Folate deficiency in alcoholism13

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1 Folate deficiency in alcoholism13 Charles H. Halsted,4 M.D. Folates are required for many metabolic functions relating to nudleoprotein synthesis and cell turnover. Thus, the most common sign of folate deficiency is megaloblastic anemia, while less obvious structural and functional changes are found in the small intestinal mucosa (1, 2). Dietary folates consist of pteroylpolyglutamates, whereas, after intestinal absorption, folates circulate in the form of reduced and methylated pteroylmonoglutamates (3). The recommended dietary allowance for folate is 400 j.tg, and the minimal amount required in the diet to maintain a normal serum folate level is 50 to 100 sg (1). Folate deficiency results from diets inadequate in sources of folate (leafy vegetahies, yeast, and liver), from the increased demands of pregnancy, from diseases of the proximal small intestinal mucosa (e.g., tropical or ceiac sprue), and in association with the chronic use of certain drugs, including alcohol. Chronic alcoholism is probably the leading cause offolate deficiency in the United States. As summarized recently, the incidence of folate deficiency in chronic alcoholic patients is as high as 87%, if low serum levels are used as the criterion of folate deficiency, with up to a 61 % incidence of megaloblastic anemia (4). Folate deficiency is most prevalent in poorly nourished derelict alcoholics and in association with alcoholic liver disease. Clinical observations In evaluating the etiology of anemia in 12 chronic alcoholic patients with cirrhosis in 1956, Jandl and Lear (5) described four patients with megaloblastic bone marrows in whom a reticulocyte response and reversion to normoblastic bone marrow occurred after the oral administration of folic acid, (pteroylmonoglutamate or PteGlu), 250 to 500 tg/ day, or to hospital diets containing 1200 zg of folate per day (5). In 1963, Herbert et al. (6) reported a large series of folate deficient alcoholics, fmding that serum folate levels were less than 5 ng/ml in 80% of 70 patients, about halfofwhom had significant associated liver disease. Low serum folate levels were significantly correlated with a history of recent binge drinking, and less well with poor dietary histories and associated liver disease. In the study by Klipstein and Lindenbaum (7) of 55 patients with liver disease, the majority ofwhom were chronic alcoholics, folate deficiency with megaloblastic anemia occurred in 42% and correlated best with a history of poor dietary intake. Sullivan and Herbert (8) demonstrated a direct relationship between the ingestion of alcohol and the availability of folate to the bone marrow in three alcoholic cirrhotic patients with severe folate deficiency and megaloblastic anemia in whom the reticulocyte and bone marrow response to oral or parenteral PtcGlu, 75.tg/day, could be suppressed by the concommitant administration of alcohol in daily amounts equivalent to a pint of whiskey per day (8). The relationship of the acute and chronic ingestion of alcohol to the development of folate deficiency was investigated in a series of studies from the University of Washington group. Low serum folate levels with megaloblastic bone marrows, found in 40% of 65 patients admitted to hospital with chronic alcoholism, were correlated with poor diets and with excessive alcohol intake in the weeks preceding admission (9). After treatment, four patients who were placed on low folate diets with ethanol in daily amounts equal to a pint of whiskey developed low serum folate levels within 4 days and megaloblastic bone marrows within 2 to 3 weeks. Subsequently, megaloblastic From the Department of Internal Medicine, University of California, Davis. 2Supported by Grant AM from the National Institutes of Health and by grants from the National Council on Alcoholism and the United States Brewers Association. 3Address reprint requests to: Charles H. Halsted, M.D., TB 192, School of Medicine University of California, Davis, California Professor of Internal Medicine The American Journal ofclinical Nutrition 33: DECEMBER 1980, pp Printed in U.S.A.

2 FOLATE DEFICIENCY IN ALCOHOLISM 2737 anemia was induced in 6 to 10 weeks in two of the same subjects by the administration of a folate-deficient diet without added alcohol (10). The time required for induction of megaloblastosis by folate-deficient diet in the chronic alcoholic subjects contrasted with Herbert s (1 1) previous observation that nineteen weeks were required to induce megaloblastosis by diet in a normal healthy subject. Another study suggested that ethanol acutely limits the availability of circulating folate, since raising blood ethanol to intoxicating levels in normal volunteers was followed by an abrupt fall in serum folate levels (12). To summarize, these clinical observations have shown a frequent association of folate deficiency and chronic alcoholism in patients with histories of poor diet and recent excessive drinking. Folate deficiency is less predictably correlated with the presence of liver disease in the chronic alcoholic patient. The studies suggest that the greater risk for folate deficiency in the drinking alcoholic may be partly attributed to decreased folate stores and to an acute limiting effect of ethanol on serum folate levels, and hence on the availability of folate to the tissues. Pathogenesis of folate deficiency in chronic alcoholism. Possible causes of folate deficiency in alcoholic patients include inadequate diet with resultant decreased body storage, intestinal malabsorption, altered serum protein binding and tissue affinity, and altered hepatobiliary metabolism. Dietary deficiency and decreased body stores offolate Studies correlating poor diets with folate deficiency in alcoholics are described above. Prolonged dietary folate deficiency should predictably decrease body stores of folate and, conversely, the presence oflimited folate stores increases the risk for developing folate deficiency with continued ingestion of a marginal diet. The total body storage of folates is estimated at 7 to 10 mg, most of which is present in the liver (1). Normal man requires about 19 weeks to develop a megaloblastic bone marrow while ingesting a folate-deficient diet, and the minimal daily requirement for folate, obtained by dividing stores by this time period in days, is therefore 50 gig/day (1 1). By applying this same equation, and assuming the same minimal daily folate requirement, the shorter time observed for the dietary induction of folate deficiency in the alcoholic patient should be directly proportional to the decrease in hepatic folate reserve (13). However, this hypothesis is not consistently supported by measurements of folate levels in alcoholism with liver disease. Although Leevy et al. (14) correlated low serum folate levels with low hepatic folate concentrations in chronic alcoholic patients (14) there was no significant decrease in hepatic folate concentration in patients with alcoholic cirrhosis in another study (15). Intestinal malabsorption Clinical studies have demonstrated that binge drinking alcoholism is associated with diarrhea and malabsorption of several water soluble vitamins, including folic acid (pteroylmonoglutamate or PteGlu)(l6). Additionally, severe folate deficiency is known to affect jejunal histology, with reversible megaloblastosis of the crypt epithelium (17). Our experiments suggested that malabsorption of PteGlu is conditioned by the combination of folate deficiency and prolonged ethanol intake, factors typically found in the chronic alcoholic patient. In an initial study, the intestinal absorption of 3H-PteGlu, measured as the percentage recovery of the isotope in the serum after an oral dose (1.5 pg/kg) and a flushing parenteral dose of PtcGlu, 15 mg, was decreased in a group of 10 binge-drinkers as compared to a control group of 23 nonalcoholics (18). Subsequently, the jejunal uptake of3h-pteglu from the perfused jejunum was found decreased in eight poorly nourished derelict alcoholics as compared to results obtained in the same patients after 2 weeks of hospital diet ( 19). A third study evaluated prospectively the jejunal uptake of 3H-PteGlu in four chronic alcoholic patients (20). After obtaining base-line studies, two patients were fed a folate deficient diet with ethanol (256 g/day) and a third patient was fed a folate-deficient diet without ethanol until induction ofmegaloblastosis in the bone marrow after 7 to 9 weeks. A fourth patient was fed a regular hospital diet to which was added ethanol, 300 g/day for 3 weeks. Jejunal histology was unchanged by any of the regi-

3 2738 HALSTED mens. Compared to initial values, the jejunal uptakes of :)HpteGlu glucose and sodium were decreased in the two folate-deficient patients fed ethanol. In the other two patients, either folate deficiency or 3 weeks administration of ethanol resulted in decreased water and sodium uptake from the perfused jepnum, but no changes in the uptake of H- PteGlu or glucose. Thus, the combination of dietary folate deficiency with chronic intake of ethanol impaired a variety of transport processes in the jejunum including the absorption of H-PtcGlu, whereas water and electrolyte transport were affected separately by folate deficiency and chronic ethanol intake. These data suggested a vicious cycle in which folate malabsorption contributes to folate deficiency which, at the same time, aggravates intestinal folate malabsorption. The effect of acute or chronic alcoholism in the initial digestion of dietary polyglutamyl folates and on the absorptive process of methylated or reduced monoglutamyl folates has not been established. Serum binding, tissue affinity, and renal excretion Folates circulate as 5-methyltetrahydrofolate (CH3H4PteG1u), most of which is bound nonspecifically to a variety of proteins and specifically to a B-globulin. Normally this specific folate binding protein is two-thirds saturated. Although the serum concentration of folate binding protein is unchanged in alcoholic cirrhosis, its saturation is significantly decreased (2 1), a circumstance that could result in increased urinary excretion of unbound folate (see below). Tissue affinity for folate has been measured by the uptake of a tracer dose and the subsequent displacement of the label by a large nonradioactive flusing dose (22). Tissue affinity for folate may be decreased by ingestion of ethanol or in alcoholic cirrhosis. The rate of disappearance from the circulation of tracer doses of 3H-PteGlu or 4CH3H4 PtcGlu was unchanged, whereas the subsequent displacement ofthe label by a flushing dose of PteGlu was significantly increased in normal volunteers studied before and after the daily ingestion of ethanol, 8 oz/day for 6 days (23). Cherrick et al. (24) found that the hepatic uptake of :IH..pteGlu measured as the difference in brachial artery and hepatic vein radioactivity after a parenteral tracer dose, was similar in six normal subjects and in six chronic alcoholic patients with biopsy proven cirrhosis. However, the hepatic affmity for labeled folate was significantly less in the cirrhotics, in whom the increase in hepatic venous tritium after a flushing dose of PteGlu was 10 times greater than the control subjects (24). The effect of alcoholism on urinary folate excretion has not been studied. However, two groups showed increased urinary folate excretion in viral hepatitis (25, 26), suggesting that hepatocellular injury from other causes, such as alcoholism, may be associated with urinary loses of folate. Hepatic metabolism and biliary excretion of folates Hepatic folate metabolism includes the steps of transport of circulating CH3H4PteG1u across the hepatocyte membrane (27), binding to intracellular proteins (28), synthesis of polyglutamyl folates (PteGlu0) (29), and exit to the hepatic vein or biliary ductules (30). Studies of the effects of acute or chronic alcoholism on hepatic folate metabolism include the following. In vitro, the uptake of CH3H4PteGIu by the isolated rat hepatocyte was found significantly enhanced by the presence of ethanol, 40 mm (27). In one study the synthesis of polyglutamyl folate, measured as the incorporation of injected 3H-PteGlu in the liver as 3H-PteGlu, was significantly decreased in rats fed 8% ethanol in their drinking water for 14 days (3 1 ). However, the studies in the rat by Hillman et al. (32) suggest an opposite effect of ethanol on hepatobiliary metabolism. After the administration of 10% ethanol in drinking water for 3 days, they observed a sharp decrease in serum folate levels, and, after intravenous injection of3h-ptcglu, decreased biliary excretion of the label but increased incorporation of the label in the hepatic pool of PteGlu0 (32). These data suggest that the fall in serum folate after short-term exposure to ethanol is due to trapping of folate in the hepatic pool and its decreased circulation in the enterohepatic folate pool. Ongoing studies In consideration of the pathogenesis of folate deficiency in alcoholism, it is important

4 FOLATE DEFICIENCY IN ALCOHOL1SM 2739 to distinguish the effects on folate metabolism of the acute ingestion ofethanol from chronic alcoholism associated with liver injury. To study the effects of chronic alcoholism on folate metabolism, our laboratory developed a primate model (Macacca radiata) in which the feeding of liquid diets containing 50% of energy as ethanol (33) was followed in 2 years by mild hepatic injury and decreased levels of hepatic folate (34, 35). Over a 2-year period, four monkeys in group E consumed 6.1 ± g of ethanol per kg body weight per day in diets which were isocaloric and were offered as 100 kcal/kg body weight per day. Each of four control monkeys (group C) was offered the same amount ofenergy consumed by his pair on the previous day. Each diet contained 50 tg/kcal folic acid. Weight gain was similar in each group over 2 years. There were no differences with respect to hematological fmdings, serum folate levels, fecal fat excretion, nitrogen balance, or d-xylose absorption. Using tissue removed at laparotomy after 24 months of feeding, we found normal jejunal histology by light and electron microscopy in group E, and no differences between the groups with respect to the activities ofjejunal sucrase, lactase, and folate conjugase. Liver histology in each ethanol-fed monkey was characterized by steatosis, increase in smooth endoplasmic reticulum, and enlarged mitochondria, with no evidence of hepatocellular necrosis or inflammation. Measured by microbiological assay after hog kidney conjugase treatment, liver folate levels were significantly decreased in group E when expressed on the basis of wet weight, protein or DNA. The intestinal absorption of folic acid was assessed in each monkey by measuring urinary and fecal excretion of tritium after the intragastric administration of 3H-PteGlu, 33 tg/kg body weight (34). The data suggested intestinal malabsorption of folic acid in group E, since in 5-day collections the ethanol-fed monkeys excreted significantly greater tritium in the feces and significantly less tritium in the urine than their pair-fed controls. Hepatic folate metabolism was measured in open liver biopsies obtained three days after an intramuscular injection of a tracer dose of 3H-PteGlu after 2 years of feeding (35). The recovery oftritium was significantly less in the liver tissue from the ethanol-fed animals. Chromatographic analysis of liver homogenates demonstrated that the processes of reduction, methylation and formylation of monoglutamyl folate derivatives, and the synthesis ofpolyglutamyl folates was not affected by chronic ethanol feeding. However, since the concentration of tritiated folates was less in the livers of the ethanol-fed monkeys, the calculated incorporation of the injected dose as newly synthesized polyglutamyl folates was significantly decreased in the ethanol-fed animals. To summarize these experiments, our data suggest that the chronic ingestion of ethanol with a nutritious diet in this species is followed after 2 years by mild hepatic injury and decreased hepatic folate concentrations. Factors contributing to decreased hepatic folate levels include intestinal malabsorption of folic acid and decreased capacity of the liver to retain folates. Possible mechanisms that could account for decreased retention of folates in the alcoholic liver include decreased transport into the hepatocyte, decreased ccllular binding capacity of folate, or increased excretion of folate into the biliary system. Conclusions Folate deficiency, the most common sign of malnutrition in chronic alcoholism, is multifactorial in etiology. The pathogenesis of folate deficiency in alcoholism includes dietary inadequacy, intestinal malabsorption, acute effects of ethanol on the delivery of circulating folate to the tissues, decreased hepatic retention, and probable altered enterohepatic cycling. In assessing the etiology of folate deficiency in this disease, it is important to distinguish between the effects of acute ethanol intake and ofchronic alcoholism with associated liver injury. a References 1. Recommended Dietary Allowances. Washington, D.C.: National Academy ofsciences, 1980, pp HALSTED, C. H. Folate deficiency and the small intestine. In: Folic Acid in Neurology, Psychiatry, and Internal Medicine, edited by M. I. Botez and E. H. Reynolds. New York: Raven Press, 1979, pp HALSTED, C. H. Intestinal absorption and malabsorption offolates. Ann. Rev. Med. 31: 79, 1980.

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