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Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada
2To whom correspondence should be addressed. E-mail: hweiler{at}ms.umanitoba.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • essential fatty acids • piglets • bone • growth
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
), dietary intakes of the essential fatty acids (EFA),3
linoleic acid [LA; 18:2(n-6)] and 
-linolenic acid [ALA; 18:3(n-3)], have been manipulated experimentally to reveal their role in growth and development. Recommended (n-6):(n-3) ratios have since been established for human infants. In Canada, the recommended ratio of (n-6):(n-3) EFA for infant formula is between 16:1 and 4:1 (3
). This is similar to the recommendations in the United States (16:1 to 6:1) (4
) and in Europe (15:1 to 5:1) (5
). The EFA are the base structure for synthesis of the long-chain polyunsaturated fatty acids (LC-PUFA), arachidonic [AA; 20:4(n-6)] and docosahexaenoic acid [DHA; 22:6(n-3)]. Manipulation of the EFA ratio alters LC-PUFA status after as little as 6 d in weanling rats (6
).
Similar to the development of our understanding of the role of EFA in growth and development, research into the role of these fatty acids in bone biology has indicated that adequacy is required for bone health. For example, 16 d of EFA inadequacy in neonatal rats resulted in fractures of the long bones (7
). The dietary intake required to optimize bone growth, however, remains elusive. Over the last decade, the role of (n-3) and (n-6) fatty acids in bone biology has become more clear with experiments conducted in chicks, rats of various ages, piglets, rabbits and humans. Each of these experiments was based on the addition of at least one preformed LC-PUFA in the form of fish oil or as purified and semipurified fatty acids from a variety of sources. Studies conducted by Watkins et al. (8
–10
) included predominantly the addition of fish oils to the dietary fat mixture with the consistent result of enhanced bone formation in chicks and rats. In contrast, the addition of very high amounts of fish oil (10 g/100 g) to diets fed to neonatal rabbits reduced growth of the long bones (11
). The addition of 
-linolenic and eicosapentaenoic acids to diets fed to healthy (12
,13
) or ovariectomized rats (14
,15
) suppressed bone resorption and enhanced bone mass. The same fatty acids, used as an experimental treatment for osteoporosis for 3 y, reduced bone turnover and elevated bone mass in 80-y-old women (16
). However, no effect was observed in pre- and perimenopausal women when consumed for 1 y (17
). In our laboratory, the addition of semipurified AA and DHA to piglet formula enhanced growth and bone mass over 14–15 d (18
,19
). Thus, early and late in life or during menopause, LC-PUFA may modulate bone metabolism and mass. However, the responses of bone mass and metabolism to elevated dietary ALA only have not been reported.
To date, the recommendations for fat content and essential (n-6):(n-3) fatty acid ratios have not been tested for their effect on neonatal bone. Whether the addition of AA and DHA to formula or manipulation of the total (n-6):(n-3) ratio suppresses bone resorption (12
) is unclear. Because AA and DHA are susceptible to oxidation, the addition of only the precursors may be of benefit to manufacturers of infant formula. Thus, the objective of this research was to define the response of whole-body growth, fatty acid status and bone mass to a reduced dietary (n-6):(n-3) fatty acid ratio. In our previous studies of the relationship between LC-PUFA and bone in piglets, two levels of fat were used, 37 (18
) and 60 g/L (19
), as well as two (n-6):(n-3) ratios, 4.9:1 (18
) and 9.1:1 (19
). The recommended amount of dietary fat in human infant formula ranges from 
30 to 44 g/L depending on energy density, similar to human milk with 31 to 53 g/L (5
). Excess (n-3) LC-PUFA impairs bone growth (11
). Therefore, to explore the relation of the amount of fat to bone mass, a secondary objective of the study was to determine whether the amount of dietary fat affects bone mass and metabolism and whether there is an interaction between the amount of dietary fat and the (n-6):(n-3) fatty acid ratio.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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At 3 d of age, male piglets (n = 32), were randomly assigned to receive 21 d of treatment to commence at 5 d of age. The trial was 6 d longer than our earlier 14- to 15-d studies (18
,19
) based on a study in rats in which fatty acid status was changed in response to diet after only 6 d (6
). All piglets were > 1.3 kg at birth; they were from 8 litters consisting of 10–12 piglets each with at least 4 males. Treatments were diets with (n-6):(n-3) ratios of 9:1 or 4.5:1 plus either 30 or 60 g/L of fat: group 1: [30 g fat/L + (n-6):(n-3) ratio 4.5:1]; group 2: [30 g fat/L + (n-6):(n-3) ratio 9.0:1]; group 3: [60 g fat/L + (n-6):(n-3) ratio 4.5:1]; and group 4: [60 g fat/L + (n-6):(n-3) ratio 9.0:1]. Piglets were transported on d 3 of life from the Glenlea Swine Research Unit, Manitoba, to the University of Manitoba where the light:dark cycle was 16 h:8 h in the housing facility. The piglets were taught to lap the standard liquid formula according to the method previously described (20
) between d 3 and 5 of life and permitted this period of time to adapt to the formula. Diluted formula was fed on d 3 by mixing formula with sterile distilled water in a 50:50 ratio of formula to water followed by 75:25 formula/water on d 4 of life and no dilution by d 5 of life. All procedures were in agreement with Canada Council guidelines (21
) and approved by the University of Manitoba Protocol Management and Review Committee.
All formulas were equal in energy (4389 kJ, 1050 kcal/L) and contained 50 g/L protein, 2.1 g/L calcium and 1.4 g/L phosphorous. Formula with 30 g/L of dietary fat was kept isocaloric by the addition of dextrose (68 g/L). The lower amount of dietary fat is similar to that used in an earlier study (18
) in which dietary fat (37 g/L) was selected to approximate the quantities in formulas designed for prematurely born infants (Enfalac Premature 20 with 34 g fat/L and Enfalac Premature Plus with 41 g fat/L, Mead Johnson Nutritionals, Ottawa, Canada). The higher amount of fat was more consistent with the amount of fat in sows’ milk, 62 g/L, as measured in milk 5 d postpartum from the Glenlea Research Unit and the same as the amount used in another study conducted in our laboratory (19
). The components of the diets are outlined in Table 1
. The amount of LA ranged from 7.02 to 14.30 g/L of formula and ALA ranged from 0.80 to 3.09 g/L. Formulas were fed at 400 mL/(kg · d) divided into equal amounts at 0900, 1500 and 2100 h for the duration of the study. Any formula not consumed from a feed was measured and discarded, but an equal volume was added to the next feed to ensure full volume was reached. Piglets were housed individually in stainless steel cages, but group housed for 3–5 h during feeding; ambient temperature was maintained at 29–30°C.
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3 h/block, piglets were without food and water for 12–15 h before sodium pentobarbital (30 mg/kg) anesthesia and sample collection on d 22. Growth.
Weight was measured daily to the nearest gram in piglets that had not been fed using a scale with an animal weighing program (Mettler-Toledo, Hightstown, NJ) and mean daily weight gain calculated [g/(kg · d)]. Snout-to-tail length was measured to the nearest millimeter using a nonstretchable measuring tape at the end of study while pigs were anesthetized.
Tissue samples.
Blood was taken into heparinized syringes followed by sodium pentobarbital overdose. Plasma was obtained by centrifugation at 2000 x g for 20 min at 4°C. Plasma was centrifuged again under the same conditions to remove any remaining RBC and stored under nitrogen gas at -80°C until analysis of fatty acids. Urine was obtained using bladder puncture and stored at -20°C until analysis of minerals and N-telopeptide.
Fatty acid analysis.
Total lipids in plasma and tissues were extracted using 1:2 methanol/chloroform according to the method of Folch et al. (22
). An internal standard, 17:0, was added to each sample. Crude lipid extracts were transmethylated in 1 mL of methanolic HCl (3N, Supleco, Bellefonte, PA) at 100°C for 15 min. Brain was extracted and methylated using the same method but methylation occurred over 90 min. Fatty acid methyl esters (FAME) were separated by gas-liquid chromatography (Varian Star 3400, Mississauga, Canada) with hydrogen as the carrier gas. The gas chromatograph was equipped with a 30-m long capillary column made of fused silica and coated with DB225 (25% cyanopropylphenyl; J&W Scientific, Folsom, CA), 8100 autosampler, an integrator and flame ionization detector. Samples were injected (0.5 µL) at an initial temperature of 180°C and the oven temperature was increased to a final temperature of 220°C at a rate of 3°C/min. FAME were identified by comparison with retention times of Supelco 37 component FAME mix (Supelco) and expressed as a percentage of total fatty acids (g/100 g fatty acids).
Biochemical assessment of bone metabolism.
Plasma osteocalcin was analyzed using a RIA (DiaSorin, Stillwater, MN) This assay is based on rabbit antiserum to bovine osteocalcin that has been proven to be a valid approach for measuring porcine osteocalcin (23
) Urinary N-telopeptide was measured in urine using an ELISA (Osteomark, Ostex, Seattle, WA). Although this assay is designed for use in human samples, it has been validated for use in samples from growing pigs (24
) N-Telopeptide, calcium and phosphorous were corrected to creatinine as determined by the Jaffe method (procedure no. 555; Sigma-Aldrich, Oakville, Canada). Ex vivo release of prostaglandin E2 (PGE2) was measured in an 1-g segment of tibia that was cleaned of muscle tissue, periosteum and marrow followed by incubation in Hank’s Balanced Salt Solution for 2 h at 37°C according to the method of Dekel et al. (25
). The released PGE2 was analyzed in diluted samples using an ELISA (Cedarlane Laboratories, Hornby, Canada) and corrected to the weight of the tibia segment studied. This assay crossreacts with PGE1 (100%) and PGE3 (17%) according to manufacturer’s specifications.
Bone mineral content.
After tissues were removed, the abdominal cavity was closed with suture to maintain tissue depth. Piglet carcasses were then transported to the dual energy X-ray absorptiometer (QDR4500W; Hologic Waltham, MA). Single scans were completed to determine bone mineral content (BMC) of the whole body (software version V8.16a:5), lumbar 3, using the low density spine program, and left femur, using the subregion array hip program. The whole-body scan also provides an assessment of fat mass (g/100 g body). The low density spine program was used because it has been validated by Braillon et al. (26
) for measuring bone of infants smaller (2.1–3.8 kg) than the piglets in this study (9–10 kg). Both whole-body and femur measurements have been validated for use in piglets (27
) All scans were performed with the piglet in the anterior-posterior position with limbs extended as described by Brunton et al. (27
). Whole-body BMC was corrected to body weight (g/kg) to account for potential differences in size.
Statistical analysis.
The sample size required to observe a significant difference in whole-body BMC of 25 ± 13 g with power of 0.80 and of 0.05 was calculated to be 8 per group on the basis of data from Weiler (18
). All data are means ± SD unless otherwise stated. Differences between groups were detected using two-way ANOVA; the factors were (n-6):(n-3) ratios of 9:0 or 4.5:1 and amount of fat (30 or 60 g/L). Differences with P < 0.05 were considered significant. The Levene Median test for equal variance was used before conducting the ANOVA; no differences in variation were detected among groups. Post-hoc analysis with Student-Newman-Keuls all-pair-wise test was used to identify differences among the four treatment groups only when a significant ratio x amount of fat interaction occurred. Correlation analysis using Pearson correlation coefficients was used to detect relationships among tissue fatty acids (AA and DHA), and between plasma fatty acids and biochemical markers of bone metabolism.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Whole-body bone formation, determined by analysis of plasma osteocalcin, was not affected by the diets (group 1, 20.1 ± 4.5 µmol/L; group 2, 24.9 ± 6.3 µmol/L; group 3, 24.7 ± 4.6 µmol/L; and group 4, 23.8 ± 5.2 µmol/L). Whole-body bone resorption, determined with urinary N-telopeptide (group 1, 6.0 ± 3.1 µmol:mmol creatinine; group 2, 5.8 ± 1.9 µmol:mmol creatinine; group 3, 5.1 ± 2.7 µmol:mmol creatinine; and group 4, 6.1 ± 3.1 µmol:mmol creatinine) and bone PGE2 (group 1, 12.2 ± 5.2 ng/g; group 2, 14.2 ± 7.7 ng/g; group 3, 9.2 ± 4.9 ng/g; and group 4, 14.7 ± 10.1 ng/g) were not affected by diet treatments.
In contrast to the lack of diet effects on growth and bone, the interventions affected fatty acid status in liver, plasma, brain and adipose tissues (Table 2
). Piglets fed diets 1 and 3 had elevated ALA but reduced AA in liver. The dietary (n-6):(n-3) ratio had no effect on these variables in plasma and brain. Piglets fed diet 3 had greater proportions of ALA in adipose tissue than did the other 3 groups. In brain, a significant ratio x amount of fat interaction was observed for AA but differences among groups were not significant. Piglets fed diets 1 and 3 [i.e., (n-6):(n-3) ratio of 4.5:1] had the greatest proportions of DHA in brain and adipose tissue.
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Liver and plasma fatty acids were significantly related. Liver DHA was positively correlated with plasma DHA (Fig. 1
) and liver AA was positively correlated with plasma AA (Fig. 2
). Plasma and liver AA were not correlated with brain and adipose tissue AA. However, DHA in liver was significantly associated with DHA in brain (r = 0.42, P = 0.02) and adipose tissue (r = 0.56, P < 0.001). Plasma DHA was also associated with DHA in brain (r = 0.50, P = 0.004) and adipose tissue (r = 0.47, P = 0.008). The only biochemical marker of bone metabolism that was correlated with AA or DHA was urinary N-telopeptide. This marker was negatively associated with plasma DHA (Fig. 3
). When the outlier at 3.55 g DHA/100 fatty acids was removed, the relationship remained significant (P = 0.04). The only correlation among the biochemical markers of bone was the negative association between plasma osteocalcin and bone PGE2 (Fig. 4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), alterations in LC-PUFA as a result of manipulations in the essential (n-6):(n-3) ratio do not appear to do so. In our earlier study, the elevation in whole-body BMC due to dietary LC-PUFA supplementation was 22%. In the present study, the maximum difference observed between groups 1 and 3 was only 6%. This difference was eliminated by correction to body weight. Nonetheless, higher plasma DHA was related to lower rates of bone resorption as assessed by urinary N-telopeptide (see Fig. 3
). This is consistent with other studies conducted in rats in which elevations in dietary (n-3) LC-PUFA using fish oil reduced bone resorption as assessed by histomorphometry (12
).
The mechanisms underlying the response of bone to dietary fatty acids are not fully understood. Thus far, alterations have been reported in eicosanoid metabolism, insulin-like growth factor (8
–10
), as well as enhanced calcium absorption and balance (12
,13
). Bone formation, as indicated by plasma osteocalcin, was suppressed in the piglets in this study when ex vivo release of bone PGE2 was elevated (Fig. 4)
. This observation parallels that of Watkins et al. (8
,10
) in which bone formation, assessed by histomorphometry, was negatively associated with ex vivo release of PGE2. As a whole, these results suggest that reduction in the (n-6):(n-3) ratio using flax seed oil as the source of ALA does not affect piglets’ bone in the short term, i.e., over 21 d. Higher plasma DHA concentrations due to lower dietary (n-6):(n-3) ratios were associated with reduced bone resorption, which may alter bone growth and mass over the longer term.
The proportion of AA and DHA in bone was not determined in this study. Previously, in piglets (18
) and chicks (9
), dietary alterations in LC-PUFA, such as AA or DHA, were reflected in liver and bone tissues. After feeding diets high in (n-3) fatty acids, release of PGE2 from bone is reduced in chicks and rats (9
,10
). Although the (n-3) content of the diet fed to piglets in this study was elevated by the addition of flax seed oil to the fat mixture, no reduction in PGE2 release from bone was observed and bone metabolism did not differ among the diet groups. Whether bone fatty acids were altered by the diets similarly to those in liver remains to be determined in subsequent investigations. The additional measurement would help clarify whether the fatty acid composition of bone is sensitive only to preformed AA, DHA or eicosapentaenoic acid (8
,18
) compared with endogenously synthesized LC-PUFA. This is important to clarify because Watkins et al. (10
) clearly showed in bone that reduced AA is associated with reduced PGE2 accompanied by elevated formation rates. In that study, the cortical bone polar lipids were reduced by 3–4 g/100 g fatty acids and in our piglets, the reduction observed only in liver was 1.5–2.5 g/100 g fatty acids. Thus, the reduction in AA via elevation in dietary ALA was likely not sufficient to affect either PGE2 or bone.
The piglets in this study grew at a rapid rate [60–65 g/(kg · d)] regardless of the dietary (n-6):(n-3) ratios. Skeletal mass was appropriate to body size, and bone metabolism was within expected limits (18
). The amount of dietary fat (30 or 60 g/L) did not influence any of these outcomes. Piglets fed diet 4 had the highest AA status in liver but those fed diet 3 had the most DHA in liver, brain and adipose tissues. Therefore, a reduction in the (n-6):(n-3) fatty acid ratio using flax seed oil appears to be safe in terms of growth and increases the proportions of DHA in tissues. Other studies in which flax seed meal and its purified lignan were fed to neonatal rats (28
,29
) demonstrated potentially negative effects of flax seed on bone. However, in our study, only the oil was used and no negative effects were detected. Whether longer-term use of flax seed oil continues to enhance LC-PUFA status with no consequences for growth or bone metabolism requires substantial investigation.
Arbuckle et al. (30
) also studied piglets fed varied ratios of LA to ALA. The ratios of LA to ALA were 4:1 and 8:1, similar to those used in the present study at 4.5:1 and 9:1. Only one level of dietary fat was investigated, 59 g/L, consistent with the concentration in sows’ milk. The amount of ALA, as opposed to the (n-6):(n-3) ratio, was considered to be very important for accretion of DHA in brain (30
). However, infant formula has variable amounts of fat depending on the type of formula and target population. The present study included two concentrations of fat, 30 and 60 g/L, to address whether the amount of LC-PUFA (within recommended levels) and total fat, as well as the (n-6):(n-3) ratio were important. Piglets with the highest brain DHA were those fed diets 1 and 3 with the 4.5:1 ratio. On the basis of this result, the amount of dietary fat and the amount of LA and ALA (see Table 1
) are not as critical to brain composition as is the ratio of LA:ALA.
Upon examination of the two human trials in infants in which only LA:ALA ratios were manipulated (31
,32
), only one group reported the amount of dietary fat and the resulting weight and length outcomes (32
). Formula containing 36 g fat/L and a ratio of LA:ALA of 10:1 or 5:1 were fed for 34 wk after birth. Both formulas supported growth equally but the lower ratio resulted in better DHA status (32
). This is similar to our results for the piglets when absolute weight and length were examined. However, a significant ratio x amount of fat interaction was observed with the highest rate of growth in group 3 [60 g fat/L and (n-6):(n-3) ratio of 4.5:1]. Sows’ milk contains 62 ± 18 g fat/L. The 30 g fat/L formula was based on the lower limit in human milk and the lower limit of the recommended amount of fat (5
). It is thus possible that when dietary fat is adequate, the lower (n-6):(n-3) ratio does not impair growth.
In summary, reduction in the (n-6):(n-3) fatty acid ratio from 9:1 to 4.5:1 using flax seed oil as a source of ALA supports normal somatic and bone growth in piglets. The lower ratio appears to be optimal for DHA status. It is not a negative observation that bone mass and metabolism were similar among the four treatment groups because the goal is to optimize brain and retinal DHA composition without adverse effects on other systems such as bone. However, as a group, higher DHA status was associated with reduced bone resorption. This may be a detriment to bone development if modeling is inappropriately limited or of benefit when resorption is too high relative to formation.
For the preterm infant, DHA status is compromised when formula devoid of DHA is fed (33
). In addition, bone resorption was considered too high to permit normal modeling (34
), suggesting that diets designed to support brain DHA and development may also support bone development. Whether DHA status and bone resorption could be normalized with manipulation of the (n-6):(n-3) fatty acid ratio in the diet rather than addition of DHA to the milk remains to be investigated.
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FOOTNOTES |
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3 Abbreviations used: AA, arachidonic acid; ALA, -linolenic acid; BMC, bone mineral content; DHA, docosahexaenoic acid; FAME, fatty acid methyl esters; LA, linoleic acid; LC-PUFA, long-chain polyunsaturated fatty acid; PGE2, prostaglandin E2.
Manuscript received 29 April 2002. Initial review completed 27 May 2002. Revision accepted 12 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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