Excess Choline Research Articles

Hepatic Betaine-Homocysteine Methyltransferase Activity in the Chicken Is Influenced by Dietary Intake of Sulfur Amino Acids, Choline and Betaine

There is much interest in the metabolism of homocysteine, because elevated plasma homocysteine [hyperhomocyst(e)inemia] is an independent risk factor for the development of cardiovascular disease. Four chick assays were conducted to determine the effects of varying dietary sulfur amino acids, choline and betaine on the activity of hepatic betaine-homocysteine methyltransferase (BHMT), an enzyme likely to be important in modulating plasma homocysteine. In Experiment 1, chicks were fed a purified crystalline amino acid diet containing adequate sulfur amino acids and choline. Excess dietary methionine, or the combination of excess cystine with choline or betaine, caused a small increase (P < 0.05) in BHMT activity. In Experiment 2, use of a methionine-deficient purified diet resulted in a threefold increase (P < 0.05) in BHMT activity, and addition of choline or betaine further increased (P < 0.05) BHMT activity. In Experiment 3, use of a methionine-deficient corn-peanut meal diet increased BHMT (P < 0.05) relative to that of chicks supplemented with adequate methionine, and addition of surfeit choline to the methionine-deficient basal diet caused a further increase (P < 0.05). In Experiment 4, addition of both surfeit choline and surfeit betaine to the methionine-deficient corn-peanut meal diet caused an increase (P < 0.05) in BHMT activity relative to that observed in chicks fed the methionine-deficient basal diet. These assays show that large increases in BHMT activity can be produced under methionine-deficient conditions, especially in the presence of excess choline or betaine.

NIMG-46. TOWARDS PREDICTING TUMOR AGGRESSIVENESS WITH RADIOPATHOMIC ANALYSIS OF MULTI-PARAMETRIC ANATOMICAL, DIFFUSION-WEIGHTED, AND METABOLIC MRI IN PATIENTS WITH NEWLY-DIAGNOSED GLIOMAS

Abstract INTRODUCTION Pathologically aggressive tumor biology can extend beyond the contrast-enhancing or non-enhancing anatomical lesions in patients with glioma. Identification of malignant regions can help guide diagnosis and subsequent treatment planning. This study leverages a unique multi-parametric MRI dataset with tissue samples of known spatial coordinates to noninvasively predict cellular proliferation (KI-67) and a novel index of tumor aggressiveness (TAI), that combines proliferation, cellularity, and tumor-score. METHODS 420 tissue samples were collected from 162 patients with newly-diagnosed glioma (47% IDH-wildtype). Clinical imaging consisted of T2-weighted, T2-FLAIR, T1-weighted pre- and post-contrast images, and apparent diffusion coefficient (ADC) and fractional anisotropy (FA) from diffusion-weighted imaging. Mean normalized imaging metrics were quantified from 5mm spheres centered at the location of the tissue sample. A single spectrum was reconstructed at the location of each tissue sample from 3D 1H-MR Spectroscopic Imaging (MRSI) before quantifying normalized metabolite peak-heights for choline, creatine, NAA, lactate/lipid, and relative indices. Univariate mixed-effects linear regression models were employed and features with p&amp;lt; 0.2 were selected for subsequent model building. Support vector machine (SVM), random forest, and gradient boosting machine-learning algorithms were trained and tested on a ⅔-⅓ train-test split with 4-fold cross-validation in training to predict a high/low KI-67 and TAI. RESULTS Although none of the individual imaging metrics were significantly associated with KI-67 in the univariate analysis, all diffusion and several MRSI metrics (ncholine, nNAA, CNI, excess choline and creatine) were significantly associated with cellularity. Preliminary multivariate analyses to date suggest that the best radiopathomic model performance is achieved when an SVM was used along with T1-precontrast, nADC, and all metabolite levels (mean cross-validation AUC=0.73 and accuracy=.77). CONCLUSION Our results suggest that multi-parametric physiologic and metabolic MRI are useful for radiopathomic-mapping of tumor aggressiveness and are currently being optimized in a larger cohort.

Choline Supplementation Promotes Hepatic Insulin Resistance in Phosphatidylethanolamine N-Methyltransferase-deficient Mice via Increased Glucagon Action

Biosynthesis of hepatic choline via phosphatidylethanolamine N-methyltransferase (PEMT) plays an important role in the development of type 2 diabetes and obesity. We investigated the mechanism(s) by which choline modulates insulin sensitivity. PEMT wild-type (Pemt(+/+)) and knock-out (Pemt(-/-)) mice received either a high fat diet (HF; 60% kcal of fat) or a high fat, high choline diet (HFHC; 4 g of choline/kg of HF diet) for 1 week. Hepatic insulin signaling and glucose and lipid homeostasis were investigated. Glucose and insulin intolerance occurred in Pemt(-/-) mice fed the HFHC diet, but not in their Pemt(-/-) littermates fed the HF diet. Plasma glucagon was elevated in Pemt(-/-) mice fed the HFHC diet compared with Pemt(-/-) mice fed the HF diet, concomitant with increased hepatic expression of glucagon receptor, phosphorylated AMP-activated protein kinase (AMPK), and phosphorylated insulin receptor substrate 1 at serine 307 (IRS1-s307). Gluconeogenesis and mitochondrial oxidative stress were markedly enhanced, whereas glucose oxidation and triacylglycerol biosynthesis were diminished in Pemt(-/-) mice fed the HFHC diet. A glucagon receptor antagonist (2-aminobenzimidazole) attenuated choline-induced hyperglycemia and insulin intolerance and blunted up-regulation of phosphorylated AMPK and IRS1-s307. Choline induces glucose and insulin intolerance in Pemt(-/-) mice through modulating plasma glucagon and its action in liver.

The effect of choline and cystine on the utilisation of methionine for protein accretion, remethylation and trans-sulfuration in juvenile shrimpPenaeus monodon

This 35-d feeding experiment examined in juvenile shrimp Penaeus monodon (3·3g initial body weight) the effects of methionine (Met), choline and cystine on protein accretion and the activity of two key enzymes of remethylation (betaine-homocysteine methyltransferase; BHMT) and trans-sulfuration (cystathionine β-synthase; CBS). The interaction between Met and choline was tested using semi-purified diets either adequate or limiting (30 or 50%) in total sulphur amino acid (SAA) content with a constant cystine:Met ratio. The diets contained either basal or excess choline (3 v. 7g/kg feed). Cystine was added to two other 30 and 50% Met-limiting diets to adjust the SAA supply to that of the control diet in order to evaluate the interaction between Met and cystine. As expected, N accretion was significantly lower with the SAA-limiting diets but increased back to control levels by the extra choline or cystine, demonstrating their sparing effect on Met utilisation for protein accretion. We show, for the first time, the activities of BHMT and CBS in shrimp hepatopancreas. Only BHMT responded to the SAA deficiencies, whereas the extra choline and cystine did not stimulate remethylation or down-regulate trans-sulfuration. Our data also suggest the capacity of P. monodon to synthesise taurine, being significantly affected by the cystine level in the 30% SAA-limiting diets. Further research is warranted to better understand the metabolic regulation of taurine synthesis in shrimp and of the observed Met-sparing effects.

Perinatal choline supplementation improves cognitive functioning and emotion regulation in the Ts65Dn mouse model of Down syndrome.

In addition to mental retardation, individuals with Down syndrome (DS) also develop the neuropathological changes typical of Alzheimer's disease (AD) and the majority of these individuals exhibit dementia. The Ts65Dn mouse model of DS exhibits key features of these disorders, including early degeneration of cholinergic basal forebrain (CBF) neurons and impairments in functions dependent on the two CBF projection systems; namely, attention and explicit memory. Herein, we demonstrate that supplementing the maternal diet with excess choline during pregnancy and lactation dramatically improved attentional function of the adult trisomic offspring. Specifically, the adult offspring of choline-supplemented Ts65Dn dams performed significantly better than unsupplemented Ts65Dn mice on a series of 5 visual attention tasks, and in fact, on some tasks did not differ from the normosomic (2N) controls. A second area of dysfunction in the trisomic animals, heightened reactivity to committing an error, was partially normalized by the early choline supplementation. The 2N littermates also benefited from increased maternal choline intake on 1 attention task. These findings collectively suggest that perinatal choline supplementation might significantly lessen cognitive dysfunction in DS and reduce cognitive decline in related neurodegenerative disorders such as AD.

Streptococcus pneumoniaeAutolysis Prevents Phagocytosis and Production of Phagocyte-Activating Cytokines

Streptococcus pneumoniae is a major pathogen in humans. The pathogenicity of this organism is related to its many virulence factors, the most important of which is the thick pneumococcal capsule that minimizes phagocytosis. Another virulence-associated trait is the tendency of this bacterium to undergo autolysis in stationary phase through activation of the cell wall-bound amidase LytA, which breaks down peptidoglycan. The exact function of autolysis in pneumococcal pathogenesis is, however, unclear. Here, we show the selective and specific inefficiency of wild-type S. pneumoniae for inducing production of phagocyte-activating cytokines in human peripheral blood mononuclear cells (PBMC). Indeed, clinical pneumococcal strains induced production of 30-fold less tumor necrosis factor (TNF), 15-fold less gamma interferon (IFN-gamma), and only negligible amounts of interleukin-12 (IL-12) compared with other closely related Streptococcus species, whereas the levels of induction of IL-6, IL-8, and IL-10 production were similar. If pneumococcal LytA was inactivated by mutation or by culture in a medium containing excess choline, the pneumococci induced production of significantly more TNF, IFN-gamma, and IL-12 in PBMC, whereas the production of IL-6, IL-8, and IL-10 was unaffected. Further, adding autolyzed pneumococci to intact bacteria inhibited production of TNF, IFN-gamma, and IL-12 in a dose-dependent manner but did not inhibit production of IL-6, IL-8, and IL-10 in response to the intact bacteria. Fragments from autolyzed bacteria inhibited phagocytosis of intact bacteria and reduced the in vitro elimination of pneumococci from human blood. Our results suggest that fragments generated by autolysis of bacteria with reduced viability interfere with phagocyte-mediated elimination of live pneumococci.

Review of the carcinogenic activity of diethanolamine and evidence of choline deficiency as a plausible mode of action

Diethanolamine (DEA) is a chemical used widely in a number of industries and is present in many consumer products. Studies by the National Toxicology Program (NTP) have indicated that lifetime dermal exposure to DEA increased the incidence and multiplicity of liver tumors in mice, but not in rats. In addition, DEA was not carcinogenic when tested in the Tg.Ac transgenic mouse model. Short-term genotoxicity tests have yielded negative results. In view of these apparent inconsistencies, we have critically evaluated the NTP studies and other data relevant to assessing the carcinogenic potential of DEA. The available data indicate that DEA induces mouse liver tumors by a non-genotoxic mode of action that involves its ability to cause choline deficiency. The following experimental evidence supports this hypothesis. DEA decreased the hepatic choline metabolites and S-adenosylmethionine levels in mice, similar to those observed in choline-deficient mice. In contrast, DEA had no effect in the rat, a species in which it was not carcinogenic at a maximum tolerated dose level. In addition, a consistent dose–effect relationship had been established between choline deficiency and carcinogenic activity since all DEA dosages that induced tumors in the NTP studies were also shown to cause choline deficiency. DEA decreased phosphatidylcholine synthesis by blocking the cellular uptake of choline in vitro, but these events did not occur in the presence of excess choline. Finally, DEA induced transformation in the Syrian hamster embryo cells, increased S-phase DNA synthesis in mouse hepatocytes, and decreased gap junctional intracellular communication in primary cultured mouse and rat hepatocytes, but all these events were prevented with choline supplementation. Since choline is an essential nutrient in mammals, this mode of action is qualitatively applicable to humans. However, there are marked species differences in susceptibility to choline deficiency, with rats and mice being far more susceptible than other mammalian species including humans. These differences are attributed to quantitative differences in the enzyme kinetics controlling choline metabolism. The fact that DEA was carcinogenic in mice but not in rats also has important implications for human risk assessment. DEA has been shown to be less readily absorbed across rat and human skin than mouse skin. Since a no observed effect level for DEA-induced choline deficiency in mice has been established to be 10 mg/kg/d, this indicates that there is a critical level of DEA that must be attained in order to affect choline homeostasis. The lack of a carcinogenic response in rats suggests that exposure to DEA did not reach this critical level. Since rodents are far more sensitive to choline deficiency than humans, it can be concluded that the hepatocarcinogenic effect of DEA in mice is not predictive of similar susceptibility in humans.

Species Differences in the Induction of Hepatocellular DNA Synthesis by Diethanolamine

Diethanolamine increased the incidence and multiplicity of liver tumors in the mouse following chronic exposure. Diethanolamine is known to inhibit cellular choline uptake. Since choline deficiency produces tumors in rodents, diethanolamine, through choline depletion, may result in tumor development in rodents. The potential for diethanolamine to function through this mode of action in humans is not known. The present studies examined the effect of diethanolamine (0-500 mug/ml) and choline depletion on DNA synthesis and changes in expression of genes involved in cell growth pathways in primary cultures of mouse, rat, and human hepatocytes. In mouse and rat hepatocytes DNA synthesis was increased following treatment with 10 mug/ml diethanolamine and higher (3- to 4-fold over control). In contrast, diethanolamine failed to increase DNA synthesis in human hepatocytes. Incubation of hepatocytes in medium containing reduced choline (1/10 to 1/100 of normal medium; 0.898 to 0.0898 mg/l vs. 8.98 mg/l) increased DNA synthesis (1.6- and 1.8-fold of control in mouse and rat hepatocytes, respectively); however, choline depletion did not induce DNA synthesis in human hepatocytes. Mouse and rat hepatocytes incubated in medium supplemented with 2- to 50-fold excess choline reduced diethanolamine-induced DNA synthesis to control levels or below. Gene expression analysis of mouse and rat hepatocytes following diethanolamine treatment showed increases in genes associated with cell growth and decreases in expression of genes involved in apoptotic pathways. These results support the hypothesis that choline depletion is central to the mode of action for the induction of rodent hepatic neoplasia by diethanolamine. Furthermore, since diethanolamine treatment or choline depletion failed to induce DNA synthesis in human hepatocytes, these results suggest that humans may not be at risk from the carcinogenic effects of diethanolamine.

Cell cholesterol esters and high-density lipoprotein plasma levels during liver hyperplasia in choline-fed male and female rats.

Sexual dimorphism exists in the response of rats to lead nitrate, liver hyperplasia occuring earlier and being more pronounced in males. Excess dietary choline in females shifted the growth pattern towards that of males. To determine whether phosphatidylcholine-induced growth modulations could be related to a derangement of cholesterol metabolism, liver accumulation of cholesterol esters and plasma lipoprotein patterns were investigated. In males, lead-induced liver hyperplasia was associated with increased total cholesterol hepatic content, accumulated cholesterol esters and reduced concentration of plasma High Density Lipoprotein (HDL) cholesterol. Females were less responsive to the liver mitogenic signal of lead nitrate; there was no elevation of cholesterol content nor any marked accumulation of cholesterol esters. This is consistent with the lack of change in the plasma levels of HDL cholesterol. Continuous choline feeding displaced the liver cholesterol ester pattern and plasma HDL cholesterol levels in females, and in parallel that of DNA synthesis, towards those of males. Choline was not observed to have any effect in males. These results suggest that the derangement of phosphatidylcholine metabolism induces growth-related changes in cholesterol turnover; they are consistent with the proposal that the intracellular content of cholesterol esters may have a role in regulating liver growth rates.

Choline supplementation inhibits diethanolamine-induced morphological transformation in syrian hamster embryo cells: evidence for a carcinogenic mechanism.

DEA, an amino alcohol, and its fatty acid condensates are widely used in commerce. DEA is hepatocarcinogenic in mice, but shows no evidence of mutagenicity or clastogenicity in a standard testing battery. However, it increased the number of morphologically transformed colonies in the Syrian hamster embryo (SHE) cell morphologic transformation assay. The goal of this work was to test the hypothesis that DEA treatment causes morphologic transformation by a mechanism involving altered cellular choline homeostasis. As a first step, the ability of DEA to disrupt the uptake and intracellular utilization of choline was characterized. SHE cells were cultured in medium containing DEA (500 microg/ml), and (33)P-phosphorus or (14)C-choline was used to label phospholipid pools. After 48 h, SHE cells were harvested, lipids were extracted, and radioactive phospholipids were quantified by autoradiography after thin layer chromatographic separation. In control cells, phosphatidylcholine (PC) was the major phospholipid, accounting for 43 +/- 1% of total phospholipid synthesis. However, with DEA treatment, PC was reduced to 14 +/- 2% of total radioactive phospholipids. DEA inhibited choline uptake into SHE cells at concentrations > or = 50 microg /ml, reaching a maximum 80% inhibition at 250-500 microg/ml. The concentration dependence of the inhibition of PC synthesis by DEA (0, 10, 50, 100, 250, and 500 microg/ml) was determined in SHE cells cultured over a 7-day period under the conditions of the transformation assay and in the presence or absence of excess choline (30 mM). DEA treatment decreased PC synthesis at concentrations > or = 100 microg/ml, reaching a maximum 60% reduction at 500 microg/ml. However, PC synthesis was unaffected when DEA-treated cells were cultured with excess choline. Under 7-day culture conditions, (14)C-DEA was incorporated into SHE lipids, and this perturbation was also inhibited by choline supplementation. Finally, DEA (10-500 microg/ml) transformed SHE cells in a concentration-dependent manner, whereas with choline supplementation, no morphologic transformation was observed. Thus, DEA disrupts intracellular choline homeostasis by inhibiting choline uptake and altering phospholipid synthesis. However, excess choline blocks these biochemical effects and inhibits cell transformation, suggesting a relationship between the two responses. Overall, the results provide a plausible mechanism to explain the morphologic transformation observed with DEA and suggest that the carcinogenic effects of DEA may be caused by intracellular choline deficiency.

Diethanolamine Inhibits Choline Uptake and Phosphatidylcholine Synthesis in Chinese Hamster Ovary Cells

Diethanolamine (DEA), an alkanolamine used widely in industry, is hepatocarcinogenic in mice. The goal of this work was to determine whether DEA altered choline homeostasis in cultured cells, so as to ascertain whether the liver tumor response may be related to choline deficiency. CHO cells were cultured in Ham's F-12 medium containing DEA (0-1000 μg/ml) and [33P]-phosphorus was used to label phospholipid pools. After 48 hours incubation, lipids were extracted and [33P]-labeled phospholipids were quantified by autoradiography after thin layer chromatographic separation. In control cells, phosphatidylcholine (PC) accounted for 51 ± 0.7% of the total lipid 33P incorporation. DEA had no effect on cell number or total phospholipid biosynthesis, but it significantly decreased the incorporation of 33P into PC at concentrations ≥50 μg/ml. DEA (≥20 μg/ml) also inhibited the uptake of [3H]-choline into CHO cells, with 95% inhibition observed at 250 μg/ml. To determine whether supplemental choline prevented PC synthesis inhibition by DEA, CHO cells were cultured with or without excess choline (30 mM) and DEA (500 μg/ml). DEA reduced PC synthesis to 27 ± 3% of total phospholipids, but had no effect on PC synthesis in choline-supplemented cells. When [14C]-DEA was incubated with CHO cells, it was also incorporated into the phospholipid fraction. Collectively, these results indicate that DEA reversibly inhibits PC synthesis by blocking choline uptake and competing for utilization in the CDP-choline pathway in CHO cells.

Formation of N-methylnicotinamide in the brain from a dihydropyridine-type prodrug : Effect on brain choline

The enhancement of brain choline levels is a possible therapeutic option in neurodegenerative diseases; however, brain choline levels are held within narrow limits by homeostatic mechanisms including the rapid clearance of excess choline from the brain. The present study tests whether N-methylnicotinamide (NMN), an inhibitor of the outward transport of choline from the brain, can elevate brain choline levels in vivo. As NMN does not cross the blood–brain barrier, we synthesized and administered the brain-permeable prodrug, 1,4-dihydro- N-methyl-nicotinamide (DNMN), and tested its effect on the levels of NMN and choline in brain extracellular fluid, using the microdialysis procedure. Administration of DNMN (1 mmol/kg s.c.) caused a 4- and 9-fold increase in plasma and liver NMN levels, respectively, as determined by HPLC. Concomitantly, the brain tissue levels of NMN were increased by a factor of twenty. In brain extracellular fluid, the injection of DNMN (1–3 mmol/kg s.c.) elevated NMN levels by 3- to 10-fold to maximum levels of >10 μM. In spite of these enhanced NMN levels, the choline concentrations in the brain extracellular fluid and in the cerebrospinal fluid (4.7 μM) remained unchanged or were even slightly decreased. Microsomal incubations of DNMN indicated that cytochrome P-450 3A isoforms may be involved in NMN formation in the liver, but not in the brain. We conclude that DNMN, a brain-permeable prodrug of NMN, is efficiently oxidized to NMN in the brain, but a 10-fold increase in extracellular NMN levels is not sufficient to reduce the clearance of choline from the brain.

Brain choline has a typical precursor profile

Choline is product and precursor to both acetylcholine and membrane phospholipids, and, in the brain, is ultimately provided by the circulation. The brain is protected from excess choline and choline deprivation by a refined system of homeostatic mechanisms that maintain a level of extracellular choline that, for its role as precursor, meets saturation criteria under normal conditions. The kinetic and activity profiles of choline are typical for a biosynthetic precursor.

Excess choline availability: A transient effect on spermatogenesis in the rat

The reproductive effects of choline (trimethyl-β-hydroxyethylammonium) are unknown. Excess dietary intake of choline may occur in humans. Adult male rats were administered i.p. aqueous choline chloride (25 mg/rat, daily for 12 or 24 days). Administration of excess choline for 12 days did not significantly alter spermatogenesis. Administration for 24 days depleted pachytene spermatocytes until posttreatment day 5, while slight proliferation of spermatogonia was noted from day 5 onwards. By day 12, the tubules showed almost normal cellular associations. It is suggested that perhaps a prolonged administration of excess choline may prove to be toxic to male reproduction.

Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high-choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (greater than 96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet-induced changes of the net uptake and release of choline.

Choline-mediated aggregation and dissociation of autolysin of Streptococcus pneumoniae.

The low molecular weight, inactive form of pneumococcal amidase called E-amidase showed the full activity and a high molecular mass (> 500 kDa) when the enzyme was incubated with high concentrations of choline followed by the removal of excess choline. Before the removal of choline, the enzyme showed a low molecular mass-material (about 36 kDa) similar to that of the original E-amidase, suggesting that the aggregation of the protein took place upon the removal of choline. Furthermore, the addition of choline to the native C-amidase (active form) which had a high molecular mass (> 500 kDa) caused dissociation to a low molecular mass enzyme (about 36 kDa) in the presence of choline. These dissociated enzymes reaggregated upon the removal of choline.

Effect of chronic choline availability on lung and lymph nodes of rat

Male albino rats were given intraperitoneal injections of choline chloride (0. 1,0.33 or 0.5 × lethal dose 50) for a total period of one month and then killed at the end of 30,90 and 240 days for the study of pathotoxicokinetics of choline. Chronic choline administration in rats caused a decrease in growth rate, a dose dependent modulating effect on the somatic tissue indices of lung and lymph nodes, as well as cellularity of lymph nodes. In another experiment, the effect of choline on mica induced pulmonary lesions was studied. The combined effect of choline and mica caused adenocarcinoma of bronchiolar epithelium and marked lymphadenopathy with abnormal cells in the lymph nodes at the termination of experiment (330 days). The results of the present investigation suggest that excess choline availability not only produces pulmonary pathological lesions by itself but it also further enhances the lung lesions in altered pulmonary conditions

The incorporation of solubilized choline-transport activity into liposomes.

The choline-transport system has been solubilized from synaptic plasma membrane by using either sodium cholate or Triton X-100, and re-incorporated into unilamellar liposomes by using the technique of cholate dialysis. The criteria of choline-transport activity were saturability by excess choline, inhibition by hemicholinium-3, and trans-activation (i.e. stimulation of the uptake of [3H]choline into liposomes by preloading them with non-radioactive choline). Liposomes prepared from detergent extracts of synaptic plasma membrane and added lipid showed uptake of [3H]choline fulfilling these three criteria. Data on choline-transport activity of liposomes at various choline concentrations could be interpreted as implying that the transport system has two apparent Km values (2-5 microM and 50-100 microM), or alternatively that the system is composed of two or more negatively co-operating subunits (or units). It was shown by t.l.c. that the transported radioactivity was choline and that it was not significantly acetylated. Replacing Na+ by K+ on the outside of these liposomes partially inhibited uptake, and the formation of a potential gradient (inside negative) with valinomycin increased the total but not the saturable components of uptake when liposomes were prepared in a K+ medium, and transferred to an Na+ medium.

Regulation of body cholesterol pools: Influence of cholesterol input and excretion in an animal model

Biliary cholesterol excretion closely parallels lecithin excretion in the rat and may be increased by feeding an excess of choline and decreased by choline deficiency. To determine the relative influence of cholesterol input and excretion on whole body cholesterol metabolism, we have measured by compartmental analysis rates of cholesterol transport and pool sizes when both input and choline-mediated biliary excretion were increased and diminished. In choline-deficient animals with impaired excretion, plasma cholesterol was reduced. However, in deficient animals more cholesterol was transported into the slowly exchanging pool to increase pool size, and, when input was increased (by addition of cholesterol to diet), the slowly exchanging pool was even more markedly enlarged. In contrast, when excess choline was fed, plasma cholesterol was increased but excretion so exceeded transport into the slowly exchanging pool that pool size was actually reduced. Furthermore, in choline-fed animals with unimpaired excretion, addition of cholesterol to the diet to increase input did not result in pool expansion. Thus, in this model, cholesterol excretion and tissue deposition were reciprocally related, and, regardless of plasma cholesterol concentration and cholesterol input, stores were found to increase only when biliary excretion was impaired.

Determination of glycogen in small tissue samples.

Determination of glycogen in small tissue samples.