Urinary pyridinium crosslink concentrations at wk 3 and 5 of urine collection were greater (P 0.05). Serum α-1,25-dihydroxycholcalciferol was elevated by 33% (P

Femur mineral analysis revealed that the calcium-restricted group had decreased calcium and phosphorus compared to the other 3 groups (P

Whole-body bone mineral density (BMD) at 3 wk was decreased for the calcium-restricted group compared to the other 3 groups (Table 2). By the end of the study, the BMD of the calcium-restricted group was the lowest, followed by the iron-deficient group. The iron- deficient group had a lower BMD than both the pair-fed and the control groups, but the BMD of the pair-fed group also was significantly lower than that of the control group. The control and pair-fed groups had greater BMD at wk 5 compared to wk 3 (P

TABLE 2

Bone mineral content and morphometric measures of rats fed control, calcium-restricted, iron-deficient, and PF diets for 5 wk1

Morphometric analysis of the radiograms of the femurs revealed several differences among the different dietary treatments (Table 2). Femur total area was reduced in the iron-deficient and pair-fed groups (P

Femur BMD and bone mineral content (BMC) were lowest in the calcium-restricted group (P

Bone strength was compromised in femurs from rats fed calcium- restricted and iron-deficient diets (P

Image analysis of the L-3 vertebrae by microcomputer tomography revealed that the control and pair-fed groups differed from the iron- and calcium-restricted groups. The iron-deficient group had the greatest reduction in trabecular number (P 0.05), but the calcium restricted group had lower trabecular numbers than the control group (P 0.05). The reduction in trabecular thickness was greatest in the calcium- restricted group, followed by the iron-deficient group, both of which differed from the control and pair-fed groups (P 0.05). The structural model index is an indicator of whether the bone structure is more plate-like or more rod-like. Plate-like structures have values near zero and those that are completely rod-like have values approaching 3. Clearly, the bones of the calcium-restricted group had the most rod-like microarchitecture, followed by the bones of the iron-deficient rats (P

FIGURE 1 Trabecular bone morphology of L-3 vertebrae of rats given control, calcium-restricted, iron-deficient, and pair-fed diets for 5 wk as determined with micro-CT imaging. (A) trabecular number, (B) trabecular bone volume/total volume, (C) trabecular thickness, (D) structural model index, (E) trabecular separation. Values are means SEM, n = 8. Bars without a common letter differ (P ≤ 0.05).

Three-dimensional reconstructions of typical L-3 trabecular bone from each of the 4 groups are presented in Fig. 2. The most severe change was in the calcium-restricted group, but the iron-deficient group also had visual alterations compared to the control and pair- fed groups.

Finite element analysis revealed that trabecular cores from vertebrae of calcium-restricted rats required less total force for complete compression than all other groups. The force required to compress vertebrae from the iron-deficient group also was lower than the pair-fed and control groups. The pair-fed group required less total force to compress the vertebrae compared to the control group (Fig. 3). Bone stiffness and size-adjusted stiffness revealed similar patterns, with the calcium-restricted group having the lowest val\ues, but there were no differences between control and pair-fed groups. In simulating von Mises stresses, a 20.18-N force was utilized for all vertebrae. The von Mises force represents the amount of stress within the bone when that force is applied. The calcium-restricted group had the greatest stress within it when this force was applied, followed by the iron-deficient group; both groups differed from each other as well as from the control and pair-fed groups. The control and pair-fed groups did not differ from one another.

FIGURE 2 Images of L-3 trabecular bone of rats given (A) control, (B) calcium-restricted, (C) iron-deficient, and (D) pair-fed diets for 5 wk.

DISCUSSION

We previously reported compromised bone biology in rats fed iron- deficient diets (11,12). However, the dramatic decrease in body weight and corresponding decrease in food intake observed in iron deficiency could contribute to these changes in bone biology. Severe food restriction in mature and young rats results in decreased cortical bone area and mineral content (16,17). Moderate food restriction of ~25% results in decreased mineralization, cortical bone area, and breaking strength (18). In this study, the iron- deficient rats had changes in femurs that differed from the pair- fed group. However, in some measures, the pair-fed group differed from the control group. For femur measures, such as BMD, BMC, and bone breakage, the values of the iron-deficient group were decreased compared to the pair-fed group, but the values of the pair-fed group were also decreased relative to the control group. This may suggest that some of the alteration in bone biology of the iron-deficient rats could be confounded by body weight or food intake. Similarly, the whole-body BMD at 5 wk revealed a similar pattern. However, with respect to the L-3 vertebrae, no such confounding was apparent. The iron-deficient and calcium-restricted groups differed from the control and pair-fed groups for most measures, and the latter 2 groups generally did not differ from each other.

The use of micro-CT imaging gave persuasive results that iron deficiency has a substantial and consistent negative impact upon trabecular bone biology. The indicators presented suggested that there was an increase in bone porosity and that the bone in both calcium-restricted and iron-deficient rats became more rod-like in contrast to the normal plate-like appearance of bone.

Finite element analysis has been used in engineering fields, and biomedical applications with respect to bone biomechanics have gained acceptance. Several reports have validated this technique experimentally (19-21), including the Scanco Medical finite element analysis software (22). Finite element analysis suggested that the force required to compress the vertebrae was the least for the calcium-restricted group, but that the iron-deficient group was significantly lower than the control and pair-fed groups. This is consistent with the femur data, but this is the first report, to our knowledge, with regard to iron deficiency and vertebrae strength. The consistency of the estimates for stiffness and von Mises stresses and the minimal variation of these measures within each treatment group gave us confidence in concluding that iron deficiency has a negative impact upon bone biomechanics. The decrease in stiffness for the bones of calcium-restricted and iron- deficient rats means they are more compliant than the control and pair-fed groups, which is common for bones that are less mineralized (23).

Like our previous study (11), reduced width and area of cortical bone were apparent in iron-deficient rats. The degree of reduction was greater in calcium-restricted rats. Malecki et al. (24) claimed that iron deficiency did not affect the mechanical properties of bone in mice but their animals were not truly iron deficient in that hematocrit levels were normal. Iron-replete, hypotransferrinemic mutated mice differed significantly from those fed an iron- deficient diet. However, the hematocrit was 0.40 in the iron- deficient mice, which is not considered physiological anemia. They also used mice and the current study evaluated rats. On the other hand, Campos et al. (25) reported that iron-deficient rats had decreased femur mineralization that was accompanied by increased cortisol and parathyroid hormone. In humans, serum ferritin levels and bone density of skulls of young women were significantly related (26). Recently, dietary iron in postmenopausal women was reported to be positively associated with increased bone mineral density in those with low to moderate calcium intakes (27).

FIGURE 3 Finite element analysis of trabecular L-3 bone of rats given control, calcium-restricted, iron-deficient, and pair-fed diets for 5 wk. (A) Force required for compression of vertebrae, (B) stiffness, (C) size-independent stiffness, and (D) von Misses stresses assuming an applied force of 20.18 N. Values are means SEM, n = 8. Bars without a common letter differ (P ≤ 0.05).

Urinary deoxypyridinium crosslinks and serum osteocalcin were markedly increased in calcium-restricted rats compared to all other groups, but there were no differences for the iron-deficient rats. This may suggest a different mechanism for the changes in bone physical strength and density in the iron-deficient group. The increased deoxypyridinoline crosslinks represent bone breakdown and the increased serum osteoclacin suggests greater bone turnover in the calcium-restricted rats but not in iron-deficient rats. We also measured the active form of cholcalciferol, α-1,25- dihydroxycholcalciferol, because the final hydroxylation is iron dependent (28). In rats fed the calcium-restricted diet, there was an almost 33% increase in 1,2 5-dihydroxycholcalciferol compared to the other 3 groups, which was expected. However, the iron-deficient group had levels similar to those of control and pair-fed rats, suggesting that iron deficiency did not affect circulating levels of this form of cholcalciferol.

Type 1 collagen is an important component of bone. Several studies demonstrated that decreased collagen crosslinking leads to bone pathology. Lysyl oxidase is a copper-containing enzyme that catalyzes the crosslinking of the e-amino groups of lysine and hydroxyproline between adjacent collagen fibrils, thereby increasing the mechanical strength of the protein. Copper deficiency was shown to result in decreased breaking strength in femurs of rats (11). Jonas et al. (29) reported that femurs from copper-deficient rats had decreased maximal torque, angular distortion, and toughness compared to pair-fed controls. Ash weight and calcium content did not differ between the 2 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 bones from copper-deficient chicks. Opsahl et al. (31) reported that lysyl oxidase was impaired in copper-deficient chicks, which resulted in decreased torsion strength when levels of dietary copper dropped below 1 mg/kg diet. Injection of the lathrogen, β-aminoproprionitrile (BAPN), an inhibitor of lysyl oxidase, resulted in decreased hydroxypyridinnium crosslinks and decreased mechanical strength of femoral diaphyses (32). Others reported that BAPN administration to rats can impair ligaments of teeth (33,34). Iron is a cofactor for prolyl and lysyl hydroxylases, enzymes that catalyze an ascorbate-depcndent hydroxylation of prolyl and lysyl residues, essential steps prior to crosslinking by lysyl oxidase (10). Using a scorbutic rat model, Ellender and Gazelakis (35) reported reduced physical strength of the caudal vertebrae. There is no consensus on the importance of crosslinks to bone strength. One school of thought suggests that crosslinks increase toughness but do not have a profound impact upon the stiffness or strength of bone (36). Osteoporotic women have fewer crosslinks in bone collagen, as reviewed by Burr and Turner (23).

Further studies on marginal dietary iron intake as it relates to bone biology are warranted. Basic studies on how iron deficiency may impact endocrine aspects of bone biology and osteoblastic function may give further insight to the mechanism(s) responsible for these observations.

0022-3166/04 $8.00 2004 American Society for Nutritional Sciences.

Manuscript received 20 May 2004. Initial review completed 22 June 2004. Revision accepted 20 August 2004.

1 Supported by funds from the Kansas Attorney General’s Office, K- State Research and Extension and the Oklahoma Agriculture Experiment Station.

3 Abbreviations used: 1,25D, 1,25-dihydroxy vitamin D; AAS, atomic absorption spectrophotometry; BAPN, β- aminoproprionitrile; BMC, bone mineral content; BMD, bone mineral density; BV, bone volume; DEXA, dual-energy X-ray absorptiometry; PF, pair-fed; TV, total volume.

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Denis M. Medeiros,*2 Barbara Stoecker,[dagger] Aaron Plattner,* Dianne Jennings,* and Mark Haub*

* Department of Human Nutrition, 213 Justin Hall, Kansas State University, Manhattan, KS 66506 and [dagger] Department of Nutritional Sciences, Oklahoma State University, Stillwater, OK 74078

2 To whom correspondence should be addressed. E-mail: Medeiros@ksu.edu.

Copyright American Institute of Nutrition Nov 2004




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