Significantly inverse correlations were found between dietary choline and betaine intakes, with all obesity measurements: total percent body fat (%BF), percent trunk fat (%TF), percent android fat (%AF), percent gynoid fat (%GF) and anthropometrics: weight, body mass index, waist circumference, waist-to-hip ratio in both women and men (r range from -0.13 to -0.47 for choline and -0.09 to -0.26 for betaine, p<0.001 for all). Dietary choline intake had stronger association than betaine. Moreover, obese subjects had the lowest dietary choline and betaine intakes, with overweight subjects in the middle, and normal weight subjects consumed the highest dietary choline and betaine (p<0.001). Vice versa, when subjects were ranked according to dietary choline and betaine intakes, subjects with the highest intake of both had the lowest %TF, %AF, %GF, %BF and highest %LM among the groups in both sexes.

The beneficial effect of choline and betaine on reducing body fat has been demonstrated in animals, including rats [ 20 ], pigs [ 21 , 22 ], chickens [ 23 , 24 ]. Choline and betaine are widely used as dietary additives to promote growth and to improve carcass characteristics (reducing percentage body fat and increasing relative lean mass) in livestock industry [ 25 , 26 ]. However, studies on the effect of dietary choline and betaine intakes on body composition and furthermore on the development of obesity in humans are scarce, and the limited results are contradictory as well. A cross sectional study in examining Norwegian middle aged and elderly men and women showed that plasma betaine was inversely associated with body mass index (BMI), body fat percentage and waist circumference (WC), while plasma choline had the opposite effect [ 19 ]. Similarly, a recent study found that higher serum concentration of betaine rather than choline was associated with better profiles of body fat and fat distribution in Chinese adults [ 27 ]. Few studies have investigated the effect of betaine supplementation on body weight and body composition in small samples. A study consisting of 23 lean males reported that, 11 of whom received a 2.5 g/day betaine supplementary with simultaneous 6-week progressive resistance training program significantly improved their body composition by both increasing lean mass and reducing fat mass compared with placebo treatment subjects [ 28 ]. However, another study in 42 obese subjects with 12 weeks of 6 g/day betaine supplementation did not improve body composition [ 29 ]. Similarly, no change of body composition was found in a study involving 34 young males after 10 days of 2 g/day betaine supplementation [ 30 ].

Choline and betaine are quaternary ammonium compounds, which are obtained from diet or by de novo synthesis in tissues. Both of them are found in a wide variety of foods. Among the most concentrated sources of dietary choline are eggs, beef, pork, liver, seafood and milk, whereas betaine is mainly obtained from grains, cereal, beets and spinach [ 9 – 13 ]. Choline is recognized as an essential nutrient and plays an important role in neurotransmitter synthesis, cell-membrane signaling, lipid transport in lipoproteins and methyl-group metabolism by serving as a precursor for acetylcholine, sphingolipid and phospholipids head groups, and the methyl donor betaine [ 14 , 15 ]. Betaine serves as a compatible osmolyte and a methyl donor in many pathways, including the side-chain methylation of the homocysteine sulfhydryl to form methionine [ 16 ]. Growing evidence shows that choline and betaine are involved in the pathogenies of various diseases, including fatty liver [ 17 ], cardiovascular diseases [ 18 ], metabolic syndrome [ 19 ], and various cancers [ 12 ].

Obesity is widely recognized as a chronic disease associated with many serious health problems including type 2 diabetes, cardiovascular disease, hypertension and at least a dozen of cancers [ 1 , 2 ]. In the past three decades, the prevalence of obesity and overweight has risen substantially and affects over 600 million people globally [ 3 ]. Due to the high prevalence of obesity and its associated health consequences, effective weight reduction and maintenance strategies are needed. Excessive weight gain is influenced by a range of factors including genetic predisposition, improper nutrition, individual behaviour, environmental and social factors [ 4 ]. The role of nutrients in diet, especially micronutrients in obesity has been long debated [ 5 – 8 ].

According to the criteria recommended by World Health Organization (WHO) [ 41 ], obesity status was categorized based on BMI into normal weight (18.5–24.9 kg/m 2 ), overweight (25.0–29.9 kg/m 2 ) and obese groups (≥30.0 kg/m 2 ). As the number of underweight subjects (BMI<18.5 kg/m 2 ) was too small (n = 35, female/male = 33/2) to perform any effective statistical analysis, they were excluded from analyses. Obesity status was also categorized by percent body fat according to age and gender specific criteria recommended by Bray [ 42 ]. Subjects less than 20 years old were excluded due to unavailability of criteria for this age group by Bray criteria. Dietary choline, betaine intakes and body composition measurements were compared among adiposity groups by using ANCOVA controlling for age, total calorie intake and physical activity level. In order to further explore the relationship between dietary choline, betaine intakes and obesity phenotypes, participants were divided into tertiles (low, medium, or high) based upon dietary choline or betaine intakes expressed in mg/kg/day. Differences of obesity measurements, such as weight, BMI, WC, WHR, %TF, %AF, %GF, %BF and %LM among the three groups of choline or betaine intakes were assessed using ANCOVA controlling for age, total calorie intake and physical activity level.

Statistical interaction between dietary choline, betaine intakes and gender on the main outcomes was tested by analysis of covariance (ANCOVA). Partial correlation analysis controlling for age, total calorie intake, and physical activity level was used to evaluate the correlations of dietary choline, betaine intakes with weight, BMI, WC, WHR, %TF, %AF, %GF, %BF and %LM within female and male groups. To overcome the possible influence of smoking, medication use, drinking and menopausal status, participants were also divided into subgroups according to smoking status (yes or no), medication use (yes or no) and alcohol consumption (yes or no). Women were further divided into pre- or post-menopausal groups according to their menopausal status.

Dual Energy X-Ray Absorptiometry (DXA; Lunar Prodigy; GE Medical Systems, Madison, WI) was used for the measurement of percent trunk fat (%TF), percent android fat (%AF), percent gynoid fat (%GF), total percent body fat (%BF) and total percent lean mass (%LM). The Lunar Prodigy software system determines automatically the regions. Trunk fat region is from the top of the shoulders to the top of the iliac crest, while the android fat region is the top of the second lumbar vertebra to the top of the iliac crest and the gynoid fat region extends down iliac crest twice the height of the android area. The enCORE (Ver 12.2, 2008, GE Medical Systems, Madison, WI) software package was used for DXA data acquisition. Quality assurance was performed on the DXA scanner daily and the typical CV during the study period was 1.3% [ 33 , 34 ].

Anthropometrics, body composition measurements were collected following a 12-hour fast. Trained personnel conducted the anthropometric measurements of each subject using standard procedures. Standing height was measured using a fixed stadiometer (to the nearest 0.1 cm). After urinating to empty their bladders, subjects wore standard hospital gowns for all weight measurements using a platform manual scale balance (Health O Meter, Bridgeview, IL; to the nearest 0.1 kg). BMI (kg/m 2 ) was calculated as weight in kilograms divided by height squared in meters. WC was defined as the midway point between the iliac crest and the lower rib, and hip by the maximum circumference over the buttocks below the iliac crest. Waist-to-hip ratio (WHR) was division of WC by hip circumference.

All participants were from the CODING (Complex Diseases in the Newfoundland population: Environment and Genetics) study [ 31 – 35 ]. Eligibility of participants for the CODING study was based upon the following inclusion criteria: 1) ≥19 years of age; 2) at least a third generation Newfoundlander; and 3) without serious metabolic, cardiovascular, or endocrine diseases. A total of 3214 participants (2295 females and 919 males) were employed. Among them, 160 individuals were excluded for having incomplete data, including: 8 individuals missed weight and height, 38 individuals missed waist and hip circumference, 12 individuals missed the results of body scan, 156 individuals missed the results of Food Frequency Questionnaire (FFQ), and 69 individuals missed physical activity questionnaire. All participants provided written and informed consent and the study received ethics approval from the Health Research Ethics Authority (HREA), Memorial University, St. John’s, Newfoundland, Canada, with Project Identification Code #10.33 (latest date of approval: 21 January 2015).

Subjects were divided into tertiles (low, medium, or high) according to dietary choline and betaine intakes (mg/kg/day). As shown in Tables 5 , 6 and S2 Fig , a significant ‘dose-dependent’ declining in weight, BMI, WC, WHR, %TF, %AF, %GF and %BF was found with the increase of dietary choline and betaine intakes in both females and males after controlling for age, total calorie intake, and physical activity. %LM presented a significantly ‘dose dependent’ increasing trend. Compared to low dietary choline intake group, female subjects of high dietary intake group had significantly lower weight, BMI, WC, WHR (-11.85 kg, -4.24 kg/m 2 , -11.79 cm, -0.02), and %TF, %AF, %GF, %BF(-7.59%, -8.89%, -4.65%, -6.75%) but higher %LM (6.30%). Male subjects of high dietary choline intake group had significantly lower weight (-11.19 kg), BMI (-3.87 kg/m 2 ), WC (-12.82 cm), WHR (-0.03), %TF (-10.56%), %AF (-12.66%), %GF (-5.94%), %BF (-8.35%), but higher %LM (8.25%) compared with low choline intake group. While, the average differences of obesity measures between high and low dietary betaine intake (mg/kg/day) groups were -7.42 kg (weight), -2.77 kg/m 2 (BMI), -7.31 cm (WC), -0.02 (WHR), -6.19% (%TF), -6.24% (%AF), -3.13 (%GF), -4.55% (%BF), 4.00% (%LM) in females, and -8.60 kg (weight), -3.01 kg/m 2 (BMI), -8.81 cm (WC), -0.03 (WHR), -7.77% (%TF), -8.92% (%AF), -4.19% (%GF), -6.19% (%BF), 6.19 (%LM) in males.

When subjects were divided by %BF, as shown in Table 4 and S1 Fig , similar significant relationship was found among these three adiposity groups as seen in BMI groups. Compared to normal weight female and male groups, dietary choline intake (mg/kg/day) was significantly lower by 17.9%, 32.9%, 29.3%, and 38.6% in female overweight, female obese, male overweight and male obese groups, respectively. Correspondingly dietary betaine intake (mg/kg/day) was significantly lower by 13.3%, 33.0%, 29.7%, and 41.7% in female overweight, female obese, male overweight and male obese groups, respectively, compared to normal weight groups. Furthermore, betaine intake expressed as mg/day was also significantly lower with the increase of %BF in both female and male subjects (p<0.05) but significant difference was not found for choline.

According to BMI, as shown in Table 3 and S1 Fig , weight, BMI, WC, WHR, %TF, %AF, %GF and %BF were significantly higher, while %LM was significantly lower in obese and overweight subjects than normal weight subjects (p<0.001). Dietary choline and betaine intakes expressed in mg/kg/day were significantly lower as adiposity status increased. Compared to the normal weight female and male groups, dietary choline intake (mg/kg/day) was significantly lower by 21.4%, 35.8%, 27.6%, and 44.2% in female overweight, female obese, male overweight and male obese groups, respectively. In addition, dietary betaine intake (mg/kg/day) was similarly lower by 22.8%, 36.0%, 21.6%, and 46.6% in female overweight, female obese, male overweight and male obese groups, respectively, compared to normal weight female and male groups. However, there is no significant difference among the three groups when dietary choline and betaine intakes were expressed as mg/day.

Significant interactions between dietary choline, betaine intakes and gender on weight, BMI, WC, WHR, %TF, %AF, %GF, %BF and LM% were found (p<0.05 for all). The correlations between dietary choline, betaine intakes and weight, BMI, WC, WHR, %TF, %AF, %GF, %BF and %LM in different gender groups are presented in Table 2 . In both female and male subjects, dietary choline, betaine intakes (mg/kg/day) were negatively correlated with weight, BMI, WC, WHR, %TF, %AF, %GF, %BF and positively correlated with %LM. The correlations remained significant after adjusting for age, total calorie intake, and physical activity level. Dietary choline intake had the most pronounced correlation with body weight among all measures in females (r = -0.47, p<0.001), while with %AF in males (r = -0.35, p<0.001). However, the association between dietary betaine intake and BMI was the most pronounced in females (r = -0.25, p<0.001), while TF% had the most prominent correlation with dietary betaine intake in males (r = -0.26, p<0.001).

Demographic, physical and dietary characteristics of the entire population and different gender groups are presented in Table 1 . Female subjects were on average 3.4 years older than male subjects. Weight, BMI, WC, WHR, %LM and physical activity were significantly higher in males than females (p<0.001). %TF, %AF, %GF, and %BF were significantly lower in males as compared to females (p<0.001). In terms of dietary intake, male participants consumed significantly higher daily calorie (kcal/day) and choline intake (mg/day) than females (p<0.001), while there was no significant difference for daily betaine intake (mg/day) between different gender groups. When daily choline and betaine intakes were expressed in per kilogram body weight (mg/kg/day), female participants consumed significantly more betaine than males but there was no significant difference for choline intake.

Discussion

To the best of our knowledge, this is the first large cross-sectional study specifically designed to investigate the association between dietary choline, betaine intakes and DXA-derived body composition. We found significant associations of higher dietary choline and betaine intakes with better body composition profile (lower %BF, higher %LM). Furthermore, the inverse correlation of dietary choline intake with obesity measures was stronger than that of betaine.

Association study is relatively easy to do but hard to do well, especially in large population based study. The first challenge is the accurate measurement of physiological and biological markers because sophisticated equipment is required, and long period of time is needed to recruit large number of subjects. In the present study, body composition in all subjects was measured using the same DXA system by certified technicians over 12 years. DXA produces accurate measurement of adipose tissue in the body with a low margin of error and is considered to be one of the most reliable measurements of adiposity [33]. The second challenge lies in the identification and measurements of numerous confounding factors that can potentially affect the relationship of the major factors under investigation. In most studies, unfortunately, information on these confounding factors is largely unavailable [43, 44]. There is no doubt that age is a primary important factor that affects the development of obesity [45]. Total dietary calorie intake is a critical factor in maintaining energy balance [46]. In addition, physical activity level is likely the most important non-dietary factor that can influence energy consumed, amount of choline and betaine intakes and ultimately body composition [46, 47]. In the present study, all of these confounding factors have been properly adjusted. The beneficial effect of dietary choline and betaine intakes on obesity was demonstrated in every obesity measurement in both women and men independent of these factors. Other confounding factors, menopause, smoking, medication and alcohol consumption were taken into consideration as well. Menopause is an important factor related to changes in body composition as menopause is usually accompanied by dramatic changes in sex hormones that can predispose women to weight gain [48]. We found that the associations between dietary choline, betaine intakes and body composition are equally profound in both pre- and post-menopausal women (S1 Table). Smoking status, medication use and alcohol consumption are potentially important covariates known to affect appetite and body weight regulation [49–51]. After separating subjects according to these covariates, the beneficial association remained significant (S2–S4 Tables).

We used the unit of mg/kg/day to express choline and betaine intakes in all analyses because dietary intake of both is highly correlated with body weight. It would be physiologically inappropriate not to account for body mass when comparing dietary intake among subjects with large difference of body weight. Physiological dynamics of nutritive intake is in many ways similar to that of pharmacodynamics where in the case of choline and betaine, the same dose may be adequate for a person with 60 kg body weight, but insufficient for a person with a body weight of 100 kg or more. The use of mg/kg/day in the present study eliminates the bias of body weight variation in the population and reveals the true relationship between dietary choline, betaine intakes and body composition. Otherwise, we also analyzed the data for mg/day and the negative associations were also significant but not strong (S5 Table). Furthermore, same findings were obtained when obesity status was classified according to either BMI or %BF by Bray criteria. Obese subjects had the lowest dietary choline and betaine intakes. Vice versa, subjects with the highest dietary choline or betaine intakes had the lowest obesity measurements. And the associations were stronger with dietary choline than dietary betaine. Moreover, the associations seem different among fat distribution. The associations were stronger between dietary choline and betaine intakes with %TF, %AF than %GF. Similar findings were reported on the association of serum rather than dietary betaine with body fat percentage and fat distribution [19, 27]. However, an opposite relationship between plasma choline and obesity measurements was reported [19], while chen et al found a negative association between serum choline and body weight and BMI [27]. The relationship between dietary intakes and serum levels of choline and betaine is not clear. A small study consisting of children with autism reported a positive correlation between dietary intakes and plasma levels of the nutrients [52]. It is reported that the serum choline level is lower than betaine, although dietary choline intake is higher than betaine in the general populations [12, 19, 27, 53]. This difference between dietary intakes and serum levels highlights the importance to perform independent studies and suggesting that relationship between dietary choline and betaine with obesity might differ from that with serum levels.

To date, a few small interventional studies have examined the effect of betaine supplementation on body fat but no study is available on the effect of choline supplementation in humans. A significant decrease in fat mass and percent body fat was observed among 11 male participants received a periodical training program [28]. However, the positive effect may have been due to the greater physical activity levels of participants. Two other studies failed to replicate the results [29, 30]. Animal studies have provided the main evidence of the beneficial effect of dietary choline and betaine on body fat and muscle. Choline or betaine supplementation on the growth performance and carcass characteristics of gilts, other pigs, meat ducks, chickens and mice were well examined [20–26]. These studies have generally indicated that choline and betaine supplementation can promote growth and reduce fat deposition in various animals. Our findings have filled the knowledge gap of the relationship between dietary choline, betaine intakes and body composition in humans. The effect of dietary choline might be more complex. Jacobs et al found that a choline-deficient diet decreased fat mass in mice with high fat diet or genetic defect induced obesity [54, 55]. Another study showed that high fat feeding led to weight gain in choline-replete mice, whereas choline deficiency did not affect body or adipose depot weights [56]. However, these animal experiments were performed under extreme condition: choline depletion which virtually does not exist in general human population.

The exact mechanisms by which choline and betaine improve body composition are unclear, while several mechanisms have been suggested. It has been postulated that choline and betaine supplementation could promote fatty acid β-oxidation in pigs and humans by enhancing muscle carnitine accretion and thereby increase carnitine palmitoyl transferase I-mediated free fat acid translocation into the mitochondria [57–61]. Choline can also be metabolized to betaine and plays a role in altering body composition. The betaine-related methylation of homocysteine to methionine may reduce the availability of substrate (acetyl-coenzyme A) for fatty acid synthesis [60]. Betaine supplementation could decrease the capacity for fatty acid and triglyceride synthesis by decreasing the activity of acetyl-CoA carboxylase, fatty acid synthase and malic enzyme and their mRNA expression in abdominal adipose tissue [61, 62]. Betaine may also reduce uptake of triglycerides from circulating lipoproteins by decreasing the mRNA expression of lipoprotein lipase [62]. Dietary betaine in pigs mobilized fat degradation in adipose tissue by increasing hormone sensitive lipase activity [63]. Betaine supplementation may promote protein synthesis by stimulating growth hormone secretion and improving insulin and IGF-1 receptor signaling [64–66]. Accumulation of betaine in cells resulting in increased sarcoplasmic osmolality may also contribute to the increase in muscle mass [67, 68].

Although our study provides clear evidence on association of increased dietary choline and betaine intakes with favorable body composition, a number of limitations of the work should be considered. Our use of FFQ to evaluate patterns of dietary intake raises the possibility of recall bias by subjects [38]. However, FFQ is the most widely used questionnaire for the assessment of dietary intake at the population level and has been successfully validated and used in a Canadian populations [69–71], including in the Newfoundland population [72]. Thus, we consider the Willett FFQ to be a reasonable method for the assessment of dietary intake patterns in this study. Another potential weakness in the present study is related to its cross-sectional design. Cross sectional study alone does not establish a cause effect relationship. Direct intervention studies or clinical trials with reasonable sample size and well matched subjects between treatment and control groups are warranted. Finally, although multiple factors, including socio-demographic characteristics, lifestyle habits, physical activities and energy intake were comprehensively adjusted in our analysis, genetic factors, dietary nutrient factors absorbed when consuming choline or betaine-rich foods and unknown or poorly measured factors could not be completely ruled out.