Dietary docosahexaenoic acid contributes to increased bone mineral accretion and strength in young female Sprague-Dawley rats

Prostaglandins, Leukotrienes and Essential Fatty Acids 144 (2019) 32–39

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Original research article

Dietary docosahexaenoic acid contributes to increased bone mineral accretion and strength in young female Sprague-Dawley rats☆ Zahra Farahnak, Marie Therese Freundorfer, Paula Lavery, Hope A. Weiler

T

⁎

School of Human Nutrition, McGill University, 21111 Lakeshore Rd, Ste-Anne-de-Bellevue, QC H9 × 3V9, Canada

ARTICLE INFO

ABSTRACT

Keywords: Dose-response DHA Peak bone mass Growth

Growing evidence suggests that docosahexaenoic acid (DHA: 22:6n-3) enhances bone mineral content (BMC) and bone mineral density (BMD) in adulthood and during aging, however the effects during and after sexual maturation are unclear. The aim of this study was to examine the dose-response of BMC, BMD and microarchitectural properties of bone to dietary DHA in healthy growing female rats during acquisition of peak bone mass (PBM). Female Sprague-Dawley rats (n = 12/diet) were randomized to receive a control diet (AIN-93 M, 60 g soybean oil/kg diet) or an experimental diet containing 0.1, 0.4, 0.8 and 1.2% DHA (w/w of total diet) for 10 weeks. Dietary DHA increased the whole body, lumbar spine and long bone BMC compared to the control, in addition to higher aBMD and also BMD. Additionally, an increase in cortical bone microarchitecture parameters of lumbar spine as well as peak force were observed in dietary DHA diet groups. Dietary DHA contributes to PBM when consumed during and after sexual maturation, however higher doses of DHA do not provide further benefits.

1. Introduction Osteoporosis, known as the most prevalent bone disorder in the world, is characterized by low bone mass, microarchitectural deterioration of bone tissue and decreased bone strength [1]. An imbalance in bone homeostasis, either from an increase in bone resorption and/or a decrease in bone formation results in bone loss [2], which increases the risk of fracture with an inevitable increased financial burden for healthcare systems [3]. Based on 2000 census data, the prevalence of osteoporosis is estimated to increase from ∼10 million to >14 million people in 2020 in the United States [4]. At present, methods for prevention of osteoporosis and fractures focus on the achievement of optimal peak bone mass (PBM) during growth and early adulthood, and maintenance of bone during menopause and aging [5]. Not achieving optimal PBM during skeletal maturity contributes to an increased risk for osteoporosis due to insufficient bone mass to maintain structural integrity, emphasizing the importance of maximizing bone mass during growth [6,7]. Nutrition is considered one of main modifiable factors that affects PBM [8]. The n-3 series of dietary long chain polyunsaturated fatty acids (n-3 LCPUFA) play important roles in the regulation of a variety of biological processes including bone metabolism [9]. Increasing evidence suggests that

n-3 LCPUFA, particularly docosahexaenoic acid (DHA: 22:6n-3), increase bone mineral content (BMC) and bone mineral density (BMD) and inhibit bone resorption [2,10,11] through various mechanisms including reduction of receptor activated nuclear factor κβ ligand (RANKL)-induced production of pro-inflammatory cytokines and inhibition of RANKL-induced osteoclastogenesis and osteoclast differentiation [12,13]. However, there is still insufficient data to determine the impact of n-3 LCPUFA on PBM. In a population-based cross-sectional study, Chen et al. [14] found that postmenopausal women with higher sea fish intake (quintile 5) with a mean intake of 64 g/d compared to women with lowest intake (quintile 1) showed greater BMC, BMD and lower risk of osteoporosis. In a case-control study of Japanese elderly, fish intake of 3–4 times per week in adulthood was associated with the reduction of the risk of hip fracture in adults over 65 years of age [15]. While these studies did not consider DHA in particular, knowing that the richest dietary source of DHA is fish, the findings point towards beneficial actions of DHA on bone health. To date, studies investigating the effect of DHA in particular on bone health outcomes have revealed conflicting results. For instance, Moon et al. [16] observed a weak positive association between erythrocyte DHA percentage and BMD (r = 0.193, P = 0.019) in postmenopausal Korean women. Fish oil supplementation (180 mg EPA and 120 mg DHA) concomitant with aerobic exercise for 6 months increased both lumbar spine

This work was supported by Natural Sciences and Engineering Research Council of Canada [Grant no. RGPIN-2015-03854], Canada Foundation for Innovation [Grant no. 217519], and Canada Research Chairs Program [Grant no. 950-230633]. ⁎ Corresponding author. E-mail address: [email protected] (H.A. Weiler). ☆

https://doi.org/10.1016/j.plefa.2019.04.005 Received 20 November 2018; Received in revised form 17 April 2019; Accepted 17 April 2019 0952-3278/ © 2019 Published by Elsevier Ltd.

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BMD (25%) and femoral head BMD (38%) in post-menopausal Iranian women [17]. On the other hand, higher plasma phosphotidylcholine concentrations of DHA were associated with loss of femoral neck BMD in women over 4 years, although the DHA was protective against bone loss in men [18]. The conflicting findings may be due to differences in experimental designs, biomarkers reflecting DHA status, baseline characteristics of samples/participants (e.g. sex, age, bone health status) and source and amount of DHA consumption. It is also important to note that bone physiology is very different in the aging population than in youth and early adulthood when PBM is achieved. Long-term studies pertaining to bone health outcomes are costly to conduct, it is therefore prudent to first test whether DHA improves PBM in an animal model by using multiple doses from a single source of DHA. Thus, the aim of the present study was to examine the dose-response effects of DHA on BMC, BMD and bone microarchitecture properties in growing healthy female rats.

Baxter Corporation, Mississauga, ON, Canada) and placed in the prone position, with hind-legs extended distally and secured in a standardized position with a velcro strap. Micro-CT scans included a whole body scan (120 µm voxel size, 1020 µm pitch, 360° rotation angle, artifact removal of the soft tissue), and regional scans of the lumbar spine (L2-4) and of the legs from the femoral head to mid-tibia (both settings at 120 µm voxel size, 1020 µm pitch, 360° rotation angle, artifact removal of the soft tissue). The region of interest (ROI) for whole body analysis included from the bottom of the atlas to tail-tip (excluding the skull). Femurs were analyzed as a whole. The ROI used for bone mass and cortical microarchitecture analysis for the lumbar spine vertebrae was L2-L4. Cortical microarchitectural analysis was conducted to determine cortical area (Ct.Ar.), total cross-sectional area (Tt.Ar.), cortical area ratio (Ct.Ar./Tt.Ar) and average cortical thickness (Ct.Th.). 2.3.3. Ex vivo micro-computed tomography After thawing and wrapping the bones in parafilm, ex vivo µCT (1174 Skyscan, Bruker, Madison, WI, USA) analysis was conducted on the left femur, left tibia and L3 vertebra. Left femurs were scanned at 11.7 μm resolution with a 5500 ms exposure time (0.4 mm Al filter, 50 kV, 800 µA). Trabecular bone was analyzed for 150 slices of the distal femoral metaphysis starting 150 slices proximal to the growth plate. L3 vertebra were scanned at 14.2 um resolution with an 8000 ms exposure time (0.2 mm Al filter, 50 kV, 800 µA). Trabecular bone was analyzed for the vertebral body, the region of interest excluding the primary spongiosa. Left tibias were scanned at 11.7 µm resolution with an 8000 ms exposure time (0.4 mm Al filter, 50 kv, 800 µA). Trabecular bone was analyzed for 350 slices of the proximal tibia metaphysis starting 75 slices distal to the growth plate.

2. Materials and methods 2.1. Animals and diets The study design, time course and diet composition have been published in detail [19]. Briefly, 6-week-old female Sprague-Dawley rats were randomized to receive a control (AIN-93 M, 60 g soybean oil/kg diet) [20] or experimental diet for 10 weeks. Experimental diets contained 0.1, 0.4, 0.8 and 1.2% DHA (w/w of total diet). The source of DHA was DHASCO (inkind, DSM, Columbia, MD, USA) which contained 44.8% DHA. All diets were isocaloric and controlled for vitamin D, calcium, phosphorous and protein content to prevent possible confounding factors of these nutrients on peak bone mass (PBM). The ages of 7 to 17 wk of age were selected to represent stages of bone maturation from early reproductive ages [7] to an age when PBM is reached [21].

2.3.4. Three-point flexure testing Bone strength was assessed using the right femur and the threepoint bending test at mid-diaphysis on an Instron 5544 (Norwood, MA, USA). Femurs were positioned anterior side up on fulcrum with a 16.4 mm span length. A 0.1 N load was applied at a preloading rate of 1 mm/s until contact was made with the bone, followed by a rate of 0.1 mm/min until breakpoint. The bones were thawed, cleaned of all soft tissue and kept hydrated in saline solution for 1 h minimum at 25 °C before the test was performed. The femur length (from greater trochanter to intercondylar fossa) and anterior-posterior diameter at middiaphysis were measured before the test with a digital caliper (Fisher Scientific, St. Laurent, QC, Canada). Bluehill version 2.6 was used to generate load-deformation curves to determine peak load, peak extension and energy at breakpoint. Maximum stress (force per unit area), and Young's modulus (stress/strain for linear portion of deformation curve) were calculated using the following equations:

2.2. Blood and bone sampling Blood samples (800–1200 µL) were collected in the non-fed state (8 h) into one blood gas tube, and serum and lithium heparin tubes via saphenous vein at study weeks 0, 5 and 10. Serum and plasma were stored at −80 °C until analysis. At study endpoint, directly after the imaging scans were acquired, deep anesthesia was induced, followed by exsanguination by cardiac puncture and excision. Cardiac blood was saved and used as quality control samples for biochemical analyses. Necropsy was conducted immediately postmortem. The left tibia and L4 vertebra were cleaned then immediately placed in 10% formalin (Fisher Scientific, St. Laurent, QC, Canada), switching after 24 h to 70% ethanol to fix the bone for future analyses. 2.3. Bone assessment

Maximum stress =

2.3.1. Dual-energy X-ray absorptiometry Three sets of scans were conducted using dual-energy x-ray absorptiometry (DXA, QDR 12.3, 4500A Elite series; Hologic Inc, Bedford, MA, USA) at study weeks 0, 5 and 10 while the rats were under anesthesia (AErrane; Baxter Corporation, Mississauga, ON, Canada) and in the prone position with femurs and tibia positioned at 90° angles. One scan was taken for the whole body, and then high resolution scans were taken of each hind leg and the lumbar spine (L1-L4) to determine BA, BMC, and areal BMD (aBMD). For quality control, scans were conducted in triplicate on different rats at midpoint for the whole body (BA CV = 0.89%, BMC CV = 0.86%, aBMD CV = 0.35%), the lumbar spine (BA CV = 3.8%, BMC CV = 2.7%, aBMD CV = 1.2%), and femurs (BA CV = 2.6%, BMC CV = 2.4%, aBMD CV = 1.1%).

peak load*span length*(anterior posterior diameter)/2 4*moment of inertia

Young ’s modulus =

peak load*(span length)3 48*peak extension*moment of inertia

Maximum strain (relative displacement to original position) was estimated from Young's modulus and maximum stress. 2.4. Blood biochemical markers Ionized calcium, potassium, sodium, chloride and hematocrit were measured within 3 h following blood collection by radiometry (ABL 80 Co-ox Flex, Radiometer Medical ApS, Brønshøj, Denmark; CV < 2.1%). Plasma osteocalcin and parathyroid hormone (PTH) concentrations were quantified at study weeks 0, 5 and 10 using Milliplex kits (Cat. # RBN3-31 K, EMD Millipore, Billerica, MA, USA). The intra- and interassay CVs were less than 15% for the pooled cardiac plasma from the control group. Serum osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-Β ligand (RANKL) concentrations were quantified

2.3.2. In vivo micro-computed tomography In vivo µCT (Latheta LCT-200, Hitachi Aloka Medical, Tokyo, Japan) was used to determine volumetric BMD (vBMD) with high resolution scanning at study weeks 0, 5 and 10 while the rats were under anesthesia (AErrane; 33

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Table 1 Whole body bone assessment of female Sprague-Dawley rats fed diets containing different doses of DHA at weeks 0, 5 and 10. Variable

Time

Diet Groups Control

BA (cm2)

BMC (g/kg)

aBMD (g/cm2)

vBMD (mg/cm3)

wk0 wk5 wk10 All

43.5 65.7 73.2 60.8

± ± ± ±

0.7 1.7 2.2 2.3

Main effects

0.1% DHA

0.4% DHA

0.8% DHA

1.2% DHA

44.0 64.3 72.4 60.2

44.6 64.6 71.9 60.4

45.2 66.4 73.1 61.6

44.5 66.9 75.5 62.3

± ± ± ±

0.6 1.1 1.6 2.1

± ± ± ±

1.0 1.4 1.7 2.1

± ± ± ±

1.1 1.3 1.7 2.2

± ± ± ±

All

1.0 2.0 2.6 2.5

44.4 ± 0.4C 65.6 ± 0.7B 73.2 ± 0.9A

wk0 wk5 wk10 All

27.0 ± 0.5 30.4 ± 0.4 31.3 ± 0.5 29.6 ± 0.4ab

27.1 ± 0.3 32.5 ± 1.2 33.5 ± 1.4 31.0 ± 0.8a

27.6 ± 0.5 31.2 ± 0.5 32.9 ± 0.5 30.6 ± 0.5ab

27.5 ± 0.5 30.8 ± 0.6 31.5 ± 0.4 29.9 ± 0.4ab

26.7 ± 0.2 30.5 ± 0.5 30.8 ± 0.7 29.3 ± 0.4b

27.2 ± 0.2B 31.0 ± 0.3A 32.0 ± 0.4A

wk0 wk5 wk10 All

0.12 ± 0.00 0.15 ± 0.00 0.16 ± 0.00 0.14 ± 0.00b

0.12 ± 0.00 0.16 ± 0.00 0.17 ± 0.00 0.15 ± 0.00a

0.12 ± 0.00 0.16 ± 0.00 0.17 ± 0.00 0.15 ± 0.00a

0.12 ± 0.00 0.16 ± 0.00 0.17 ± 0.00 0.15 ± 0.00a

0.12 ± 0.00 0.16 ± 0.00 0.17 ± 0.00 0.15 ± 0.00a

0.12 ± 0.00C 0.16 ± 0.00B 0.17 ± 0.00A

wk0 wk5 wk10 All

464.8 ± 5.9 593.0 ± 9.0 641.5 ± 9.9 566.4 ± 13.5b

460.3 ± 5.4 596.7 ± 7.1 650.2 ± 7.9 569.1 ± 14.1bc

471.5 ± 4.7 608.2 ± 6.9 658.8 ± 6.9 579.5 ± 13.8ac

471.5 ± 5.5 614.0 ± 5.5 664.0 ± 5.3 583.1 ± 14.1ac

472.0 ± 5.1 617.7 ± 5.8 668.7 ± 7.2 586.2 ± 14.5a

468.0 ± 2.4C 605.9 ± 3.3B 656.6 ± 3.5A

Interactions

p value Diet

p value Time

p value Diet x Time

0.34

<0.0001

0.95

0.021

<0.0001

0.67

0.002

<0.0001

0.27

0.0002

<0.0001

0.89

Values are means ± SEMs, n = 12/group. wk0: baseline, wk5: midpoint, wk10: endpoint. Means in a row (diet) without a common superscript letter differ, P < 0.05, a > b > c. Means in a column (time) without a common superscript letter differ, P < 0.0001, A > B > C. No differences were observed among groups at baseline. BA, bone area; BMC, bone mineral content; aBMD, areal bone mineral density; vBMD, volumetric bone mineral density.

at study week 10 using Milliplex magnetic kits (Cat. # RBN1MAG-31 K, Cat. # RRANLMAG-31 K, EMD Millipore, Billerica, MA, USA); OPG intra-assay CV=11.9%, RANKL intra-assay CV=2.8% for the pooled cardiac serum from the control group. All samples were analyzed using Luminex 200 (Luminex Corp., Austin, TX, USA).

prior to analyses. Statistical analysis of data was conducted using mixed model ANOVA (SAS version 9.4, SAS Institute Inc., Cary, NC, USA) for fixed effects of time and diet, and random effects of study block. Covariates such as food intake and body weight were evaluates, but they did not improve the model and thus were not included in the analysis. For all long bone microarchitecture analyses and bone strength testing, bone length was included in the statistical model as a covariate. The mixed model was originally designed with the assumptions that when random parameters are included in the model, the distribution of the data is either not normal, or the mean is not linearly related to the model parameters, or the data mean is related to the variances [18]. Post hoc testing for significant effects of diet, time or diet-by-time interactions were assessed using the all pairwise Bonferroni's test with a P < 0.05. Correlations between serum RANKL/OPG ratio and whole body BMC accretion and aBMD were tested using Pearson's correlation coefficients. All results are expressed as means ± SEMs.

2.5. Red blood cell fatty acid analysis Fatty acid profiling was conducted on red blood cells (RBC) using the LePage-Roy direct fatty-acid methylation protocol as described previously [22] and in accordance with international recommendations [23] with heptadecanoic acid (Sigma-Aldrich, Bellefonte, PA, USA) as the internal standard. Fatty acid methyl esters (FAME) for each sample were identified using a Varian CP-3800 (Lake Forest, CA, USA) gas chromatograph (GC) with a flame ionization detector (FID). FAME were separated using a capillary column (VF-23 ms, 60 m by 0.25 mm i.d., Varian, Lake Forest, CA, USA). Hydrogen was used for the carrier gas with a column flow rate of 0.8 mL/min. The oven temperature was set at 120 °C and held for 2 min, then increased at 3 °C/min to 250 °C to give a total run time of 40 min. Sample FAME peaks were identified by comparing their retention times with those of a standard 37 component FAME mix (Supelco, Sigma-Aldrich, Bellefonte, PA, USA) and quantified using Galaxie Workstation 1.9.3.2 and Microsoft Excel. Pooled cardiac RBC from the control group was tested in triplicate for quality control (Inter-assay CV = 5.4%, Intra-assay CV < 6.5%, 72% recovery 17:0). Fatty acid analysis was also conducted on the experimental diet using the same protocol and procedure as explained above for RBC [19]. Fatty acids were expressed as% of total fatty acids from 14:0 to 22:0 for RBC.

3. Results 3.1. Anthropometric measurements and food intake There were no differences among diet groups for body weight or length at baseline, or food intake and body length at any time-point. Over time, all diet groups showed an increase in body weight and length (weight at wk0: 194.9 ± 2.3, wk5: 332.1 ± 5.5, wk10: 386.0 ± 7.3 g; length at wk0: 35.1 ± 0.2, wk5: 41.3 ± 0.2, wk10: 43.0 ± 0.2 cm) whereas food intake was reduced (wk0: 107.2 ± 1.0, wk5: 64.2 ± 0.8, wk10: 50.5 ± 0.8 g/kg/wk); with no interactions between time and diet. Rats in the 1.2% DHA diet group had higher weight (321.9 ± 18.1 g) compared to the 0.1% and 0.4% DHA diet (292.4 ± 13.9, 297.4 ± 13.6 g, respectively) groups.

2.6. Statistical analyses Using previous whole body BMD data for 12 week old females, it was estimated that 11 rats were needed per group to detect 5% differences in a 2-sided test (α = 0.05 and β = 80). Twelve rats were used per group to provide coverage in the event of loss of an animal or smaller size effect. All data were tested for normality and homogeneity of variances with the Shapiro-Wilk test and Levene's test respectively

3.2. Whole body bone assessment No differences were observed among diet groups at baseline for whole body measures of BA, BMC, aBMD, and vBMD (Table 1). Diet had no effect on whole body BA however whole body BMC was 5.8% 34

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Table 2 Regional bone assessment of lumbar spine, left femur of female Sprague-Dawley rats fed diets containing different doses of DHA at weeks 0, 5 and 10. Variable

Time

Diet Groups Control

BA (cm2)

BMC (g)

aBMD (g/cm2)

vBMD (mg/cm3)

BA (cm2)

BMC (g)

aBMD (g/cm2)

vBMD (mg/cm3)

0.1% DHA

Main effects

0.4% DHA

0.8% DHA

1.2% DHA

All

Lumbar spine wk0 wk5 wk10 All

0.97 ± 0.02 1.39 ± 0.02 1.42 ± 0.02 1.26 ± 0.04b

0.95 ± 0.02 1.40 ± 0.02 1.46 ± 0.02 1.27 ± 0.04ab

0.96 ± 0.02 1.42 ± 0.03 1.48 ± 0.03 1.29 ± 0.04 ab

0.99 ± 0.02 1.40 ± 0.02 1.43 ± 0.02 1.27 ± 0.04 ab

1.00 ± 0.02 1.43 ± 0.03 1.51 ± 0.03 1.31 ± 0.04a

0.97 ± 0.01C 1.41 ± 0.01B 1.46 ± 0.01A

wk0 wk5 wk10 All

0.19 ± 0.00 0.40 ± 0.01 0.44 ± 0.01 0.35 ± 0.02b

0.20 ± 0.01 0.43 ± 0.01 0.49 ± 0.01 0.37 ± 0.02a

0.20 ± 0.01 0.43 ± 0.01 0.50 ± 0.02 0.37 ± 0.02a

0.20 ± 0.01 0.42 ± 0.01 0.47 ± 0.01 0.36 ± 0.02ab

0.20 ± 0.01 0.43 ± 0.01 0.49 ± 0.01 0.37 ± 0.02a

0.20 ± 0.00C 0.42 ± 0.00B 0.48 ± 0.01A

wk0 wk5 wk10 All

0.20 ± 0.00 0.29 ± 0.01 0.31 ± 0.01 0.27 ± 0.01b

0.21 ± 0.00 0.30 ± 0.00 0.33 ± 0.01 0.28 ± 0.01a

0.21 ± 0.00 0.30 ± 0.00 0.34 ± 0.01 0.28 ± 0.01a

0.21 ± 0.00 0.30 ± 0.00 0.33 ± 0.00 0.28 ± 0.01a

0.20 ± 0.00 0.30 ± 0.00 0.32 ± 0.00 0.27 ± 0.01ab

0.20 ± 0.00C 0.30 ± 0.00B 0.33 ± 0.00A

wk0 wk5 wk10 All

400.9 ± 5.9 504.8 ± 10.4 534.5 ± 12.8 480.0 ± 11.2b

403.4 ± 4.2 519.3 ± 7.3 551.3 ± 9.6 491.3 ± 11.5ab

407.9 ± 5.4 525.8 ± 8.1 559.2 ± 8.6 497.6 ± 11.8a

404.8 ± 4.8 529.6 ± 5.2 561.4 ± 5.6 498.6 ± 11.8a Femur

405.1 ± 4.4 531.4 ± 5.5 567.2 ± 7.3 501.2 ± 12.2a

404.4 ± 2.2C 552.2 ± 3.5B 554.7 ± 4.2A

wk0 wk5 wk10 All

0.81 ± 0.01 0.97 ± 0.01 1.03 ± 0.01 0.93 ± 0.02b

0.82 ± 0.01 1.01 ± 0.02 1.11 ± 0.02 0.98 ± 0.02a

0.84 ± 0.02 1.02 ± 0.02 1.11 ± 0.02 0.99 ± 0.02a

0.82 ± 0.02 1.01 ± 0.02 1.06 ± 0.02 0.97 ± 0.02ab

0.81 ± 0.01 1.01 ± 0.02 1.04 ± 0.01 0.95 ± 0.02ab

0.82 ± 0.01C 1.00 ± 0.01B 1.07 ± 0.01A

wk0 wk5 wk10 All

0.20 ± 0.01 0.38 ± 0.01 0.43 ± 0.01 0.34 ± 0.02b

0.21 ± 0.01 0.42 ± 0.01 0.48 ± 0.01 0.37 ± 0.02a

0.22 ± 0.01 0.42 ± 0.01 0.47 ± 0.01 0.37 ± 0.02a

0.21 ± 0.01 0.41 ± 0.01 0.46 ± 0.01 0.36 ± 0.02ab

0.21 ± 0.01 0.42 ± 0.01 0.48 ± 0.01 0.37 ± 0.02a

0.21 ± 0.00C 0.41 ± 0.01B 0.46 ± 0.01A

wk0 wk5 wk10 All

0.25 ± 0.01 0.39 ± 0.01 0.42 ± 0.01 0.35 ± 0.01b

0.25 ± 0.01 0.41 ± 0.01 0.43 ± 0.01 0.36 ± 0.01ab

0.26 ± 0.01 0.41 ± 0.01 0.43 ± 0.01 0.36 ± 0.01ab

0.25 ± 0.01 0.40 ± 0.01 0.43 ± 0.01 0.36 ± 0.01ab

0.26 ± 0.01 0.42 ± 0.01 0.46 ± 0.01 0.38 ± 0.02a

0.25 ± 0.00C 0.41 ± 0.00B 0.43 ± 0.00A

wk0 wk5 wk10 All

473.8 ± 7.3 645.8 ± 10.4 696.3 ± 12.0 605.3 ± 17.1b

466.0 ± 7.2 648.6 ± 10.1 704.5 ± 10.2 606.3 ± 18.0b

476.1 ± 5.4 659.2 ± 10.0 717.6 ± 11.2 617.6 ± 18.1ab

470.9 ± 7.3 664.7 ± 6.8 717.6 ± 5.6 617.7 ± 18.3ab

483.8 ± 5.3 679.1 ± 6.8 736.8 ± 7.4 633.2 ± 18.7a

474.1 ± 2.9C 659.5 ± 4.2B 714.5 ± 4.5A

Interactions

p value Diet

p value Time

p value Diet x Time

0.03

<0.0001

0.65

0.005

<0.0001

0.28

0.001

<0.0001

0.53

0.004

<0.0001

0.75

0.001

<0.0001

0.50

0.0003

<0.0001

0.49

0.008

<0.0001

0.69

0.0001

<0.0001

0.75

Values are means ± SEMs, n = 12/group. wk0: baseline, wk5: midpoint, wk10: endpoint. Means in a row (diet) without a common superscript letter differ, P < 0.05, a > b > c. Means in a column (time) without a common superscript letter differ, P < 0.0001, A > B > C. No differences were observed among groups at baseline. BA, bone area; BMC, bone mineral content; aBMD, areal bone mineral density; vBMD, volumetric bone mineral density.

greater (P < 0.05) in the 0.1% DHA diet group compared to the 1.2% DHA diet group. Rats in all DHA diet groups showed greater (7.1%) whole body aBMD (P < 0.05) compared to the control group. Whole body vBMD was also increased (P < 0.05) in rats in the 0.4, 0.8 and 1.2% DHA diet groups compared to the control diet group and in rats in the 1.2% DHA diet compared to the 0.1% DHA diet group. Whole body BA, BMC, aBMD, and vBMD increased (P < 0.001) over time in all diet groups.

1.2% DHA diet groups compared to the control diet group. Left femur BA was increased (P < 0.05) in the 0.1 and 0.4% DHA diet group, 5.4% and 6.5% respectively compared to the control diet group. Furthermore, rats in the 0.1, 0.4 and 1.2% DHA diet groups all showed 8.8% higher left femur BMC than rats in the control group (P < 0.01). Left femur aBMD was also increased 8.6% in the 1.2% DHA diet group compared to the control diet group (P < 0.01). Moreover, higher left femur vBMD was observed in the 1.2% DHA diet group (∼ 4.5%) compared to the 0.1% DHA and control diet groups (P < 0.05).

3.3. Regional bone assessment of lumbar spine, femur and tibia

3.4. Trabecular microarchitecture assessment

There were no significant differences at baseline among diet groups for BA, BMC, aBMD, and vBMD of lumbar spine, left femur assessments (Table 2). Rats in the 1.2% DHA diet group showed greater (4.0%) lumbar spine BA compared to rats in the control diet group (P < 0.05). Lumbar spine BMC was also increased (P < 0.05) in the 0.1, 0.4 and 1.2% DHA diet group (5.7%) compared to the control diet group. A 3.6% greater lumbar spine aBMD was observed in rats in the 0.1, 0.4 and 0.8% DHA diet groups compared to the control diet group. Lumbar spine vBMD was 3 to 4% higher (P < 0.05) in rats in the 0.4, 0.8 and

No differences among diet groups were observed for lumbar spine (L3) in the ratio of bone volume to tissue volume, trabecular thickness and trabecular separation at the endpoint of the study (Table 3). However trabecular number of L3 vertebral body was decreased (P < 0.05) in the 1.2% DHA compared to the 0.1% diet group. Similarly, trabecular microarchitecture at the distal femur metaphysis was not altered by diet except for bone volume to tissue volume ratio which was higher (P < 0.05) in the 0.4% DHA diet group compared to the control 35

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Table 3 Trabecular microarchitecture of lumbar spine (L3), femur distal metaphysis and tibia proximal metaphysis of female Sprague-Dawley rats fed diets containing different doses of DHA for 10 wk. Variable

Lumbar Spine BV/TV (%) Tb.N. (mm−1) Tb.Th. (mm) Tb.S. (mm) Femur BV/TV (%) Tb.N. (mm−1) Tb.Th. (mm) Tb.S. (mm) Tibia BV/TV (%) Tb.N. (mm−1) Tb.Th. (mm) Tb.S. (mm)

Diet Groups

p value

Control

0.1% DHA

0.4% DHA

0.8% DHA

1.2% DHA

17.4 ± 0.4 1.92 ± 0.05ab 0.09 ± 0.00 0.24 ± 0.00

18.8 ± 0.5 2.05 ± 0.05a 0.09 ± 0.00 0.23 ± 0.00

18.2 ± 0.5 1.96 ± 0.05ab 0.09 ± 0.00 0.23 ± 0.00

17.1 ± 0.7 1.83 ± 0.07ab 0.09 ± 0.00 0.24 ± 0.00

17.0 ± 0.5 1.82 ± 0.06b 0.09 ± 0.00 0.24 ± 0.00

0.08 0.046 0.24 0.11

19.9 ± 1.5b 2.77 ± 0.20 0.07 ± 0.00 0.19 ± 0.01

26.5 ± 2.4ab 3.47 ± 0.23 0.08 ± 0.00 0.16 ± 0.01

27.0 ± 2.4a 3.47 ± 0.26 0.08 ± 0.00 0.17 ± 0.01

23.0 ± 0.9ab 3.01 ± 0.11 0.08 ± 0.00 0.17 ± 0.01

25.2 ± 1.5ab 3.28 ± 0.18 0.08 ± 0.00 0.16 ± 0.01

0.048 0.05 0.07 0.12

24.7 ± 2.7 3.20 ± 0.30 0.08 ± 0.00 0.22 ± 0.02

27.2 ± 2.6 3.54 ± 0.27 0.08 ± 0.00 0.19 ± 0.02

28.7 ± 2.7 3.52 ± 0.27 0.08 ± 0.00 0.19 ± 0.02

27.9 ± 1.9 3.45 ± 0.21 0.08 ± 0.00 0.19 ± 0.01

31.2 ± 2.7 3.76 ± 0.24 0.08 ± 0.00 0.17 ± 0.01

0.23 0.37 0.07 0.32

Values are means ± SEMs, n = 12/group. Means in a row without a common superscript letter differ, P < 0.05, a > b > c > d. BV/TV, bone volume to tissue volume ratio; Tb.N., Trabecular Number; Tb.Th., Trabecular Thickness; Tb.S., Trabecular Separation.

the 0.8% DHA diet (66.2 ± 11.7 pg/ng) in comparison to the 1.2% DHA diet group (37.2 ± 6.1 pg/ng).

group. 3.5. Cortical microarchitecture assessment

3.8. RBC fatty acid profile

There were no significant differences among diet groups for total cross-sectional area, cortical area, cortical area ratio and cortical thickness of lumbar spine (L2-L4) cortical microarchitecture assessment at baseline (Supplemental Table 1). Lumbar spine (L2-L4) total cross-sectional area was 4.5% greater (P < 0.05) in the 0.1% DHA diet group than the control diet group. Moreover, cortical area of lumbar spine (L2-L4) was also increased (P < 0.05) in the 0.1, 0.4 and 1.2% DHA diet groups compared to the control diet group. Rats in the 0.8 and 1.2% DHA diet groups showed an 3.4% increase (P < 0.01) in lumbar spine cortical area ratio compared to the control diet group. The cortical thickness of lumbar spine was higher (P < 0.05) in the three highest doses of DHA diet compared to the control diet. All cortical microarchitecture assessment of lumbar spine (L2-L4) were elevated (P < 0.05) over time in all diet groups except for cortical area ratio which was higher in both weeks 5 and 10 compared to week 0 (Supplemental Table 1).

RBC linoleic acid (LA, 18:2n-6) showed higher (P < 0.001) percentage in rats in the 0.4, 0.8 and 1.2% DHA compared to the control and 0.1% DHA diet groups (Table 5). Unlike linoleic acid, RBC alphalinolenic acid (ALA, 18:3n-3) percentage decreased (P < 0.01) in rats fed the 1.2% DHA compared to rats in the control and 0.1% DHA diet group, 27.3% and 20% respectively. Arachidonic acid (AA, 20:4n-6) decreased (P < 0.001) among the diet groups dose-dependently as the dose of DHA increased. Rats in the 0.4, 0.8 and the 1.2% DHA diet groups had a significant dose-dependent increase (P < 0.001) of EPA (20:5n-3) compared to rats in the control and the 0.1% DHA diet groups. DHA percentage increased (P < 0.001) between each diet group in a dose-dependent manner (Table 5). In agreement with RBC EPA and DHA%, omega 3 index (EPA+DHA), increased (P < 0.001) between each diet group dose-dependently as the dose of DHA increased. The n6/n3 fatty acid ratio decreased (P < 0.001) among the diet groups dose-dependently as the dose of DHA increased except between rats in the control and 0.1% DHA diet groups which showed no significant difference. RBC linoleic acid and alpha-linolenic acid percentages were significantly different in all time points with higher value in midpoint compared to baseline and the study endpoint. RBC EPA, DHA and EPA+DHA% showed increases (P < 0.001) over time in all diet groups, by contrast, RBC arachidonic acid% was lower (P < 0.001) at the study weeks 5 and 10 compared to baseline. RBC n6/n3 fatty acid ratio also decreased (P < 0.001) overtime. Except for alpha-linolenic acid, the interactions between diet and time were found for RBC fatty acids, omega 3 index and n6/n3 fatty acid ratio (P < 0.001). Profile of all fatty acids examined in RBC are presented in Supplemental Table 3.

3.6. Femoral bone strength Femoral length and anterior-posterior diameter at mid-diaphysis were not different among the diet groups (Table 4). Rats in the 0.4, 0.8 and 1.2% DHA diet groups showed higher (P < 0.05) peak force than rats in the control group. There were no significant differences among diet groups for peak stress, peak strain, energy at break and Young's modulus. 3.7. Blood biochemical markers of bone and mineral metabolism There were no significant differences among diet groups at baseline for any of ionized blood biomarkers measured. No differences were observed among diet groups for ionized calcium, ionized potassium, ionized sodium, hematocrit, plasma osteocalcin, plasma PTH. However, ionized chloride was decreased (P < 0.05) in the 1.2% DHA diet group compared to the 0.1% DHA diet and the control diet group (Supplemental Table 2). Serum OPG was not changed by diet. Serum RANKL was elevated (P < 0.05) in the 0.8% DHA diet (43.9 ± 6.4 pg/mL) compared to other DHA diet groups on average of 24.9 ± 2.5 pg/mL, but not to the control diet group. RANKL:OPG ratio was also increased (P < 0.05) in

3.9. Association between serum RANKL/OPG ratio and whole body BMC accretion and aBMD There was a negative correlation between serum RANKL/OPG ratio and whole body BMC accretion (from weeks 0 to 10) (r = −0.35, p = 0.008, n = 60). Serum RANKL/OPG ratio was also negatively correlated with whole body aBMD (r = −0.31, p = 0.018, n = 60) at weeks 10 of the study (Fig. 1). 36

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Table 4 Bone strength of right femur at mid-diaphysis of female Sprague-Dawley rats fed diets containing different doses of DHA for 10 wk. Variable

Length (mm) Mid-diaphyseal diameter (mm) Peak force (N) Peak stress (N/mm2) Peak strain (%) Energy at break (mJ) Young's modulus (N/mm2)

Diet Groups

P value

Control

0.1% DHA

0.4% DHA

0.8% DHA

1.2% DHA

33.9 ± 0.2 3.10 ± 0.05 137.4 ± 3.7b 59.8 ± 3.1 11.7 ± 0.6 53.9 ± 4.3 105180 ± 7450

34.0 ± 0.2 3.25 ± 0.04 150.3 ± 3.9ab 61.7 ± 2.3 13.1 ± 1.0 72.5 ± 5.5 96286 ± 5175

34.3 ± 0.4 3.24 ± 0.06 155.1 ± 5.6a 64.8 ± 3.0 13.4 ± 0.7 70.0 ± 4.8 109651 ± 5408

34.0 ± 0.3 3.24 ± 0.04 156.7 ± 3.3a 64.5 ± 3.9 12.4 ± 0.6 67.1 ± 4.9 110481 ± 7642

33.7 ± 0.2 3.23 ± 0.04 154.4 ± 4.0a 60.3 ± 2.8 12.9 ± 0.6 65.3 ± 3.3 102862 ± 5403

0.58 0.10 0.012 0.62 0.48 0.06 0.49

Values are means ± SEMs, n = 12/group. Means in a row without a common superscript letter differ, P < 0.05, a > b > c > d.

3.10. Association between RBC DHA percentages and left femur vBMD

spine and long bone in the order of 5.7 to 8.8% higher BMC compared to the control, in addition to 3.6% to 10.0% higher aBMD and vBMD. An increase in cortical bone microarchitecture parameters of lumbar spine as well as peak force by dietary DHA was a further indication of improvement in bone strength and structural properties. These observations were complemented by a negative association between serum RANKL/OPG ratio and whole body BMC accretion and aBMD. The present study demonstrates that DHA is capable of increasing bone mineral accretion and strength in female rats when consumed during and after sexual maturation, therefore positively contributing towards optimal PBM. Achieving optimal PBM during growth is a preventive strategy to

There was a positive correlation between RBC DHA and left femur vBMD (r = 0.28, p = 0.034, n = 60) at weeks 10 of the study (Fig. 2). 4. Discussion To our knowledge, this is the first study investigating the dose-response of BMC and BMD to dietary DHA in female rats from early sexual maturity to mature adult ages that would reflect acquisition of PBM. The current study implies that dietary DHA had an augmenting effect on BMC both at the whole body and regional levels including lumbar

Table 5 Red blood cell fatty acids profile of female Sprague-Dawley rats fed diets containing different doses of DHA at weeks 0, 5 and 10. RBC Fatty Acids (% of total fatty acids)

LA (18:2n-6)

ALA (18:3n-3)

AA (20:4n-6)

EPA (20:5n-3)

DHA (22:6n-3)

EPA + DHA (20:5n-3 + 22:6n-3)

n6/n3 fatty acid ratio

Time

Diet groups

Main effects

Control

0.1% DHA

0.4% DHA

0.8% DHA

1.2% DHA

All

wk0 wk5 wk10 All

8.3 ± 0.2 8.5 ± 0.2 7.4 ± 0.3 8.1 ± 0.2b

8.0 ± 0.2 8.8 ± 0.3 7.8 ± 0.3 8.2 ± 0.2b

8.2 ± 0.2 10.1 ± 0.2 8.9 ± 0.2 9.1 ± 0.2a

8.1 ± 0.2 10.3 ± 0.2 9.1 ± 0.3 9.2 ± 0.2a

8.3 ± 0.2 10.4 ± 0.3 9.5 ± 0.3 9.4 ± 0.2a

8.2 ± 0.1C 9.6 ± 0.2A 8.6 ± 0.2B

wk0 wk5 wk10 All

0.06 ± 0.01 0.15 ± 0.01 0.12 ± 0.02 0.11 ± 0.01a

0.06 ± 0.00 0.13 ± 0.01 0.12 ± 0.02 0.10 ± 0.01a

0.06 ± 0.00 0.13 ± 0.01 0.10 ± 0.01 0.10 ± 0.01ab

0.06 ± 0.01 0.11 ± 0.01 0.08 ± 0.01 0.09 ± 0.01ab

0.06 ± 0.00 0.10 ± 0.01 0.08 ± 0.01 0.08 ± 0.01b

0.06 ± 0.00C 0.12 ± 0.00A 0.10 ± 0.01B

wk0 wk5 wk10 All

24.4 ± 0.9 27.7 ± 0.3 27.8 ± 0.2 26.7 ± 0.4a

24.2 ± 0.8 25.4 ± 0.4 26.4 ± 0.4 25.3 ± 0.4b

25.4 ± 0.6 22.0 ± 0.3 22.0 ± 0.5 23.1 ± 0.4c

26.0 ± 0.4 18.4 ± 0.2 17.9 ± 0.3 20.7 ± 0.7d

25.6 ± 0.5 14.8 ± 0.3 13.5 ± 0.3 18.0 ± 0.9e

25.1 ± 0.3A 21.7 ± 0.6B 21.5 ± 0.7B

wk0 wk5 wk10 All

0.29 ± 0.02 0.50 ± 0.03 0.49 ± 0.02 0.43 ± 0.02d

0.29 ± 0.02 0.59 ± 0.03 0.62 ± 0.02 0.50 ± 0.03d

0.31 ± 0.02 1.16 ± 0.08 1.21 ± 0.09 0.89 ± 0.08c

0.31 ± 0.02 2.24 ± 0.10 2.50 ± 0.11 1.68 ± 0.17b

0.31 ± 0.02 3.50 ± 0.23 4.48 ± 0.30 2.76 ± 0.32a

0.30 ± 0.01C 1.60 ± 0.16B 1.86 ± 0.20A

wk0 wk5 wk10 All

3.1 ± 0.2 3.7 ± 0.1 3.8 ± 0.1 3.5 ± 0.1e

3.2 ± 0.2 4.9 ± 0.1 5.2 ± 0.1 4.4 ± 0.2d

3.5 ± 0.1 7.0 ± 0.2 7.5 ± 0.2 6.0 ± 0.3c

3.7 ± 0.1 9.0 ± 0.1 9.5 ± 0.2 7.4 ± 0.5b

3.4 ± 0.1 10.7 ± 0.3 11.7 ± 0.3 8.6 ± 0.6a

3.4 ± 0.1C 7.1 ± 0.4B 7.5 ± 0.4A

wk0 wk5 wk10 All

3.4 ± 0.2 4.2 ± 0.1 4.3 ± 0.1 4.0 ± 0.1e

3.5 ± 0.2 5.5 ± 0.2 5.9 ± 0.1 4.9 ± 0.2d

3.8 ± 0.1 8.2 ± 0.3 8.7 ± 0.3 6.9 ± 0.4c

4.0 ± 0.1 11.3 ± 0.2 12.0 ± 0.2 9.1 ± 0.6b

3.7 ± 0.1 14.2 ± 0.3 16.1 ± 0.4 11.4 ± 0.9a

3.7 ± 0.1C 8.7 ± 0.5B 9.4 ± 0.6A

wk0 wk5 wk10 All

7.1 ± 0.2 6.5 ± 0.2 6.0 ± 0.1 6.5 ± 0.1a

6.8 ± 0.3 5.2 ± 0.1 4.9 ± 0.1 5.6 ± 0.2b

6.5 ± 0.2 3.5 ± 0.1 3.2 ± 0.1 4.4 ± 0.3c

6.2 ± 0.2 2.4 ± 0.0 2.1 ± 0.1 3.6 ± 0.3d

6.7 ± 0.2 1.7 ± 0.0 1.4 ± 0.0 3.3 ± 0.4e

6.7 ± 0.1A 3.9 ± 0.2B 3.5 ± 0.2C

Interactions

P value Diet

P value Time

P value Diet x Time

<0.0001

<0.0001

<0.0001

0.0034

<0.0001

0.09

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

<0.0001

Means ± SEMs, n = 12/group. Means in a row (diet) without a common superscript letter differ, P < 0.05, a > b > c. Means in a column (time) without a common superscript letter differ, P < 0.0001, A > B > C. No differences were observed among groups at baseline. RBC, red blood cell; ALA, alpha-linolenic acid; LA, Linoleic acid; AA, Arachidonic acid; EPA, Eicosapentaenoic acid; DHA, Docosahexaenoic acid; n6/n3 fatty acid ratio, Total n-6 fatty acid/total n-3 fatty acid. 37

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reduce the risk of osteoprosis. The beneficial role of DHA in bone mass and bone mineral accretion in the current study aligns with some human research. For instance, Hogstrom et al. [24] found a positive relationship between serum DHA concentration and total and spine BMD in 17-year-old Caucasian healthy males over a period of 7 years. Furthermore, serum DHA concentration was positively associated with lumbar spine aBMD in 8 year old Caucasian children [25]. However, these studies did not consider dietary consumption of DHA necessary to achieve these results in addition to serum DHA levels. On the other hand, the increases in BMC, BMD and BA in lumbar spine are contrary to previous animal studies that showed DHA had no impact on this region [26,27]. Dietary intervention with 0.6% DHA from menhaden oil for 5 weeks did not affect lumbar spine bone mineral accretion in male and female Sprague-Dawley rats. The existence of other fatty acids like eicosapentaenoic acid along with DHA in diet mixtures can cause conflicting results compared to using diets containing semi-purified DHA, as used in the present study. In addition to that, the study time course of 5 weeks was likely insufficient to produce a significant effect [27]. Furthermore, Li's study [26] was conducted in male rats which may not be comparable to the current study due to the sexual dimorphism in the action of DHA on bone [28]. To expand our knowledge beyond bone density assessments, this study explored the effect of DHA on lumbar spine microarchitecture using a growing rat model. An aging mouse model similarly found that the caudal vertebra bone to tissue volume ratio and trabecular number were significantly higher after 5 months of a diet with 0.1% DHA compared to controls, however all significance disappeared after an additional 9 months feeding [29]. Perhaps during advanced aging and an estrogen deficient state higher doses of DHA are required as presented in this study. Whether the changes in lumbar spine microarchitecture as observed in the present study persist over a longer period of time and after menopausal ages remain open for investigation. The beneficial effects of DHA on BMC and aBMD of long bone are consistent with Lukas et al's study which found a significant increase in femur bone mass of growing female Sprague-Dawley rats in the 0.3% DHA diet group with DHA provided in the form of tuna oil [30]. In addition to increases in femur microarchitecture properties in response to dietary DHA, an elevation of peak load at the femoral mid-diaphysis was also observed in DHA doses ranging from 0.4 to 1.2%. This suggests that the main effects of DHA on bone mass translate to increased bone strength. The positive correlation between circulating DHA levels and higher BMC and femoral peak load was also found in young fat-1 mice [31], implying that DHA is implicated in the prevention of fractures. Interestingly, we observed that the same increase in BMC in response to dose ranges of 0.1, 0.4 and 1.2% of dietary DHA in lumbar spine and long bone in addition to higher whole body BMC in the 0.1% DHA in comparison to 1.2% DHA diet, suggesting that higher doses do not necessarily display greater beneficial effects during growth. In line with present findings, we have recently indicated 0.4% dietary DHA as an effective dose in enhancing lean mass [19]. It now remains to be tested whether this dose of DHA is an optimal dose for improving overall musculoskeletal health in other species. In addition to study the overall effect of DHA on bone mineral accretion and bone strength, it was also of interest to examine how dietary DHA affects RBC fatty acid profiles as cells originating from bone marrow. The present study showed that DHA was effectively incorporated into RBC in a dose-dependent manner. Furthermore, we observed a positive association between RBC DHA and left femur vBMD which is in agreement with Atkinson's study that showed an increase in the amount of femoral bone marrow as well as the number of bone marrow cells in Fischer rats fed 4.3% dietary DHA [32]. The higher number of bone marrow cells can increase the potential for osteoblastogenesis [33]. Moreover, DHA accumulation within the marrow and periosteum of the femur and its positive correlation with femur BMC were also reported in growing rats fed milk supplemented with 1% DHA [26]. Accumulation of DHA with the osteoblast-rich and nerve-

W B B M C A c c r e tio n w e e k s 0 to 1 0 (g /w k )

A 1 .0

0 .8

0 .6 C o n tro l d ie t 0 .1 % D H A d ie t

0 .4

0 .4 % D H A d ie t

r = - 0 .3 5

0 .8 % D H A d ie t

P = 0 .0 0 8

1 .2 % D H A d ie t

0 .2 0 .0 0

0 .0 5

0 .1 0

0 .1 5

R A N K L /O P G r a t io

B

0 .1 8

2

W B a B M D ( g /c m )

0 .1 9

0 .1 7

0 .1 6 C o n tro l d ie t 0 .1 % D H A d ie t

0 .1 5

0 .4 % D H A d ie t

r = - 0 .3 1

0 .8 % D H A d ie t

P = 0 .0 1 8

1 .2 % D H A d ie t

0 .1 4 0 .0 0

0 .0 5

0 .1 0

0 .1 5

R A N K L /O P G r a t io

Fig. 1. Correlation between serum RANKL/OPG ratio and whole body BMC accretion (weeks 0 to 10) (A) and whole body aBMD (B) of female SpragueDawley rats (n = 60) fed diets containing different doses of DHA for 10 wk. RANKL/OPG ratio, receptor activator of nuclear factor κβ ligand/ osteoprotegerin ratio; BMC, bone mineral content; aBMD, areal bone mineral density; WB, whole body.

3

L e f t F e m u r v B M D ( m g /c m )

800

750

700

C o n tro l d ie t

650

0 .1 % D H A d ie t r = 0 .2 8

0 .4 % D H A d ie t

P = 0 .0 3 4

0 .8 % D H A d ie t 1 .2 % D H A d ie t

600 3

6

9

12

15

R B C D H A (% o f to ta l fa tty a c id s )

Fig. 2. Correlation between RBC DHA percentages and left femur vBMD of female Sprague-Dawley rats (n = 60) fed diets containing different doses of DHA for 10 wk. RBC, red blood cell; vBMD, volumetric bone mineral density.

38

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abundant periosteum of femur suggests that DHA is available and perhaps necessary for healthy bone modeling [26]. It appears that DHA by increasing osteoblastogenesis and therefore bone formation, contributes to achieve PBM. Although this dose-response study using a semi-purified DHA diet provides new insight into how DHA affects bone mineralization and strength, future molecular investigation is required to identify the mechanisms of DHA action by targeting molecular RANKL/RANK/OPG pathway. Furthermore, the semi-purified DHA experimental diets also included small amounts of other fatty acids (e.g. 12:0, 14:0) and the diet had less LA and ALA [19]. Future studies should also account for these fatty acids in the control diet. Conducting the study only in female rats due to the sexual dimorphism in DHA action on the bone is a strength of the present study, which however needs future studies to examine whether the same dose response pattern is observed in males. Dietary DHA ranging from 0.1 to 1.2% of diet by weight enhanced various biomarkers of bone health, including bone mineral accretion and strength, that are well known as protective against osteoporosis and fracture. These alterations contribute to achieve optimal PBM during growth, however whether it ultimately improves final PBM and retention with aging requires extended studies of longer durations. The overall importance of the findings of this study is that DHA can help to improve PBM when consumed during and after sexual maturation.

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Conflict of interest All authors report no conflicts of interest. Author contributions HW, MTF and ZF: designed the research study; HW, MTF, PL and ZF: were involved in conducting the research; ZF and HW: performed the statistical analyses; ZF and HW: interpreted the data and critically evaluated and revised the final manuscript; ZF: drafted the manuscript; ZF, MTF, PL, and HW: contributed to the intellectual content; All authors: read and approved the final manuscript. Acknowledgement We thank Sherry Agellon for her contribution in fatty acid analyses. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.plefa.2019.04.005. References [1] T. Coughlan, F. Dockery, Osteoporosis and fracture risk in older people, Clin. Med. (Lond.) 14 (2014) 187–191. [2] M.C. Kruger, M. Coetzee, M. Haag, H. Weiler, Long-chain polyunsaturated fatty acids: selected mechanisms of action on bone, Prog. Lipid Res. 49 (2010) 438–449. [3] R. Burge, B. Dawson-Hughes, D.H. Solomon, J.B. Wong, A. King, A. Tosteson, Incidence and economic burden of osteoporosis-related fractures in the United States, 2005-2025, J. Bone Miner. Res. 22 (2007) 465–475. [4] N.O. Foundation, America's Bone Health: the State of Osteoporosis and Low Bone Mass in Our Nation, National Osteoporosis Foundation, Washington, DC, 2002, pp. 1–55. [5] D. Cech, Prevention of osteoporosis: from infancy through older adulthood, Hong Kong Physiother. J. 30 (2012) 6–12. [6] R. Heaney, Achieving the protection of high peak bone mass, Osteoporos. Int. 27 (2016) 1279–1280. [7] R. Heaney, S. Abrams, B. Dawson-Hughes, A. Looker, R. Marcus, V. Matkovic, C. Weaver, Peak bone mass, Osteoporos. Int. 11 (2000) 985–1009. [8] A. Prentice, Diet, nutrition and the prevention of osteoporosis, Public Health Nutr. 7

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