Reduced bone mineral density is not associated with significantly reduced bone quality in men and women practicing long-term calorie restriction with adequate nutrition
Luigi Fontana, Washington University School of Medicine, Campus Box 8113, 4566 Scott Avenue, St. Louis, MO 63110, USA. Tel.: +1 314 747 1485; fax: +1 314 362 7657; e-mail: email@example.com
Calorie restriction (CR) reduces bone quantity but not bone quality in rodents. Nothing is known regarding the long-term effects of CR with adequate intake of vitamin and minerals on bone quantity and quality in middle-aged lean individuals. In this study, we evaluated body composition, bone mineral density (BMD), and serum markers of bone turnover and inflammation in 32 volunteers who had been eating a CR diet (∼35% less calories than controls) for an average of 6.8 ± 5.2 years (mean age 52.7 ± 10.3 years) and 32 age- and sex-matched sedentary controls eating Western diets (WD). In a subgroup of 10 CR and 10 WD volunteers, we also measured trabecular bone (TB) microarchitecture of the distal radius using high-resolution magnetic resonance imaging. We found that the CR volunteers had significantly lower body mass index than the WD volunteers (18.9 ± 1.2 vs. 26.5 ± 2.2 kg m−2; P = 0.0001). BMD of the lumbar spine (0.870 ± 0.11 vs. 1.138 ± 0.12 g cm−2, P = 0.0001) and hip (0.806 ± 0.12 vs. 1.047 ± 0.12 g cm−2, P = 0.0001) was also lower in the CR than in the WD group. Serum C-terminal telopeptide and bone-specific alkaline phosphatase concentration were similar between groups, while serum C-reactive protein (0.19 ± 0.26 vs. 1.46 ± 1.56 mg L−1, P = 0.0001) was lower in the CR group. Trabecular bone microarchitecture parameters such as the erosion index (0.916 ± 0.087 vs. 0.877 ± 0.088; P = 0.739) and surface-to-curve ratio (10.3 ± 1.4 vs. 12.1 ± 2.1, P = 0.440) were not significantly different between groups. These findings suggest that markedly reduced BMD is not associated with significantly reduced bone quality in middle-aged men and women practicing long-term calorie restriction with adequate nutrition.
Long-term calorie restriction (CR) without malnutrition extends health span and lifespan in rodents and monkeys (Anderson et al., 2009). In humans, long-term CR without malnutrition protects against obesity, type 2 diabetes, hypertension and atherosclerosis, which are leading causes of morbidity, disability and mortality (Fontana & Klein, 2007). However, low body mass index (BMI) is a well-documented risk factor for future fragility fractures (an important cause of morbidity and mortality among the elderly) in the general population eating typical Western diets (Slemenda, 1995; Ravn et al., 1999). Whether long-term CR with adequate micronutrient intake affects the risk of developing bone fractures is not known. In particular, we are not aware of any study that has evaluated the effects of long-term CR with adequate nutrition on risk factors for bone fractures (e.g. bone mineral density (BMD), bone turnover, and bone quality) in middle-aged lean humans practicing CR.
Data from experimental animal studies indicate that long-term 30–50% CR can have a significant impact on bone metabolism, BMD, bone microarchitecture, and bone strength in rodents, depending on the age when CR is started, the severity and duration of CR, and the micronutrient content of the diet (Saville & Lieber, 1969; Kalu et al., 1984; McCay et al., 1989; Talbott et al., 2001; Lamothe et al., 2003; Lambert et al., 2005; Westerbeek et al., 2008). The majority of studies published so far indicate that 30–50% CR reduces BMD in rodents and monkeys, independent of the age when CR is started (Saville & Lieber, 1969; Kalu et al., 1984; McCay et al., 1989; Talbott et al., 2001; Lamothe et al., 2003; Lambert et al., 2005; Westerbeek et al., 2008). On the other hand, it has been reported that long-term 40% CR in animals improves bone quality and strength through a reduction of bone turnover and a prevention of secondary hyperparathyroidism (Kalu et al., 1984; Tatsumi et al., 2008). Moreover, one study showed that 40% CR preserves trabecular bone mass in the mouse skeleton (Hamrick et al., 2008).
As major species differences between rodents and humans exist in bone composition, bone metabolism, BMD, and bone quality (Aerssens et al., 1998), the availability of individuals practicing long-term CR with adequate nutrition (i.e. adequate intake of vitamin D, calcium and other micronutrients that may influence bone health) made it possible for us to investigate bone mass and quality in people on a low calorie diet. In this article, we report data on BMD, markers of bone turnover and inflammation, in individuals who have been practicing a CR diet for periods ranging from three to 20 years. We also report novel data on bone quality, which is an important predictor of bone fractures (Seeman & Delmas, 2006), specifically trabecular microarchitecture as measured by high resolution magnetic resonance microimaging.
Body composition and bone mineral density
Body weight and BMI were significantly lower in the CR group than in the Western diets (WD) group (Table 1). Total body fat and lean mass were also lower in the CR group than in the WD group (Table 1). The mean BMD and T scores in the CR group were significantly lower than in the WD group at the lumbar spine, total hip, and femoral neck and trochanter sites (Table 1). None of the participants had clinical evidence of bone fractures.
Table 1. Subject characteristics, body composition, bone mineral density, and markers of bone turnover and inflammation
|Age (years)||52.7 ± 10.3||53.4 ± 9.4||0.761|
|Sex (M/F)||28/4||28/4|| |
| Body weight (kg)||57.5 ± 5.3||84.8 ± 11.4||0.0001|
| Body mass index (kg m−2)||18.9 ± 1.2||26.5 ± 2.2||0.0001|
| Fat mass (%)||10.6 ± 6.6||25.4 ± 7.7||0.0001|
| Lean mass (kg)||49.2 ± 7.3||60.0 ± 9.3||0.0001|
|Bone mineral density|
| Lumbar spine (g cm−2)||0.870 ± 0.111||1.138 ± 0.120||0.0001|
| T score||−2.1 ± 1.0||0.36 ± 1.0||0.0001|
| Total hip (g cm−2)||0.806 ± 0.118||1.047 ± 0.118||0.0001|
| T score||−1.47 ± 0.78||0.12 ± 0.73||0.0001|
| Femoral neck (g cm−2)||0.677 ± 0.110||0.856 ± 0.118||0.0001|
| T score||−1.8 ± 0.8||−0.53 ± 0.83||0.0001|
| Trochanter (g cm−2)||0.611 ± 0.102||0.809 ± 0.123||0.0001|
| T score||−1.3 ± 0.80||0.26 ± 0.90||0.0001|
|Markers of bone turnover and inflammation|
| S-CTX (ng mL−1)||0.595 ± 0.288||0.508 ± 0.179||0.152|
| S-BSAP (U L−1)||17.6 ± 5.8||18.1 ± 4.5||0.691|
| HsCRP (mg L−1)||0.19 ± 0.26||1.46 ± 1.56||0.0001|
Among the simple topological parameters measured using high resolution magnetic resonance imaging (hr-MRI), only trabecular thickness was significantly lower in the CR group compared with the WD group, whereas there were no significant between-group differences across all other parameters (BV/TV fraction, volume, and skeleton density). More importantly, there were no significant group-differences in the composite topological parameters of TB microarchitecture, surface-to-curve ratio, and erosion index (the sample sizes required for significant differences are equal to 137 and 687, respectively), indicating intact trabecular network (Table 2).
Table 2. Hr-MRI Parameters
|BV/TV||0.092 ± 0.019||0.102 ± 0.022||0.318|
|Trabecular thickness (mm)||0.089 ± 0.007||0.097 ± 0.008||0.023|
|Volume (cc)||1.706 ± 0.314||1.877 ± 0.424||0.320|
|Skeleton density||0.051 ± 0.011||0.056 ± 0.012||0.353|
|Surface-to-curve ratio||10.3 ± 4.3||12.2 ± 6.7||0.440|
|Erosion index||0.917 ± 0.276||0.876 ± 0.278||0.739|
Markers of bone turnover and inflammation
The serum CTX- and bone-specific alkaline phosphatase (BSAP) concentrations in the CR group were not significantly different from those of the WD group (the sample sizes required for significant differences are equal to 124 and 1699, respectively) (Tables 1 and 3). Serum high sensitive CRP concentration was markedly lower in the CR group than in the WD group (Table 1).
Table 3. Subject characteristics, body composition, bone mineral density, and markers of bone turnover and inflammation in the subgroup of subjects who underwent Hr-MRI analyses
|Age (years)||57.1 ± 9.8||57.7 ± 7.2||0.878|
|Sex (M/F)||7/3||7/3|| |
| Body weight (kg)||56.8 ± 7.4||83.1 ± 17.2||< 0.001|
| Body mass index (kg m−2)||19.3 ± 0.8||28.1 ± 5.1||< 0.001|
| Fat mass (kg)||9.6 ± 3.2||25.4 ± 1.3||0.002|
| Fat-free mass (kg)||47.2 ± 8.4||57.8 ± 12.2||0.037|
|Bone mineral density|
| Lumbar spine (g cm−2)||0.820 ± 0.113||1.186 ± 0.138||< 0.001|
| T score||−2.4 ± 0.9||1.0 ± 1.2||< 0.001|
| Total hip (g cm−2)||0.724 ± 0.118||1.007 ± 0.138||< 0.001|
| T score||−2.0 ± 0.7||0.0 ± 0.6||< 0.001|
| Femoral neck (g cm−2)||0.599 ± 0.083||0.834 ± 0.104||< 0.001|
| T score||−2.4 ± 0.6||−0.6 ± 0.7||< 0.001|
| Trochanter g cm−2)||0.558 ± 0.070||0.780 ± 0.112||< 0.001|
| T-score||−1.7 ± 0.5||0.2 ± 0.8||< 0.001|
| Ultra distal radius (g cm−2)||0.397 ± 0.021||0.483 ± 0.064||0.009|
| T-score||−1.5 ± 0.8||−0.1 ± 0.8||< 0.001|
|Markers of bone turnover|
| S-CTX (ng mL−1)||0.702 ± 0.285||0.507 ± 0.192||0.423|
| S-BSAP (U L−1)||22.6 ± 5.7||23.1 ± 4.7||0.837|
| HsCRP (mg L−1)||0.3 ± 0.1||3.2 ± 1.3||0.047|
Calorie and nutrient intakes differed significantly between the CR and WD groups. The CR practitioners designed their diets in order to reduce the caloric intake by eliminating the consumption of energy-dense foods and increasing the intake of a wide variety of nutrient-dense foods that supply more than 100% of the Recommended Daily Intake (RDI) for all the essential nutrients. The total caloric intake of the CR group was ∼35% lower than the caloric intake of the WD group (1758 ± 369 kcal per day vs. 2699 ± 459 kcal per day, respectively). The CR practitioners eat a wide variety of vegetables, fruits, nuts, low-fat dairy products, egg whites, wheat and soy proteins, fish and meat (∼23.7% calories from protein, ∼28.5% from fat, ∼48% from complex carbohydrates and ∼1% from alcohol). All of the participants in the CR group strictly avoided refined and processed foods containing trans-fatty acids and high glycemic foods (e.g. refined carbohydrates, potato, white bread, white rice, sweets and soft drinks). Their mean daily dietary intakes of calcium and vitamin D were 1339 ± 366 mg per day and 1036 ± 516 U per day, respectively. The WD group ate typical Western diets containing ∼16.5% calories from protein, ∼34.7% from fat, ∼49% from carbohydrates and ∼3.6% from alcohol. Their mean daily dietary intakes of calcium and vitamin D were 1082 ± 382 mg per day and 358 ± 186 U per day, respectively. Accordingly, in a subgroup of CR (n = 18) and WD subjects (n = 18), the serum vitamin D levels were 85.4 ± 36.3 nmol L−1 and 41.8 ± 28.5 nmol L−1, respectively (P < 0.05).
In this study, we compared BMD, TB bone microarchitecture, and markers of bone turnover in healthy weight-stable lean men and women, who were consuming a self-imposed CR-diet, containing more than 100% of the RDI for all essential nutrients (including calcium and vitamin D), for three–20 years, with age- and sex- matched sedentary individuals, who were consuming Western diets. Our data show that BMD at the lumbar spine and hip sites was significantly lower in the CR group than in the WD group. However, serum CTX-1 and BSAP, two well accepted markers of bone resorption and formation (Ross et al., 2000; Johnell et al., 2002), respectively, were not significantly different between the two groups, which suggest that long-term CR with adequate micronutrient intake does not significantly increase the rate of bone turnover. In addition, one of the key and novel findings from this study was that TB microarchitecture of the distal radius, as indicated by surface-to-curve ratio and erosion index assessed by hr-MRI, was not significantly different between the CR and WD groups, despite markedly low BMD.
Nutrition has a unique role in the maintenance of bone health. Dietary macro- and micronutrients affect bone metabolism, bone density, and strength through changes in body weight, hormonal, and inflammatory status (Muhlbauer & Li, 1999; Ravn et al., 1999; Ilich & Kerstetter, 2000). In general, data from epidemiological studies conducted in Europe and North America indicate that a low body weight is associated with low bone mass and an increased risk of fragility fractures (Slemenda, 1995; Ravn et al., 1999). However, correlation is not the same as causation, and cross-national analyses have shown that the age-adjusted incidence rates of hip fracture among Asian populations seems to be 30–70% of those observed among Caucasians, despite lower BMI and BMD values, suggesting that other factors may be involved (Silverman & Madison, 1988; Ross et al., 1991; Blaum et al., 2005). In our study, body weight, body fat, and BMD of the spine and hip were markedly low in both men and women in the CR group, but markers of bone turnover and TB microarchitecure were not significantly different between the CR and the WD groups.
Evidence that bone turnover and bone quality play a key role in determining fracture risk is provided by the finding that in osteoporotic patients, bisphosphonate therapy results in a rapid reduction in markers of bone turnover and bone remodelling, and in a 30–40% reduction in fracture risk, even in the absence of any change in bone mass (Bjarnason et al., 2001; Cummings et al., 2002; Sarkar et al., 2002). Moreover, type 2 diabetics with a high BMI have increased bone fracture risk, despite a high bone mass, probably because of poorer bone quality (Verhaeghe et al., 1994; Reddy et al., 2001; Schwartz, 2003). Our findings of not significantly altered rates of bone turnover and TB microarchitecture despite markedly low BMD in CR subjects reinforce these previous findings (Verhaeghe et al., 1994; Bjarnason et al., 2001; Reddy et al., 2001; Cummings et al., 2002; Sarkar et al., 2002; Schwartz, 2003) of the lack of concordance between BMD and bone quality. In addition, these findings appear consistent with the known effects of CR in suppressing inflammation, reducing protein glycosylation, and other metabolic/hormonal changes that may reduce the slope of decline of age-related bone loss (Fontana & Klein, 2007; Tatsumi et al., 2008). Indeed, long-term CR in humans results in very low levels of markers of systemic inflammation (e.g. CRP and TNF-α), and because inflammatory cytokines are potent bone resorptive agents, (Bertolini et al., 1986; Schett et al., 2006) our data suggest that a low state of inflammation in the bone microenvironment may partially mediate the lack of increased bone turnover in individuals practicing long-term CR. We measured CRP in the present study because it is a sensitive marker of systemic inflammation, and is an inexpensive and simple test that has been used to assess the risk for atherosclerosis (Cesari et al., 2003). Numerous studies have found a negative association between CRP and BMD (Koh et al., 2005; Ding et al., 2008) and bone fracture (Ma et al., 1996), and bone turnover (Oelzner et al., 1999). However, it is not known whether CRP is a direct mediator of bone loss or whether it is a surrogate marker for other factors directly associated with osteoporosis. CRP is predominantly produced in the liver, and IL-1, IL-6, and TNF-α have been identified as regulators of CRP production. It is possible that inflammatory processes can upregulate these cytokines, which strongly stimulate CRP production from the liver as well as induce bone resorption, and increased bone resorption may result in increased bone turnover and decreased BMD. The discovery of the importance of osteoprotegerin and the RANK ligand in modulating osteoclast formation and activity provides further support for the important role that inflammation plays in determining the rate of bone turnover and quality (Kong et al., 1999; Theill et al., 2002; Takayanagi, 2007).
Trabecular bone microarchitecture is also a major determinant of bone strength and a good predictor of the risk of developing fragility fracture (Dempster, 2003; Seeman & Delmas, 2006). A number of studies have shown that patients with fragility fractures have poorer TB microarchitectural integrity than age-, sex- and BMD-matched non-fractured control subjects (Kleerekoper et al., 1985; Aaron et al., 2000; Legrand et al., 2000; Homminga et al., 2002). Interestingly, aging is associated with a conversion from a honeycomb-like TB microarchitectural structure to a network of interconnected rods with higher structural and mechanical anisotropy (Dempster, 2003; Seeman & Delmas, 2006). Studies using high-resolution peripheral quantitative computed tomography have shown that by age 30, TB architecture starts to deteriorate with a similar pattern at the femur neck, distal radius, and distal tibia in both men and women (Riggs et al., 2004). However, vital information on TB microarchitecture is not routinely available because normally it requires invasive bone biopsy or exposure to a high dose of X-ray radiation. In this study, we used the technique of hr-MRI to determine whether the trabecular architecture of individuals practicing long-term CR with adequate vitamin D and calcium intake differs from that of age- and sex-matched individuals eating a typical WD diet. This novel and non-invasive technique allowed us to calculate the degree to which trabecular plates have deteriorated to become rods in the distal radius (Wehrli et al., 2002; Wehrli, 2007). We found that parameters such as the erosion index and the ratio of surface voxels to curve voxels were not significantly different between groups, indicating that the low BMD in CR subjects is accompanied by preserved TB microachitecture, consistent with normal rates of bone turnover, and suppressed inflammation. It has been shown that hr-MRI parameters differentiate subjects who have vertebral fractures from those who do not, better than by bone densitometry (Wehrli et al., 2001; Wehrli, 2007). Therefore, given the markedly decreased BMD in the CR subjects (T score approximately −2.0), we would have expected that they would have even worse TB microarchitecture as evaluated by the sensitive hr-MRI technique but clearly this was not the case (e.g. no significant differences in erosion index and surface-curve ratios from the WD subjects with normal BMD).
Some limitations in our study must be acknowledged. One is the fact that the CR volunteers are a heterogeneous group, and therefore differences in macro- or micronutrient content of their diets may be responsible, at least in part, for the differences in BMD, independent of caloric intake. Although we think it is likely that the low bone mass is due to bone loss after institution of CR diet, because this is a cross-sectional study we cannot completely exclude the possibility that this could also be due to low peak bone mass. Finally, our sample size was small; therefore, larger studies are needed to confirm our preliminary findings that low BMD may not be associated with reduced bone quality in men and women practicing long-term CR.
In conclusion, the results of this cross-sectional study on 32 individuals practicing severe CR without malnutrition provide preliminary evidence that a CR diet is associated with low bone mass at clinically important skeletal regions but without evidence of significant increase in bone turnover and impaired bone quality. Clearly, it will be necessary to follow a large number of people practicing severe CR with adequate nutrition for a sufficiently long period. Long-term follow up is necessary to determine whether or not people practicing CR have an increased risk of developing fractures.
Thirty-two individuals who strictly adhere to a CR diet were recruited through the Calorie Restriction Society. They had been practicing CR with adequate nutrition for an average of 6.8 ± 5.2 years (range 3–20 years). None of the CR group was physically trained; ∼50% of them do 30–45 min of weight training or interval training per week to try to maintain muscle mass. They do not exercise more because this would necessitate increasing their calorie intake. Thirty-two sedentary (regular exercise < 1 h per week) individuals eating typical Western diets matched with the CR group in terms of age, sex, and height, served as a sedentary comparison group. The characteristics of the study participants are shown in Table 1. None of the subjects had a history or clinical evidence of bone fractures or any other chronic disease (including cardiovascular, lung, gastrointestinal, and autoimmune disease, type 2 diabetes, and cancer) based on medical history, complete physical exam, routine biochemistries, hematological evaluation, and urinalysis. They were all non-smokers. All of the women in the CR and WD groups were postmenopausal. None of the participants in this study were taking drugs that affect bone metabolism (e.g. biphosphonates, hormone replacement, steroids), or other medications that could affect the variables that were measured. All of the study participants were weight stable, i.e. < 2 kg weight change in the preceding 6 months. Informed consent was obtained from all subjects. This study was approved by the Human Studies Committee of Washington University School of Medicine.
Anthropometric, body composition, and bone density measurement
Height was measured without shoes to the nearest 0.1 cm. Body weight was obtained on a balance scale in the morning after a 12-h fast. Body mass index (BMI) was calculated by dividing body weight (in kilograms) by the square of height (in meters). Bone mineral density (BMD) of the total body, lumbar spine (L1-L4), and proximal femur, were measured by dual-energy X-ray absorptiometry (DXA) using a Hologic QDR 4500 (Hologic Inc, Waltham, MA, USA). Assessments of test-retest reliability of BMD measurements yielded intraclass correlation coefficients that were greater than 0.98 for all sites of interest. Regarding precision, the coefficients of variation for BMD were all < 1.5%. DXA was also used to estimate body composition using v5.71 of the enhanced whole body analysis software. The precision of measuring total mass, fat mass, bone mineral mass, and non-bone fat- free mass (lean mass) was 0.96 ± 4%, 1.66 ± 0%, 0.86 ± 3%, and 1.86 ± 9%, respectively.
Bone quality measurement with high resolution MRI in vivo
High resolution magnetic resonance imaging (hr-MRI) was performed in a subgroup of 10 subjects in the CR group and 10 subjects in the WD group. Hr-MRI utilizes a patented algorithm that converts data into a highly detailed three dimensional (3-D) model of TB microstructure as previously described (Wehrli et al., 1998). Briefly, the components of the hr-MRI are the (i) radiofrequency (RF) pulse sequence software, (ii) the imaging RF coil and immobilization device, and (iii) the post processing software algorithm. We used the fast large-angle spin echo (FLASE) pulse sequence, which is specifically designed for structural imaging of trabeculae and provides artifact-free images of signal to noise ratio adequate for the processing algorithms to operate reliably (Ma et al., 1996). A custom designed receive only RF phased array surface coil was used to assess the distal radius using the Siemens Sonata 1.5 Tesla Scanner (Siemens, Iselin, NJ, USA). The distal radius was used because signal-to-noise limitations dictate the use of a peripheral site and because the presence of distinct anatomic landmarks facilitates the precise location of the scan and analysis volume (Wehrli, 2007). Analyses of the scans were done by MicroMRI Inc (Langhorne, PA, USA) in a blinded manner using software algorithms that include processing the image, enhancing the resolution, and providing visualizations and quantitative metrics regarding 3-D TB architecture. The technique permits quantification of the degree to which trabecular plates (surfaces) have deteriorated to become rods (curves), a change that highlights osteoporosis (Wehrli et al., 2001). The hr-MRI indices include: (i) simple topological parameters, such as the bone volume to total volume ratio (BV/TV), trabecular thickness, skeleton density, and volume; and (ii) two composite parameters that have been found to be very sensitive to bone loss (Wehrli et al., 2001) and response to treatment (Benito et al., 2005; Greenspan et al., 2010), such as the surface-to-curve ratio where higher values indicate a more intact trabecular network and lower values indicate a network that has deteriorated; and an erosion index, a ratio of parameters expected to increase when bone trabeculae deteriorate, where higher values indicate greater deterioration. The average coefficients of variation of hr-MRI-based parameters of TB architecture in the distal radius are 4.6%, 10.0%, and 6.9% for BV/TV, surface-to-curve ratio, and erosion index, respectively (Gomberg et al., 2004). The validity of hr-MRI has been established by performing hr-MRI on TB obtained from autopsy specimens at ‘gold standard’in vitro resolution (39 μm), and then the images were resampled to yield images of in vivo resolution (156 μm) (Wehrli et al., 2001). In all parameters of trabecular architecture, the results at the two resolutions highly correlated with each other (r = 0.684 to 0.928). Moreover, hr-MRI parameters of TB architecture have been shown to differentiate women who have vertebral fractures from those who do not, better than in bone densitometry (i.e. subjects with fractures had lower surface-curve ratios (P < 0.001) and higher erosion index (P = 0.001) than subjects without fractures, whereas BMD did not show significant differences between groups (all P > 0.05) (Wehrli et al., 2001; Wehrli, 2007).
A venous blood sample was taken after subjects had fasted for at least 12 h. Commercial ELISA kits were used to measure serum C-terminal cross -linking telopeptide of type-I collagen (CTX-1) (Nordic Bioscience Diagnostics, Herlev, Denmark), bone-specific alkaline phosphatase (BSAP) (Quidel Corporation, San Diego, CA, USA), and high sensitivity C-reactive protein (ALPCO Diagnostics, Windham, NH, USA) concentrations. Serum hsCRP concentrations from 28 of 32 CR volunteers were reported previously (Fontana et al., 2008). Serum vitamin D level was measured using commercial enzyme-linked immunosorbent assay kits (Immunodiagnostic Systems Limited, Boldon, UK).
The study participants were instructed by a research dietician to record for seven consecutive days all foods and beverages consumed, preparation methods, and approximate portion sizes in food diaries at the time of consumption. To assist with portion size determinations, measuring spoon and cup sets were provided to all participants, and all food diaries had a ruler imprinted on the back cover. The food record was analysed using the NDS-R program (version 4.03_31), which is the Nutrition Data System for research from the Nutrition Coordinating Center at the University of Minnesota. The database has 117 nutrients. The nutrients of interest are calories, total fat, total carbohydrate, total protein, animal protein, vegetable protein, calcium, vitamin D, soluble fiber, insoluble fiber, folate, all of the amino acids, and phytic acid.
The unpaired Student’s t-test was used for normally distributed variables with approximately equal SD. For variables not normally distributed or with unequal SD the Wilcoxon two-samples test was used. Statistical significance was set at P ≤ 0.05. Data were analyzed by using SPSS FOR WINDOWS software, version 17.0 (SPSS Inc, Chicago, IL, USA). Values are expressed as means ± SD.
Financial disclosures: Pamela Seeman, Allon Shahar, and Michael Kleerekoper are employees of MicroMRI, Inc. Funding/Support: Supported by grants from the National Center for Research Resources (UL1 RR024992; a component of the National Institutes of Health and NIH Roadmap for Medical Research), the National Institute of Diabetes And Digestive And Kidney Diseases (P30DK056341), Mallinckrodt Institute of Radiology, Barnes Jewish Hospital Foundation, Alliance for Better Bone Health, Istituto Superiore di Sanità/National Institutes of Health Collaboration Program Grant, the Longer Life Foundation (an RGA/Washington University Partnership), and a donation from the Scott and Annie Appleby Charitable Trust. Role of the sponsor: The funding agencies had no role in the analysis or interpretation of the data or in the decision to submit the report for publication.