The effects of dietary calcium (Ca) deficiency on skeletal integrity are well characterized in growing and mature mammals; however, less is known about Ca nutrition during the neonatal period. In this study, we examined the effects of neonatal Ca nutrition on bone integrity, endocrine hormones, and mesenchymal stem cell (MSC) activity. Neonatal pigs (24 ± 6 h of age) received either a Ca-adequate (1.2 g/100 g) or an ~40% Ca-deficient diet for 18 d. Ca deficiency reduced (P < 0.05) bone flexural strength and bone mineral density without major differences in plasma indicators of Ca status. There were no meaningful differences in plasma Ca, phosphate (PO4), parathyroid hormone, or 1,25-dihydroxycholecalciferol due to Ca nutrition throughout the study. Calcium deficiency also reduced (P < 0.05) the in vivo proliferation of MSC by ~50%. In vitro studies utilizing homologous sera demonstrated that MSC activity was affected (P < 0.05) by both the Ca status of the pig and the sera as well as by their interaction. The results indicate that neonatal Ca nutrition is crucial for bone integrity and suggest that early-life Ca restriction may have long-term effects on bone integrity via programming of MSC.

Quercetin (Q), a naturally occurring flavonoid with antioxidant and anti-inflammatory properties, has been reported to have an ergogenic effect on aerobic performance and to increase VO2max. PURPOSE: Compare effects of 7 days Q supplementation on aerobic performance and metabolic markers during two days of acute strenuous exercise. METHODS: Sixteen men (22 ± 3 yrs, 177 ± 7 cm, 86 ± 10 kg) completed a 3-day trial in a randomized, double-blinded crossover design. VO2max was measured on day 1 using an uphill treadmill running protocol. Days 2 & 3 involved 75 minutes of treadmill walking at 50-55% VO2max while wearing a 15 kg vest, followed by a 200kJ cycle ergometer time trial (TT). Volunteers completed the battery after consuming 1000 mg Q or placebo (P) bars for 7 days. Blood was drawn four times: before a standard meal (REST), pre-TT (PRE), immediately post-TT (POST), and one hour post-TT (+60). Plasma was analyzed for Q, glycerol, glucose, non-esterified fatty acids (NEFA) and triglycerides. Repeated measures ANOVA and t-tests were used as appropriate. RESULTS: Q supplementation resulted in elevated plasma Q concentrations (Q: 231 ± 41 ng/mL, P: 24 ± 5 ng/mL; p<0.01). Q had no effect on TT duration (Q: 1086 ± 42 sec, P: 1097 ± 47 sec), VO2max (Q: 48.9 ± 0.8 ml/kg/min, P: 49.4 ± 0.8 ml/kg/min), or plasma metabolic markers (see Table 1). Plasma metabolic markers were altered by exercise (p<0.05). CONCLUSION: 7 days of supplementation with quercetin did not improve aerobic performance, change VO2max, or alter plasma markers of metabolism in response to exercise.Table

Effects of dietary calcium (Ca) deficiency on skeletal integrity and endocrine parameters are well characterized in growing and mature mammals; however, little work has examined Ca nutrition during the neonatal period. In this study, we examined the effects of neonatal Ca nutrition on bone integrity, endocrine parameters, and in vivo mesenchymal stem cell (MSC) activity. Neonatal pigs (24 ± 6h post‐partum) were pair‐fed either a Ca adequate or a 30% Ca deficient liquid formula diet for 18 days. There were no differences in growth rate or feed conversion efficiency based on dietary Ca level and all pigs grew at a rate similar to sow‐reared pigs. As anticipated, dietary Ca deficiency reduced (P < 0.05) both BMD and bone flexural strength. The anticipated increase (P < 0.05) in plasma PTH levels in pigs fed the Ca deficeint diet was not evident until the end of the study. Surprisingly, dietary Ca level did not affect plasma Ca or 1,25(OH) 2 vitamin D concentrations throughout the study. Calcium deficiency reduced (P < 0.05) the in vivo proliferation of MSC isolated from bone marrow by approximately 50%. These results indicate that neonatal Ca nutrition is crucial for bone integrity and suggest that early life Ca restriction may have long‐term effects on bone integrity via its effects on MSC activity.

Quercetin (Q), a naturally occurring flavonoid, has antioxidant and anti-inflammatory properties and has been reported to have an ergogenic effect during prolonged strenuous exercise. PURPOSE: To compare the effects of Q supplementation on measures of fatty acid metabolism and perceived exertion and discomfort during two days of strenuous exercise. METHODS: Nine men (22 ± 3 yrs, 176 ± 5 cm, 83 ± 10 kg) completed a two day battery of loaded treadmill marching in a randomized, double-blinded crossover design. Each day involved 75 minutes of loaded marching (LM) at 50%-55% VO2max while wearing a 15 kg vest. During LM respiratory exchange ratio (RER) was measured using closed circuit spirometry. Heart rate (HR), rating of perceived exertion (RPE), and rating of perceived pain, soreness and discomfort (PSD) were also recorded at these times. Volunteers first completed a baseline two day battery with no experimental treatment (B) and then after being given 500 mg Q or placebo (P) at 8 AM and 8 PM via a First StrikeTM bar for 7 days. Energy intake was monitored via food records. Blood was drawn before volunteers ate a standard meal prior to LM. Blood was collected again post-LM. Plasma was analyzed for Q and glycerol. ANOVA for repeated measures was used for all analyses. RESULTS: Supplementation with Q elevated plasma Q concentration (B=35.2 ng/ml, P= 41.8 ng/ml, Q=332 ng/ml). Plasma glycerol was similar between B, P and Q (p=0.27) as well as across testing days (day 1 vs. day 2) (p=0.23). RER, HR, RPE and PSD values were similar for all conditions.Table 1: Plasma glycerol and RER at baseline testing and after supplementation with placebo or Q (n=9).CONCLUSIONS: Our results suggest Q does not affect fatty acid metabolism, as measured by glycerol concentrations and indirect calorimetry, RPE or perceived discomfort from submaximal steady state exercise.

PURPOSE: To assess the effects of 7 days of quercetin ingestion on aerobically demanding Soldier performance. METHODS: In a double-blind crossover study, 9 male Soldiers (age =22±3 yrs, height =176±5 cm, weight=83±10 kg) were trained to perform 3 days of aerobic exercise under 3 conditions: Baseline (B), Placebo (P) and Quercetin (Q). Day 1 was an uphill treadmill running VO2peak test. Days 2 and 3 were identical and consisted of both loaded marching (LM) and a cycling time trial (TT). For LM Soldiers wore a 15 kg vest and walked for 75 min at 50% VO2peak. The subsequent TT was performed on a Lode Excaliber with the linear mode set to elicit 50% treadmill VO2max at 60 rpm. Soldiers completed 200kJ of work as quickly as possible. Following the B measurement, Soldiers consumed an energy bar at 8 AM and 8 PM, each containing 500 mg of quercetin (Q) or placebo (P) for a total intake of 1000mg per day for 8.5 days. After 6 days of consumption, Soldiers performed the 3 exercise days. Following a washout period, the process was repeated with the second bar. Order of bar presentation was randomized across subjects. Diet and activity levels were monitored and controlled for 3 days prior to and during each testing period. Repeated measures ANOVAs were run to examine performance following supplementation. RESULTS: There was a significant (p<0.05) increase in plasma Q following Q supplementation. There were no differences after P or Q supplementation in VO2peak, nor in TT time on Day 1 or Day 2 as compared to B. In addition, Q did not alter maximal heart rate or RPE at VO2peak or during the TT.TABLE 1: VO2 peak and Time Trial Time at Baseline and after Placebo or Quercetin SupplementationCONCLUSION: Supplementation of 1000 mg/day of Quercetin for 7+ days increased the plasma Q eight-fold, but had no effect on aerobically demanding Soldier performance.

Dietary zinc (Zn) is important for bone remodeling. Phytase (P) increases the bioavailability of Zn and other micronutrients. How modeling adaptations of the lumbar spine may be affected by a P supplemented low Zn diet has not been investigated.PURPOSEDetermine the effects of a low Zn diet and supplemental P on the 4th lumbar vertebra (L‐4) in exercising rats.METHODS30 Male Sprague‐Dawley rats were housed in cages fitted with voluntary running wheels and fed either a 5ppm Zn (ZnLo), 5ppm Zn + 1,500 FTU/kg P (ZnLo+P) or a 30ppm Zn (ZnAd) diet for 9 wks, (n=10 per group). In vivo peripheral quantitative computed tomography scans were performed at 40, 50 and 60% of the total L‐4 length. Cortical content (CNT) (mg), area (A) (mm2) and bone mineral density (BMD) (mg/cm3) were measured. Plasma concentrations (PC) (µg/ml) of Zn were determined. A one‐way ANOVA was used to compare the data.RESULTSZnLo+P exhibited increased CNT (11.6 ± 1.5 vs. 9.9 ± 0.7, p=0.006), A (12.0 ± 1.4 vs. 10.5 ± 0.7, p=0.007) and BMD (959.5 ± 15.9 vs. 938.8 ± 7.9 p=0.012) compared to ZnLo and increased BMD (959.5 ± 15.9 vs. 942.5 ± 18.9, p=0.043) compared to ZnAd. ZnAd exhibited increased PC of Zn (1.2 ± 0.2 vs. 0.8 ± 0.1 and 0.5 ± 0.2, p<0.001) compared to ZnLo+P and ZnLo respectively. ZnLo+P exhibited increased PC of Zn (0.8 ± 0.1 vs. 0.5 ± 0.2, p=0.01) compared to ZnLo.CONCLUSIONP supplementation increased PC of Zn and increased mid‐vertebral cortical CNT, A and BMD in a ZnLo fed sample. The greatest L‐4 cortical modeling was observed in rats fed a ZnLo+P diet.The study was funded by the U.S. Army MRMC

Vertical jump testing is a field expedient method for evaluating whole-body peak power output. While Vertec (VER) is one of the most widely used vertical jump test methods, there are limited data confirming that VER results are comparable to lab biomechanical methods to assess power output. PURPOSE: Compare the VER method of measuring jump height and peak power output with two lab-based methods. METHODS: 74 male US Army Soldiers (mean±SD: age 24.2±5.1 yrs, body mass 81.3±12.6 kg, stature 176.5±7.3 cm, body mass index 26.2±3.6) performed a series of 3 maximum effort vertical jumps. Jump performance was simultaneously measured using 3 different methods. Subjects jumped from a Leonardo (LEO) dual-forceplate with a VER vertical jump test device as their jump and reach target. These jumps were recorded for motion analysis with a digital video camera interfaced with Dartfish (DF) motion analysis software. The LEO provided a direct measurement of peak power output and a calculated estimate of jump height. Peak power output estimations from the VER and DF were made using the equation of Harman et al. (1991) which uses body mass and jump height as inputs. Results were compared between methods using a one-way ANOVA with Tukey HSD. Effect sizes were calculated to express differences between means relative to a pooled SD. RESULTS: For jump height (cm) there were no differences between LEO (52.6±8.0) and DF (53.0±7.3) methods. However, VER jump height (49.5±8.0) was significantly less than LEO and DF (p<0.011). There were no differences in peak power output (kw) between the 3 methods (VER = 4.2±0.6, LEO = 4.4±0.6 and DF = 4.3±0.8). Jump height and peak power output showed strong positive correlations between all methods (r≥0.81). While significant differences in jump height were observed between VER and the other methods, effect sizes for these comparisons (.058 -.450) indicate weak practical differences. CONCLUSION: VER is a robust and valid method for determining jump height and peak power output in the field.

Adding vitamin D to a low calcium diet is known to effect trabecular but not cortical bone. Although adding phytase (P) to a low zinc (Zn) diet improves overall bone strength by increasing the bioavailability of Zn and other micronutrients, it is unknown if this supplementation has a differential effect on cortical and trabecular bone.PurposeExamine the effect of P‐supplementation to a low Zn diet in cortical and trabecular bone in both, L‐4 vertebrae and tibiae of rats.MethodsMale Sprague‐Dawley rats were fed a diet containing either 5ppm Zn (ZnLo) (n=10), 5ppm Zn+1,500FTU/kg P (ZnLo+P) (n=10), or 30ppm Zn (ZnAd) (n=10) for 9 wks. In vivo peripheral quantitative computed tomography was used to scan right tibiae at 10% (trabecular parameters) and 50% (cortical parameters) of total. The L‐4 vertebrae were scanned at the 40, 50, and 60% of total length; an average of these represented the mid‐20% vertebral region. One way ANOVA determined group differences for cortical and trabecular bone mineral content (BMC), bone area (BA) and bone mineral density (BMD).ResultsCortical parameters at the L‐4 were significantly higher in rats fed ZnLo+P compared to ZnLo diets. There was no significant difference between groups in trabecular parameters.ConclusionIn contrast to the vitamin D:calcium relationship, P‐supplementation of a low Zn diet exhibited a positive effect on cortical BMD, BMC and BA at the L‐4 site, but had no effect on trabecular or tibial parameters.The study was funded by the U.S.Army MRMC

It is not clear how the adaptations to chronic exercise may be affected by a phytase (P) supplemented low zinc (Zn) diet. Phytic acid (PA) is known to form insoluble complexes with nutritionally essential minerals; including Zn. Phytases are enzymes that aid the removal of essential minerals from PA, thus increasing their bioavailability. PURPOSE: To determine the effects of a low Zn diet and supplemental P on bone and muscle in voluntary exercising rats. METHODS: 30 male Sprague-Dawley rats were housed in cages fitted with voluntary running wheels and fed either a 5ppm Zn (ZnLo), 5ppm Zn+1,500 FTU/kg P (ZnLo+P) or a 30ppm Zn (ZnAd) diet for nine weeks, (n=10 per group). In vivo peripheral quantitative computed tomography scans were performed at 10 and 50% of the total tibia length. Cortical content (CCNT) (mg), cortical area (CA) (mm2) and polar strength strain index (SSIp) (mm3) were investigated at the 10 and 50% regions of interest (ROI). Muscle area (MA) (mm2) was additionally investigated at the 50% ROI. A one-way ANOVA and Tukey post-hoc were used to analyze the data. RESULTS: At the 10% ROI, ZnAd exhibited increased CCNT and greater SSIp as compared to ZnLo and ZnLo+P. At the 50% ROI, ZnAd exhibited increased CCNT, CA and greater SSIp as compared to ZnLo and increased MA as compared to ZnLo and ZnLo+P. Additionally at the 50% ROI, ZnLo+P exhibited increased CCNT and MA as compared to ZnLo.TableCONCLUSION: P-supplementation increased mid-shaft CCNT and MA in a moderately Zn deficient sample but did not match the superior indices of bone strength and lean mass that were observed by consuming a 30ppm Zn diet.

To assess metabolic defects resulting from moderate zinc (Zn) deficiency, we observed the effects of dietary Zn on body composition and bone structure in voluntary exercising rats. Sprague-Dawley rats (n=72) were fed moderately Zn-deficient (5ppm, ZnLO) or Zn-adequate (30ppm, ZnAD) diets ad libitum, and divided into exercise (Ex) or sedentary (NoEx) groups, in a 2×2 design. NoEx rats were significantly heavier from the 2nd week, and no catch-up growth occurred in Ex rats. Feed intakes (24hr) were not different between groups throughout the study. After 9 weeks, Ex rats had decreased whole body bone mineral content (BMC) and bone area (BA), with no change in bone mineral density (BMD), as determined by in vivo DXA analysis. In Ex rats, the ZnLO diets exacerbated the significantly reduced fat mass indices. In vivo pQCT analysis demonstrated that Ex rats had higher cortical BMD and smaller medullary BA in mid-shaft tibial region (C50), indicative of stronger bones. These indices were Zn-insensitive. Conversely, to accommodate the significantly heavier body mass, sedentary ZnLO rats exhibit increased total BA at C50, to include increased endosteal and periosteal circumferences, signifying a thinner cortical shell. These indices of bone health are reversed when dietary Zn is provided at the recommended 30ppm to sedentary rats, as there were no differences between NoEx-ZnAD, Ex-ZnLO, and Ex-ZnAD rats. The findings confirm that bone quality is vulnerable to disruption by moderate Zn deficiency in sedentary animals.

Bone size and mineralization have been shown to contribute to stress fracture, a bone overuse injury. Characterization of these parameters of bone strength in new recruits may help to understand observed gender differences in the incidence of stress fracture. PURPOSE: To identify gender differences in density and geometry of the tibia, a frequent site of stress fracture. METHODS: Fifty-eight Israeli Defense Forces (IDF) basic training soldiers (11 men, 47 women; age 18–21 yr) volunteered to participate in this study. Peripheral quantitative computed tomography (pQCT) scans were taken at 4% and 38% proximal to the tibial endplate prior to entering basic combat training. Measures included bone mineral content (BMC), bone area, bone mineral density (BMD), and a cortical polar strength strain index (SSIp). A one-way ANOVA was used to analyze gender differences in trabecular parameters at the 4% site and cortical parameters at the 38% site. RESULTS: As seen in the table below, women had lower BMC and smaller area at the 4% and 38% sites compared to the men, and a decreased SSIp at the 38% site. Women had higher cortical BMD than men, while trabecular BMD did not differ between genders.TableDISCUSSION: Gender adequately differentiated bone mineral and geometric properties in our sample. The comparatively increased density at the 38% site in women in the context of the decreased area at the 4% and 38% sites suggests that midshaft cortical density increases to compensate for a more slender bone. Gender differences in size, rather than BMD, may provide an explanation for the higher incidence of stress fracture seen in women.

The ability of short-term periodized exercise programs to influence parameters of bone strength has not been well studied. PURPOSE: To determine if 13-week exercise interventions influence parameters of bone strength in untrained young women. METHODS: 57 healthy female college students (age 22±2 yrs) were randomized to one of four groups: control; resistance; aerobic; or combined aerobic and resistance training. Exercise groups performed supervised exercise sessions for 70–90 min, 3 times per week for 13 weeks. Peripheral quantitative computed tomography (pQCT) scans were taken at baseline (pre), 7 weeks (mid), and after 13 weeks of training (post). Bone mineral and structural properties of the non-dominant tibia were measured using pQCT 4% and 38% proximal to the tibial endplate. All variables were analyzed using repeated measures ANOVA. Post hoc analyses were conducted using Tukey's method. RESULTS: The aerobic group demonstrated a small, but significant increase in trabecular bone mineral density (TBMD) at the 4% site (p<0.01) and a decrease in the medial/lateral strength strain index (bending strength of bone; SSIx) at the 38% site (p<0.05) (see table).Table: Mean and (SE) reported: † pre to post; * mid to postCONCLUSIONS: A 13-wk aerobic training program resulted in site-specific adaptive responses of bone, specifically trabecular bone formation (modeling) and cortical bone turnover (remodeling). These changes may have resulted from ballistic loading forces associated with running. Changes were not seen in the combined group despite similar aerobic training volume, indicating a mitigating response of resistance exercise on bone adaptation due to ballistic loading.

The bone adaptation response to exercise is specific to the site being loaded, and may differ between regions of trabecular and cortical bone. It has been suggested that strenuous physical training may result in decreased bone mineral density (BMD) at regions susceptible to stress fracture. Additionally, individuals may respond uniquely to comparable exercise programs. PURPOSE: 1) To determine if regional BMD of the tibia decreases following 16 weeks of recruit training, and 2) to assess whether a change in tibial BMD is related to onset of lower extremity stress fracture. METHODS: 105 women (18.9 ± 0.5 yr) underwent DXA scanning before and after a 16-wk recruit training program in the Israeli Defense Forces. 57 women participated in a physically rigorous combat training program and did not sustain a stress fracture (no stress fracture), 10 participated in rigorous training and were diagnosed with a stress fracture (stress fracture), and 38 completed an academic program with light physical activity requirements (control). BMD was assessed at 5 regions of interest (ROI) at points 4, 33, 50, 66, and 95% from the distal end plate of the tibia. The 4 and 95% regions reflect predominantly trabecular bone; the 33, 50, and 66% sites reflect cortical bone. The 33 and 95% sites are the most frequent site of stress fracture. A total tibial BMD value was calculated as the mean of the five ROIs. Data were analyzed using a repeated measures ANOVA and Tukey post hoc testing. RESULTS: Stress fracture incidence was 14.9% in the combat training group, whereas there were no stress fractures in the control group. The following table presents tibial BMD (g/cm2) compared across groups for each ROI (mean±SD):Table 1Only the control group exhibited changes in tibial BMD over the study period, with increases evident at the 33% and 95% regions. CONCLUSION: Sixteen weeks of strenuous training did not result in decreased tibial BMD, whereas light physical training over the same period increased BMD at two regions, and resulted in a small but significant overall increase in tibial density.

Dual X-ray Absorptiometry (DXA) is considered the gold standard when assessing bone mineral properties, but is limited by its inability to quantify bone geometry. Anthropometric variables reflect parameters of body composition, which are thought to influence bone geometry. Peripheral quantitative computed tomography (pQCT) allows direct measurement of bone geometry including the Strength Strain Index (SSI), an index of bone strength PURPOSE: To determine if anthropometric measurements, parameters of bone measured by DXA Regions of Interest (ROI), or both could be used to estimate tibial bone strength as measured by SSI in healthy females. METHODS: 69 healthy college women (age 21.5 ± 1.9 years) were measured for height, weight, body mass index, calf circumference, and tibial length of their non-dominant leg. Subsequently, all subjects underwent DXA and pQCT scans of the non-dominant tibia at ROI 38% and 66% proximal to the tibial endplate. SSI was calculated at each site. Correlation coefficients were calculated to evaluate the association of anthropometric and DXA data with SSI measurements at these sites. Parameters demonstrating a Pearson's r > 0.50 were subsequently used in linear and stepwise regression analyses to determine the best-fit regression line. RESULTS: Bone mineral content (BMC) and bone area (BA) measured by DXA were the only parameters that met the criteria to be included in the regression analyses. Anthropometric variables and DXA derived bone mineral density (BMD) failed to correlate strongly enough with SSI to be included in these analyses. Regression equations are included in the table.Table 1

U.S. Army initial entry training (IET) requires soldiers to undergo a vigorous nine week basic training course designed to enhance physical strength, endurance and function to prepare them for combat operations. However, there is little known about the effect of this type of training on bone parameters. PURPOSE To observe the response of bone and lean tissue mass (LM) to strenuous physical training in a cohort of IET soldiers, and to explore the association between LM and bone area (BA), bone mineral content (BMC), and bone mineral density (BMD). METHODS 151 female soldiers (age 21.4 ± 3.4 years) underwent dual x-ray absorptiometry scanning before and after nine weeks of basic training. A total body scan provided BA, BMC, BMD and LM measures for the total body (TB), the upper extremities (UE), and the lower extremities (LE). Data were analyzed using paired sample t-tests and Pearson's correlation coefficients. RESULTS Total body BMD decreased from 1.20 ± 0.08 to 1.19 ± 0.08 g/cm2 (p<0.001). Total body LM increased from 39.38 ± 4.30 to 41.84 ± 4.42 kg (p<0.001), and was not significantly correlated with parameters of bone. Total body BMC and BA did not change significantly. Extremity data are presented in the following table:TableThe observed changes in LM were positively correlated with changes in BA for the UE (r=.52) and LE (r=.32) (both p<0.001). CONCLUSION This study provides evidence for the influence of physical training on bone acquisition in the skeletally mature female. Lower extremity bone area increases in response to short term, strenuous, weight bearing activity without a concomitant increase in BMD or BMC. Increases in bone area in the extremities are positively associated with increases in lean tissue mass. At this time it is unknown if significant changes in soft tissue composition result in artifactual variability in measures of bone.