This is the first study of choline requirements that included women. We found that most men and postmenopausal women developed organ dysfunction when deprived of choline, whereas most premenopausal women did not. Susceptibility to choline deficiency was not altered by supplementation with folic acid.
We identified a group of rapid depleters who developed organ dysfunction when fed the AI of choline (550 mg choline · 70 kg−1
). We presume that these men, when free-living, consumed diets that had a higher choline content; this would explain the decreased mean plasma phosphatidylcholine concentrations that we observed after 10 d of the 550-mg choline diet. Similarly, our experimental diet was likely higher in betaine than was the subjects’ ad libitum diets, perhaps explaining the increased mean plasma betaine concentration observed. These rapid depleters developed fatty liver, muscle damage, or both that reversed when they consumed a high-choline diet (825 mg · 70 kg−1
diet) or an ad libitum diet. Five of 6 subjects developed especially high CPK activities in plasma. These men are worthy of further study; it is possible that these men have a defect in CTL1
, the mammalian homolog of the yeast choline transporter gene responsible for choline uptake into muscle (32
). Because the ad libitum diet differed from the repletion diet in more nutrients than just choline, we cannot be sure that the reversal of organ dysfunction observed was solely due to choline restoration. For this reason, we segregated the analysis of data from these subjects and did not include them in the analyses of the other 51 subjects studied.
Liver and muscle dysfunction occurred in response to a low-choline diet in both men and women. Fatty liver (hepatosteatosis) is common in human and animal models of choline deficiency (2
) because a specific lack of phosphatidylcholine limits the export of excess triacylglycerol from the liver (34
). Leakage of enzymes (eg, AST, ALT, AP, LD, and CPK) from tissues of liver and muscle into blood likely occurs because choline deficiency induces apoptotic pathways in these cells (24
). Because choline deficiency is associated with DNA damage and apoptosis (37
), it is not surprising that choline-depleted humans have hyperuricemia because uric acid, a product of purine metabolism, is released into blood after tissue lysis (40
The glomerulus of the kidney uses 2 metabolites of choline (betaine and glycerophosphocholine) as osmolytes (41
), and choline-deficient rodents have renal dysfunction (1
). We did not observe dietary choline-related changes in urinalysis or urine-specific gravity in our subjects (data not shown).
Choline is derived not only from the diet but from de novo synthesis of phosphatidylcholine catalyzed by PEMT. As discussed earlier, PEMT activity is increased by estrogen in animal models. We hypothesize that this is the reason why premenopausal women were more resistant to developing signs of organ dysfunction when fed a low-choline diet. We are currently examining whether the human PEMT gene has an estrogen response element in its promoter. Only 2 postmenopausal women in our study were treated with hormone replacement therapy; 1 became deplete and 1 did not during the low-choline diet. The subject who became deplete was using a transdermal patch and was receiving a dose lower than that of the other subject who took hormone replacement therapy orally. Further studies are needed before we can determine whether the choline requirement in postmenopausal women is decreased by estrogen therapy.
The requirement for choline in the diet is quite variable. A portion of the men and women we studied required more than the recommended AI for choline, whereas others required <50 mg choline · 70 kg−1
. Some subjects became deplete quickly and some took almost 7 wk to develop organ dysfunction when fed a low-choline diet. Estrogen status accounts for much of this variability. In addition, we recently reported that genetic polymorphisms may account for the rest of the variability in dietary choline requirements. We identified a single nucleotide polymorphism in the promoter region of the PEMT
gene (rs12325817), for which 18 of the 23 female carriers of the variant C allele (78%) developed organ dysfunction when fed a low-choline diet (odds ratio: 25; P
= 0.002); the variant C allele was relatively common: 18% of subjects were CC, 56% were GC, and 26% were GG genotype (44
). In addition, premenopausal women who were carriers of the common 5,10-methylenetetrahydrofolate dehydrogenase-1958A (MTHFD1
) gene allele were >15 times as likely as were noncarriers to develop signs of choline deficiency (P
< 0.0001) during the low-choline diet unless they were also treated with a folic acid supplement (45
We previously published that choline metabolism is interrelated to homocysteine metabolism (18
). It is interesting that premenopausal women, whatever the response group, had a lower plasma homocysteine concentration at baseline (with signs: 5.7 ± 0.4 nmol/mL; without signs: 4.6 ± 0.2 nmol/mL) than did men (with signs: 7.4 ± 0.3 nmol/mL; without signs: 6.9 ± 0.6 nmol/mL); however, when subjects were fed the low-choline diet, homocysteine concentrations uniformly increased 20% in men (with signs: 8.8 ± 0.6 nmol/mL; without signs: 8.6 ± 0.7 nmol/mL), premenopausal women (with signs: 6.9 ± 0.6 nmol/mL; without signs: 5.8 ± 0.2 nmol/mL), and postmenopausal women (data not shown). Also, we report that plasma concentrations of methylated end products of choline and methionine metabolism changed in predicted directions ().
We observed no effect of folic acid supplementation on susceptibility or on mode of presentation. Previously published studies suggesting that folic acid supplementation might decrease requirements for choline used diets much higher in choline (150−300 mg choline/d) than ours (< 50 mg/d) (20
). Perhaps the effects of folate become apparent only when marginally adequate amounts of choline are supplied and not when diets are almost devoid of choline.
Plasma concentrations of choline, betaine, and phosphatidylcholine decreased when subjects were fed a low-choline diet, but the decrease was not highly correlated with susceptibility to developing organ dysfunction while this diet was being consumed. Thus, decreased plasma concentrations of choline metabolites are a necessary, but not sufficient, criterion for predicting choline deficiency–associated organ dysfunction. Plasma concentrations likely do not fully reflect intracellular concentrations of these metabolites.
This study, in combination with our previous work, establishes a panel of measurements that can be used to define individuals who are sufficiently deplete of choline to develop liver and muscle dysfunction. Factors that we identified that increase susceptibility to developing organ dysfunction in humans fed low-choline diets, such as menopausal status and genetic polymorphisms, are likely to be of clinical importance. Humans fed intravenously with solutions low in choline (total parenteral nutrition) often develop liver dysfunction that sometimes resolves when a choline source is added to their feeding solution (46
); it may be that susceptible individuals can be identified based on the factors we identified and treatment appropriately modified. Poor dietary intake of choline contributes to adverse outcomes during pregnancy, a time when choline demand is high (47
). Deficient maternal dietary intake of choline during pregnancy in humans is associated with a 4-fold increased risk of having a baby with a neural tube defect (47
); it is possible that susceptible mothers can be identified through examination of the genetic polymorphisms we describe. In rodent models, maternal dietary choline intake influences brain development. Rodents fed choline-deficient diets during late pregnancy have offspring with diminished progenitor cell proliferation and increased apoptosis in fetal hippocampus (49
), insensitivity to long-term potentiation when they were adult animals (51
), and decremented visuospatial and auditory memory (52
). For these reasons, understanding sources of variability in human choline requirements is important.
Of the 57 subjects fed the low-choline or baseline diets, the current AI for choline was sufficient to prevent or reverse organ dysfunction associated with choline deficiency in 46 individuals (81%); the remainder needed 825 mg choline · 70kg−1
or the amount of choline in an ad libitum diet (>550 mg · 70 kg−1
). This data should help inform the Institute of Medicine as they refine estimates for DRIs for choline.