Four-week dietary supplementation with 10- and/or 15-fold basal choline caused decreased body weight in Sprague Dawley rats

Abstract

Choline is an essential nutrient utilized for phosphatidylcholine biosynthesis and lipoprotein packaging and secretion. Recently, choline supplementation has been used by athletes and the public for weight loss. However, the potential toxicological impact of choline dietary supplementation requires further investigation. This study examined the effects of choline dietary supplementation in Sprague Dawley rats for 4 weeks. Rats were fed diets containing basal choline levels (control) or 5-, 10-, or 15-fold (5×, 10×, or 15×) basal diet concentration. In groups fed choline-supplemented diets, there were no toxicologically relevant findings in clinical observations, food intake, clinical chemistry, liver weights, or liver histopathology. However, decreased mean body weights (8.5–10.2%) and body weight gains (24–31%) were noted for the 10× choline-supplemented (females only) and 15× choline-supplemented (both sexes) groups relative to the control groups from day 3 onward. These body weight effects were not related to a persistent reduction in average food intake. Serum cholesterol was increased in the 15× choline-supplemented male rats relative to the controls, an expected effect of choline supplementation; however, there were no changes in the serum cholesterol of female rats. Serum choline concentrations were increased in female rats relative to the male rats across all treatment groups. The maximum tolerated dose for male and female rats were the 15× and 10× choline supplements, respectively, based on decreased mean body weight and body weight gains. This study supported the conclusions of a clinical trial that showed a high choline diet can decrease body weight in humans.

Keywords

Introduction

Choline is an essential dietary nutrient found in leafy green vegetables, eggs, and meat (Zeisel and da Costa, 2009). Choline is critical for hepatic biosynthesis of phosphatidylcholine that is used as a surface molecule in lipoprotein packaging (Li and Vance, 2008). Recently, commercially available choline supplements have been used by athletes attempting to decrease body weight (Elsawy et al., 2014; Grunewald and Bailey, 1993). However, there is limited information on the toxicity of dietary choline supplementation. For example, the Institute of Medicine tolerable upper limit of 3500 mg/day was selected based on a single case report of hypotension following ingestion of 7500 mg/day choline for 2 weeks and vomiting, salivation, sweating, diarrhea, and fishy body odor at 16,000 mg/day choline for 2 weeks (Institute of Medicine, 1998). The Institute of Medicine recommended additional animal studies to investigate the effect of increased dietary choline intake on organ systems including the liver.

The available data in rats are limited as well. Rats administered intraperitoneal injection of 150 or 225 mg/kg/day choline chloride for 5 days/week for 5 weeks had decreased body weights through 26 weeks post-dose with splenic and thymic cellular depletion (Sahu et al., 1986). Some rats were reported to have died during the course of the experiment, but the timing and conditions prior to death were not reported. In addition, rats fed diets containing 5-fold (5×) the choline nutritional requirement of 750 mg choline/kg diet (National Research Council, 1995) for 12 weeks (Moreno et al., 2013) demonstrated no evidence of toxicity. However, the potential for choline toxicity following dietary intake at higher levels has not been investigated in rats since 1945 (Hodge, 1945), and that study had deficiencies that limited its utility.

Rats were fed diets containing choline at 0.01%, 1.0%, 2.7%, 5.0%, and 10.0% by weight (w/w) for 4 months (Hodge, 1945). Standard rodent growth diets, such as AIN-93G purified diet (Reeves et al., 1993), contain 0.1% w/w choline (1000 mg choline/kg diet), thus the diets used by Hodge contained choline at 0.1–100 times (0.1× to 100×) the choline concentration in modern rat diets. The groups that received ≥27× choline had decreased body weight with no changes in liver weight or histopathology. However, there were deficiencies in this study: (1) only limited descriptions of the study methods were provided; (2) a basal diet control group was not included; (3) the sex and strain of rats were not reported; and (4) the range of choline concentrations was too wide at low doses with only one group (i.e. 10×) between the 0.1× and 27× choline-supplemented diets.

Because the available data set does not provide a good basis to understand the toxicological effects of choline dietary supplementation at increased levels of exposure (e.g. up to 15× basal choline), this study was undertaken to fill this gap in knowledge. Briefly, rats were fed basal diet (control) or diets containing 5×, 10×, or 15× supplemental choline for 4 weeks. This investigation focused on histopathology and clinical chemistry effects on the liver because the liver is important for choline metabolism and lipoprotein production. The maximum tolerated dose for choline dietary supplementation in rats was determined and could be useful for assessing effects of human choline supplementation.

Methods

Diets

Modified pellet diets based on AIN-93G purified diet (Reeves et al., 1993) were obtained from Harlan Teklad (Indianapolis, IN, USA)containing basal choline (control, 2.5 g choline bitartrate/kg diet, 0.1% w/w choline, undyed), 5× choline (12.5 g choline bitartrate/kg diet, 0.57% w/w choline, red dyed), 10× choline (25.0 g choline bitartrate/kg diet, 1.15% w/w choline, yellow dyed), and 15× choline (37.5 g choline bitartrate/kg diet, 1.7% w/w choline, green dyed). Food-grade dyes were added to the diets by the vendor to assure diet identification. The corn starch was reduced to account for the weight balance of the increased choline bitartrate. Caloric content was similar across the diets. All diets were stored at 4 ± 1°C per vendor instructions.

The diets were analyzed for choline content and homogeneity using a chromophore method adapted from Method 999.14 (AOAC International, 2005) with a lower limit of quantitation (LLOQ) of 0.0067% w/w choline.

Animal selection and husbandry

Animal procedures complied with animal welfare guidelines (National Research Council, 1996). Six- to eight-week-old adult Sprague Dawley rats (23/sex) were obtained from Harlan Laboratories (Indianapolis, Indiana, USA) at body weights ranging from 170.7 to 193.9 g (males) and from 134.0 to 158.6 g (females). The rats were individually housed in hanging wire cages (changed weekly) with enrichment (i.e. Nylabone® chewables and marbles) in a room maintained at 22 ± 2°C, relative humidity of 30–70%, 10 air exchanges/hour, and a 12-h light/12-h dark cycle. Tap water was available ad libitum. All rats were acclimated and fed the basal diet for 7 days prior to study initiation. Rats were randomly assigned to the basal diet (control, 5/sex) and the 5×, 10×, and 15× choline-supplemented groups (6/sex/group). There were no differences in the initial group mean body weights (Figure 1(a) and (b)). The rats were fed the control or experimental diets ad libitum for 4 weeks.

In-life procedures

Clinical observations, body weights, and food intake (accounting for spillage) were recorded daily. The rats were fasted overnight and euthanized on day 28 by carbon dioxide asphyxiation. Blood was collected in plain BD Vacutainer®tubes (Becton Dickinson and Company, San Jose, CA, USA) by exsanguination via the abdominal aorta. The clotted blood was centrifuged at 1500 × g for 15 min at 10°C in an Avanti J30I centrifuge (Beckman Coulter Life Sciences, Indianapolis, IN, USA). The serum was stored at −70°C until analysis for choline content and clinical chemistry. All organs were subjected to gross evaluation. Liver weights were recorded. Liver samples were collected from the right major lobe and left lateral lobe and fixed in neutral-buffered formalin for histopathology.

Serum choline analysis

Trimethyl-d9-choline standards (5–500 ng/mL; Sigma Aldrich, St. Louis, MO, USA), blank (deionized water purified through Milli-Q® (Millipore Corporation, Billerica, MA, USA) and a C-18 HPLC column (Phenomenex, Torrence, CA, USA)), and thawed rat serum samples were diluted at 100 µL of sample in 500 µL acetonitrile. After 10 min, the samples were vortexed for 15–20 s, 600 µL of 0.1 N formic acid was added, and the solution was centrifuged at 3000 × g for 10 min. The organic layer was collected and the serum choline concentration was determined using a Sciex API 5000 mass spectrometer (Applied Biosystems/MDS-Sciex Instrument Corporation, Framingham, MA, USA) with turbo ion spray in the positive ion mode. Separation was completed on a Phenomenex polar HPLC column (150 mm × 3.0 mm, 4 µm, Phenomenex, Torrence, California, USA) with a gradient elution and a flow rate of 0.25 mL/min. Buffer solutions of acetonitrile (VWR International, Chicago, IL, USA) and 0.1 N formic acid (J.T. Baker, Fisher Scientific, Hanover Park, IL, USA) were run with initial conditions of 10% acetonitrile and 90% 0.1 N formic acid, and then the concentrations were adjusted to 90% acetonitrile and 10% 0.1 N formic acid with an analysis time of 11.0 min. The transition ions were monitored at:

Choline: 104 amu transitioned to 60 amu

Trimethyl-d9-choline: 113 amu transitioned to 69 amu

Peak areas were integrated and the ratio of choline to the internal standard was calculated.

All standards were 1/× weighted for the regression analysis and reviewed for the best regression fit. The LLOQ for choline content analysis was 5 ng/mL (i.e. 5 ppb). The quadratic regression R ² for the standard curve in this study was 0.9995.

Clinical chemistry

Serum glucose, aspartate aminotransferase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALKP), bilirubin (total, direct, and indirect), total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein, triglycerides (TGs), total protein, blood urea nitrogen, creatinine, phosphorus, calcium, sodium, potassium, chloride, bicarbonate, anion gap, creatine kinase, lactate dehydrogenase, amylase, lipase, albumin, globulin, albumin/globulin ratio, urea/creatinine ratio, and sodium/potassium ratio were measured using a Beckman Coulter AU 5800 Clinical Chemistry System (Brea, California, USA).

Histopathology

Formalin-fixed liver samples were embedded in paraffin, 5 µm sections were cut using a microtome, and stained with hematoxylin and eosin for histopathology evaluation. The hematoxylin and eosin stained slides were examined using light microscopy. Flash-frozen samples were prepared from the formalin-fixed liver samples, and 5 µm sections were cut using a microtome and stained with Oil Red O for fat content evaluation. The Oil Red O-stained samples were assessed for the degree of fat accumulation.

Statistical analyses

Statistical differences between groups within sex were evaluated by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. Sex-related differences in choline intake and serum choline concentrations were evaluated by two-way ANOVA with Bonferroni’s multiple comparisons test. Measured choline diet concentrations were compared to target choline diet concentrations by one-sample t-tests. All statistics were performed using GraphPad Prism®, version 6.04, and results were significantly different at p ≤ 0.05.

Results

Clinical signs

No adverse clinical signs were observed. All 15× choline rats had green feces from day 6 to termination; likely arising from the green food-grade dye in that diet. Minor scabs were noted sporadically on the neck of rats of the 15× choline group (1/sex, data not shown). These were incidental findings and were not choline-supplement related.

Body weights and body weight gain

Male 15× choline rats had decreased mean daily body weight from day 3 through termination compared to the control (8.5–10.2%), 5× (5.9–7.9%), and 10× (4.8–7.0%) groups (Figure 1(a)). There were no effects on mean daily body weight of male rats in the 5× and 10× group compared to the controls. Female rats in the 15× group had decreased mean daily body weight (10.3–12.8%) from day 3 through termination compared to the control group (Figure 1(b)). The female 5× and 10× body weights were not different from the control group throughout the study with the exception of day 18, where the 10× group was decreased by 9% relative to the control group. There were no significant body weight differences between the female 5×, 10×, and 15× choline-supplemented groups.

The mean weekly body weight gain in male 15× choline rats was decreased in weeks 1 and 2 by 56% and 16%, respectively, relative to the control group and relative to the 5× and 10× to a lesser degree (Figure 1(c)). There were no other differences between the male groups during weeks 1–4. Mean overall body weight gain of the male 15× choline rats was decreased by 26% relative to the control group. There were no other differences between the overall body weight gains of the other male groups. In week 1, mean female body weight gains were decreased in the 10× and 15× groups by 45% and 66%, respectively, relative to the control group (Figure 1(d)). In week 2, mean female 15× choline rat body weight gain was decreased by 33% relative to the control group. The mean body weight gain in the female 5× choline rats was similar to the control group throughout the study. There were no differences in body weight gain on weeks 3 and 4 for female rats of any group. Overall mean body weight gain was decreased in the female 10× and 15× groups relative to the control group (24% and 31%, respectively) with no differences between the female choline-supplemented groups (Figure 1(d)).

Food intake

Mean food intake was decreased in male rats of the 10× (26% decrease) and 15× groups (up to 39% decrease) relative to the control group and/or the 5× group until day 5 (Figure 2(a)). Mean food intake was also decreased in female rats of the 10× group (19% decrease relative to the 5× group) and the 15× group (up to 40% decrease relative to all groups) until day 5 (Figure 2(b)). From day 5 onward, there were no differences in food intake for any group of either sex.

Dietary choline concentration and choline intake

The choline diets were homogenous and the choline concentrations were similar to the target concentrations. Mean choline intake for the control, 5×, 10×, and 15× groups, respectively, was approximately 99, 434, 868, and 1168 mg/kg/day for males and 107, 468, 922, and 1273 mg/kg/day for females (data not shown).

Serum choline concentrations

A sex-related difference in mean serum choline concentration was observed without regard to choline supplement status (Figure 3). All female groups had increased mean serum choline concentrations relative to the similarly treated male group. There were no differences in mean serum choline concentrations between the control or choline-supplemented groups of the same sex.

Clinical chemistry

Blood for clinical chemistry analysis could not be obtained from two females in the 10× choline group because of technical difficulties during blood collection. The remaining four females in the 10× choline group provided sufficient data for evaluation of this group’s clinical chemistry response.

Most clinical chemistry parameters were within the historical reference ranges for rats (Marshfield Labs, 2016). Incidental significant differences in mean serum total cholesterol, HDL, creatinine, and albumin values slightly exceeded the historical control ranges for male and/or female rats fed up to 15× choline in the diet (Table 1 and Online Supplemental Tables S1 and S2). None of these differences were considered toxicologically relevant. There were no other significant differences in any other clinical chemistry parameters for either sex including all parameters correlated with potential liver toxicity (i.e. ALT, AST, ALKP, bilirubin, GGT, or TG).

Table 1.

Selected mean (±SD) clinical chemistry results from rats that were fed control diet or diets supplemented with 5×, 10×, or 15× basal choline for 4 weeks.^a,b

Sex	Male				Female^c
Parameter (units)	Control (basal choline)	5× Choline	10× Choline	15× Choline	Control (basal choline)	5× Choline	10× Choline	15× Choline
Aspartate aminotransferase (U/L)	107.8 ± 21.7	97.8 ± 11.1	97.0 ± 10.1	98.5 ± 22.7	103.2 ± 13.0	109.5 ± 7.7	100.8 ± 9.5	98.8 ± 6.9
Alanine transaminase (U/L)	35.6 ± 1.1	33.7 ± 3.7	32.8 ± 6.9	39.8 ± 8.9	34.4 ± 6.8	34.5 ± 2.2	33.8 ± 6.3	34.8 ± 10.8
Total cholesterol (mg/dL)	80.8 ± 5.9	92.3 ± 10.3	87.5 ± 7.7^@	105.2 ± 11.3*^,$	86.4 ± 9.4	94.5 ± 9.9	100.0 ± 24.7	89.2 ± 14.9
High-density lipoprotein (mg/dL)	27.6 ± 1.5	27.0 ± 2.0^@	29.7 ± 2.3	34.7 ± 5.1*^,#	27.8 ± 2.7	32.7 ± 1.2*	33.5 ± 3.7*	32.3 ± 3.2
Low-density lipoprotein (mg/dL)	8.2 ± 1.6	9.5 ± 1.6	9.0 ± 2.4	10.7 ± 1.9	6.4 ± 0.9	6.3 ± 0.8	6.8 ± 1.0	7.3 ± 1.0
Blood urea nitrogen (mg/dL)	16.4 ± 1.5	16.3 ± 2.1	16.3 ± 1.5	16.8 ± 2.1	19.2 ± 1.8	17.3 ± 2.4	18.0 ± 1.6	17.2 ± 2.7
Creatinine^d (mg/dL)	0.3 ± 0.1	0.3 ± 0.0	0.3 ± 0.1*	0.3 ± 0.1*	0.4 ± 0.1	0.3 ± 0.1	0.3 ± 0.1	0.3 ± 0.1*
Albumin (g/dL)	3.6 ± 0.0	3.6 ± 0.2	3.6 ± 0.1	3.5 ± 0.2	3.6 ± 0.2	3.6 ± 0.1^d	3.5 ± 0.1	3.3 ± 0.1*^,#

ANOVA: analysis of variance.

^aSignificantly different parameters are indicated by (*) Versus control, (#) versus 5×, ($) versus 10×, and (@) versus 15×. Evaluated by group within sex using one-way ANOVA with Tukey’s multiple comparisons test (p < 0.05, GraphPad Prism®, versus 6.04).

^bMean (±SD) values for all evaluated clinical chemistry parameters are provided in Online Supplemental Tables S1 (males) and S2 (females).

^c N = 5 control males and 6 males/choline supplement group. Not enough blood was not successfully collected from two females of the 10× choline group for clinical chemistry analysis due to technical difficulties. Sufficient animals remained for evaluation of this group. N = 4 female rats for the 10× choline group, 5 control females, and 6 females/group for the remaining choline supplement groups.

^dThere was a greater incidence of 0.2 mg/dL creatinine values in the groups that were indicated as statistically significantly different from the control group. However, this minor difference was not toxicologically relevant, because it was within the historical reference range (Marshfield Labs, 2016).

Mean serum total cholesterol was increased in the male 15× rats relative to the control group and the 10× group and exceeded the reference range by 14%. The increase was influenced by a significant increase in mean serum HDL in male 15× rats relative to the control and 5× groups; however, this increase in serum HDL did not exceed the historical control range. There were no significant differences in female serum total cholesterol concentrations. Female serum HDL was significantly increased in the 5× and 10× groups; however, this increase was within the reference range and the lack of an increase in the 15× female rats suggested that this was not a treatment-related effect. The increased serum total cholesterol in male rats was not considered toxicologically relevant because it was influenced by elevated HDL cholesterol levels.

Terminal fasted body weights and absolute and relative liver weights

The 15× group fasted body weights were significantly decreased by 10–13% relative to the control group (both sexes) and/or the 5× group (males only) (Table 2). Liver weight was significantly decreased by 13% in the male 15× group relative to the control group. There were no significant differences in the liver weight of females or liver-to-body weight ratios of both sexes for any choline-supplemented group.

Table 2.

Fasted body weights, absolute liver weights, and relative liver weights for male and female Sprague Dawley rats fed control diet or diets supplemented with 5×, 10×, or 15× basal choline for 4 weeks.^a

Sex	Male				Female
Parameter (units)	Control (basal choline)^b	5× Choline	10× Choline	15× Choline	Control (basal choline)	5× Choline	10× Choline	15× Choline
Fasted body weight (g)	301.8 ± 8.9	294.0 ± 10.0^$	291.3 ± 9.5	271.4 ± 17.7*^,#	209.3 ± 19.9	197.8 ± 12.4	190.6 ± 8.2	182.4 ± 6.0*
Liver weight (g)	10.3 ± 0.8	9.5 ± 0.5	9.7 ± 0.6	8.9 ± 0.9*	6.4 ± 1.0	6.4 ± 0.5	6.2 ± 0.5	5.8 ± 0.4
Liver-to-body weight ratio	0.034 ± 0.002	0.032 ± 0.0009	0.033 ± 0.001	0.033 ± 0.002	0.030 ± 0.002	0.032 ± 0.001	0.032 ± 0.002	0.032 ± 0.002

ANOVA: analysis of variance.

^aSignificantly different parameters are indicated as follows: (*) Versus control, (#) versus 5×, ($) versus 15×. Evaluated by group within sex using one-way ANOVA with Tukey’s multiple comparisons test (p < 0.05, GraphPad Prism®, v. 6.04). There were no significant differences between the 10× groups and any other group for any parameter.

^b N = 5 control rats/sex and 6 choline supplement rats/sex/group.

Histopathology

Liver histopathology findings in all choline-supplemented groups were similar to the control groups (Table 3) with the exception of minimal hepatocellular hypertrophy in two of six of the 15× choline males and one of six of the 10× choline females. No other histopathology changes or correlative clinical pathology changes (i.e. increased ALT) were noted; thus, the hepatocellular hypertrophy was considered adaptive and not adverse based on a recent publication (Hall et al., 2012). Minimal inflammation was observed in the control and 5× groups (both sexes) and one male of the 10× group but was absent in the 15× group. This finding was only present in a small portion of each section examined and was considered incidental. Oil Red O staining showed no clear changes in liver fat content in males and females across the treatment groups (Table 3); however, there was a trend of decreased liver fat content correlated to increased choline supplementation.

Table 3.

Incidence and severity of liver histopathology results for male and female Sprague Dawley rats fed control diet or diets supplemented with 5×, 10×, or 15× basal choline for 4 weeks.

Sex	Male				Female
Group	Control (basal choline)	5× Choline	10× Choline	15× Choline	Control (basal choline)	5× Choline	10× Choline	15× Choline
Number of animals	5	6	6	6	5	6	6	6
Parameter (severity)
Inflammation	3	3	1	0	2	1	0	0
Minimal	3	3	1	–^a	2	1	–	–
Necrosis, focal, hepatocytes	1	1	0	0	0	0	0	0
Minimal	1	0	–	–	–	–	–	–
Vacuolation, hepatocellular, periportal	0	0	0	0	2	2	0	0
Minimal	–	–	–	–	1	2	–	–
Mild	–	–	–	–	–	–	–	–
Moderate	–	–	–	–	1	–	–	–
Hypertrophy, hepatocellular	0	0	0	2	0	0	1	0
Minimal	–	–	–	2	–	–	1	–
Oil Red O Staining	5	6	6	6	5	6	6	6
Minimal	–	2	3	4	–	–	1	4
Mild	5	4	3	2	3	5	5	2
Moderate	–	–	–	–	2	1	–	–

^aThere were no observations for this group at this severity.

Discussion

The main effects of choline dietary supplementation were decreased body weight and body weight gain in the 10× (females only) and 15× (both sexes) groups. These effects may have been influenced by an acute reduction in dietary intake for these groups from study initiation through day 5. However, there was no subsequent increase in dietary intake following day 5 and no indication that the body weights were normalizing to control levels. Mean daily body weights were decreased by 8.5–12.8% in the male and female 15× rats from day 3 to termination. Overall, body weight gain was decreased relative to the control in the 10× female rats (24%) and the 15× male (26%) and female rats (31%). A 10% decrease in body weight gain in a 90-day study is considered a maximum tolerated dose (Eaton and Gilbert, 2008). Thus, these body weight changes were considered suggestive of a maximum tolerated dose.

The decreased body weights in the present study are generally consistent with previous data (Hodge, 1945). After 4 weeks on choline-supplemented diet in the Hodge study, body weights and body weight gains of the 10× group were similar to the 0.1× group and body weights of the 27×, 50×, and 100× groups were decreased by 20%, 50%, and 67%, respectively, relative to the 0.1× group. After 4 months, the 27×, 50×, and 100× groups did not recover to body weights similar to the 0.1× group. A lack of body weight recovery was also observed in this study where the 10× (females only) and the 15× (both sexes) did not attain control body weight levels by termination. When considered with the results from Hodge, the present study demonstrates that there is a clear dose-dependent correlation between decreased body weight and choline dietary supplementation, even at lower choline supplement concentrations.

There was no evidence of adverse effects on the liver of male or female rats based on the results for multiple clinical chemistry parameters (e.g. ALT, AST, ALKP, bilirubin, and TG), mean liver weights, and liver histopathology. Similarly, Hodge reported no treatment-related histopathology findings in the liver (Hodge, 1945). Mean serum total cholesterol was increased in the male 15× group and exceeded the reference range. Increased serum cholesterol is expected in the presence of a high choline diet based on the well-known importance of choline for phosphatidylcholine formation and cholesterol packaging (Vance, 2008). It is likely that the increased availability of choline led to an increase in phosphatidylcholine production and thus increased lipoprotein packaging and secretion. This is supported by the trend of decreased hepatic lipid content, as shown by Oil Red O staining, correlated to increased choline supplementation. However, the lack of significant differences in serum cholesterol concentrations in female rats suggests a difference in choline handling in female rats relative to the male rats.

Baseline serum choline concentrations were higher in female rats relative to male rats (Figure 3). There were no differences between treatment groups within each sex. The sex-related difference may indicate a greater choline reservoir in female rats. It is possible that females have a higher capacity for choline storage and production. For example, female mice can mobilize choline from extrahepatic tissue in response to choline deprivation (Li and Vance, 2008). Similarly, when humans were fed a choline-deficient diet for up to 42 days, 77% of men and 80% of postmenopausal women developed a choline deficient phenotype (i.e. fatty liver and/or muscle damage) while only 44% of premenopausal women had this phenotype (Fischer et al., 2007). This suggests that females have a greater capacity than males to address choline deprivation and the serum choline results in this study would point to a larger homeostatic serum choline reservoir in female rats than male rats. However, a sex-related difference in serum choline has not been reported in humans (Melse-Boonstra et al., 2005), and, given the limited data set in rats, the observed difference may be a species-specific effect and warrants further investigation.

The choline intakes in this study (i.e. 434–1273 mg/kg/day across all choline-supplemented groups) greatly exceed the tolerable upper limit in humans of 3500 mg/day (Institute of Medicine, 1998) or 60 mg/kg/day in a 58-kg woman (International Organization for Standardization (ISO), 2002). However, even at the high intakes in this study, the main observed effect was decreased body weights with no adverse clinical observations or liver toxicity. Administration of 2500 mg/day choline to 10–12 female martial artists/group for 1 week caused a 10.2% decrease in body fat versus a 4% decrease for the control group (placebo) and a 12.2% decrease in body mass index versus 7.9% decrease for the control group (Elsawy et al., 2014). Data from the present study indicate that choline dietary supplementation up to 15× basal levels can result in decreased body weight with no adverse effects on the liver in rats, which would support the limited data in humans (Elsawy et al., 2014).

In the absence of other clear toxicological effects, the body weight changes, although not necessarily adverse, were considered evidence of a maximum tolerated dose at 10× (females) and 15× (males) of the basal choline dietary levels. These levels can be used to guide future research where dietary choline supplementation is part of the experimental procedure.

Footnotes

Acknowledgements

The authors would like to thank Jill A. Hart and Trina John of 3M Company for their assistance during in-life procedures, Kara Andres of 3M Company for her advice on statistical methods, and Dr Anthony Kiorpes for his consultation during the authoring of this document.

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Dr. Bagley, Dr. Chang, and Mr. Eveland are current employees of 3M Company; Mr. Ehresman and Dr. Butenhoff are former employees of 3M Company; and Drs. Parker and Peters have no conflicts of interest.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by 3M Company, Saint Paul, MN, USA.

Supplemental material

Supplementary material for this article is available online.

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