Choline Status Research Articles

Choline status in newborns, infants, children, breast-feeding women, breast-fed infants and human breast milk

This study assessed the choline status in newborns, infants, children, breast-feeding women, breast milk, infant formula, breast-fed and formula-fed infants. The serum free choline level was 35.1±1.1 μmol/L at birth and decreased to 24.2±1.6, 18.1±0.8, 16.3±0.9, 14.3±0.8, 12.9±0.6 or 10.9±0.6 μmol/L at 22–28, 151–180, 331–365, 571–730, 731–1095 or 4016–4380 days after birth, respectively. The serum phospholipid-bound choline level was 1997±75 μmol/L at birth and increased gradually to 2315±190 or 2572 ±100 μmol/L at 571–730 or 4016–4380 days after birth, respectively. In breast-feeding women, serum free and phospholipid-bound choline levels were doubled at 12–28 days after birth, they decreased toward the control values with time. Free choline, phosphocholine and glycerophosphocholine were major choline compounds in breast milk. Their concentrations in mature milk were much greater than in colostrum and serum. Choline contents of breast milk varied greatly between mothers, and milk free choline levels were correlated with serum free choline ( r=.541; P<.001), phospholipid-bound choline ( r=.527; P<.001) and glycerophosphocholine ( r=.299; P<.01) concentrations and lactating days ( r=.520; P<.001). In breast-fed infants, serum free choline concentrations were correlated with free choline ( r=.47; P<.001), phosphocholine ( r=.345; P<.002), glycerophosphocholine ( r=.311; P<.01) and total choline ( r=.306; P<.01) contents of breast milk. Serum free choline concentration in formula-fed infants was lower than breast-fed infants. These data show that (a) circulating choline status is elevated during infancy and lactation, (b) choline contents of breast milk vary between mothers and milk free choline contents are influenced by maternal circulating choline status, and (c) the choline contents of breast milk can influence infants' circulating choline status.

Declines in serum free and bound choline concentrations in humans after three different types of major surgery

We examined the changes in circulating choline status in humans in response to major surgery by measuring serum free and phospholipid-bound choline concentrations before, during and 1-72 h after total abdominal hysterectomy, off-pump coronary artery graft surgery or brain tumor surgery. Preoperatively, the mean serum free and phospholipid-bound choline concentrations in patients scheduled for abdominal hysterectomy (n = 26), off-pump coronary artery grafting surgery (n = 34) or brain tumor surgery (n = 24) were 12.3 +/- 0.5, 12.1 +/- 0.4 and 11.4 +/- 0.4 micromol/l, and 2495 +/- 75, 2590 +/- 115 and 2625 +/- 80 micromol/l, respectively. Serum free choline and phospholipid-bound choline concentrations decreased from these baseline values to 8.8 +/- 0.7 (p < 0.001), 8.8 +/- 0.5 (p < 0.001) and 8.2 +/- 0.4 micromol/l (p < 0.001), and 2050 +/- 108 (p < 0.001), 2166 +/- 59 (p < 0.001) and 1884 +/- 104 micromol/l (p < 0.001) at 1 h after hysterectomy, off-pump bypass graft surgery or brain tumor surgery, respectively. They remained at these low levels for 24 h and then gradually increased towards the preoperative values at 48-72 h postoperatively. Serum cortisol increased postoperatively in all surgical patients for 24 h and its levels were inversely correlated with serum free and bound choline concentrations. These results show that circulating free and bound choline concentrations decrease for 72 h after total abdominal hysterectomy, off-pump coronary artery graft surgery or brain tumor surgery in humans.

Phosphatidylethanolamine-N-methyltransferase Activity and Dietary Choline Regulate Liver-Plasma Lipid Flux and Essential Fatty Acid Metabolism in Mice

Phosphatidylethanolamine-N-methyltransferase (PEMT) catalyzes the methylation of phosphatidylethanolamine to form phosphatidylcholine (PC) and represents one of the two major pathways for PC biosynthesis. Mice with a homozygous disruption of the PEMT gene are dependent on the 1,2-diacylglycerol cholinephosphotransferase (CDP-choline) pathway for the synthesis of PC and develop severe liver steatosis when fed a diet deficient in choline. The present study used quantitative lipid metabolite profiling to characterize lipid metabolism in PEMT-deficient mice fed diets containing varying concentrations of choline. Choline supplementation restored liver, but not plasma PC concentrations of PEMT-deficient mice to levels commensurate with control mice. Choline supplementation also restored plasma triglyceride concentrations to normal levels, but did not restore plasma cholesterol ester concentrations in the PEMT-deficient mice to those equal to control mice. PEMT-deficient mice also had substantially diminished concentrations of docosahexaenoic acid [22:6(n-3)] and arachidonic acid [20:4(n-6)] in plasma, independent of choline status. Thus, choline supplementation rescued some but not all of the phenotypes induced by the knockout. These findings indicate that PEMT activity functions beyond its recognized role as a compensatory pathway for PC biosynthesis and that, in contrast, PEMT activity is involved in many physiologic processes including the flux of lipid between liver and plasma and the delivery of essential fatty acids to blood and peripheral tissues via the liver-derived lipoproteins.

Plasma choline in normal newborns, infants, toddlers, and in very-low–birth-weight neonates requiring total parenteral nutrition

Choline deficiency is associated with hepatic abnormalities in adult volunteers and patients administered total parenteral nutrition (TPN). Preliminary investigation has suggested that plasma-free choline concentration (PFCh) is greater in neonatal animals, including humans, than in adults. The aims of this study were to determine the normal PFCh and phospholipid-bound choline concentration (PPLBCh) for newborns, infants, and toddlers and to determine the change during TPN. We also sought to determine the degree of fetal choline extraction, the relation between maternal and newborn plasma choline concentrations, and the relation between plasma choline status and normal newborn length, weight, and gestational age. Blood samples were obtained from 104 full-term newborns in two centers (Ben Taub and Maimonides), 25 mothers, 21 normal infants aged 20.3 ± 11.8 wk, 12 normal infants aged 62.4 ± 3.9 wk, and 14 preterm infants (gestational age = 28.9 ± 2.2 wk) who required TPN. The vein PFChs were 28.1 ± 13.0 nmol/mL (Ben Taub) and 68.1 ± 16.9 nmol/mL (Maimonides). The artery PFChs were 27.1 ± 13.0 nmol/mL (Ben Taub) and 57.9 ± 11.6 nmol/mL (Maimonides). The vein PPLChs were 1004.7 ± 246.6 nmol/mL (Ben Taub) and 1121.2 ± 289.6 nmol/mL (Maimonides). The artery PPLChs were 1065.7 ± 469.3 nmol/mL (Ben Taub) and 1106.9 ± 285.8 nmol/mL (Maimonides). The vein-minus-artery differences for PFCh were 1.0 ± 9.7 nmol/mL (Ben Taub) and 10.2 ± 10.9 nmol/mL (Maimonides). The vein-minus-artery differences for PPLCh were −51.9 ± 398.2 nmol/mL (Ben Taub General Hospital, Houston, Texas) and 14.4 ± 254.3 nmol/mL (Maimonides, New York, New York). Maternal venous PFCh was 8.4 ± 3.1 nmol/mL. Maternal venous PPLCh was 2592.1 ± 584.0 nmol/mL (range = 1227.8–3729.0). Maternal venous PFCh correlated with newborn arterial PFCh ( r = 0.53, P < 0.05) but not with newborn venous PFCh. No correlation was seen between maternal venous and newborn PPLCh. No significant differences were seen in PPLCh or choline extraction in Ben Taub versus Maimonides patients, although PFCh was significantly greater in the newborns from Maimonides ( P < 0.05). The mean venous PFCh and PPLCh in the preterm infants before beginning TPN was 21.2 ± 6.3 and 1366.8 ± 339.1 nmol/mL, respectively. Just before initiation of tube feeding (4.0 ± 2.7 d after TPN had been started), mean venous PFCh and PPLCh was 18.4 ± 5.3 and 2251.8 ± 686.9 nmol/mL, respectively. When TPN was discontinued and tube feeding increased to goal, after 10.8 ± 10.4 d, venous PFCh and PPLCh was 22.6 ± 8.7 and 2072.5 ± 540.6 nmol/mL, respectively. Venous PFCh and PPLCh was 13.4 ± 2.5 and 1827.5 ± 327.0 nmol/mL, respectively in the older infant group. In conclusion, newborn PFCh is significantly greater than PFCh in adults but falls to adult levels within the first year of life. Low maternal PFCh may be associated with low newborn PFCh. Normal newborn plasma choline status has no bearing on intrauterine growth, although the role of maternal choline deficiency in underweight newborns is unknown. Newborn PPLCh is substantially below that of adults, which suggests its use in membrane synthesis during growth.

Plasma Free, Phospholipid-Bound and Urinary Free Choline All Decrease During a Marathon Run and May Be Associated with Impaired Performance

Background: Previous investigations have shown that plasma free choline decreases during long distance running.Objective: This study was undertaken to determine if body choline status changes during a marathon run and whether performance is thereby adversely affected.Design: Twenty-three accomplished marathon runners 25 to 49 years of age were studied before and after the 1997 Houston-Methodist Marathon. Fasting blood and five-hour urine samples were obtained in the morning, 14 days prior to the race, immediately after the race and approximately 48 hours after completion of the race. Runners were asked to predict their finish times two weeks prior to the race. Performance was indicated by the ratio of predicted to actual time.Results: Both plasma free and phospholipid-bound choline concentrations as well as urinary free choline concentration decreased immediately following the race (19.2±4.5 to 14.6±4.2 nmol/mL, p=0.005, and 2565.2±516.4 to 2403.4±643.0 nmol/mL, p=0.068, respectively) and, except for the phospholipid-bound choline, rebounded towards baseline after 48 hours (15.6±3.2 and 2299.9±426.7 nmol/mL), although plasma concentrations remained significantly below baseline. Plasma free and phospholipid-bound choline concentrations were significantly correlated (r=0.46, p=0.0001), although urinary free choline concentration was not correlated with either. There was no correlation between plasma free, phospholipid-bound or urinary free choline concentration and actual finish time or the ratio of predicted to actual finish time. However, the percent decrease in urinary free choline concentration was significantly correlated with the ratio of predicted to actual time (r=0.47, p=0.036). No relationship was seen between this ratio and the percent decrease in either plasma free or phospholipid-bound choline concentrations immediately after the race.Conclusion: Our finding of both decreased free and phospholipid-bound choline suggests the decrease in choline status is related to accelerated choline metabolism or enhanced choline uptake by tissues rather than decreased hepatic choline release. The role of choline supplementation during endurance running requires further investigation.

Folate Nutriture Alters Choline Status of Women and Men Fed Low Choline Diets

Choline and folate share methylation pathways and, in studies of rats, were shown to be metabolically inter-related. To determine whether choline status is related to folate intake in humans, we measured the effect of controlled folate depletion and repletion on the plasma choline and phosphatidylcholine concentrations of 11 healthy men (33–46 y) and 10 healthy women (49–63 y) fed low-choline diets in two separate metabolic unit studies. Total folate intake was varied by supplementing low folate (25 and 56 μg/d for men and women, respectively) and low choline (238 and 147 mg/d for men and women, respectively) diets with pteroylglutamic acid for 2–6 wk following folate-depletion periods of 4–5 wk. The low folate/choline intakes resulted in subclinical folate deficiencies; mean plasma choline decreases of 28 and 25% in the men and women, respectively; and a plasma phosphatidylcholine decrease of 26% in the men (P < 0.05). No functional choline deficiency occurred, as measured by serum transaminase and lipid concentrations. The decreases in choline status measures returned to baseline or higher upon moderate folate repletion and were more responsive to folate repletion than plasma folate and homocysteine. Feeding methionine supplements to the men did not prevent plasma choline depletion, indicating that folate is a more limiting nutrient for these methylation pathways. The results indicate that 1) choline is utilized as a methyl donor when folate intake is low, 2) the de novo synthesis of phosphatidylcholine is insufficient to maintain choline status when intakes of folate and choline are low, and 3) dietary choline is required by adults in an amount > 250 mg/d to maintain plasma choline and phosphatidylcholine when folate intake is low.

Dietary choline supplementation in pregnant rats increases hippocampal phospholipase D activity of the offspring.

Supplementation with choline during pregnancy in rats causes a long-lasting improvement of visuospatial memory of the offspring. The biochemical mechanism of this effect may be related to the function of choline as a precursor of phosphatidylcholine (PC), the substrate of a receptor-stimulated enzyme, phospholipase D (PLD). PLD activation initiates the sequential formation of two intracellular messengers, phosphatidic acid and 1,2-sn-diacylglycerol. We hypothesized that prenatal choline status may cause long-term modulation of PLD-catalyzed PC hydrolysis in the hippocampus, a brain region implicated in visuospatial memory functions. PLD activity was determined in hippocampal slices prelabeled with [3H]glycerol or [3H]oleic acid by measuring the PLD-catalyzed formation of [3H]phosphatidylpropanol in the presence of 1-propanol. Slices were obtained from male pups born to mothers consuming a control diet, a choline-supplemented diet, or a choline-free diet from days 11 to 17 of pregnancy. The radiolabeling of phospholipid classes was unaffected by the treatments. Prenatal choline supplementation significantly increased basal PLD activity in [3H]glycerol-labeled slices [by 46% of controls on postnatal day (P) 7 and by 36% on P21], and [3H]oleate-labeled slices (by 91% on P7), as well as glutamate-stimulated PLD activity in [3H]oleate-labeled slices (by 60% on P7). Prenatal choline deficiency failed to alter PLD activity. The actions of choline apparently required intact cells because in vitro assays of PLD activity in hippocampal homogenates, using fluorescent NBD-PC as substrate, revealed no differences between groups. The results show that prenatal choline supplementation up-regulates basal and receptor-stimulated PLD activity in the hippocampus during postnatal development.

Bioavailability of choline and choline esters from milk in rat pups

Previously, we had shown that human milk and infant formulas contained choline, phosphocholine (PCho), glycerophosphocholine (GPCho), and phosphatidylcholine (PtdCho). The relative bioavailability of these choline-containing compounds in milk has not previously been studied. Using a rat pup model, infant formula (S.M.A. TM, Wyeth-Ayerst) containing either [ 14C-methyl]-choline chloride ( 14C-Cho) [ 14C-methyl]-PCho, [ 14C-methyl]-GPCho), or [L-α-dipalmitoyl- 14C-methyl]-PtdCho was fed intragastrically by a single intubation into 15-day-old postnatal rat pups. Label from the water-soluble metabolites of choline (choline, phosphocholine, and glycerophosphocholine) appeared rapidly within blood and liver, reaching peak levels within 1 to 5 hr, and label in brain continued to increase for more than 24 hr. Label from the lipid soluble metabolite, phosphatidylcholine, took much longer to appear in blood and liver (5 to 8 hr) and label remained elevated in blood for at least 24 hr. Label in brain continued to increase for more than 24 hr, but always remained lower than that attained after treatment with the labeled water-soluble choline metabolites. The liver is a major storage site for choline metabolites, and provides a sensitive indicator of dietary choline status. In liver, a large portion of the label derived from the water-soluble choline metabolites was in the form of betaine at 4 hr post dose. At the same time, most of the PtdCho-derived label was still present as PtdCho in liver. At 24 hr after dose, most of the label derived from choline and PCho in liver was present as betaine (85%) and PtdCho (15%), label derived from GPCho was found as betaine (54%), PtdCho (15%), PCho (11%), GPCho (2%), and choline (18%). Label derived from PtdCho was found as betaine (13%) PCho (2%), and PtdCho (85%). We conclude that 15-day-old postnatal rat pups can absorb the various choline compounds in milk. Choline and PCho appear to be essentially identical in their absorption and metabolic fate. GPCho and PtdCho have different rates of absorption and/or metabolism. Thus, we conclude that there are significant differences in bioavailability, tissue uptake and metabolism among the choline compounds that are present in milk.

Nicotinamide Megadosing Increases Hepatic Poly(ADP-Ribose) Levels in Choline-Deficient Rats

Previous work in our laboratory has shown that dietary megadoses of nicotinamide, used in the prevention of diabetes, cause increases in hepatic poly(ADP-ribose). Poly(ADP-ribose) is synthesized from NAD+ by a nuclear enzyme, poly(ADP-ribose)poly-merase, which is activated by DNA strand breaks. The nicotinamide-induced increase in poly(ADP-ribose) could result from an increase in substrate, NAD+, or the induction of strand breaks in DNA. Strand breaks may result from the depletion of single carbon groups, through the excretion of methylated derivatives of nicotinamide. To differentiate between these mechanisms, a 3 ՠ3 factorial experiment was conducted in which rats were fed diets containing various supplements of choline bitartrate (0, 2, 20 g/kg diet) and nicotinamide (0, 1, 2 g/kg diet). At the conclusion of treatments, blood NAD+ and liver lipid, NAD+ and poly(ADP-ribose) levels were determined. Choline deficiency caused the characteristic accumulation of fat in the liver at all levels of nicotinamide. In choline deficient rats, nicotinamide supplements further increased liver lipid concentration. Blood and liver NAD+ concentrations were increased by nicotinamide supplementation, irrespective of choline status. In contrast, liver poly(ADP-ribose) levels were increased by nicotinamide supplementation only in choline deficient rats. These results show that nicotinamide-induced increases in poly(ADP-ribose) levels appear to be dependent on decreased methyl donor status and suggest that adequate choline status is important for preventing some deleterious effects of nicotinamide treatment.

Choline-betaine requirements of rainbow trout ( Oncorhynchus mykiss)

A series of experiments with rainbow trout ( Oncorhynchus mykiss) were conducted to quantify the dietary requirement for choline as well as to determine whether betaine or methionine could spare the trout's choline requirement. Fish weighing 1.4 and 3.2 g, which were acclimated to a choline-deficient semipurified diet for 14 days before the start of the experiments, were shown to have a choline requirement of 813 and 714 ppm, respectively. When an excess of betaine was added to the choline-deficient basal diet, the choline requirement was reduced to about 400 ppm. These results suggest that about half of the trout's choline requirement must be met by choline itself, whereas the other half can be supplied by betaine. However, the requirement for choline itself must first be met before a response to betaine can be obtained. The addition of betaine to a choline-deficient diet resulted in no improvement in weight gain. Likewise, the addition of L-methionine, isomethyl to 1000 ppm choline, was without effect on growth in a basal diet marginally adequate in sulfur amino acids — suggesting that the trout's requirement for choline is not replaceable by methionine. Because the effects of dietary choline on liver lipid were variable and difficult to interpret, it was demonstrated that growth was more sensitive than liver lipid as an indicator of choline status.

Effect of Choline Deficiency on Lung Phospholipid Concentrations in the Rat

Lung surfactant and nonsurfactant phospholipid concentrations were analyzed in relation to choline status in male rats fed a choline-deficient (CD) or choline-supplemented (CS) diet over an 8-d period. On the first day plasma choline concentrations were significantly lower (11.5 ± 0.9 µM) in rats fed the CD diet than in those fed the CS diet (16.1 ± 1.2 µM). This relationship continued for the duration of the study. Hepatic phosphatidylcholine/phosphatidylethanolamine (PC/PE) ratios were significantly lower after d 1 of the CD diet, further decreased on d 2 (1.09 ± 0.04) and remained low through d 8. Only on d 4 were lung surfactant PC and total phospholipid concentrations lower in rats fed the CD diet than in those fed the CS diet. The composition of surfactant, determined by the ratio of PC to total phospholipids, did not change. On both d 4 and d 8 the PC/PE ratios in the non-surfactant fraction were lower in rats fed the CD diet than in those fed the CS diet. This shift in PC/PE ratio in the lung is similar to the PC/PE shift in the liver associated with dietary choline deficiency. The altered lung phospholipid concentrations in the nonsurfactant (residual) fraction on d 4 and d 8 suggest an adaptation in the lung’s phospholipid metabolism to replenish the physiologically essential surfactant PC.

Measurement of free choline concentrations in maternal and neonatal blood by micropyrolysis gas chromatography

The development of a micropyrolysis gas Chromatographie technique for the determination of free choline in plasma and erythrocytes is reported as a means to clinically assess choline status. A micropyrolyzer syringe unit was fabricated and the method was standardized through repeated trials with a single plasma specimen. The interassay coefficient of variance was 1.3% for the standardized trials on plasma and erythrocytes. Choline status was assessed in control women, mothers and premature infants at birth and up to 14 days of life. At birth, plasma choline levels in the infants (32.2 ± 5.5 μmol/l) were significantly higher than those in the mothers (12.9 ± 2.6 μmol/l) and the control women (16.9 ± 1.6 μmol/l). The infants' plasma choline concentrations had decreased significantly by 7 days of life and remained at the lower level at day 14, independent of the nutritional intervention administered.

Lung Surfactant Phospholipids as Related to Hydration and Choline Status of Fasted Rats

Hydration status and choline nutrition were evaluated relative to the concentration of lung surfactant phosphatidylcholine (PC) in fasted rats. Rats deprived of food for 72 h showed lower voluntary water consumption and consistently lower levels of both PC and total phospholipid (TPL) present in isolated pulmonary surfactant than ad libitum-fed controls, although the ratio of surfactant PC to TPL and the residual PC and TPL concentrations were not different. Higher hematocrit values observed in the fasted animals were not altered by the administration of water or saline by orogastric tube nor was the low surfactant PC level corrected by fluid therapy. Evidence of choline deficiency was demonstrated in the fasted rats as there was a significant shift in hepatic PC:phosphatidylethanolamine; however, plasma choline levels did not change. The administration to fasted animals of up to 2.4 mmol of choline chloride via the drinking water (containing 60 mM choline) or an orogastric tube did not affect the plasma choline concentrations or the production of lung surfactant PC.

The Methionine and Choline Status of Rat Diets and Their Effects on Nutrition and Myocardial Lesions

This study evaluated for rats the nutritional adequacy of casein-based diets routinely used to test the cardiopathogenicity of vegetable oils. Diets were formulated containing 20% by weight casein, 20% soybean oil and graded levels of choline with and without methionine and were fed to male Sprague-Dawley rats for 16 weeks. Rats fed methionine-supplemented diets had improved growth and food consumption, increased liver lipids mainly in the form of triglycerides and normal amino acid metabolism. On the other hand, choline supplementation reduced liver lipids but had no effect on growth and feed consumption. The results would indicate that diets including 20% casein and 20% oil require methionine supplementation to assure the nutritional adequacy of this high caloric diet. However, in this study there was no evidence to indicate that choline or methionine supplementation affected the heart lesion incidence in male rats. It was therefore concluded that the amount and type of fat in the diet, not the choline and methionine status of the diet, is related to heart lesions in male rats.

Precursor (Amino Acid): Product (Vitamin) Interrelationship for Growing Chicks as Illustrated by Tryptophan-Niacin and Methionine-Choline

Experiments were conducted with crossbred chicks to examine the growth response to a vitamin when its precursor amino acid was in either deficient or adequate supply in the diet and vice versa. The tryptophan-niacin and methionine-choline interrelationships were thus examined. No growth responses to dietary niacin or choline were observed during severe tryptophan or methionine deficiency, respectively. However, when adequate levels of the precursor amino acids were present, marked gain and gain/feed responses to these vitamins occurred. In contrast, gain and feed efficiency responses to tryptophan or methionine occurred regardless of the chick's niacin or choline status, respectively.

Assay for free and total choline activity in biological fluids and tissues of rats and man with Torulopsis pintolopessi

The sensitive, specific growth response to choline activity of the thermophilic enteric yeast Torulopsis pintolopessi enables estimation of free and bound choline activity in rat and human fluids and tissues—as little as 10 ng/ml of choline is measurable. Unlike other microbial assays, estimation of unbound (free) choline activity is not influenced by methionine or phospholipids. The method also distinguishes differences in choline activity of fluids and tissues from choline-deficient and choline-replete rats. Free and bound choline activity in blood, red blood cells, plasma, and liver from choline-deficient rats were almost 2-fold lower than from choline-supplemented animals. Free and bound choline activity in whole brain from choline-deficient rats were significantly higher (more than 2-fold). The application of the T. pintolopessi method in studying choline status in man and reasons for high choline activity in brain of choline-deficient rats are suggested.