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,3
Departments of
* Human Nutritional Sciences and
Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, R3T 2N2 Canada
3To whom correspondence should be addressed. E-mail: hweiler{at}cc.umanitoba.ca.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.38, P 
0.05) with whole body and femur BMC and BMD and lumbar spine BMC. Liver AA:EPA ratio correlated negatively (r –0.039, P 0.05) with whole body, femur, and lumbar spine BMC plus whole body and femur BMD. Dietary 1.0:0.2 g AA:DHA/100 g reduced NTx relative to 2.0:0.4 g AA:DHA/100 g of fat (P = 0.039). The diets did not affect the other biochemical variables measured. Low levels of dietary AA:DHA (0.5:0.1 g/100 g of fat) elevate bone mass, but higher amounts are not beneficial.
KEY WORDS: • bone mass • long-chain polyunsaturated fatty acids • piglets • prostaglandin E2, bone modeling
At present, the majority of infant formula companies throughout the world add arachidonic acid (AA)4 and docosahexaenoic acid (DHA) to at least one of their product lines even though they are not considered essential fatty acids. The bases for this addition are as follows: 1) the potential inadequate synthesis of AA and DHA relative to requirements of the neonate (1–3) and 2) potential benefits to retinal and cognitive development (4–7). In previous years, our research group investigated the effects of these long-chain PUFA (LCPUFA) on bone mass and observed higher values with dietary supplementation of AA and DHA in a (n-6):(n-3) ratio of 5:1 to 7.5:1 (8–10).
A number of other research groups also reported that LCPUFA influence bone mass in various animal models (11–13). Part of the mechanism may involve modification of bone tissue LCPUFA concentrations, as demonstrated in chicks and rats (11,14), and the resulting changes in eicosanoids (14). Although various eicosanoids act on bone, the prostaglandins (PG) seem to be the primary mediators of bone cell function (15). Prostaglandin E2 (PGE2) is the principal PG affecting bone metabolism (15). PGE2 exhibits biphasic effects on bone, stimulating formation at low concentrations but inhibiting formation at high concentrations (16). The anabolic effect of PGE2 may occur through stimulation of insulin-growth factor-1 (IGF-1) production in bone or by increased responsiveness to IGF-1 in bone (17,18).
Another PG in bone, PGE3, is synthesized from eicosapentaenoic acid [EPA, 20:5(n-3)]. The amounts of the specific precursors and PG required for optimal bone growth and mineralization are currently not known. Very few studies have investigated the potential for AA and DHA to influence bone cell function and modeling during periods of rapid growth, and no reports exist for human infants.
Supplementation of AA + DHA (0.5:0.1 g/100 g of fat as AA:DHA) with a total (n-6):(n-3) ratio of 5:1 was associated with higher total body and regional bone mineral density (BMD) in neonatal piglets (8). Subsequently, supplementation of AA and DHA in the same proportions, but with a total ratio of 9:1, in neonatal piglets significantly elevated bone mineral content (BMC) in femur compared with control formula (10). Feeding 0.6 and 0.75 g/100 g of fat as AA with a constant amount of DHA (0.1 g/100 g of fat) elevated BMC in piglets compared with those fed lower amounts of AA (0.3 and 0.45 g/100 g of fat) (9).
In our model, the mechanism responsible for increased bone mass is not linked with PGE2. Only one of our studies showed an elevation in bone PGE2 concentrations relative to control due to supplementation of AA and DHA (0.5:0.1 g/100 g of fat); this occurred in piglets treated with glucocorticoids (10). Similarly, in piglets fed AA and DHA, plasma IGF-1 is not altered (9). In chicks, IGF-1 was elevated after they consumed a fish oil–based diet for 14 d; however, a soybean oil-based diet did not affect bone formation rate (11). In rats fed a fish oil–based diet, bone formation rate was negatively associated with PGE2 (12). In addition to changes in PGE2 or IGF-1 with dietary LCPUFA supplementation in rats, bone resorption was reduced in piglets by dietary LCPUFA (AA at 0.8:0.1 g/100 g of fat) (19). Thus, the mechanisms responsible for the LCPUFA-induced elevations in bone mass require further clarification.
Ratios of AA:DHA (0.5–0.75:0.1) and total (n-6):(n-3) 9:1 support normal growth and elevate bone mass during periods of rapid growth. These ratios are similar to those used by Watkins et al. (12,14) and Claassen et al. (20). For example, in growing rats, supplementation of (n-6):(n-3) fatty acids as 
-linolenic acid [GLA, 18:3(n-6)] and EPA + DHA in ratios of 3:1 also reduced bone resorption and elevated bone calcium content (20). However, the optimal amount of specific LCPUFA, including AA and DHA, is still unknown because the amounts used in our piglet studies were always <1 g/100 g of fat and were very low in contrast to studies in chicks and rats (12,14,20). Watkins et al. (11,12,14) supplemented fish oil at 35–50 g/kg diet in chicks and rats. Claassen et al. (20) altered the (n-6):(n-3) ratio by supplementing rats with varying amounts of GLA and EPA + DHA. The amount of GLA ranged from 5.2 to 7.4 g/100 g, EPA ranged from 1.8 to 9.2 g/100 g, and DHA ranged from 0.7 to 3.2 g/100 g. The ratios of AA:DHA that best support bone in neonatal piglets in studies conducted in our laboratory ranged from 5:1 to 8:1, but whether this applies when higher amounts are supplemented in the same ratio is unclear. A ratio of AA:DHA of 5:1 (8,10) and a total (n-6):(n-3) ratio of 9:1 (9,10) were chosen for this study because 2 previous studies also used this ratio and demonstrated higher bone mass over 15 d in piglets. The objectives of this study were to determine the amount of dietary AA + DHA that best influences bone mass and biomarkers of formation and resorption plus bone PGE2 and plasma IGF-1 concentrations.
This study was conducted in piglets to build upon previous work from our laboratory. In addition, the study was designed to test infant formula, and piglets normally suckle for as little as 20–25 d (21). Piglets respond to lipid nutrition in a manner similar to that of human infants (22), have nutrient requirements similar to those of human term infants (21), and have been used to validate the software used to measure infant bone mass (23).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1.4 kg were selected from 10 litters consisting of 10–12 piglets each with at least 4 males. Piglets arrived on d 3 of life and were taught to lap liquid formula. Piglets were fed by a combination of gavage feeding, plus lapping of formula to ensure that enough formula was consumed to continue growth for 2 d of adaptation. The strength of the control formula was progressively increased from half-strength on d 3 to full strength by the end of d 4 of life, followed by experimental or control formula on d 5. Piglets were housed individually in stainless steel cages; room temperature was maintained at 29–30°C. Based on 0900 h weight, the piglets were offered 350 mL/kg of liquid formula/d. This amount was divided into 3 equal portions provided at 0900, 1500, and 2100 h for 15 d (from d 5 to 21 of life) as per Weiler and Fitzpatrick-Wong (10). Food intake was monitored at each feeding, but formula was readily consumed without waste.
Piglets were randomly assigned to receive 1 of 4 dietary treatments (n = 9 per group). Treatments were standard formula (control) containing no AA:DHA or the same formula but made with oil containing with 0.5:0.1 g AA:DHA, 1:0.2 g AA:DHA, or 2:0.4 g AA:DHA/100 g of fat (Table 1). Supplementation was at a constant (n-6):(n-3) ratio of 5:1 (Table 1). The AA was provided in the form of RBD-ARASCO® (40.6 g/100 g of fatty acids as AA) and DHA in the form of RDB-DHASCO® (40.0 g/100 g of fatty acids as DHA). AA was derived from a common soil fungi and DHA was derived from a marine microalgae (Martek Biosciences). These sources were chosen because they were used previously in our laboratory and because they are used in the manufacturing of many North American infant formula products. Formulas were isocaloric with equal amounts of fat. The formula was based on nutritional requirements for healthy growing piglets between 3 and 10 kg as set by the NRC (25) and currently proven to support growth in our laboratory (10) (Table 2). Formula contained 1050 kcal/L (4.39 MJ/L), 60 g fat/L, 50 g protein/L, 2.1 g calcium/L, and 1.4 g phosphorous/L. Piglets were allowed 
1 h of exercise before each feed.
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Growth.
Weight (kg) was measured daily at 0900 h by digital scale (Mettler-Toleto) before feeding from d 1 to 16 of the study with an animal weighing program (mean of 3 weights).
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Length (cm) was determined by measuring from the tip of the snout to the base of tail (not including the tail) to the nearest millimeter using a nonstretchable measuring tape while the piglets were anesthetized on d 16 of study.
Sample collection. Urine was collected between 2100 h on d 14 and 0900 h on d 15 using metabolic cages and measured as two 24-h pooled samples. To ensure no sample breakdown, urine was collected between 0900 and 1500 h, 1500 and 2100, and 2100 and 0900 h the next day. Samples were stored at –20°C. Blood (5 mL) was taken from all piglets using the internal jugular blind stab technique on d 16 between 0900 h and 1000 h to remove circadian rhythm as a variable in the biomarkers of bone. Anticoagulated blood (heparin) was separated into plasma and erythrocyte fractions. Plasma and erythrocytes were obtained by centrifugation at 2000 x g for 10 min at 4°C and stored at –80°C until analysis of other outcome measurements including erythrocyte fatty acids.
After blood collection, piglets were anaesthetized by i.p. injection of sodium pentobarbital (30 mg/kg, 65 g/L concentration) or isoflurane gas and then killed by a sodium pentobarbital overdose (180 mg/kg). The liver was excised and weighed to the nearest 0.1 g. A section of white adipose tissue was removed from between the shoulders. The liver and adipose tissue were flash frozen in liquid nitrogen before storage at –80°C until analyzed for fatty acids.
Fatty acid analysis. Liver and adipose tissue fatty acids were measured to reflect body stores of AA and DHA in response to diet. Erythrocyte fatty acids were measured to enable future comparison to human neonates for whom only measurement of plasma or erythrocyte fatty acids is feasible in vivo. Total lipids from tissues were extracted according to a method adapted from Folch et al. (26). Erythrocytes were extracted in chloroform containing 0.01% BHT and adipose and liver tissue were extracted in chloroform:methanol 2:1 containing 0.01% BHT. An internal standard, heptadecaenoic acid (17:0) was added to each erythrocyte and liver sample. The liver and adipose tissues were homogenized. Crude lipid extracts were transmethylated in 1.2 mL of methanolic HCl (3 mol/L, Supleco) at 80°C for 1 h. FAME were separated by GLC (Varian Star 3400, Varian), equipped with a 30-m capillary column (J&W Scientific, Folsom) and a flame ionization detector; hydrogen was used as the carrier gas. The column, which is made of fused silica coated with DB225 (25% cyanopylphenyl), runs at 180–220°C with a between-sample temperature of 240°C to clean the column. The detector oven is set at 300°C and produces a sensitivity of 1–5 µg/L. FAME (C12–24) were identified by comparison with retention times of Supelco 37 component FAME mix and expressed as a proportion of total fatty acids (g/100 g of fatty acids).
Bone mass and biochemistry.
Small sections of the tibia cortex were excised from the mid-diaphysis (1.0 g), cleaned of marrow, and rinsed with 0.9% NaCl before bone organ culture as described by Dekel et al. (27). Tibia sections were incubated in 1X HBSS (Sigma Chemical) for 2 h at 39°C in a shaking water bath, followed by the removal of bone and rapid freezing of the solution. This method was chosen because it has been used in chicks (14), rats (12), and piglets (9) to assess PG metabolism after dietary treatment. Samples were stored at –20°C until duplicate analysis of PGE2 by ELISA (R&D Systems) and corrected to the weight of the tibia segment studied. To minimize interference of the HBSS with the alkaline phosphatase enzyme, standards were reconstituted using this solution as opposed to the buffer provided with the kit. In addition, the kits cross-reacts with PGE1 (70%) and PGE3 (16.3%). For PGE2, the CV was <15%.
After removal of tissues at the time of killing, the abdominal cavity was closed with suture to maintain tissue depth. The piglet carcasses were then transported to be scanned by dual energy X-ray absorptiometry (DXA; QDR 11.2, 4500A series, Hologic) and assessed using infant whole body, lumbar spine, and hip subregion software. All scans were performed with the piglet in the anterior-posterior position with limbs extended. Whole body and lumbar spine bone area (BA), BMC, and BMD were measured. Whole body and lumbar spine BMC were corrected to length and weight to account for potential size differences. Right femurs were then excised and freed of soft tissue for determination of weight, length, BMC, BA, and BMD. For the DXA scan, femurs were placed in a small plastic water bath (tested for interference with the scan accuracy), 3 cm in depth, and aligned in an anterior-posterior position. DXA was shown to be a simple, accurate, and precise technique for measuring BMC and BMD in isolated small animal bones (23,28). Only water should be used for determination of BMC and BMD by DXA (28). These DXA measurements are precise with CV < 4% in piglets of a similar size (8–10).
Plasma osteocalcin was measured in duplicate using an 125I RIA (DiaSorin). This assay is based on rabbit antiserum to bovine osteocalcin that was proven to be a valid approach for measuring porcine osteocalcin (29). Plasma IGF-1 was measured in duplicate using an ELISA (Quantikine, R&D Systems). Cross-linked N-telopeptides of type 1 collagen (NTx) in urine was measured in duplicate by a competitive inhibition ELISA (Osteomark, Ostex). Although it is a human assay, it was validated for use in samples from growing piglets (30). Urinary NTx sample values were corrected to creatinine as determined by the Jaffe method (procedure no. 555; Sigma-Aldrich) to account for urinary dilution. For NTx, the CV was <20% and for osteocalcin and IGF-1, the CV was <15%. Creatinine in urine was measured colorimetrically (Sigma Chemical). The CV for triplicate analysis of creatinine in all samples was <10%.
Statistical analysis.
The estimated sample size required to detect a difference in whole body BMC of 25 ± 13 g (8) with a power of 0.80 and an 
value of 0.05 was calculated to be 8/group. Main effects were tested using 2-way ANOVA (diet and litter, general linear model) using SigmaStat statistical software (Jandel Scientific) Litter was included in the statistical analysis as a main effect because previous research demonstrated that litter has a strong effect on bone parameters (9). Post hoc analysis using Bonferroni t tests was conducted when main effects were identified by the 2-way ANOVA. Relations between variables were assessed by Pearson Correlation analysis using GraphPad Prism Version 3.02 software (GraphPad Software). Correlations between PUFA in liver, selected to reflect whole body PUFA status, and bone outcomes were determined with the control group excluded because it was not a component of the dose-response relation. A P-value < 0.05 was accepted as significant. Data are expressed as means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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LCPUFA supplementation affected BMD in the whole body, (P = 0.028) (Fig. 1a) and lumbar spine (P = 0.043) (Fig. 1b). The group supplemented with 0.5:0.1 g/100 g of fat as AA:DHA had significantly higher whole body (6%) and lumbar spine (9%) BMD than control (Fig. 1a,b). Dietary treatment did not affect BA or BMC of the whole body or lumbar spine (Table 3). There was a significant main effect of LCPUFA supplementation on whole body BMC corrected to final length; however, post hoc analysis did not show significant differences among the groups (Table 2). LCPUFA supplementation did not affect femur BA, BMC (Table 2), BMD (Fig. 1c), weight, or length (data not shown).
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The proportion of erythrocyte (Table 4), liver (Table 5) and adipose tissue (Table 6) LCPUFA varied according to the amount of AA and DHA in the diet. The control group had the lowest proportion of AA and DHA in all tissues; as the dietary AA and DHA increased, the concentration of AA and DHA increased significantly. In erythrocytes, the proportions of linoleic acid (LA), -linolenic acid (ALA), EPA, AA:DHA, AA:EPA + DHA, and total (n-6):(n-3) were not affected, whereas total (n-6), total (n-3) and AA:EPA increased with supplementation. In liver, the proportion of LA and ALA plus total (n-6):(n-3) decreased, total (n-6), AA:DHA and AA:EPA + DHA were not affected, and total (n-3) and AA:EPA increased with supplementation. Liver EPA proportions did not decrease in the group receiving 0.5:0.1 g/100 g of fat as AA:DHA, but were significantly lower in the groups supplemented with larger amounts. Liver fatty acid concentrations (mg/g) were altered similarly to the proportional values (data not shown). In adipose tissue, the proportion of total (n-3) increased significantly with supplementation of AA:DHA at 1.0:0.2 g and 2.0:0.4 g/100 g of fat. EPA was not detected in adipose tissue. A dietary main effect was observed for total (n-6) fatty acid proportions in adipose tissue; however, post hoc analysis did not identify differences among the groups. The rest of the fatty acid proportions were not altered in adipose tissue with supplementation
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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20–22 g food/d and had a final weight of 350 g. Given the proportion of LCPUFA in the diets, the rats consumed 0.3g GLA/kg, 0.07 g EPA/kg, and 0.02 g DHA/kg in the 3:1 (n-6):(n-3) group. In our piglet study, the amounts of LCPUFA were 0.11 g AA/kg and 0.02 g DHA/kg throughout the study. NTx was the only marker of bone metabolism that differed among the groups; piglets fed 1.0:0.2 g/ 100 g of fat as AA:DHA had lower urinary NTx:creatinine ratios than those fed 2.0:0.4 g/100 g of fat as AA:DHA, whereas the other 2 groups had intermediate concentrations. Alterations in urinary NTx:creatinine did not affect bone mass over the short duration of the study. Similarly, piglets fed 0.8:0.1 g/100 g of fat as AA:DHA had lower urinary NTx:creatinine concentrations without alterations in bone mass (19). It is possible that bone mass would reflect resorption if the diets were consumed for a longer period or to adulthood. The highest level might be limiting over the longer term due to elevation of bone resorption, whereas the intermediate diet might enhance bone mass due to reduced resorption. In healthy (20) and ovariectomized rats (32), dietary GLA and EPA reduced resorption and enhanced bone mass. Dietary EPA alone improves bone strength in ovariectomized rats (33), and fish oil decreases osteoclastogenesis and loss of bone mass in ovariectomized mice (34). Even in humans, GLA and EPA reduced bone turnover and enhanced bone mass in 80-y-old women (35). Thus, on the basis of our research, the optimal amount of LCPUFA supplementation during growth might lie between 0.5 and 1.0 g/100 g fat. After growth and the attainment of peak bone mass, continued moderated bone resorption and turnover could improve bone mass, and reduce risk of fracture and osteoporosis.
The mechanisms by which LCPUFA enhance bone mass cannot be explained by the biochemical data obtained in this study. Only the lowest level of supplement was associated with enhanced BMD yet urinary NTx, plasma osteocalcin and IGF-1, and bone PGE2 were not affected in piglets that consumed this formula. Similarly, these measurements did not differ in piglets fed dietary AA (0.3–0.75 g/100 g of fat) with DHA held constant (0.1 g/100 g of fat) and an overall (n-6):(n-3) ratio of 9:1 (9). Although NTx and osteocalcin are accepted markers of bone metabolism, it is not known whether changes in circulating concentrations of IGF-1 reflect bone tissue concentration, and the amount in bone may be more important for bone formation (36). It is not surprising that no change in PGE2 was observed in our piglets because the (n-6):(n-3) ratio was held constant. Researchers showed that dietary PUFAs, and specifically the (n-6):(n-3) ratio, comprise a major factor in determining bone tissue content of AA and EPA, which in turn affects the capacity to synthesize PGE2 (12,14). The overall (n-6):(n-3) ratio of our formula was 9:1 for all of our diets and was not significantly reduced by the addition of AA and DHA to the formula. However, a limitation of the current research is that the concentration of ex vivo PGE2 measured may not reflect the true levels of PGE2 in bone; in addition, the ELISA kit used cross-reacts with PGE1 (70%) and PGE3 (16.3%).
In all tissues measured, AA and DHA increased significantly in proportion to the diet concentrations, indicating that the diets were capable of enriching multiple tissues. Supplementation of AA:DHA at 0.5:0.1 g/100 g of fat elevated the proportions of AA (11%) and DHA (25%) yet it did not alter the proportion of EPA or the AA:EPA ratio in the liver. Supplementation of higher amounts of AA and DHA may not be beneficial to bone due to resulting reductions in liver EPA and elevations in AA:EPA. The ratio of AA:EPA in liver was negatively correlated with whole body, femur, and lumbar spine BMC, plus whole body and femur BMD. These data suggest that reduced EPA in tissues relative to AA represents a less than optimal condition for bone mineralization. A positive effect on bone mass might have resulted if EPA had been supplemented in low amounts with AA and DHA to prevent the decline in the tissues. Whether EPA has a specific role to play in bone modeling during growth is unclear. Similar to our results, Watkins et al. (12) found that the ratio of AA:EPA in bone was negatively correlated with the bone formation rate in rats. From studies in (n-3)–deficient rats, it is clear that (n-3) PUFA play a role in normal bone modeling (37). Together, these data underscore the delicate balance between (n-6) and (n-3) PUFA that is required for a benefit to be observed in bone.
There was no effect of fatty acid supplementation on growth measured by final body weight, final body length, rate of weight gain, femur weight, or femur length. Previously, in piglets of the same age, growth was also unaffected by AA and DHA (9,19), but one study demonstrated enhanced growth (10,38). In a safety study in piglets of the same age, supplemental AA (0.62–0.96 mg/g formula vs. 0.30–1.20 mg/g in this study) and DHA (0.37–0.55 mg/g formula vs. 0.06–0.24 mg/g in this study) did not alter growth (38). Thus, although the mechanism(s) for enhanced growth in some but not all studies using very similar formulas are unclear, the discrepancy might be related to fatty acid status at study inception due to maternal diets. Future studies should include assessment of fatty acid status of the sows or the gestation diets.
In summary, LCPUFA supplemented at 0.5:0.1 g/100 g of fat as AA:DHA elevated bone mass, and greater amounts had no additional benefit. In fact, the highest level might be limiting over the longer term due to an increase in bone resorption. Also, the mechanism by which AA and DHA enhance bone mass does not seem to be altered bone turnover or PGE2 release from bone. Increases in bone mass early in life may be retained throughout life, resulting in attainment of higher peak bone mass and reduced risk of fracture and osteoporosis. Future research is warranted to determine the following: 1) how dietary LCPUFA affect bone; 2) whether the elevations in bone mass continue with longer-term supplementation; and 3) whether supplementation of these LCPUFA at other points in the life cycle could improve bone mass and/or slow the loss of bone that occurs naturally later in life.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by The Natural Sciences and Engineering Council of Canada. The Children’s Hospital Foundation of Manitoba purchased and maintains the densitometer research facilities of the Manitoba Institute of Child Health used in this study. R.C.M. was supported by a University of Manitoba fellowship and the Natural Sciences and Engineering Research Council of Canada. H.A.W. is in receipt of a New Investigator Salary Award from the Canadian Institutes of Health Research.
4 Abbreviations used: AA, arachidonic acid; ALA, -linolenic acid; BA, bone area; BMC, bone mineral content; BMD, bone mineral density; DHA, docosahexaenoic acid; DXA, dual energy X-ray absorptiometry; EPA, eicosapentaenoic acid; GLA, -linolenic acid; IGF-1, insulin-like growth factor-1; LA, linoleic acid; LCPUFA, long-chain PUFA; NTx, cross-linked N-telopeptides of type 1 collagen; PG, prostaglandin.
Manuscript received 5 August 2004. Initial review completed 10 September 2004. Revision accepted 28 December 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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