Choline deficiency-related multi-omics characteristics are susceptible factors for chemotherapy-induced thrombocytopenia.

The XELOX chemotherapy protocol that includes capecitabine and oxaliplatin is the routine treatment for colorectal cancer (CRC), but it can cause chemotherapy-related adverse events such as thrombocytopenia (TCP). To identify predictive biomarkers and clarify the mechanism of TCP susceptibility, we conducted integrative analysis using normal colorectal tissue (CRT), plasma, and urine samples collected before CRC patients received adjuvant XELOX chemotherapy. RNA-sequencing and DNA methylation arrays were performed on CRT samples, while liquid chromatography-mass spectrometry was performed on CRT, plasma, and urine samples. Differentially expressed features (DEFs) from each uni-omics analysis were then subjected to integrative analysis using Multi-Omics Factor Analysis (MOFA). Choline-deficiency in plasma and CRT was found as the most critical TCP-related feature. Based on bioinformatic analysis and literature research, we further concluded that choline-deficiency was the possible reason for most of the other TCP-related multi-omics DEFs, including metabolites representing reduced sphingolipid de novo synthesis and elevated solute carrier-mediated transmembrane transportation in CRT and plasma, DNA hypermethylation and elevated expression of genes involved in neuronal system genes. In terms of thrombocytopoiesis, these TCP-related DEFs may cause atypical maintenance and differentiation of megakaryocyte, resulting a suppressed ability of thrombocytopoiesis, making patients more susceptible to chemotherapy-induced TCP. At last, prediction models were developed and validated with reasonably good discrimination. The area under curves (AUCs) of training sets were all > 0.9, while validation sets had AUCs between 0.778 and 0.926. In conclusion, our results produced reliable marker systems for predicting TCP and promising target for developing precision treatment to prevent TCP.

Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.

Methyl-donor deficiency is a risk factor for neurodegenerative diseases. Dietary deficiency of the methyl-donors methionine and choline [methionine-choline-deficient (MCD) diet] is a well-established model of nonalcoholic steatohepatitis (NASH), yet brain metabolism has not been studied in this model. We hypothesized that supplemental betaine would protect both the liver and brain in this model and that any benefit to the brain would be due to improved liver metabolism because betaine is a methyl-donor in liver methylation but is not metabolically active in the brain. We fed male Sprague-Dawley rats a control diet, MCD diet, or betaine-supplemented MCD (MCD+B) diet for 8 wk and collected blood and tissue. As expected, betaine prevented MCD diet-induced NASH. However, contrary to our prediction, it did not appear to do so by stimulating methylation; the MCD+B diet worsened hyperhomocysteinemia and depressed liver methylation potential 8-fold compared with the MCD diet. Instead, it significantly increased the expression of genes involved in ß-oxidation: fibroblast growth factor 21 and peroxisome proliferator-activated receptor α. In contrast to that of the liver, brain methylation potential was unaffected by diet. Nevertheless, several phospholipid (PL) subclasses involved in stabilizing brain membranes were decreased by the MCD diet, and these improved modestly with betaine. The protective effect of betaine is likely due to the stimulation of ß-oxidation in liver and the effects on PL metabolism in brain.-Abu Ahmad, N., Raizman, M., Weizmann, N., Wasek, B., Arning, E., Bottiglieri, T., Tirosh, O., Troen, A. M. Betaine attenuates pathology by stimulating lipid oxidation in liver and regulating phospholipid metabolism in brain of methionine-choline-deficient rats.

Choline Supplementation in Cystic Fibrosis-The Metabolic and Clinical Impact.

BACKGROUND: Choline is essential for the synthesis of liver phosphatidylcholine (PC), parenchymal maintenance, bile formation, and lipoprotein assembly to secrete triglycerides. In choline deficiency, the liver accretes choline/PC at the expense of lung tissue, thereby impairing pulmonary PC homoeostasis. In cystic fibrosis (CF), exocrine pancreas insufficiency results in impaired cleavage of bile PC and subsequent fecal choline loss. In these patients, the plasma choline concentration is low and correlates with lung function. We therefore investigated the effect of choline supplementation on plasma choline/PC concentration and metabolism, lung function, and liver fat. METHODS: 10 adult male CF patients were recruited (11/2014⁻1/2016), and orally supplemented with 3 × 1 g choline chloride for 84 (84⁻91) days. Pre-/post-supplementation, patients were spiked with 3.6 mg/kg [methyl-D9]choline chloride to assess choline/PC metabolism. Mass spectrometry, spirometry, and hepatic nuclear resonance spectrometry served for analysis. RESULTS: Supplementation increased plasma choline from 4.8 (4.1⁻6.2) µmol/L to 10.5 (8.5⁻15.5) µmol/L at d84 (p < 0.01). Whereas plasma PC concentration remained unchanged, D9-labeled PC was decreased (12.2 [10.5⁻18.3] µmol/L vs. 17.7 [15.5⁻22.4] µmol/L, p < 0.01), indicating D9-tracer dilution due to higher choline pools. Supplementation increased Forced Expiratory Volume in 1 second percent of predicted (ppFEV1) from 70.0 (50.9⁻74.8)% to 78.3 (60.1⁻83.9)% (p < 0.05), and decreased liver fat from 1.58 (0.37⁻8.82)% to 0.84 (0.56⁻1.17)% (p < 0.01). Plasma choline returned to baseline concentration within 60 h. CONCLUSIONS: Choline supplementation normalized plasma choline concentration and increased choline-containing PC precursor pools in adult CF patients. Improved lung function and decreased liver fat suggest that in CF correcting choline deficiency is clinically important. Choline supplementation of CF patients should be further investigated in randomized, placebo-controlled trials.

Butein protects the nonalcoholic fatty liver through mitochondrial reactive oxygen species attenuation in rats.

One of the worldwide metabolic health dilemma is nonalcoholic fatty liver diseases (NAFLD). Researchers are searching effective drug to manage NAFLD patients. One of the best way to manage the metabolic imperfection is through natural principal isolated from different sources. Butein, a natural compound known to have numerous pharmacological application. In the current study we assessed the therapeutic effect of butein administration on liver function tests, oxidative stress, antioxidants, lipid abnormalities, serum inflammatory cytokines, and mitochondrial reactive oxygen species levels, in rats with methionine-choline deficient (MCD) diet induced NAFLD. Male Wistar rats were treated with MCD diet with/without butein (200 mg/kg body wt. orally) for 6 weeks. The protective effect of butein, were evident from decreased transaminase activities, restoration of albumin, globulin, albumin/globulin ratio, and oxidants in serum (P < 0.01), further it improved liver antioxidant status (P < 0.01). Butein significantly lowered lipid profile parameters (P < 0.01), suppressed inflammatory cytokines (P < 0.01), and improved liver histology. Further to understand the possible mechanism behind the hepatoprotective and lipid lowering effect of butein, the activities of heme oxygenase (HO1), myeloperoxidase (MPO), and mitochondrial reactive oxygen species (ROS) were measured. We found that butein supplementation significantly decreased the activity of HO1 (P < 0.001), and increased the activity of MPO (P < 0.001). Furthermore butein attenuated mitochondrial ROS produced in NAFLD condition. Present study shows that butein supplementation restore liver function by altering liver oxidative stress, inflammatory markers, vital defensive enzyme activities, and mitochondrial ROS. In summary, butein has remarkable potential to develop effective hepato-protective drug. © 2018 BioFactors, 44(3):289-298, 2018.

Baicalin attenuates non-alcoholic steatohepatitis by suppressing key regulators of lipid metabolism, inflammation and fibrosis in mice.

AIMS: Baicalin (BA), an active flavonoid compound originating from the herb of Scutellaria baicalensis Georgi, has been previously shown to exert anti-inflammation and anti-oxidant effects in liver diseases. However, the potential role of BA in the regulation of non-alcoholic steatohepatitis (NASH) remains elusive. In this study, we newly explored the hepatoprotective effects of BA in MCD diet-induced NASH by ameliorating hepatic steatosis, inflammation, fibrosis and apoptosis. MAIN METHODS: NASH was induced in mice fed a methionine and choline-deficient (MCD) diet for 4weeks. The mice were simultaneously treated with or without BA for 4weeks. Serum liver functional markers and inflammatory indicators were assessed by biochemical and ELISA methods, respectively. The livers were histologically examined using H&E, Oil Red O and Masson's trichrome staining methods. The qRT-PCR, IHC and Western blotting assays were applied to analyze mechanisms underlying BA protection. KEY FINDINGS: BA treatment significantly attenuated MCD diet-induced hepatic lipid accumulation partly through regulating the expression of SREBP-1c, FASN, PPARα and CPT1a. BA treatment dramatically suppressed MCD diet-induced hepatic inflammation, which was associated with decrease in serum TNF-α, IL-1ß and MCP-1 production, macrophage influx and suppression of nuclear factor-κB activation. Additionally, BA was proved to prevent liver fibrosis, which appears to be mediated by inhibition of α-SMA, TGF-ß1 and Col1A1. Furthermore, BA markedly inhibited hepatocyte apoptosis and cleaved caspase-3 protein expression in MCD diet-induced mice. SIGNIFICANCE: These results provide a possible basis of the underlying mechanism for the application of BA in the treatment of NASH.

Immunohistochemical determination of the extracellular matrix modulation in a rat model of choline-deprived myocardium: the effects of carnitine.

Choline has been identified as an essential nutrient with crucial role in many vital biological functions. Recent studies have demonstrated that heart dysfunction can develop in the setting of choline deprivation even in the absence of underlying heart disease. Matrix metalloproteinases (MMPs) are responsible for extracellular matrix degradation, and the dysregulation of MMP-2 and MMP-9 has been involved in the pathogenesis of various cardiovascular disorders. The aim of the study was to investigate the role of MMPs and their inhibitors (TIMPs), in the pathogenesis of choline deficiency-induced cardiomyopathy, and the way they are affected by carnitine supplementation. Male Wistar Albino adult rats were divided into four groups and received standard or choline-deficient diet with or without L-carnitine in drinking water (0.15% w/v) for 1 month. Heart tissue immunohistochemistry for MMP-2, MMP-9, TIMP-1, and TIMP-2 was performed. Choline deficiency was associated with suppressed immunohistochemical expression of MMP-2 and an increased expression of TIMP-2 compared to control, while it had no impact on TIMP-1. MMP-9 expression was decreased without, however, reaching statistical significance. Carnitine did not affect MMP-2, MMP-9, TIMP-1 or TIMP-2 expression. The pattern of TIMP and MMP modulation observed in a choline deficiency setting appears to promote fibrosis. Carnitine, although shown to suppress fibrosis, does not seem to affect MMP-2, MMP-9, TIMP-1 or TIMP-2 expression. Further studies will be required to identify the mechanism underlying the beneficial effects of carnitine.

Purified anthocyanins from bilberry and black currant attenuate hepatic mitochondrial dysfunction and steatohepatitis in mice with methionine and choline deficiency.

The berries of bilberry and black currant are a rich source of anthocyanins, which are thought to have favorable effects on nonalcoholic steatohepatitis (NASH). This study was designed to examine whether purified anthocyanins from bilberry and black currant are able to limit the disorders related to NASH induced by a methionine-choline-deficient (MCD) diet in mice. The results showed that treatment with anthocyanins not only alleviated inflammation, oxidative stress, steatosis, and even fibrosis but also improved depletion of mitochondrial content and damage of mitochondrial biogenesis and electron transfer chain developed concomitantly in the liver of mice fed the MCD diet. Furthermore, anthocyanins treatment promoted activation of AMP-activated protein kinase (AMPK) and expression of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α). These data provide evidence that anthocyanins possess significant protective effects against NASH and mitochondrial defects in response to a MCD diet, with a mechanism maybe through affecting the AMPK/PGC-1α signaling pathways.

Taurine supplementation does not decrease homocysteine levels and liver injury induced by a choline-deficient diet.

AIMS: The aim of this study is to examine the effects of taurine supplementation on homocysteine (Hcy) metabolism and liver injury in rats fed a choline-deficient diet. MAIN METHODS: Thirty rats were divided into three groups (n=10), to receive one of the following diets for 4 weeks: control diet (C), choline-deficient diet (CDD), or choline-deficient diet supplemented with taurine (CDDT). The CDD and the CDDT consisted of AIN-93 without the recommended choline content of 2.5%, and the CDDT was supplemented by the addition of 2.5% taurine. KEY FINDINGS: Four weeks of ingesting a CDD resulted in a significant increase in plasma Hcy (50%) as well as a decrease in liver S-adenosylmethionine (SAM) concentration and S-adenosylmethionine/S-adenosylhomocysteine ratio. No changes were found in plasma methionine and cysteine plasma levels compared to control group. Four weeks of ingesting a CDD also caused a significant (P<0.05) increase in hepatic total fat, hepatic malondialdehyde (MDA), and plasma alanine aminotransferase (ALT) levels. In addition, reduced hepatic glutathione (GSH) levels and reduced/oxidized glutathione ratios (GSH/GSSG) were found in rats fed a CDD compared to controls. Taurine supplementation of the CDD normalized genes involved in the remethylation pathway, BHMT and CHDH, which were impaired by CDD alone. However, taurine supplementation failed to prevent CDD-induced Hcy metabolism disturbances and hepatic injury. Also, taurine added to CDD caused decreased expression of PEMT, CHKa, and CHKb, key genes involved in phosphatidylcholine (PC) synthesis and liver fat accumulation. SIGNIFICANCE: Taurine supplementation failed to ameliorate impaired Hcy metabolism and liver injury caused by CDD intake.

Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after choline supplementation.

Nonalcoholic steatohepatitis (NASH) is characterized by combined pathology of steatosis, lobular inflammation, fibrosis, and hepatocellular degeneration, with systemic symptoms of diabetes or hyperlipidemia, all in the absence of alcohol abuse. Given the therapeutic importance and conflicting findings regarding the potential for healing the histopathologic features of NASH in humans, particularly fibrosis, we investigated the reversibility of NASH-related findings in Wistar rats fed a choline-deficient and iron-supplemented l-amino acid-defined (CDAA) diet for 12weeks, with a recovery period of 7weeks, during which the diets were switched to a choline-sufficient and iron-supplemented l-amino acid-defined (CSAA) one. Analysis showed that steatosis and inflammation were significantly resolved by the end of the recovery period, along with decreases in AST and ALT activities within 4weeks. In contrast, fibrosis remained even after the recovery period, to an extent similar to that in continuously CDAA-fed animals. Real-time reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical investigations revealed that expression of some factors indicating oxidative stress (CYP2E1, 4-HNE, and iNOS) were elevated, whereas catalase and SOD1 were decreased, and a hypoxic state and CD34-positive neovascularization were evident even after the recovery period, although the fibrogenesis pathway by activated α-SMA-positive hepatic stellate cells via TGF-ß and TIMPs decreased to the CSAA group level. In conclusion, persistent fibrosis was noted after the recovery period of 7weeks, possibly due to sustained hypoxia and oxidative stress supposedly caused by capillarization. Otherwise, histopathological features of steatosis and inflammation, as well as serum AST and ALT activities, were recovered.

The protective effect of menhaden oil in the oxidative damage and renal necrosis due to dietary choline deficiency.

Weanling rats fed a choline-deficient diet develop kidney oxidative damage, tubular and cortical kidney necrosis, renal failure and animal death. The effect of dietary menhaden oil was assayed on the mentioned sequence correlating oxidative stress with renal structure and function. Rats were fed ad libitum 4 different diets: (a) a choline-deficient diet with corn oil and sunflower hydrogenated oil as a source of fatty acids; (b) the same diet supplemented with choline; (c) a choline-deficient diet with menhaden oil as a source of fatty acids; and (d) the previous diet supplemented with choline. Animals were sacrificed at days 0, 2, 4 and 7. The histopathological study of the kidneys showed that renal necrosis was only observed at day 7 in choline-deficient rats receiving the vegetable oil diet, simultaneously with increased creatinine plasma levels. Homogenate chemiluminescence (BOOH-initiated chemiluminescence) and phospholipid oxidation indicate the development of oxidative stress and damage in choline-deficient rats fed vegetable oils as well as the protective effect of menhaden oil. Rats fed with the fish oil diet showed that oxidative stress and damage develop later, as compared with vegetable oil, with no morphological damage during the experimental period.

Effect of dietary supplementation with folate on choline deficiency-induced hyperhomocysteinemia in rats.

The effects of dietary supplementation with folate (20 mg/kg diet), 2.5% serine, or both on choline deprivation-induced hyperhomocysteinemia were investigated in rats fed a 10% casein diet (10C) or 25% soybean protein diet (25S) to determine whether folate supplementation with or without serine can suppress choline deficiency-induced hyperhomocysteinemia. Choline deprivation-induced enhancement of plasma homocysteine concentration was significantly suppressed by supplementation with folate, serine, or both, but the effects of these supplements were partial or limited in the rats fed both 10C and 25S. The extents of suppression of plasma homocysteine increments by folate, serine, or both were 29.6, 37.8, and 46.2%, respectively, in rats fed 10C and 27.2, 36.6, and 42.8%, respectively, in rats fed 25S. There was no significant additive effect between folate and serine, a source of C1 units. Folate supplementation with or without serine significantly increased or tended to increase hepatic 5-methyltetrahydrofolate concentration together with methionine synthase (MS) and cystathionine ß-synthase (CBS) activities and MS mRNA level in both rats fed 10C and rats fed 25S. Hepatic betaine-homocysteine S-methyltransferase activity was unaffected by folate with or without serine. Supplementation with serine alone significantly increased hepatic serine concentration and increased or tended to increase CBS activity slightly. It is thought that the suppressive effect of folate on choline deficiency-induced hyperhomocysteinemia was due to increased metabolism of homocysteine via the MS pathway and that the suppressive effect of serine was due to increased metabolism of homocysteine via cystathionine formation. One of the reasons for the insufficient effect of folate alone or in combination with serine is thought to be that the capacity of the MS pathway for homocysteine metabolism is less enhanced by supplementation with folate and serine.

Interleukin-6 is associated with liver lipid homeostasis but not with cell death in experimental hepatic steatosis.

Hepatic steatosis is a risk factor for the progression of non-alcoholic fatty liver disease. The role of pro-inflammatory interleukin (IL)-6 in hepatic steatosis etiology is controversial. We investigated in vivo and in primary hepatocyte cultures whether IL-6 has a modulator role in liver and mitochondria lipid composition and cell death in a choline-deficient (CD) diet rat model of hepatic steatosis. Dietary choline deficiency increased triglycerides and cholesterol, and reduced phosphatidylcholine (PC), phosphatidylethanolamine (PE) and the membrane integrity marker PC:PE ratio in liver. Choline-deficient diet enhanced systemic IL-6, and IL-6 receptor expression and cell death vulnerability in hepatocytes. Derangement of the mitochondrial electron transport chain and of its phospholipid environment was found in CD rat liver mitochondria, which exhibited elevated concentrations of triglycerides, cardiolipin and PC and elevated PC:PE ratio. The cell treatment with IL-6, but not PC, eliminated much of the CD-promoted lipid imbalance in mitochondria but not tumor-necrosis factor (TNF)-alpha-induced cell death. However, PC supplementation prevented the TNF-alpha-induced DNA fragmentation, cytochrome-c release and caspase-3 activity in control and CD hepatocytes. In conclusion, IL-6 ameliorated the mitochondria lipid disturbance in hepatocytes isolated from steatotic animals. Furthermore, PC is identified as a new survival agent that reverses several TNFalpha-inducible responses that are likely to promote steatosis and necrosis.

Polaprezinc attenuates liver fibrosis in a mouse model of non-alcoholic steatohepatitis.

BACKGROUND AND AIM: The effect of polaprezinc, a zinc-carnosine chelate compound, on the development of non-alcoholic steatohepatitis (NASH) was investigated in dietary methionine and choline deficient (MCD) mice. METHODS: Mice were fed the MCD diet with or without polaprezinc (2.2 g/kg diet) for 10 weeks. Liver histopathology, triglyceride and lipid peroxide levels, and the expression of genes linked to fibrosis were then assessed. RESULTS: MCD mice developed steatohepatitis accompanied by mild fibrosis with an increase in lipid peroxidation, hepatic stellate cell (HSC) activation, and the augmented mRNA expression of tumor necrosis factor-alpha, transforming growth factor-beta1 and procollagen alpha1(I). The mRNA expression levels of matrix metalloproteinase (MMP)-2 and tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also enhanced. Histopathologically, polaprezinc supplementation did not influence the development of steatosis but it apparently attenuated fibrosis. Polaprezinc slightly reduced lipid peroxidation and suppressed HSC activation as well as the mRNA expression of pro-inflammatory cytokines. Polaprezinc affected the MCD diet-enhanced expression of TIMP-1 even when administered relatively late. CONCLUSION: These results suggest that polaprezinc attenuates fibrosis in NASH by reducing inflammation and lipid peroxidation and, during a later phase, promoting fibrolysis via the inhibition of TIMP expression in the liver. Further investigation is required to clarify the clinical efficacy of polaprezinc in patients with NASH.

Genetic variation of folate-mediated one-carbon transfer pathway predicts susceptibility to choline deficiency in humans.

Choline is a required nutrient, and some humans deplete quickly when fed a low-choline diet, whereas others do not. Endogenous choline synthesis can spare some of the dietary requirement and requires one-carbon groups derived from folate metabolism. We examined whether major genetic variants of folate metabolism modify susceptibility of humans to choline deficiency. Fifty-four adult men and women were fed diets containing adequate choline and folate, followed by a diet containing almost no choline, with or without added folate, until they were clinically judged to be choline-deficient, or for up to 42 days. Criteria for clinical choline deficiency were a more than five times increase in serum creatine kinase activity or a >28% increase of liver fat after consuming the low-choline diet that resolved when choline was returned to the diet. Choline deficiency was observed in more than half of the participants, usually within less than a month. Individuals who were carriers of the very common 5,10-methylenetetrahydrofolate dehydrogenase-1958A gene allele were more likely than noncarriers to develop signs of choline deficiency (odds ratio, 7.0; 95% confidence interval, 2.0-25; P < 0.01) on the low-choline diet unless they were also treated with a folic acid supplement. The effects of the C677T and A1298C polymorphisms of the 5,10-methylene tetrahydrofolate reductase gene and the A80C polymorphism of the reduced folate carrier 1 gene were not statistically significant. The most remarkable finding was the strong association in premenopausal women of the 5,10-methylenetetrahydrofolate dehydrogenase-1958A gene allele polymorphism with 15 times increased susceptibility to developing organ dysfunction on a low-choline diet.

Improvement of cerebral ATP and choline deficiencies by Shao-Yin-Ren Shi-Quang-Da-Bu-Tang in senescence-accelerated mouse prone 8.

Shao-Yin-Ren Shi-Quang-Da-Bu-Tang (SDT) has been used traditionally to improve the systemic blood circulation and biological energy production in the body. The object of this study is to determine the effect of SDT extract on the decline of cerebral adenosine triphosphate (ATP) and choline content associated with learning and memory impairments in senescence-accelerated mice prone 8 (SAM P8). Twenty-four-week old mice were orally treated with SDT at 400 mg/kg body weight per day, and continued for 12 consecutive weeks. At the termination of the treatment, the body weight of SAM P8 was markedly lower than that of the equal aged senescence-resistant prone 1 (SAM R1), but this was conspicuously recovered to the level of SAM R1 by SDT treatment. SDT also significantly reduced the decline of cerebral weight (P < 0.05). By comparison with normal mice, a spontaneous decrease of cerebral ATP was observed in the SAM P8. Two- and 6-fold increases of cerebral ATP content were found in SAM R1 and SAM P8 by SDT administration, respectively. The cerebral choline content was significantly different between SAM R1 and SAM P8 aged 36-week old (P < 0.01). SDT remarkably restored the decrease of cerebral choline content in SAM P8 (P < 0.01). Taken together, these results demonstrate that SDT can reduce the decrease of cerebral weight, and restore the decline of cerebral ATP and choline content associated with an alteration of neuronal metabolism in SAM P8 brain. This suggests that pharmacological properties of SDT may participate in improvement of declined cerebral energy production and cholinergic neurotransmitter synthesis in senile dementia.

Determination of carnitine turnover in choline-deficient and cold-exposed rats.

Two experimetns were conducted to study the body pool size and turnover rate of carnitine in rats. The turnover of carnitine was determined by injection of a tracer dose of L-[methyl-14C] carnitine. In experiment 1, carnitine body pool size and turnover in rats fed a choline-deficient basal diet were compared with values obtained from rats fed the basal diet supplemented with choline. These rats were maintained at 22degrees. In experiment 2, carnitine body pool size and turnover were determined in cold-exposed (2degrees) rats fed the choline-deficient basal diet. Carnitine body pool sizes of rats maintained 22degrees and fed the choline-deficient basal diet and the choline-supplemented diet were 35.6 and 41.8 mumoles/100 g body weight, respectively. Carnitine body pool size of rats maintained at 2degrees and fed a choline-deficient basal diet were 6.6 and 56.1 days, for rats fed a choline-supplemented diet, 6.7 and 40.2 days, and for rats maintained at 2degrees and fed a choline-deficient diet, 2.9 and 36.4 days, respectively. Carnitine turnover times obtained with DL-[14C]carnitine in our previous study longer than turnover times obtained with DL-[14C]carnitine in our previous study (j. nutr. 104, 782-792). These observations suggest that D-carnitine is not metabolized the same way as L-carnitine, and that D-carnitine is not cleared from the body within 2 days after injection. The results also suggest that carnitine metabolism can be influenced by the amount of choline in the diet and by cold exposure.