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Center for Enhancing Foods to Protect Health, Lipid Chemistry and Molecular Biology Laboratory, Purdue University, West Lafayette, IN 47907 and * Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Division of Intramural Clinical and Biological Research, National Institutes of Health, Rockville, MD 20852
2To whom correspondence should be addressed. E-mail: baw{at}purdue.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-linolenic acid [LNA, 18:3(n-3), 2.6% of total fatty acids] and docosahexaenoic acid [DHA, 22:6(n-3), 1.3% of total fatty acids]. Fatty acid composition of the hindlimb tissues (bone and muscle) of chronically (n-3)–deficient rats revealed a marked increase in (n-6) PUFA [20:4(n-6), 22:4(n-6), and 22:5(n-6)] and a corresponding decrease in (n-3) PUFA [18:3(n-3), 20:5(n-3), 22:5(n-3) and 22:6(n-3)]. Measurement of bone mechanical properties (energy to peak load) of tibiae showed that (n-3) deficiency diminished structural integrity. Rats repleted with (n-3) fatty acids demonstrated accelerated bone modeling (cross-sectional geometry) and an improved second moment in tibiae compared with control (n-3)–adequate rats after 28 d of dietary treatment. This study showed that repletion with dietary (n-3) fatty acids restored the ratio of (n-6)/(n-3) PUFA in bone compartments and reversed compromised bone modeling in (n-3)–deficient rats.
KEY WORDS: • (n-3) fatty acids • rats • bone • mechanical properties • bone modeling
Western diets are characterized by a high content of total fat, saturated fat, trans fatty acids, and (n-6) PUFA (1,2). Conversely, there is a corresponding dietary deficit of (n-3) PUFA; intakes are low for -linolenic acid (LNA)3 and particularly for the long-chain eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The dietary ratio of (n-6)/(n-3) PUFA is therefore elevated above the ratio characteristic of traditional diets that supplied nourishment during the period of our genetic evolution (3–5). A disproportionately low dietary intake of (n-3) PUFA is generally reflected in tissue fatty acid profiles (6–8). As a consequence of low (n-3) intakes, cellular membranes become enriched with an excess of arachidonic acid (AA), which is a substrate for the biosynthesis of the eicosanoids [prostaglandin E2 (PGE2) and leukotriene B4 (LTB4)]. PGE2 is a robust modulator of biochemical activity in bone, and has recently been reported to modulate osteoblast functions (9). At moderate levels, PGE2 supports bone formation, but at high concentrations, it promotes bone resorption (8). In growing rats, a high dietary ratio of (n-6)/(n-3) PUFA was positively correlated with depressed bone formation rates and higher capacity for ex vivo PGE2 production in bone (8). Our laboratory confirmed that EPA not only lowered the production of PGE2, but also reduced the expression of induced cyclooxygenase-2 (COX-2), a key enzyme that catalyzes the biosynthesis of PGE2 from AA in osteoblastic bone cell cultures (10). Furthermore, (n-3) PUFA feeding maintained bone mineral content in the tibia of ovariectomized rats (10) and in the lumbar spine of ovariectomized mice (11). Generally, EPA can serve to attenuate AA conversion to prostanoids. Moreover, the relatively slow conversion rate of EPA to eicosanoids and the reduced bioactivity of the EPA-derived compounds provide an important means for modulating AA prostanoid-mediated physiologic and pathologic processes (12,13).
Bone is a highly specialized connective tissue of structural diversity, and its material hardness is a unique biological attribute conferred by a composite organic extracellular collagenous matrix that is strengthened by an impregnation of mineral deposits. Bone functions as both a structural and metabolic organ, and its competency in both capacities is significantly affected by the types of fatty acids present in the cellular membranes of constituent tissues (9,14,15). The microenvironment of bone is also influenced by a diverse compilation of growth, regulatory, and mediating factors that determine local tissue and cellular metabolic activities (9,16). For optimal bone health, mechanical strain and subsequent stimulation of osteoblastic activity in bone must be appropriate to the physiologic requirements associated with biological age and physical demands (16).
The collective behavior of skeletal elements is organized to facilitate bone modeling in children during growth and development (9). Until skeletal maturity is attained, in excess of 90% of the periosteal and endosteal bone surfaces are continuously involved in bone appositional and resorptional activities that result in morphological changes pertinent to growth and reshaping (16,17). During bone modeling (e.g., long bone appositional growth and geometrical restructuring) surface drifts occur that alter bone cortical thickness, marrow cavity diameter, external diaphyseal and metaphyseal diameters, longitudinal curvatures, total cortical mass, and cross-sectional geometries (17). These changes in the geometrical structure of bone tissue govern the quality of bone, which is determined not only by its material properties, but also by architectural, physical, and biological factors that influence its mechanical properties (16,18). Hence, bone modeling is a physiologic activity integral to skeletal competency, whereas remodeling processes throughout adult life determine bone quality that governs fracture risk commensurate with skeletal disease (16). Investigators have demonstrated that dietary factors including lipids can affect bone morphology and mechanical properties (19). Evaluation of bone mechanical properties, both structural and material, can be instrumental in elucidating the quality of bone architecture during bone modeling.
Evidence that significant correlations between elevated ratios of AA/EPA and ex vivo PGE2 production in bone accompany reduced bone formation rates in rats is highly suggestive that (n-3) PUFA play a regulatory role in bone modeling (8). Moreover, (n-3) PUFA levels in bone and osteoblastic cell cultures have been shown to coincide with the elevated levels of bone formation markers that are indicative of enhanced bone formation activity (9,10). In the current study (n-3)–deficient rats were used as a model to evaluate how effectively elevated ratios of (n-6)/(n-3) PUFA in musculoskeletal tissues could be reversed after an adequate dietary intake of (n-3) PUFA. The effects of (n-3) PUFA repletion were investigated in rapidly growing rats undergoing extensive bone modeling, and testing of mechanical properties was performed to evaluate whether initial diet-induced structural disturbances in bone modeling could be reversed with (n-3) PUFA repletion. Understanding the role of (n-3) PUFA in skeletal health is further justified by the new dietary reference intakes (DRI) for these fatty acids published by the Institute of Medicine of the National Academies (20).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Sample collection. At intervals of 0, 1, 2, 4, and 8 wk of feeding, F2 rats from each of the (n-3) Adq and (n-3) Def-Adq groups (n = 4 per dietary treatment) were killed by decapitation. At the end of 8 wk, 4 rats from the (n-3) Def group were killed. A bilateral hindlimb dissection was performed to collect hindlimb bones by disarticulating the hip joint. The tibialis caudalis muscle on each left hindlimb was removed with a scalpel, weighed free of its ensheathing fascia and rapidly submerged in methanol and held at 4°C while the remaining tissues of the limb were harvested. The excised femurs and tibiae of the left limbs were disarticulated and dissected free of all residual soft tissue. The mid-diaphyseal shaft of each femur and tibia was cut from the proximal and distal spongiosa using bone secateurs. Marrow was dislodged from the cortex of each mid-diaphyseal bone shaft using a pipette to flush methanol through, and the marrow was collected in separate sample tubes. Femoral and tibial cortical bones were weighed before being cooled by immersion in liquid nitrogen, crushed, and sonicated (Bransonic 5200) to ensure optimal particle dissociation during the lipid extraction phase (8).
Fatty acid analysis. Cortical bone and marrow of the femur and tibia as well as tibialis caudalis muscle were subjected to repeated extraction and homogenization (PT 3100 Polytron) to recover lipids. The FAME were prepared and analyzed by GC as previously described (8).
Mechanical testing
Specimen preparation and analytical procedures for mechanical properties testing. The femur and tibia of the right hindlimbs were dissected and cleaned of all soft tissue. Longitudinal length and external mid-diaphyseal diameters of the femur and tibia specimens were then measured using a digitized micrometer (Absolute Digimatic, accuracy of ± 0.01 mm). Each bone was then individually wrapped in paper towel moistened with a 9 g/L saline solution and placed in a plastic bag that was sealed and stored at -80°C before testing. It was shown that such freezing has no effect on rat bone mechanical properties (22,23). On the day of testing, the whole bones were thawed to ambient temperature and kept moist.
All mechanical properties were determined using a 3-point bending test (Model 3 Q-Test, MTS Systems Corporation running Testworks QT software), and loading continued to failure (24). A 45-kg load cell provided the appropriate force, and the crosshead-loading probe was calibrated to a constant speed of 2.00 mm/min and a deflection limit of 8 mm (25). The supports on which all specimens were placed were 15 mm apart (26). Shallow curved indentations were filed into each side to prevent sample movement during testing. A small preload was applied to each specimen before all mechanical tests to prevent rotation during loading.
Bone specimens were placed to facilitate central loading. Femur bones were placed horizontally on the two supports with the anterior aspect of the bone facing down. Tibiae were positioned so that they were loaded in a medial to lateral direction about the anterior-posterior axis. After the breaking of the bones under force, the horizontal and vertical internal mid-diaphyseal diameters of the marrow cavities were measured using the digitized micrometer.
Structural and material mechanical properties. A force-deformation curve was used to graphically determine structural properties including energy to peak load, load at failure, peak load, yield load, bending moment, and ultimate stress (24). Bone property measurements were calculated using equations applicable to an elastic rod with an elliptical annulus (27).
In interpreting the mechanical property testing results, the percentage change in second moment of area (Ix), a structural parameter that takes into account the diaphyseal cross-sectional area and its distribution around the bone shaft’s central axis, was included to illustrate the bone’s increasing capacity to resist the type of bending forces encountered during locomotion. The more distant the distribution of the material mass from the central axis is, the greater the bending stiffness and structural strength of the bone will be. Other mechanical properties of physiologic importance considered in this study included the following: 1) peak load, which is a measure of bone strength or the maximum force in newtons (N) supported by the bone in response to an externally applied load; 2) energy to peak load (N · mm) which corresponds to the area under the load deformation curve and denotes the capacity of the bone to absorb and store energy to preclude the onset of catastrophic bone failure; 3) load at failure or force (N) tolerated at the point of catastrophic failure; 4) ultimate stress, the force per unit area (N/mm2) that develops within the bone structure in response to load; and 5) bending moment, or the quantitative measure of the tendency of a force to bend a structure (N · mm).
Statistical analyses. Data were analyzed by a t test to determine differences in fatty acid compositions, bone morphologies, and mechanical properties within each type of tissue between the two dietary groups of rats fed either the (n-3) Adq or the (n-3) Def-Adq (repleted) diets at each time point. A 1-way ANOVA was implemented and Student-Newman-Keuls Test performed to determine the differences among the 3 groups after 8 wk of dietary treatments [(n-3) Adq, (n-3) Def-Adq, and (n-3) Def]. Differences were considered significant at P < 0.05. The results are expressed as the means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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After only 1 wk of repletion feeding, the concentrations of 18:3(n-3) and 22:6(n-3) in femoral cortical bone and marrow, and tibial cortical bone had increased appreciably and approached the concentrations in the (n-3) Adq rats.4 After 8 wk of dietary treatment, the (n-3) PUFA concentrations in femoral and tibial bone compartments of the (n-3) Def-Adq (repleted) rats were restored to a level at or above those of the (n-3) Adq rats. Femoral marrow exhibited a 5.5-fold increase in total (n-3) PUFA over the repletion period in (n-3) Def-Adq rats. However, although the femoral tissues appeared more resistant to (n-3) depletion at wk 0, the femoral cortical bone was not as responsive to (n-3) repletion throughout the experimental period. This was in contrast to tibial cortical bone, which demonstrated a 14.5-fold increase in (n-3) PUFA, and the tibial marrow, which acquired 2.12 g/100 g (n-3) PUFA over 8 wk, when initial (n-3) PUFA levels at wk 0 were undetectable. Compared with cortical bone, at wk 8, the (n-3) Def rats had relatively lower concentrations of 18:3(n-3), 20:5(n-3), and 22:6(n-3) in femoral and tibial marrow than the repleted and (n-3) Adq control rats.
Our experiment also revealed that 22:6(n-3) may be important in muscle tissue because it was preferentially retained compared with other (n-3) PUFA during (n-3) deficiency. Upon dietary repletion, tibialis caudalis muscle tissue (n-3) PUFA concentrations increased 22-fold from 0.66 g/100 g at 0 wk to 15.4 g/100 g fatty acids by 8 wk. In addition, a 48% reduction in the total (n-6) PUFA level was evident in the muscle of (n-3) repleted rats after 8 wk. As expected, rats fed the (n-3) Def diet for the duration of the 8-wk experiment had significantly lower levels of 22:6(n-3) compared with the (n-3) Adq and (n-3) repleted rats. These findings indicate that repletion restored the amounts of (n-3) fatty acids to more appropriate physiologic levels; however, the results do not imply that tissue concentrations of (n-3) PUFA had reached an upper level plateau.
The (n-3) fatty acid repletion diet was formulated to reduce the ratio of (n-6)/(n-3) PUFA fed to rats from 378:1 to 4:1 (21). To better reflect tissue concentrations of the desaturation/elongation products of the 18-carbon essential fatty acids, 18:2(n-6) and 18:3(n-3), specific long-chain PUFA species of (n-6), denoted by S(n-6), and those of (n-3) PUFA denoted by S(n-3), were combined in a novel way. A ratio of S(n-6)/S(n-3) was thereby derived as a specific calculation using the FAME data. The S(n-6) numerator comprises the sum of [20:4(n-6) + 22:4(n-6) + 22:5(n-6)] whereas the S(n-3) denominator is the sum of [20:5(n-3) + 22:5(n-3) + 22:6(n-3)].
During (n-3) repletion feeding, the ratio of S(n-6)/S(n-3) PUFA in the femoral tissues reached a value close to that of the (n-3) Adq rats by wk 4 (Fig. 1). Significant reductions in the amounts of 20:4(n-6), 22:4(n-6), and 22:5(n-6), and increases in 20:5(n-3), 22:5(n-3), and 22:6(n-3) concentrations contributed to this change in femoral cortical bone and marrow. Similar to femoral tissues, both the tibial cortical bone and marrow of (n-3) repleted rats were responsive to (n-3) PUFA enrichment, and both tissues reached the ratio of S(n-6)/S(n-3) level in (n-3) Adq animals by 4 wk (Fig. 2). Muscle ratios of S(n-6)/S(n-3) PUFA were maintained in a narrow range for the (n-3) Def and (n-3) Adq rats (Fig. 3) compared with bone tissues, and remarkably, the ratios were one tenth that of the bone compartments examined. However, consistent with bone tissues, the ratio of specific PUFA in (n-3) Def rats decreased to a value close to that of the adequate control rats by 4 wk (Fig. 3). In some instances (n-3) PUFA concentrations below the detectable limit for (n-3) Def rats at 0 and 8 wk prevented calculation of the ratio of S(n-6)/S(n-3) PUFA in the femoral and tibial tissues and also resulted in a large SD for the same calculations.
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Longitudinal bone growth. The mean length of the femur bone did not differ significantly between treatment and control groups of rats (range, 39–40 mm after 8 wk of dietary treatment). Most notable in the case of the tibia was the difference in bone length between 0 wk rats descendant from (n-3) Adq dams and fed the (n-3) Adq diet at weaning; these rats had an average tibia length of 39.75 mm, which was significantly longer (P = 0.03) than the 38.00-mm tibial length for rats descendant from (n-3) Def dams and fed the (n-3) Def diet at weaning. Further, the ratio of S(n-6)/S(n-3) PUFA decreased drastically within 1 wk in the (n-3) repleted rats after implementing the (n-3) Adq diet, and this change in the ratios of PUFA corresponded to an absence of significant differences in bone length.
Mid-diaphyseal morphology. The cortical and medullary diameters of long bones directly affect both cortical thickness and the second moment of area. The medullary (a1 and a2) and cortical (b1 and b2) diameters of femur and tibia mid-diaphyseal shafts increased progressively during the study for (n-3) Adq and (n-3) Def-Adq rats. Cortical bone diameters for femurs and tibiae (b1 in load direction and b2 perpendicular to load direction) revealed that (n-3) repleted rats, (diet Def-Adq), underwent a greater percentage change in bone modeling from 0 to 4 wk compared with the (n-3) Adq control rats (Fig. 4). Similarly, the percentage change for medullary expansion for the same period (data not shown) was higher in (n-3) repleted rats. The direct measurement for cortical bone thickness, taken at the site at which bone fracture and breakage occurred during mechanical testing, reflected a significant change in the femur and tibia size after 4 wk of feeding (n-3) PUFA. Average femur cortical thickness of (n-3) Adq rats at 0 wk was 0.55 ± 0.03 mm, which was greater (P = 0.013) than that for the (n-3) Def rats (0.44 ± 0.05 mm). At 8 wk, the (n-3) Adq control rats had larger diameters for tibiae (4.05 ± 0.03 mm) than (n-3) Def rats (3.67 ± 0.04 mm) (P = 0.009).
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Bone mechanical characteristics. There were no differences in the femur bone mechanical parameters between the (n-3) Adq controls and the (n-3) repleted and (n-3) Def rats during the study. In contrast, significant differences in mechanical properties were demonstrated in tibiae in which the ratio of (n-6)/(n-3) PUFA was extremely elevated in (n-3) Def rats compared with (n-3) Adq control rats (Fig. 5). Noteworthy results pertaining to rat tibiae include the lower (P = 0.03) peak load for (n-3) Def rats compared with the (n-3) Adq controls, and the loads at failure and bending moment were also significantly lower in the (n-3) Def rats (Fig. 5, Panel A). The energy to peak load for tibiae at 8 wk was also diminished (P = 0.005) in the absence of (n-3) fatty acids for (n-3) Def rats. This is an important finding and demonstrates that significantly less impact energy is absorbed by (n-3) Def and (n-3) Def-Adq rat tibiae compared with (n-3) Adq rats. Although (n-3) repletion diets did improve the energy to peak load in (n-3) Def-Adq rats compared with (n-3) Def rats, the effects of an early (n-3) deficiency were not fully compensated for after 8 wk of repletion feeding (Fig. 5, Panel B). The ultimate stress the tibiae was able to sustain before bone failure at 8 wk (Fig. 5, Panel B) was appreciably higher (P = 0.03) for (n-3) Def-Adq rats than for (n-3) Adq controls. The capacity of (n-3) repleted rats to surpass the ultimate stress parameter of (n-3) Adq controls suggests a compensatory improvement in tibial bone architecture consistent with greater bone modeling activity, which is also supported by the more rapid rate of change in second moment of area for (n-3) repleted rats (Fig. 4, Panel B). The higher values for bone stiffness/rigidity in the (n-3) Def rats and the consistently lower mechanical failure values imply that architecture is compromised in modeling bone of the rat tibiae during (n-3) PUFA deficiency states when the ratio of (n-6)/(n-3) PUFA in cortical bone is abnormally elevated.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Optimizing the dietary ratio of (n-6)/(n-3) PUFA is considered beneficial to animals and humans (29). According to Claassen et al. (30), in adequately nourished tissues, the ratio of (n-6)/(n-3) PUFA varies from 2 to 6, which corresponded to the ratio of these PUFA in the (n-3)–adequate control rats. The lowest ratio observed in (n-3) Adq rats was 2 in muscle tissue, and the highest was 7 in femoral marrow. After 8 wk of (n-3) PUFA repletion, the lowest ratio of (n-6)/(n-3) PUFA was 1 in muscle and the highest was 7 in tibial marrow. These data indicate that dietary lipids can manipulate the fatty acid composition of bone tissue compartments. Results in this experiment in which rat tibia was more sensitive to dietary manipulation than the more proximally located femur are in agreement with the findings that the adaptation of bone to dietary changes is bone specific (22).
A decrease in the ratio of S(n-6)/S(n-3) PUFA during (n-3) repletion occurred in rats fed the (n-3) Def-Adq diet. The reduction in ratios of S(n-6)/S(n-3) PUFA was achieved by inclusion of LNA and DHA in the diet. EPA was not included in the dietary treatments. Ratios of S(n-6)/S(n-3) PUFA in musculoskeletal tissue compartments decreased after elevated levels of 20:5(n-3) and 22:6(n-3), which occurred during (n-3) repletion. All tissue compartments exhibited a rapid increase in 22:6(n-3) levels, whereas the rise in 20:5(n-3) levels was marginal at best for the entire experimental period in repleted rats. Other studies showed that a dietary supply of 18:3(n-3) is effective at restoring tissue levels of 22:6(n-3) in (n-3)–deficient animals (29).
During (n-3) repletion, the accumulation of 22:6(n-3) contributed most to the elevation of total (n-3) PUFA in all tissues. Despite the inclusion of 18:3(n-3) in the (n-3) Adq diet at 0.26 g/100 g diet, this fatty acid did not appear to contribute to total (n-3) PUFA to the same extent as 22:6(n-3) when supplied at 0.13 g/100 g diet. The data indicate that 22:6(n-3) is incorporated into bone and marrow more efficiently than 18:3(n-3). Moreover, in studies on bone modeling in male rats, 22:6(n-3) accumulated in bone tissue compartments to a greater extent than 20:5(n-3) when both were administered in the diet (8,31).
The concentration of 20:4(n-6) decreased progressively in all musculoskeletal tissue compartments by 4 wk of (n-3) repletion, with the exception of cortical bone and muscle tissue, which retained the elevated levels of 20:4(n-6) for the first 2 wk; 20:4(n-6) is the precursor for two-series PGs in bone, including PGE2 and PGI2 (16). Studies spanning the last three decades provide evidence that PGE2 is the major prostanoid associated with bone formation and resorption (9,32). PGE2 actions on formation and resorption are concentration dependent and tissue specific in bone. PGE2 has also been found to affect osteoblast and osteoclast differentiation, making it a therapeutic target for improving and sustaining skeletal integrity (9,10). PGs are produced not only by bone cells, but also by cells adjacent to the bone marrow and periosteal tissues (9,16,32).
Cortical bone fatty acid profiles. Of significance in this experiment was the fact that the (n-3) PUFA, particularly 22:6(n-3), in the tibial compartments (cortical bone and marrow) were more severely depleted than that in the femur before commencement of repletion. This was reflected in the disparity between the initial ratio of total (n-6)/(n-3) PUFA of 61 in tibia and 18 in femur. Tibiae of (n-3)–deficient rats were almost devoid of 22:5(n-3) and 22:6(n-3), whereas more (n-3) PUFA was maintained in femur bones. Results from this experiment indicate that there is a preferential retention of (n-3) PUFA in femur cortical bone when an (n-3) deficiency prevails. Consequently, the tibia bone exhibited more significant differences in bone mechanical properties than femur bone.
Mechanical properties of bone. Bone modeling is a principle skeletal activity in young children and growing rats. Bone formation, appositional growth, and bone resorption activities are vigorous during the postweaning growth period. The tibia, more so than the femur in this experiment, was particularly affected by the diet-induced fatty acid alterations. This phenomenon was reflected in bone morphological and structural differences between (n-3)–deficient and control rats. The mid-diaphyseal region of the tibia and femur, in which cortical bone abounds, was the focus of analysis for the 3-point mechanical testing.
The midshaft cross-sectional dimension of long bones is a measurement used to determine the second moment of area. Compared with controls at 1 wk, the second moment of area tended to be reduced in both the femur and tibia of rats descended from (n-3)–deficient dams. After 4 wk of dietary treatment, the percentage change in second moment of area in tibia, compared with wk 1 measurements, was greater in repleted rats than in the controls, indicating a positive compensatory effect. Growth in femur bone was not affected by (n-3) deficiency; therefore, the relative abundance of the PGE2 precursor AA in femur bones must not have been sufficient to affect longitudinal or radial growth parameters in this compartment. The initially high ratio of S(n-6)/S(n-3) PUFA in the tibia of (n-3)–deficient rats coincided with reduced bone length in the early stages of the 8-wk experimental period.
The initial elevated ratio of (n-6)/(n-3) PUFA affected bone modeling predominantly at the structural level, and the (n-3)-deficient rats displayed differences that related to compromised bone structure. When ratios of (n-6)/(n-3) were at their highest in the tibia, values for peak load and bending moment were significantly decreased in (n-3)-deplete rats. The maximum deformation of tibia was also greater, indicating an additional degree of plasticity compared with control rats. The (n-3)–adequate control rats at 8 wk displayed a higher energy to peak load, indicating that more energy was absorbed by bones before breakage. Compared with the values at 8 wk, those for (n-3)–deficient rats and (n-3)–repleted rats were significantly lower.
Most differences in structural mechanical properties detected between the treatment and control groups of rats in this investigation were present exclusively in the tibia. In this (n-3) PUFA repletion study, the tibia was clearly more affected by the (n-3) PUFA deficiency than the femur, both in terms of the ratios of S(n-6)/S(n-3) PUFA in bone tissue compartments and the bone morphological changes associated with bone modeling that determine the inherent mechanical properties that contribute to bone strength. It is therefore suggested that highly elevated ratios of (n-6)/(n-3) PUFA contribute to altered bone metabolism. This can cause structural changes during the bone modeling process in rapidly growing rats, changes that ultimately compromise bone strength as demonstrated by bone mechanical property testing. Findings in this study necessitate further investigations for the role of (n-3) PUFA in bone morphology, mechanical properties, and architecture in growing and adult animals and humans.
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FOOTNOTES |
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3 Abbreviations used: AA, arachidonic acid; (n-3) Adq, group fed a diet adequate in (n-3) PUFA; COX, cyclooxygenase; (n-3) Def-Adq, group fed a diet deficient in (n-3) PUFA and then switched to (n-3) Adq diet; Def, group fed a diet deficient in (n-3) PUFA; DHA, docosahexaenoic acid; DRI, dietary reference intakes; EPA, eicosapentaenoic acid; LA, linoleic acid; LNA, -linolenic acid; LTB4, leukotriene B4; PGE2, prostaglandin E2; S, specific long-chain PUFA species.
4 The fatty acid compositions of rat femoral and tibial tissue compartments are included as supplemental tables in the online posting of this article at www.nutrition.org.
Manuscript received 21 June 2003. Initial review completed 11 July 2003. Revision accepted 10 November 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and rats fed the (n-3) Def diet throughout the experimental period (
). Only 0 and 8 wk data are available for the (n-3) Def rats. No (n-3) PUFA were detected at 8 wk for femoral marrow. Values are means ± SEM, n = 3 or 4. S = sum of specific long-chain (n-6) or (n-3) PUFA. S(n-6) = 20:4(n-6) + 22:4(n-6) + 22:5(n-6) and S(n-3) = 20:5(n-3) + 22:5(n-3) + 22:6(n-3).
