Choline-deficient Rats Research Articles

The effect of a nutritional model of chronic liver injury on the hepatic glucuronidation of morphine in rats.

The effect of a choline-deficient diet on the hepatic glucuronidation of morphine was investigated using a rat perfused liver system. Rats fed a choline-deficient diet developed a fatty liver with minimal necrosis. Despite the morphological changes, neither hepatic extraction ratio (0.51 +/- 0.02 in control; 0.45 +/- 0.04 in the choline-deficient rats) nor intrinsic clearance (0.85 +/- 0.05 in control; 0.77 +/- 0.09 in choline-deficient rats) were affected by this injury model. This finding suggests that glucuronidation is relatively resistant to this chronic liver injury.

Selectivity and sensitivity of changes in serum bile acids during induction of cirrhosis in rats, ,

Because some patients with cirrhosis have serum transaminase levels within the normal range, a prospective study was undertaken to determine whether the concentration of individual serum bile acids would be a sensitive indicator of development of cirrhosis. The choline-deficient rat has been used as a model for study of these changes. Using high-performance liquid chromatography, we measured the concentrations of individual serum bile acids at 3, 6, 10, 20 and 30 wk of dietary intake. Serum levels of total glycine- and taurine-conjugated bile acids were elevated at all stages tested as compared with levels in control groups (choline supplemented). Similarly, unconjugated bile acids and, particularly, cholic acid showed significantly higher levels at all stages except with the occurrence of cirrhosis at 30 wk, at which time there was a significantly lower level for unconjugated bile acids (0.48 +/- 0.11 vs. 1.40 +/- 0.36 in controls) and for cholic acid (0.17 +/- 0.05 vs. 0.91 +/- 0.39 in controls). The ratio of serum cholic acid to serum chenodeoxycholic acid changed in temporal relationship to progression in the histological lesions in livers of these rats. The ratio was at its highest at 78 +/- 3 at 3 wk (no histological change) and decreased with increasing time and changes in histological appearance until 30 wk, at which time it was down to 1.6 +/- 0.6. The routinely used markers of liver injury (serum ALT, alkaline phosphatase and bilirubin), however, did not match the progression of hepatic histological changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Selectivity and sensitivity of changes in serum bile acids during induction of cirrhosis in ratsselectivity and sensitivity of changes in serum bile acids during induction of cirrhosis in rats

Because some patients with cirrhosis have serum transaminase levels within the normal range, a prospective study was undertaken to determine whether the concentration of individual serum bile acids would be a sensitive indicator of development of cirrhosis. The choline-deficient rat has been used as a model for study of these changes. Using high-performance liquid chromatography, we measured the concentrations of individual serum bile acids at 3, 6, 10, 20 and 30 wk of dietary intake. Serum levels of total glycine- and taurine-conjugated bile acids were elevated at all stages tested as compared with levels in control groups (choline supplemented). Similarly, unconjugated bile acids and, particularly, cholic acid showed significantly higher levels at all stages except with the occurrence of cirrhosis at 30 wk, at which time there was a significantly lower level for unconjugated bile acids (0.48 ± 0.11 vs. 1.40 ± 0.36 in controls) and for cholic acid (0.17 ± 0.05 vs. 0.91 ± 0.39 in controls). The ratio of serum cholic acid to serum chenodeoxycholic acid changed in temporal relationship to progression in the histological lesions in livers of these rats. The ratio was at its highest at 78 ± 3 at 3 wk (no histological change) and decreased with increasing time and changes in histological appearance until 30 wk, at which time it was down to 1.6 ± 0.6. The routinely used markers of liver injury (serum ALT, alkaline phosphatase and bilirubin), however, did not match the progression of hepatic histological changes. The relationship of the increase in serum bile acids to the cirrhotic process is supported by qualitatively similar findings in a second rat model. Thus the hepatic pathological changes in cirrhotic rats were reflected by the changes in the individual serum bile acids together with the ratio of serum cholic acid to serum chenodeoxycholic acid, but not with other standard tests of liver function. (HEPATOLOGY 1993;18:1224-1231).

Accumulation of 1,2-sn-diradylglycerol with increased membrane-associated protein kinase C may be the mechanism for spontaneous hepatocarcinogenesis in choline-deficient rats.

Choline deficiency, via deprivation of labile methyl groups, is associated with a greatly increased incidence of hepatocarcinoma in experimental animals. This dietary deficiency also causes fatty liver, because choline is needed for hepatic secretion of lipoproteins. We hypothesized that fatty liver might be associated with the accumulation of 1,2-sn-diradylglycerol and subsequent activation of protein kinase C. Several lines of evidence indicate that cancers might develop secondary to abnormalities in protein kinase C-mediated signal transduction. We observed that rats fed a choline-deficient diet for 1, 6, or 27 weeks had increased hepatic concentrations of 1,2-diradylglycerol. At 1 and 6 weeks, hepatic plasma membrane from choline-deficient rats had increased concentrations of 1,2-sn-diacylglycerol and 1-alkyl, 2-acylglycerol, with the latter accounting for 20-26% of membrane 1,2-sn-diradylglycerol (as compared with only 2-5% in controls). Protein kinase C activity was increased in hepatic plasma membrane at 1 week of choline deficiency. By Western blotting there was an increase in the amount of protein kinase C zeta and a decrease in the amount of protein kinase C delta in liver at 1 week. By 6 weeks of choline deficiency, hepatic plasma membrane and cytosolic protein kinase C (PKC) activities were increased significantly, with increased amounts of hepatic plasma membrane protein kinase C alpha, and delta detected by Western blotting. Glycogen synthase activity in liver was diminished after 1 week of choline deficiency; this enzyme is inhibited by PKC-mediated phosphorylation. We suggest that choline deficiency perturbed PKC-mediated transmembrane signaling within liver and that this contributed to the development of hepatic cancer in these animals.

Accumulation of 1, 2-sn-diradylglycerol with increased membrane-associated protein kinase C may be the mechanism for spontaneous hepatocarcinogenesis in choline-deficient rats.

Accumulation of 1, 2-sn-diradylglycerol with increased membrane-associated protein kinase C may be the mechanism for spontaneous hepatocarcinogenesis in choline-deficient rats.

Dietary carotenoids influenced biochemical but not morphological changes in adult male rats fed a choline‐deficient diet

In a study of the effects of carotenoids, canthaxanthin (CA), beta-apo-8'-carotenal (BA), or beta-carotene in an extract of Spirulina-Dunaliella algae (AE) was fed at 0%, 0.1%, or 0.2% in a choline-deficient (CD) diet. In each of eight groups, 10 adult male Fischer 344 rats were fed diets with designated carotenoid sources and levels or a choline-sufficient diet for 12 weeks. Carotenoids altered some of the changes induced by the CD diet. Increases in enlargement of fatty livers and low plasma cholesterol levels occurred in rats fed 0.2% BA. Plasma retinol was further reduced 35% by BA or AE. BA and AE increased liver total vitamin A about 80% and 305%, respectively. Liver lipid peroxidation was enhanced and plasma alpha-tocopherol was reduced further by 1.0% AE. AE, BA, and CA (mg/g fat) depressed liver alpha-tocopherol about 49%, 67%, and 78%, respectively. The decreased liver alpha-tocopherol was concurrent with an increase in carotenoid stores of CA > BA > AE. Histopathological examination of sections of liver tissue by light microscopy showed fatty and cirrhotic changes in all rats fed CD diets. Histochemical evaluation based on a semiquantitative assay revealed a marked increase in peroxisome enzyme activity in the livers of all CD rats. None of the carotenoids appeared to have any effect on the development of morphological changes in the liver. Although carotenoids can function as antioxidants, they did not prevent changes observed in rats fed CD diets.

Effect of chronic choline deficiency in rats on liver folate content and distribution

We have previously reported that folate and choline metabolism are interrelated. Total folate concentration was significantly diminished (31%) when rats were fed a choline-deficient diet for 2 weeks. Distribution among species of folates (differing in pteridine ring structure and/or in the number of glutamic acid residues) was not altered by this short-term dietary deficiency. In the present study we analyzed folate content and distribution in livers after 12 months on a choline-deficient diet. We found that this long-term deficiency did not significantly alter the total folate concentration in liver, however there was a significant difference between the control and deficient groups in the distribution of folate species. Whereas the major folates in the livers of control rats consisted of penta- and hexaglutamyl derivatives, those from the livers of the choline-deficient group were of longer chain lengths including hepta- and octaglutamyl derivatives. It is believed that chain elongation of the glutamate residues reflects, among other things, increased residence time of the folate molecule in the liver. A plausible interpretation of the previous and the present studies is that choline deprivation interferes with the assimilation of newly acquired folate by the hepatic tissue. Initially, during choline deprivation hepatic folate losses due to normal turnover were not promptly replenished, hence, we observed a decrease in folate level. However, when the period of choline deficiency was extended, impairment in the assimilation of folate was probably compensated for by minimizing folate losses by decreasing folate turnover rates. Hence, we observed an elongation of the glutamate ends of folate molecules.

Choline: an important nutrient in brain development, liver function and carcinogenesis.

Choline is required to make certain phospholipids which are essential components of all membranes. It is a precursor for biosynthesis of the neurotransmitter acetylcholine and also is an important source of labile methyl groups. Much attention has been given to the effect of supplemental choline upon brain function, i.e., enhancement of acetylcholine synthesis and release. In addition, choline supplements administered to rats in utero or shortly after birth permanently after brain function. The mechanisms for this effect is unknown and under investigation at this time. Healthy humans fed diets deficient in choline, and humans fed parenterally have decreased plasma choline concentrations and develop liver dysfunction that is similar to that seen in choline-deficient animals. In experimental animals, fatty liver occurs in choline deficiency because phosphatidylcholine synthesis is required for very low-density lipoprotein secretion. This accumulation of lipids in liver may explain why choline-deficient rats spontaneously develop hepatocarcinoma. We found that choline deficiency was associated with the accumulation of 1,2-diacylglycerol, an activator of protein kinase C. Several lines of evidence indicate that cancers might develop secondary to abnormalities in protein kinase C-mediated signal transduction.

Evidence that remodeling of the fatty acids of phosphatidylcholine is regulated in isolated rat hepatocytes and involves both the sn-1 and sn-2 positions

The remodeling of the fatty acyl moieties of phosphatidylcholine (PC) has been studied in choline-deficient and choline-supplemented hepatocytes prepared from a choline-deficient rat. Choline-deficient hepatocytes were pre-labeled with [ Me- 3H]choline for 30 min and subsequently incubated for up to 12 h in the presence or absence of choline. Analysis of the molecular species of PC from choline-deficient cells showed that, at the end of the pulse, approx. 75% of the label was incorporated into palmitate-containing species and only approx. 16% of the labeled species contined stearate. During the chase period there was a redistribution of label and after 12 h approx. 56% of the total radioactivity was associated with palmitate containing species and 37% was recovered in stearate-containing species. A similar distribution of radioactivity was observed in choline-supplemented cells. Measurement of the specific radioactivity of the major molecular species of PC was consistent with a precursor-product relationship between palmitate-containing species and stearate-containing species with arachidonate or linoleate on the sn-2 position. A model is presented which takes into account remodeling of both the sn-1 and sn-2 positions of PC.

Evidence that the rate of phosphatidylcholine catabolism is regulated in cultured rat hepatocytes

The regulation of phosphatidylcholine (PC) catabolism has been studied in choline-deficient rat hepatocytes. Supplementation of choline-deficient hepatocytes, prelabeled with [ 3H]choline, with 100 μM choline increased the rate of PC catabolism by approx. 2-fold. The major product of PC degradation was glycerophosphocholine in both choline-deficient and choline-supplemented cells. Choline supplementation decreased the radioactivity recovered in lysoPC by 50%. This effect was accompanied by a 2-fold increase of labeled glycerophosphocholine. Comparable results were obtained when PC of the cells was prelabeled with [ 3H]methionine or [ 3H]glycerol. The activity of phospholipase A in cytosol, mitochondria and microsomes isolated from choline-deficient rat liver was similar to the activity in control liver, when determined with [ 3H]PC vesicles as the substrate. Measurement of the activity of phospholipase A with endogenously [ 3H]choline-labeled PC showed that the formation of lysoPC in mitochondria isolated from choline-supplemented cells was 40% lower than in choline-deficient cells. Alternatively, the formation of [ 3H]glycerophosphocholine and [ 3H]choline in microsomes from choline-supplemented cells was significantly higher (1.4-fold) than in microsomes from choline-deficient cells. These results suggest that the rate of PC catabolism is regulated in rat hepatocytes and that the concentration of PC might be an important regulatory factor.

Head group specificity in the regulation of phosphatidylcholine catabolism in rat hepatocytes

Previous studies have shown that the catabolism of PC is regulated in choline-deficient hepatocytes and the concentration of phosphatidylcholine (PC) might be an important regulatory factor (Tijburg, L.B.M., Nishimaki-Mogami, T. and Vance, D.E. (1991) Biochim. Biophys. Acta, 1085, 167–177). In the present study we investigated the head group specificity of the regulation of PC catabolism. Supplementation of choline-deficient rat hepatocytes, prelabeled with [ 3H]choline, with dimethylethanolamine increased the catabolism of PC by 1.6-fold after 6 h. This effect was accompanied by a 2.5-fold increase in the production of [ 3H]glycerophosphocholine (GPC). Radioactivity associated with lysoPC was decreased by 50% in dimethylethanolamine-treated cells. Supplementation of the cells with monomethylethanolamine had little effect on the degradation of PC. In other experiments choline-deficient cells were prelabeled with [ 3H]methionine. Treatment of the cells with dimethylethanolamine increased the formation of [ 3H]GPC by 5-fold, while the production of lysoPC was inhibited by 60%. Supplementation of the medium with monoemthylethanolamine resulted in a 2-fold increase in labeledGPC, with a concomitant decrease of [ 3H]lysoPC by approx. 25%. We conclude that the formation of phosphatidyldimethylethanolamine from its corresponding base mimics the effect of the synthesis of PC from choline in increasing PC catabolism, whereas the effect of monomethylethanolamine is much less pronounced.

CPT-cAMP and okadaic acid enhance phosphatidylcholine catabolism in choline-deficient rat hepatocytes.

The effect of CPT-cAMP and okadaic acid on phosphatidylcholine catabolism in suspension cultures of choline-deficient rat hepatocytes was investigated. Choline-deficient hepatocytes were pulse-labeled for 30 min with [methyl-3H]choline and subsequently chased for up to 60 min with choline in the absence or presence of 0.5 mM CPT-cAMP or 0.5 microM okadaic acid. Radioactivity in phosphatidylcholine and lysophosphatidylcholine were unchanged during the chase. However, the radioactivity incorporated into glycerophosphocholine was significantly increased (P less than 0.05) 59 and 77% after 60 min of chase in hepatocytes incubated with either okadaic acid or CPT-cAMP, respectively. Incubation of choline-deficient hepatocytes with both okadaic acid and CPT-cAMP produced an additive effect on radioactivity incorporated ino glycerophosphocholine. Crude mitochondrial, microsomal, and cytosolic phospholipaselysophospholipase activities, assayed in the presence of exogenously labeled phosphatidylcholine, were unchanged in both CPT-cAMP and okadaic acid treated hepatocytes compared with control. Phospholipase-lysophospholipase activity, assayed with endogenously labeled phosphatidylcholine, was increased 28 and 47% (P less than 0.05) in the crude mitochondrial fraction of hepatocytes treated with either okadaic acid or CPT-cAMP, respectively, compared with the control. Incubation of choline-deficient hepatocytes, labeled with L-[methyl-3H]methionine, with CPT-cAMP or okadaic acid caused a 31 and 20% increase (P less than 0.05) in the radioactivity incorporated into glycerophosphocholine, respectively, compared with the control. We postulate that phosphatidylcholine catabolism in choline-deficient hepatocytes may be regulated by a phosphorylation-dephosphorylation mechanism mediated through cAMP-dependent protein kinase and phosphoprotein phosphatase activities.

Differential regulation of liver P-450III cytochromes in choline-deficient rats: Implications for the erythromycin breath test as a parameter of liver function

Progressive liver fibrosis in rats develops when they are fed a diet deficient in choline. This diet also results in a pronounced and selective decrease in the liver microsomal content of a phase I drug-metabolizing enzyme belonging to the cytochrome P-450III gene family. Because P-450III cytochromes characteristically catalyze the N-demethylation of erythromycin, we believed that the production of breath CO2 from erythromycin would be dramatically reduced in choline-deficient rats. However, when 12 choline-deficient rats were compared with 9 control rats, the reduction in CO2 production from erythromycin (mean decrease 71%) was essentially identical to that from aminopyrine (mean decrease 69%), a substrate believed to be metabolized normally by the hepatocyte in fibrotic liver disease. Furthermore, we found that the relative erythromycin and aminopyrine demethylase activities were comparable when measured in vitro in liver microsomes prepared from the choline-deficient rats. To determine the molecular basis for the erythromycin demethylase activity in the choline-deficient rats, the liver microsomes were subjected to immunoblot analysis using a variety of polyclonal and monoclonal antibodies capable of distinguishing individual P-450III-related proteins. Our studies confirm that a major erythromycin demethylase belonging to the P-450III family, termed P-450p, was greatly reduced in the choline-deficient rat liver. However, the specific concentration of a second P-450p-related protein was essentially normal and that of a third P-450p-related protein was actually increased in the choline-deficient rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of choline deficiency and methotrexate treatment upon rat liver

Choline deficiency and treatment with methotrexate (MTX) both are associated with fatty infiltration of the liver. Choline, methionine, and folate metabolism are interrelated and converge at the regeneration of methionine from homocysteine. MTX perturbs folate metabolism, and it is possible that it also influences choline metabolism. We fed rats a choline deficient diet for 2 weeks and/or treated them with methotrexate (MTX; 0.1 mg/kg daily). Choline deficiency lowered hepatic concentrations of choline (to 43% control), phosphocholine (PCho; to 18% control), glycerophosphocholine (GroPCho; to 46% control), betaine (to 30% control), phosphatidylcholine (PtdCho; to 62% control), methionine (to 80% control), and S-adenosylmethionine (AdoMet; to 57% control), while S-adenosylhomocysteine (AdoHcy) and triacylglycerol concentrations increased (to 126% and 319% control, respectively). MTX treatment alone lowered hepatic concentrations of PCho (to 48% control), GroPCho (to 69% control), betaine (to 55% control), and AdoMet (to 75% control). The addition of MTX treatment to choline deficiency resulted in a larger decrease in AdoMet concentrations (to 75% control) and larger increases in AdoHcy and triacylglycerol concentrations (to 150% and 500% control, respectively) than was observed in choline deficiency alone. Livers from MTX-treated animals used radiolabeled choline to make the same metabolites as did livers from controls (most of the label was converted to PCho and betaine). In choline deficient animals, most of the labeled choline was converted to PtdCho. Therefore, MTX depleted hepatic PCho, GroPCho, and betaine by a mechanism that was different from that of choline deficiency. MTX increased the extent of fatty infiltration of the liver in choline deficient rats, and choline deficiency and MTX treatment damaged hepatocytes as measured by leakage of alanine aminotransferase activity. Our data are consistent with the hypothesis that the fatty infiltration of the liver associated with MTX treatment occurs because of a disturbance in choline metabolism.

Choline deficiency causes translocation of CTP:phosphocholine cytidylyltransferase from cytosol to endoplasmic reticulum in rat liver.

The choline-deficient rat liver has been chosen as a physiologically relevant model system in which to study the regulation of phosphatidylcholine biosynthesis. When 50-g rats were placed on a choline-deficient diet for 3 days, the activity of CTP:phosphocholine cytidylyltransferase (CT) was increased 2-fold in the microsomes and decreased proportionately in the cytosol. A low titer antibody to CT was obtained from chickens and used to identify the amount of CT protein in cytosol from rat liver. The amount of CT recovered from the choline-deficient cytosol was significantly less than in cytosol from choline-supplemented rats. When hepatocytes were prepared from choline-deficient livers, supplementation of the medium of the cells with choline caused CT to move from the membranes to cytosol within 1-2 h. The activity of another translocatable enzyme of glycerolipid metabolism, phosphatidate phosphohydrolase, was unchanged in cytosol from choline-deficient rat livers, and the microsomal activity of this enzyme was only minimally increased. When the livers were fractionated into endoplasmic reticulum and Golgi, there was a 2-fold increase in the activity on the endoplasmic reticulum from choline-deficient livers but no change in activity associated with Golgi. Thus, the increased association of CT with endoplasmic reticulum in choline-deficient livers appears to be specific to that subcellular fraction, and the subcellular location of other enzymes may not be affected.

Reduction in VLDL, but not HDL, in plasma of rats deficient in choline

We have analyzed plasma lipoprotein levels in young male rats fed a choline-deficient diet for 3 days. We confirmed previous studies that choline deficiency promotes 6.5-fold accumulation of triacyglycerol in the liver (23.9 +/- 6.0 versus 3.69 +/- 0.92 mumol/g liver) and reduction of triacylglycerol concentration in plasma by 60% (0.17 +/- 0.04 versus 0.46 +/- 0.10 mumol/mL plasma). Agarose gel electrophoresis showed that the plasma very low density lipoprotein (VLDL) levels were reduced in choline-deficient rats, but the concentration of plasma high density lipoproteins (HDL) was not affected. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis of fractionated plasma lipoproteins revealed that the concentrations of apolipoproteins (apo) BH, BL, and E in VLDL from choline-deficient rats were 37.1, 11.0, and 37.2% of normal levels, respectively. In contrast, the amount of apo A-I, the major one in HDL, was almost unchanged. Correspondingly, there were decreased lipid (mainly phosphatidylcholine and triacylglycerol) levels in VLDL from choline-deficient rats, but no change in the levels of phosphatidylcholine, cholesterol, and cholesterol ester in HDL. There were similar levels of apo B and E (components of VLDL) in homogenates of livers from normal and choline-deficient rats, as determined by immunoblotting. These results support the hypothesis that choline deficiency causes reduction of VLDL, but not HDL, levels in plasma as a consequence of impaired hepatic VLDL secretion.

The effect of choline and methionine deficiencies on the number and volume percentage of altered hepatic foci in the presence or absence of diethylnitrosamine initiation in rat liver

The ability of methyl-deficient, amino-acid-defined diets to produce enzyme-altered foci was quantitatively determined in the livers of rats treated both with and without an initiating dose of diethylnitrosamine (DEN). Male weanling F-344 rats were fed a complete, amino-acid-defined diet for 1 week. They were then injected i.p. with a single dose of DEN (20 mg/kg body weight) and fed the complete diet for an additional week. Forty animals in each dose group were then maintained for 5-38 weeks on the complete diet (diet 1) or one of the three methyl-deficient diets customarily used in this laboratory: diet 2, devoid of methionine and choline; diet 3, devoid of methionine only; and diet 4, devoid of choline only. In diets 2 and 3, methionine was replaced by equimolar amounts of its metabolic precursor, DL-homocystine. Ten animals per group were killed 8, 12, 17, 24 and 41 weeks after DEN initiation. For 2 weeks prior to being killed, each group was maintained on the complete diet to minimize the histological abnormalities due to acute toxicity of the diets. Serial sections of the livers were obtained, stained sequentially for gamma-glutamyltranspeptidase, ATPase and glucose-6-phosphatase, and the quantitation of the focal lesions scored by these markers was carried out by quantitative stereology. The results indicated that, regardless of the enzyme marker(s) examined, there was a general correspondence between the volume and number of altered hepatic foci (AHF) formed and the previously described tumor-promoting activities of each diet. Thus, while all DEN-treated groups contained significant numbers of AHF 24 weeks after initiation, only the diet-2-fed animals displayed such foci at 8 weeks. Similarly, among the uninitiated rats, only those fed diet 2 exhibited the presence of AHF throughout the experimental period. Interestingly, the livers of uninitiated, choline-deficient rats showed a small number of AHF at 24 and 42 weeks; these foci were not observed at all in the corresponding DEN-untreated animals fed diet 3, deficient in methionine only. The results provide evidence that the carcinogenic effects of the methionine- and choline-deficient diet result more from its strongly promoting effect than from any initiating activity by the diet.

Head Group Specificity in the Requirement of Phosphatidylcholine Biosynthesis for Very Low Density Lipoprotein Secretion from Cultured Hepatocytes

We have demonstrated that hepatic very low density lipoprotein (VLDL) secretion requires active phosphatidylcholine (PC) synthesis via either the CDP-choline pathway or phosphatidylethanolamine (PE) methylation pathway (Yao, Z., and Vance, D.E. (1988) J. Biol. Chem. 263, 2998-3004). In the present work, the head group specificity of phospholipid synthesis required for lipoprotein secretion was investigated in cultured hepatocytes isolated from choline-deficient rats. When N-monomethylethanolamine (0.1 mM) or N,N-dimethylethanolamine (0.1 mM) was added to the culture medium, the cells synthesized correspondingly phosphatidylmonomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). However, the synthesis of PDME could correct the impaired VLDL secretion only to a limited extent, whereas the synthesis of PMME inhibited VLDL secretion. Although dimethylethanolamine did not promote VLDL secretion as well as choline, dimethylethanolamine altered the increased triacylglycerol synthesis in the choline-deficient cells as effectively as choline. Supplementation of the culture medium with ethanolamine (0.1 mM) had little effect on cellular PE or PC levels, nor was normal VLDL secretion resumed. However, the amounts of cellular PC and PE were both decreased when the medium was supplemented with N-monomethylethanolamine or N,N-dimethylethanolamine. These results suggest that the choline head group moiety of PC is specifically required for normal VLDL secretion and cannot be replaced with ethanolamine, monomethylethanolamine, or dimethylethanolamine. In addition, the impaired VLDL secretion from the choline-deficient hepatocytes could also be corrected by supplementation of betaine (0.2 mM) and homocysteine (0.2 mM), indicating the utilization of a methyl group from betaine for PC formation via methylation of PE.

Relation of sinusoidal stenoses following hepatocyte swelling to hepatic vascular resistance in experimental liver cirrhosis.

To investigate the significance of sinusoidal stenoses and a decrease in the sinusoidal bed due to hepatic cell swelling as a factor increasing hepatic vascular resistance in liver cirrhosis, hepatic vascular resistance in choline-deficient diet-induced cirrhotic rats was measured by an isolated liver perfusion method. In the cirrhotic rats, the swollen hepatic cells resulting from accumulation of fat droplets narrowed the sinusoids and decreased the sinusoidal bed. Consequently, the hepatic vascular resistance was increased by 2.7 times normal (8.60 +/- 2.32 mm H2O.ml-1.min; controls, 3.13 +/- 0.67 mm H2O.ml-1.min), and portal hypertension was also recognized (188.1 +/- 26.3 mm H2O; controls, 114.2 +/- 12.8 mm H2O). In the cirrhotic rats fed with ordinary rat pellets for 2 months, however, the sinusoidal stenoses and the decreased sinusoidal bed recovered to nearly normal as a result of disappearance of the fat droplets in the hepatic cells. The increased hepatic vascular resistance was decreased to 1.5 times normal (4.68 +/- 0.82 mm H2O.ml-1.min), and the elevated portal vein pressure was reduced (141.5 +/- 17.4 mm H2O). These findings clearly demonstrate that an important factor leading to an increase in hepatic vascular resistance is sinusoidal stenoses and a decrease in the sinusoidal bed resulting from swollen hepatic cells.

Formation of aliphatic amine precursors of N-nitrosodimethylamine after oral administration of choline and choline analogues in the rat

Trimethylamine and dimethylamine are important precursors of N-nitrosodimethylamine, which is a potent carcinogen in a wide variety of animal species. Choline, a component of the normal human diet, is metabolized by bacteria within the intestine to form trimethylamine and dimethylamine. However, animals on a choline-free diet continue to excrete some trimethylamine and dimethylamine, suggesting that other dietary precursors of these methylamines might exist. To determine whether CN bond cleavage by the intestinal bacteria is specific to the choline molecule, we measured mono-methylamine, dimethylamine, trimethylamine and trimethylamine oxide excretion in rat urine after the administration of compounds that shared structural features with choline. Water, choline, dimethylaminoethanol, diethylaminoethanol, phosphocholine, betaine, carnitine, β-methylcholine or dimethylaminoethyl chloride were administered by orogastric intubation, and the urine was collected for 24 hr. Administration of choline (15 mmol/kg body weight) resulted in increased urinary excretion of dimethylamine, trimethylamine and trimethylamine oxide (increases of approximately twofold, 500-fold and 50-fold, respectively). Of the administered choline, 12% was converted to trimethylamine or trimethylamine oxide and excreted in the urine within 24 hr. Phosphocholine administration resulted in similar increases in dimethylamine, trimethylamine and trimethylamine oxide excretion by rats. Modification of the ethyl-backbone or quaternary amine end of the choline molecule resulted in marked suppression of methylamine formation. Though administration of some analogues of choline (methylcholine, betaine and carnitine) resulted in the formation of small amounts of trimethylamine or trimethylamine oxide, and the administration of others (dimethylaminoethanol and dimethylaminoethyl chloride) resulted in the formation of some dimethylamine, the amounts formed were minimal compared with the amounts of trimethylamine and trimethylamine oxide formed after choline administration. Thus, of the many components of foods, only choline and its esters are likely to be significant substrates for trimethylamine and dimethylamine formation. How then can we explain the persistence of trimethylamine and dimethylamine excretion observed in choline-deficient rats? We suggest that endogenous (non-bacterial) synthesis of trimethylamine and dimethylamine occurs within some tissue of the rat.