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Department of Nutritional Sciences, College of Human Environmental Sciences, Oklahoma State University, Stillwater, OK 74078 and * Osteoporosis Research Center, Creighton University, Omaha, NE 68178
2To whom correspondence should be addressed. E-mail: Bsmith62{at}aol.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • tail suspension • copper • osteoporosis • triethylenetetramine tetrahydrochloride • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–5
). The osteopenic response to skeletal unloading may be exacerbated by other factors known to result in bone loss such as drug treatments (6
,7
), declining hormone levels (8
,9
) and poor nutritional status (10
–12
). Elderly populations who experience limited weight-bearing activity are of particular concern (13
,14
).
A number of macro- and microelements are involved in maintaining skeletal health. Among the trace elements, copper is essential in collagen-crosslink formation as a component of the metalloenzyme, lysyl oxidase (15
). Collagen crosslinks provide tensile strength to bone (16
), and copper deficiency in several animal species produces skeletal abnormalities (17
–19
). In humans, there is some evidence of a relationship between copper deficiency and senile osteoporosis. Conlan et al. (20
) found reduced serum copper in elderly patients with fractures of the femoral neck compared with age- and sex-matched controls. Furthermore, copper status was reported to be a very important predictor of bone health, even more so than calcium status, in individuals with osteoporotic changes related to immobilization (21
).
Although severe copper deficiency in the United States is not common, the median dietary copper intake is < 1 mg/d (22
). In vulnerable populations such as the elderly, copper has been identified as one of three nutrients within the diet that is particularly low (23
). The best food sources of copper are legumes, nuts and seeds, but these foods are traditionally low in the diets of elderly (23
). The new Dietary Reference Intakes set the copper Recommended Dietary Allowance at <1 mg for adults, but there are few data available on copper requirements for optimal bone metabolism (24
). Over time, low copper intake may lead to marginal copper status and accelerate the deterioration of bone quality.
Given the roles of copper in bone and the potential for inadequate dietary copper intake, the two-fold purpose of this study was: 1) to investigate the effects of copper inadequacy on bone quality (i.e., bone mineral density and biomechanical strength) in mature animals, and 2) to examine whether copper depletion exacerbates the deterioration of bone in mature animals under conditions of skeletal unloading. The skeletally mature, tail-suspended (TS)3
rat (25
,26
) provides a valuable model for studying alterations in bone metabolism associated with skeletal unloading. This model prevents weight-bearing activity by the hind limbs, but allows normal loading of the forelimbs without restricting movement. Although most studies using this model have been with young growing rats, further examination of the skeletal response to unloading in mature animals is warranted.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Thirty-six 5.5-mo-old male Sprague-Dawley rats (Charles River Laboratories, Kingston, NY) were housed individually in an environmentally controlled animal laboratory, allowed to acclimate in suspension cages with free access to deionized water and fed commercial laboratory diets until the initiation of dietary treatments. In a 2 x 2 experimental design, rats were assigned to one of two diets based on the American Institute of Nutrition rodent diet (AIN-93M) with either adequate copper (Control = 5.7 mg Cu/kg diet) or no copper added (-Cu = 1.1 mg Cu/kg diet) (27
). Rats were fed their respective diets beginning 21 d before suspension and throughout the study (i.e., 49 d total dietary treatment). During the 21-d preliminary feeding period, rats fed the control diet were pair-fed to the average intake of the -Cu group. Also -Cu rats were given the copper chelator, triethylenetetramine tetrahydrochloride (TETA, at 0.83% of diet), for 5 d (i.e., d 8–12) to further promote copper depletion (28
,29
). This TETA regimen has been used previously by Cohen et al. (29
) to effectively reduce plasma copper concentration, ceruloplasmin activity and tissue copper in rats. Post-TETA tail blood was collected and ceruloplasmin assayed to verify copper depletion.
At the end of the initial feeding period, half of the rats in each diet treatment were assigned to either suspension (TS) or ambulatory (AMB) groups. Tail suspension was accomplished via the method of Wronski and Morey-Holton (30
), using orthopedic tape, a suspension mechanism and a metabolic cage especially designed for suspension studies. During the 28-d suspension period, AMB rats served as controls and were pair-fed to the average intake of the TS group consuming their respective diets. Body weights were determined weekly before suspension and every 3rd d throughout the suspension period. After 28 d of tail suspension, rats were food-deprived for 12 h, anesthetized with an intraperitoneal injection of ketamine/xylazine (100 and 5 mg/kg body weight, respectively) and exsanguinated via cardiac puncture. This study was approved by the Oklahoma State University Animal Care and Use Committee.
Sample collection.
At d 14 and 28 of suspension, 12-h urine was collected and stored at -20°C. At the time of necropsy, serum was collected via the descending aorta, separated by centrifugation at 1500 x g for 20 min at 4°C, and aliquots frozen at -20°C. The liver, femurs, humeri and the 3rd-5th lumbar vertebrae (L3-L5) were also removed, cleaned and stored at -20°C. The right femur and L4 were stored in PBS for mechanical testing and the left femur, humerus and L5 were stored for densitometry measurements. L5 and liver samples were also available for mineral analysis.
Densitometric measurements.
The excised left femur, humerus and L5 were thawed at room temperature, placed in a dish containing deionized water (2.5 cm in depth), and scanned using dual-energy X-ray densitometry (DEXA) small animal high-resolution scan module (Hologic QDR-2000, Bedford,MA) (31
). Bone mineral area (BMA), bone mineral content (BMC) and bone mineral density (BMD) were recorded. The CV for bone densitometry was 0.87%.
Mechanical strength testing.
Mechanical properties of the femur were examined by torsion testing. Before testing, femur geometry was measured using a vernier caliper (accuracy, 0.1 mm). The length of the femur was assessed from the greater trochanter to the distal condyles. Specimens were prepared for mechanical testing by potting both the proximal and distal ends of the femur in a low melting point metal, while being held with precision fixtures. The fixtures were then aligned in the torsion testing system and rotated at a speed of 1°/s. Femoral rotation was in the internal direction with torque and angular displacement data recorded until the point of fracture. Each bone was then visually inspected to ensure the characteristic spiral pattern of fracture. Maximum torque (N · m) and maximum angular displacement (°) were determined.
Compression tests of vertebral bodies were performed on an Instron 5543 material test system (Canton, MA) in stroke control at a rate of 3 mm/min. All specimens were tested at room temperature and were kept moist with saline solution during testing. Each vertebral body (L4) was prepared with parallel flat ends and placed on a fixed lower pressure plate while an upper plate was allowed to move downwards causing compression in the specimen along the anterior-posterior direction. Slight lack of parallelism between the two ends of the vertebral body was corrected by the pivot action of the lower plate. Load displacement plots were analyzed to determine material properties. After the test, vertebral bodies were cut at the fracture site and the cross sections were traced at 20X magnification on a profile projector (Nikon, V-10). Two traced sections from each vertebral body were digitized using the SECTION program (Biomechanics Laboratory, Creighton University) on a VAXstation 2000 (Digital Equipment, Maynard, MA) to measure the average cross-sectional area. The average cross section for each vertebral body was used to calculate its ultimate stresses and modulus of elasticity (32
).
Mineral analysis.
Portions of the liver, 1-g samples of diet and L5 were weighed into acid-washed borosilicate glass tubes, dried for 24 h at 100°C and dry weight recorded. Wet and dry ashing were performed to remove all organic material using a modification of the method of Hill and colleagues (33
). L5 was then analyzed for copper, iron, zinc, magnesium and calcium content. Copper, iron and zinc concentrations of the liver were determined, as well as dietary copper. All samples were analyzed using a Perkin-Elmer Zeeman 5100PC atomic absorption spectrophotometer with either flame or graphite furnace (Perkin Elmer, Norwalk, CT).
Biochemical analyses.
Plasma ceruloplasmin was measured immediately after feeding TETA (0.83% of diet) for 5 d to assess the effects of the copper depletion diet and TETA (34
). Ceruloplasmin was expressed as mg ceruloplasmin per dL of plasma. The interassay CV was 1.25%.
Serum alkaline phosphatase (ALP) and bone extracted ALP (Roche Reagents, Nutley, NJ) activities were assessed as indicators of bone formation. Extracted ALP was analyzed on L3 and on the left humerus (i.e., normal loaded bone) to identify alterations in bone formation occurring in either weight-bearing or nonweight-bearing bones (35
). Bone-specific ALP was expressed per mg of total bone protein. The intra-assay and interassay CV for both serum and bone-specific ALP was 1.9 and 2.8%, respectively.
Urinary deoxypyridinoline (DPD) crosslinks at 14 and 28 d of suspension were assayed using an enzyme-linked immunoassay, Pyrilinks-D (Metra Biosystems, Mountain View, CA). DPD crosslink excretion reflects degradation of type-I collagen and is thus an indicator of bone resorption (36
). The DPD data are presented as total nmol of DPD excreted per 12 h. The intra-assay variation for DPD was 4.3%, and interassay variation between d 14 and 28 was avoided by analyzing both samples for each individual animal in the same run.
Statistical analysis.
The data were analyzed as a 2 x 2 factorial design using the Statistical Analysis System (SAS) version 6.11 (Cary, NC). ANOVA and least-square means were calculated using the general linear model (GLM) procedure and P < 0.05 was accepted as significant. Data are reported as mean ± SEM unless otherwise indicated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Despite pair-feeding, tail suspension by d 18 reduced (P < 0.05) the body weight of the TS rats compared with the AMB controls (Fig. 1
). During the remainder of the treatment period, AMB rats regained body weight but TS rats did not.
|
|
). DEXA calculates BMD as BMC/BMA. The decrease in the femur BMD associated with TS was due to reduced BMC (P < 0.05), but in L5, both BMA and BMC were decreased (Table 2
). Copper deficiency significantly reduced L5 BMD (P < 0.01) (Fig. 2)
and tended (P = 0.086) to diminish BMD of the femur. However, neither BMC nor BMA of L5 and the femur were significantly altered by diet (Table 2)
. No change in BMA, BMC or BMD of the humerus resulted from either TS or -Cu (Table 2)
.
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|
). Tail suspension, but not -Cu resulted in a significant reduction (P < 0.05) in maximum torsional strength of the femur. Similarly, the ability of L4 to withstand a compression force was reduced by TS, but not by copper deficiency. Neither TS nor -Cu significantly altered the angle of deformation or the modulus of elasticity in the femur or L4.
|
). However, crosslink excretion was not significantly affected by copper depletion.
|
. TS significantly lowered (P < 0.05) the bone calcium and magnesium contents Table 3
). The -Cu diet lowered L5 copper content (P < 0.01) as expected, as well as decreasing the iron content (P < 0.05) and increasing the zinc content (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We observed that skeletal unloading decreased the BMD of the unloaded vertebra and femur without altering the BMD of the humerus. Dietary copper depletion also had a negative effect on the BMD of unloaded bones, while at the same time having no effect on the BMD of the normally loaded humerus. The more pronounced changes in vertebral bone in response to both unloading and copper depletion may be attributed to the higher proportion of trabecular bone in the vertebrae than the femur. The densitometry measurements in the present study are in agreement with results by Vico and colleagues (37
), who found diminished BMD of the unloaded hindlimbs in 6-mo-old tail-suspended rats. Previous studies have not examined the effects of copper depletion on vertebral BMD, although Saltman et al. (38
) found that copper supplementation in conjunction with calcium, zinc and manganese had a positive effect on the vertebral BMD of postmenopausal women. The limited number of older individuals performing weight-bearing activities combined with the poor copper intake reported among elderly populations (23
,39
) make the reduced BMD associated with copper depletion in unloaded bone a potentially major public health concern.
Mechanical strength of the femur and vertebra was also decreased by TS. Similar reductions in bone strength have been reported previously in young growing rats exposed to skeletal unloading via TS (40
), but the change in vertebral mechanical properties due to TS in mature animals has not been reported. Although vertebral and femoral BMD were reduced by copper depletion, mechanical strength was unaffected by diet. Diminished bone strength due to copper deficiency in young growing animals has been reported (19
,41
), but no studies have examined the role of copper in the mature skeleton. Researchers have attributed the diminished bone strength of young growing animals to a decrease in the number of collagen crosslinks (16
,19
,42
), but BMD has not typically been reported in conjunction with these findings. In the present study, skeletal unloading and/or copper depletion did not reduce the elastic properties of the femur or vertebra. The copper content of L5 confirmed that the combination of the -Cu diet over a 7-wk period and 5 d of TETA reduced the copper content of bone by 15%. We had anticipated that copper depletion would exacerbate the loss of mechanical strength and/or elasticity as well as diminish bone density in tail-suspended animals; however, in this study of mature rats, this was not the case. Only BMD was affected by copper depletion. The 49-d copper depletion period in this study, although long enough to produce mechanical alterations in the bones of young growing animals, may not have been a sufficient period of time in which to produce substantial collagen turnover in mature rats. The study was limited to 28 d due to the duration of tail suspension used with older animals. Also worth noting, the 45% reduction in liver copper indicated that the deficiency was not as low as in some studies of young animals in which a 65–85% decrease in liver copper was produced (19
,43
).
Biochemical markers of bone metabolism (i.e., DPD and ALP) offer further insight into the metabolic changes in bone associated with skeletal unloading and copper depletion. Although tail suspension produced a dramatic increase in markers of bone resorption, no change in bone formation was noted. Decreased bone formation in this model (i.e., 6-mo-old tail-suspended rat) may not be responsible for the bone loss, but it is possible that the alterations occurred early and normalized by the end of the 28-d suspension period when bone and serum ALP were assessed. Such trends were observed in bone resorption because skeletal unloading had a greater effect on bone resorption as indicated by DPD during the early stage of tail suspension (i.e., d 14) than at the end (i.e., d 28) (Fig. 4)
. The increase in bone resorption offers a plausible explanation for the decreased mechanical strength and BMD of the femur and vertebra due to unloading. Even though Smith et al. (44
) observed a similar increase in bone resorption in humans during bed rest and space flight, histomorphometry data from 6-mo-old TS rats have suggested that bone formation was also decreased (45
). It is apparent that the effects of skeletal unloading on the metabolism of mature bone have not been completely resolved. These discrepancies in the biochemical and histomorphometry data require further investigation to explain the alterations in osteoblast and osteoclast activity in mature animals and humans experiencing little or no weight-bearing activity.
In our study, copper depletion did not alter biochemical markers of bone formation or bone resorption. Although most effects of copper depletion on bone metabolism have been observed in young growing animals (46
), we are not aware of any reports in the literature of decreased bone formation associated with copper depletion in the mature skeleton. Recently, Baker et al. (24
) reported that when healthy adult men (age 20–59 y) switched from an 8-wk dietary copper intake of 1.6 mg/d to a copper intake of 0.7 mg/d, no change in markers of bone formation (i.e., osteocalcin) occurred, but a 25% increase in bone resorption was observed. Furthermore, in vitro studies (47
,48
) have supported a role of increased osteoclastic activity as a possible explanation for bone loss associated with copper depletion. Although the underlying mechanisms of the osteoporotic-like changes in bone with copper depletion in animals and humans are not understood, the implications of marginal copper intake on bone metabolism of the mature and aging skeleton warrant further investigation.
Our findings of reduced calcium content in the vertebra in response to skeletal unloading are in agreement with previous findings examining the tibia of young growing (49
,50
) and mature TS rats (37
). The reduced iron content and elevated zinc content of the vertebra in response to the copper depletion diet may be explained by a copper and/or iron interaction with zinc (51
). These same alterations in the iron and zinc concentrations in the liver were not observed in response to copper depletion. Further investigation into the cause of elevated zinc in the vertebral bone is warranted. Feeding TETA made copper depletion of the adult rats in this study possible in a relatively short period of time and enabled us to examine the simultaneous effects of copper deficiency and skeletal unloading. Nonetheless, TETA also made it difficult to discern whether observed changes in mineral status were a direct result of copper depletion, of chelation by TETA or of a combination of these two factors.
Most early studies using tail suspension as a model for bone loss did not report weight loss (26
,52
,53
). In mature animals, however, the body weight of TS animals was significantly less than that of AMB controls (37
,45
) despite pair-feeding. In our study, weight loss occurred during the first 9 d of suspension, but these changes did not become significant until AMB rats started regaining weight. Suspended rats failed to regain lost weight during the 28-d suspension period although their average food intake approached amounts consumed before suspension. Reduced muscle and bone mass due to the atrophic effect of tail suspension may be partly responsible for the decline in body weight (54
). Due to the 
30° angle at which the rat’s body is suspended, the cephalic fluid shift is another possible factor contributing to weight loss. Fluid that tends to pool in the head and upper torso distends the surrounding vasculature (55
), and during space flight, this distention causes a physiologic response similar to fluid-volume overload. The result is a decrease in total body water (56
). Such a decrease in total body water, in addition to atrophy of the bone and muscle, could explain the weight loss during tail suspension.
The potential roles of stress on body weight, bone loss and muscle atrophy in the adult TS rat model remains an issue. Halloran and colleagues (49
) found that decreased bone formation in young TS rats was not a consequence of increased plasma glucocorticoids or sensitivity to glucocorticoids. Dehority and colleagues (45
) recently reported that TS did not alter the thymus weight of adult rats, providing further support that glucocorticoid excess is not occurring (57
). Additionally, the humerus provides an internal comparison for distinguishing between systemic and local responses to skeletal unloading of the hind limbs. Alterations in bone parameters occurring in the femur or other unloaded bone and not in the humerus would seem to be a result of locally mediated responses.
We conclude that copper depletion has a more detrimental effect on the unloaded femur and vertebra compared with the normally loaded humerus. However, the effects of copper depletion on bone metabolism and bone mechanical properties in the mature skeleton warrant further investigation. Twenty-eight days of tail suspension was an adequate period of time in which to produce osteopenia in the unloaded bones of 5.5-mo-old rats. Biochemical markers indicated that the bone loss observed with unloading in this study resulted from increased bone resorption. The extent to which bone formation is altered in the mature animal remains to be elucidated. These data represent the mature skeletal response to unloading and seem to better model the alterations in bone quality experienced by elderly populations compared with data from young growing animals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: ALP, alkaline phosphatase; AMB, ambulatory; BMA, bone mineral area; BMC, bone mineral content; BMD, bone mineral density; DEXA, dual-energy X-ray densitometry; DPD, deoxypyridinoline; TETA, triethylenetetramine tetrahydrochloride; TS, tail suspension.
Manuscript received 16 July 2001. Initial review completed 27 July 2001. Revision accepted 19 October 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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