Division of Nutrition and Metabolism, University Hospital Clementino Fraga Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Received 28 March 2002; in revised form 28 May 2002; accepted 26 June 2002
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ABSTRACT |
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INTRODUCTION |
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Chronic alcohol intake was shown to decrease PEMT activity in the liver membranes, both in baboons and in human subjects (Duce et al., 1988; Lieber et al., 1994
). In the rabbit jejunum, chronic ethanol ingestion was reported to decrease the BBM phosphatidylcholine content (Keelan et al., 1985
). We have thus decided to examine PEMT activity in the jejunal BBM of rats fed ethanol chronically. A nutritional evaluation was also undertaken, in order to exclude a possible interference of malnutrition secondary to chronic ethanol ingestion.
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MATERIALS AND METHODS |
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Experimental design
The animals were pair-fed for 4 weeks receiving a nutritionally adequate LieberDeCarli liquid diet as the sole source of food (Dyets Inc., Bethlehem, PA, USA). Diets followed American Institute of Nutrition (AIN) recommendations for adult rodents maintenance (Reeves et al., 1993) and contained 1 kcal/ml. The EFG received the liquid diet ad libitum but cornstarch was substituted isocalorically by ethanol to provide 35% of the total calories. The PFG received the isocaloric liquid diet without ethanol in a volume equal to that ingested by rats of the EFG. Liquid diets were renewed twice a day, so the animals consumed ethanol continuously.
On experimental day 29, the rats were killed by decapitation, and blood was collected. The small intestine was rapidly removed, washed with ice-cold saline, and placed on a pre-chilled glass plate. The intestine was divided into three equal segments. The first segment was opened longitudinally along the mesenteric border and the mucosal surface was removed by gently scraping with a microscope slide, placed into pre-weighed tubes, which were immediately frozen in liquid nitrogen and stored at 80°C for later purification of the BBM.
Nutritional evaluation
Body weight was recorded twice a week and diet ingestion was quantified daily. Nitrogen balance was obtained from the difference between the calculated nitrogen intake and the measured urinary and fecal nitrogen excretion, and was evaluated during a period of three consecutive days, at the first and last weeks of the experiment. Nitrogen was determined according to Fleck and Munro (1965). The 24 h urinary creatinine excretion was evaluated weekly by a modification of the kinetic Jaffé reaction (Larsen, 1972
). Serum transferrin was determined in the blood collected at death and expressed as the percentage of serum iron/total iron-binding capacity ratio, using a colorimetric assay kit provided by Labtest (Belo Horizonte, MG, Brazil).
Isolation of BBM
The method used for isolation of the BBM from rat small intestine mucosa has already been presented in detail (Booth and Kenny, 1974). All work was performed at 04°C. Mucosa was homogenized in 4 vol. of ice-cold 300 mM mannitol 12 mM Tris buffer, pH 7.3, and the homogenate was prepared by the Mg2+ precipitation treatment. The homogenate was centrifuged at 1500 g for 12 min and the supernatant was subsequently centrifuged at 21 000 g for 12 min. The pellet was re-suspended in 8 ml of 50 mM mannitol2 mM Tris buffer, pH 7.3, and MgCl2 was added to give a concentration of 10 mM and then stirred gently for 15 min in an ice bath. This suspension was centrifuged at 2200 g for 12 min and the resulting supernatant was subsequently centrifuged at 21 000 g for 30 min. The final pellet was re-suspended in 300 µl of 50 mM mannitol2 mM Tris buffer, pH 7.3, and aliquots were stored at 80°C. Protein was determined by the method of Lowry et al. (1951)
, using bovine serum albumin as a standard.
Cytochemical method
The efficiency of the preparative method was assessed by a cytochemical method to determine the presence of alkaline phosphatase, a BBM enzyme marker. A sample of the final pellet obtained from the BBM preparation was embedded in synthetic resin (Tissue-Tek, Sakura, USA) and frozen in liquid nitrogen. The blocks obtained were stored at 80°C. Sections of 6 µm were obtained in a cryostat. After fixation in 4% paraformaldehyde buffer, the sections were prepared according to the method of Brenan and Bath (1989). In brief, slides were incubated with a substrate solution containing chloro-indoxyl phosphate dissolved in dimethylformamide and tetranitro blue tetrazolium for 30 min in a darkened humidified chamber at 37°C, washed in distilled water and mounted with Aquamount. The positive reaction, a dark blue staining, represented the presence of alkaline phosphatase in the sample.
Electron microscopy
Additionally, membrane preparations were examined in a transmission electron microscope, to assess the absence of contaminating structures. A sample of the final pellet of the BBM was fixed with 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 for 2 h, post-fixed with a 1% buffered osmium tetroxide solution (w/v), dehydrated through graded alcohols and embedded in epoxy resin (EM-Bed; Electron Microscopy Sciences, Fort Washington, PA, USA). Ultrathin sections of about 70 nm thickness were double stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (Carl Zeiss 109) operating at 80 kV.
Assay of PEMT in the BBM
PEMT activity was determined in the BBM by measuring the incorporation of [3H]-methyl groups from S-adenosyl-l-(methyl-3H)-methionine (77.0 Ci/mmol; Amersham Biosciences, Little Chalfont, Bucks., UK) into phospholipids, as described by Castaño et al. (1980). The reaction mixture contained, in a final volume of 0.5 ml, 10 mM 4,2-hydroxyethyl-1-piperazine ethanesulphonic acid (pH 7.3), 4 mM dithiothreitol, 5 mM MgCl2, 100 µM S-adenosyl-l-methionine, 2 µCi S-adenosyl-l-(methyl-3H)-methionine and the BBM (0.3 mg protein). The reaction was initiated by the addition of a mixture of the labelled and unlabelled S-adenosyl-l-methionine, incubated at 37°C, and terminated (5, 10, 20 and 40 min later) by pipetting 100 µl of the assay mixture into 2 ml of chloroform/ methanol/2 N HCl (6:3:1, by vol.), containing 33 µg of butylated hydroxytoluene as an antioxidant, for lipid extraction. The chloroform phase was washed with 1 ml of 0.5 M KCl in 50% methanol. After washing, 0.6 ml of the chloroform phase was pipetted into a counting vial, dried at room temperature, dissolved into 4 ml of scintillation liquid and counted. Specific activity of the enzyme is expressed as femtomoles of [3H]-methyl groups incorporated into phospholipids/mg of protein/incubation time, at 37°C.
Statistical analysis
The enzyme activity and urinary creatinine excretion were examined using a two-way repeated measures analysis of variance (ANOVA) and a multiple comparison Bonferroni test. The remaining nutritional parameters were analysed by the Mann Whitney test. All the results were expressed as means ± SEM. Probabilities of less than 0.05 were accepted as significant.
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RESULTS |
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The ultrastructural analysis of the final BBM preparation showed membranes without organelle contaminants (Fig. 1).
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DISCUSSION |
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The increase in jejunal PEMT activity observed suggests that chronic ethanol consumption might stimulate the synthesis of phosphatidylcholine by the transmethylation pathway, since it may be assumed that phosphatidylcholine is the main product resulting from the enzymatic methylation of phosphatidylethanolamine (Bremer and Greenberg, 1961). A previous study, however, has shown a decrease in the total phospholipid and phosphatidylcholine content in the jejunal BBM of rabbits fed ethanol chronically, which resulted in a lower jejunal BBM phospholipid/cholesterol and choline/amine phospholipid ratio (Keelan et al., 1985
). The choline/amine phospholipid ratio was also reported to be decreased in the small intestine of alcoholic subjects (Bender-Braulio et al., 1990
).
Our results differ from those of Duce et al. (1988) and Lieber et al. (1994)
, but are in agreement with those of Uthus et al. (1976)
and Carrasco et al. (2002)
. Duce et al. (1988)
showed a marked decrease in PEMT activity in liver biopsies from human patients with alcoholic or post-hepatitic cirrhosis. Nevertheless, these authors did not find any change in relative phospholipid composition. Lieber et al. (1994)
have also demonstrated a decrease in PEMT activity in liver biopsies from baboons with alcohol-induced fibrosis, prior to the development of cirrhosis. This effect was accompanied by a decrease in the hepatic content of phosphatidylcholine. In contrast, Uthus et al. (1976)
and Carrasco et al. (2002)
reported that chronic ethanol ingestion markedly increased hepatic PEMT activity.
The precise mechanism by which ethanol stimulated the jejunal BBM transmethylation pathway in our study remains to be determined. In the liver, the two major pathways for the biosynthesis of phosphatidylcholine (the CDP-choline and the transmethylation pathways) seem to be regulated in a co-ordinated way, i.e. an inhibition of the CDP-choline pathway is accompanied by a stimulation of the transmethylation route (Mato and Alemany, 1983). If a similar regulation occurs in the small intestine, it is possible that the increase in PEMT activity observed in the present study is associated with an inverse effect on the CDP-choline pathway, leading to a deficient synthesis of phosphatidylcholine by the latter pathway.
Another possibility which could explain the elevation of PEMT activity in response to chronic ethanol ingestion is the increase of phosphatidylethanolamine levels, as a consequence of ethanol metabolism within the intestinal cell. The availability of phosphatidylethanolamine has already been demonstrated to regulate the PEMT activity (Ridgway et al., 1989; Cui et al., 1993
). Indeed, Carrasco et al. (1996)
reported that the levels of phosphatidylethanolamine were elevated in the hepatocytes after their incubation in the presence of ethanol and radiolabelled ethanolamine. Furthermore, in a previous study, we have shown that chronic ethanol ingestion significantly increased phosphatidylethanolamine levels (62%) in the mucosa of human small intestine (Bender-Braulio et al., 1990
). Recently, Carrasco et al. (2002)
have clearly demonstrated in rat hepatocytes that ethanol activates the biosynthesis of phosphatidylethanolamine and the methylation pathway of phosphatidylethanolamine to produce phosphatidylcholine, after both short-term and chronic ethanol treatments. In addition, these authors have also reported the inhibitory effect of chronic ethanol intake in the liver phosphatidylcholine synthesis in the CDP-choline pathway.
In conclusion, our results show a significant increase in jejunal PEMT activity due to chronic ethanol intake. Since no protein or caloric malnutrition was detected, we may assume that ethanol independently affects the phospholipid transmethylation pathway in the small intestinal mucosa.
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FOOTNOTES |
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REFERENCES |
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Blackburn, G. L. and Thornton, P. A. (1979) Nutritional assessment of the hospitalized patient. Medical Clinics of North America 63, 1110311115.[Medline]
Booth, A. G. and Kenny, A. J. (1974) A rapid method for the preparation of microvilli from rabbit kidney. Biochemical Journal 142, 575581.[Medline]
Bremer, J. and Greenberg, D. M. (1961) Methyl transfering enzyme system of microsomes in the biosynthesis of lecithin phosphatidylcholine. Biochimica et Biophysica Acta 46, 205216.[ISI]
Bremer, J., Figard, P. H. and Greenberg, D. M. (1960) The biosynthesis of choline and its relation to phospholipid metabolism. Biochimica et Biophysica Acta 43, 477488.[ISI]
Brenan, M. and Bath, M. L. (1989) Indoxyl-tetranitro blue tetrazolium method for detection of alkaline phosphatase in immunohistochemistry. Journal of Histochemistry and Cytochemistry 37, 12991301.[Abstract]
Carrasco, M. P., Sanchez-Amate, M. C., Marco, C. and Segovia, J. L. (1996) Evidence of differential effects produced by ethanol on specific phospholipid biosynthetic pathways in rat hepatocytes. British Journal of Pharmacology 119, 233238.[Abstract]
Carrasco, M. P., Jimenez-Lopez, J. M., Segovia, J. L. and Marco, C. (2002) Comparative study of the effects of short-and long-term ethanol treatment and alcohol withdrawal on phospholipid biosynthesis in rat hepatocytes. Comparative Biochemistry and Physiology Part B 131, 491497.
Castaño, J. G., Alemany, S., Nieto, A. and Mato J. M. (1980) Activation of phospholipid methyltransferase by glucagon in rat hepatocytes. Journal of Biological Chemistry 255, 90419043.
CIOMS (1985) International guiding principles for biomedical research involving animals. Alternatives to Laboratory Animals 12, 128.
Cui, Z., Vance, J. E., Chen, M. H., Voelker, D. R. and Vance, D. E. (1993) Cloning and expression of a novel phosphatidylethanolamine N-methyltransferase. Journal of Biological Chemistry 268, 1665516663.
Duce, A. M., Ortiz, P., Cabrero, C. and Mato, J. M. (1988) S-adenosyl-l-methionine synthetase and phospholipid methyltransferase are inhibited in human cirrhosis. Hepatology 8, 6568.[ISI][Medline]
Dudeja, P. K. and Brasitus, T. A. (1987) Identification and partial characterization of phospholipid methylation in rat small-intestinal brush-border membranes. Biochimica et Biophysica Acta 919, 307310.[ISI][Medline]
Fleck, A. and Munro, H. N. (1965) The determination of organic nitrogen in biological material. Clinica Chimica Acta 11, 212.[ISI]
Harari, Y. and Castro, G. A. (1985) Phosphatidylethanolamine methylation in intestinal brush border membranes from rats resistant to Trichinella spiralis. Molecular and Biochemical Parasitology 15, 317326.[ISI][Medline]
Keelan, M., Walker, K. and Thomson, A. B. (1985) Effect of chronic ethanol and food deprivation on intestinal villus morphology and brush border membrane content of lipid and marker enzymes. Canadian Journal of Physiology and Pharmacology 63, 13121320.[ISI][Medline]
Kennedy, E. G. and Weiss, S. B. (1956) The function of cytidine coenzymes in the biosynthesis of phospholipides. Journal of Biological Chemistry 222, 193214.
Larsen, K. (1972) Creatinine assay by a reaction-kinetic approach. Clinica Chimica Acta 41, 209217.[ISI][Medline]
Lieber, C. S., Robins, S. J. and Leo, M. A. (1994) Hepatic phosphatidylethanolamine methyltransferase activity is decreased by ethanol and increased by phosphatidylcholine. Alcoholism: Clinical and Experimental Research 18, 592595.[ISI][Medline]
Lorenz-Mayer, H., Femmel, C. and Tautenberg, M. (1980) The effect of the elemental diet on the morphology and functions of the intestinal mucosa after long term exposure. Aktuel Ernahrungsmedizi 5, 7480.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265275.
Mato, J. M. and Alemany, S. (1983) What is the function of phospholipid N-methylation? Biochemical Journal 213, 110.[ISI][Medline]
Reeves, P. G., Nielsen, F. H. and Fahey, G. C. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. Journal of Nutrition 123, 19391951.[ISI][Medline]
Ridgway, N. D., Yao, Z. and Vance, D. E. (1989) Phosphatidylethanolamine levels and regulation of phosphatidylethanolamine N-methyltransferase. Journal of Biological Chemistry 264, 12031207.
Trocki, O., Mochizuki, H., Dominioni, L. and Alexander, J. W. (1986) Intact protein versus free amino acids in the nutritional support of thermally injured animals. Journal of Parenteral and Enteral Nutrition 10, 139145.[ISI][Medline]
Uthus, E. O., Skurdal, D. N. and Cornatzer, W. E. (1976) Effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities in liver microsomes. Lipids 11, 641644.[ISI][Medline]