Mechanistic insights of intestinal absorption and renal conservation of folate in chronic alcoholism

Folate mediated one-carbon metabolism is of fundamental importance for various cellular processes, including DNA synthesis and methylation of biological molecules. Due to the exogenous requirement of folate in mammals, there exists a well developed epithelial folate transport system for regulation of normal folate homeostasis. The intestinal and renal folate uptake is tightly and diversely regulated and disturbances in folate homeostasis like in alcoholism have pathological consequences. The study was sought to delineate the regulatory mechanism of folate uptake in intestine and reabsorption in renal tubular cells that could evaluate insights of malabsorption during alcoholism. The folate transporters PCFT and RFC were found to be associated with lipid rafts of membrane surfaces in intestine and kidney. Importantly, the observed lower intestinal and renal folate uptake was associated with decreased levels of folate transporter viz. PCFT and RFC in lipid rafts of intestinal and renal membrane surfaces. The decreased association of folate transporters in lipid rafts was associated with decreased protein and mRNA levels. In addition, immunohistochemical studies showed that alcoholic conditions deranged that localization of PCFT and RFC. These findings could explain the possible mechanistic insights that may result in folate malabsorption during alcoholism.     

Folate malabsorption is associated with down-regulation of folate transporter expression and function at colon basolateral membrane in rats

Folates, an essential component (important B vitamin) in the human diet, are involved in many metabolic pathways, mainly in carbon transfer reactions such as purine and pyrimidine biosynthesis and amino acid interconversions. Deficiency of this micronutrient leads to the disruption of folate-dependent metabolic pathways that lead to the development of clinical abnormalities ranging from anaemia to growth retardation. Folate deficiency due to alcohol ingestion is quite common, primarily due to malabsorption. The present study dealt with the mechanistic insights of folate malabsorption in colonic basolateral membrane (BLM). Wistar rats (n 12) were fed 1 g/kg body weight per d ethanol (20 %) solution orally for 3 months and folate transport was studied in the isolated colonic BLM. The folate exit across colon BLM shows characteristics of carrier-mediated process with the major involvement of reduced folate carrier (RFC). The chronic ethanol ingestion decreased the uptake by decreasing the affinity by 46 % (P < 0·01) and the number of transport molecules by 43 % (P < 0·001) at the colon BLM. The decreased uptake was associated with down-regulation of proton-coupled folate transporter (PCFT) and RFC expression at mRNA and protein levels. The extent of decrease was 44 % (P < 0·01) and 24 % (P < 0·05) for PCFT and 23 % (P < 0·01) and 57 % (P < 0·01) for RFC at mRNA and protein levels, respectively. Moreover, folate transporters were associated with lipid rafts (LR) of colon BLM, and chronic alcoholism decreased the association of these transporters with LR.     

Effects of chronic ethanol and diet treatment on urinary folate excretion and development of folate deficiency in the rat

Because the folate deficiency of chronic alcoholism has been proposed to result from ethanol-induced effects on metabolism or urinary excretion of folate, the present study was designed to evaluate the role of chronic ethanol-induced urinary folate loss on folate homeostasis in the rat. Male Sprague-Dawley rats were fed nutritionally sufficient liquid diets for 12 wk with or without ethanol, folate and sulfonamide. Urinary folate excretion was increased in ethanol-fed rats consuming folate-containing diets, but not in rats fed folate deficient diets. Consumption of folate-deficient diets led to a rapid decrease in urinary folate excretion, suggesting renal adaptation to conserve folate. Tissue and plasma levels of folate were mostly unaffected by ethanol ingestion in rats fed folate-containing diets. Ethanol treatment did not consistently enhance tissue folate depletion in rats fed folate-deficient diets. The results suggest that in rats consuming diets containing high levels of folate, chronic ethanol ingestion increased urinary folate excretion, but not to a sufficient magnitude to consistently affect folate homeostasis.     

Chronic alcoholism alters the transport characteristics of folate in rat renal brush border membrane.

Folic acid transport across the epithelial cell membrane of kidney tubules is an essential step for its reabsorption, conservation, and homeostasis in the body. We characterized [(3)H]-folic acid transport in renal brush border membrane vesicles after 12 weeks of chronic ethanol ingestion to rats. The results demonstrated that chronic ethanol administration decreased the renal tubular reabsorption by a mechanism which involved an increase in the value of K(m) and a decrease in V(max). Importantly, ethanol feeding also interfered with disulfide bond status, temperature sensitivity, and Na(+) and divalent cation dependency of the transport process. The transport was transmembrane pH dependent, and ethanol did not have any effect on the pH optimum of the folate transport. The reduction in uptake in the ethanol-fed group was more pronounced at pH less than 6. In addition, the binding component was found to contribute to an appreciable extent to the total folate uptake; however, the amount of folate binding was less in the ethanol-fed group. Moreover, the folic acid analog methotrexate inhibited the transport to great degrees in control and ethanol-fed rats. These findings highlight the possible mechanism of renal disturbances of folate conservation during chronic alcoholism.     

Folate absorption in alcoholic pigs: in vivo intestinal perfusion studies

To determine if impaired intestinal absorption contributes to the folate deficiency observed in chronic alcoholics, we assessed in vivo folate absorption in Hanford mini-pigs fed ethanol with an adequate diet. Sixteen minipigs were pair-fed diets supplemented with ethanol or sucrose to 60% of total calories for 11 mo. In the ethanol-fed pigs peak blood alcohol concentrations averaged 28 mmol/L, serum alanine transaminase and aspartate transaminase activities were elevated, and liver histology showed a centrilobular distribution of succinate dehydrogenase. Tissue folate concentrations were comparable in both groups. The jejunal uptake of folic acid, measured by intestinal perfusion, was similar in both groups of animals and was not affected by acute exposure to 445 mmol/L ethanol. The in vivo hydrolysis of polyglutamyl folate was reduced by 35% in one ethanol-fed minipig. Decreased hydrolysis of polyglutamyl folate may represent an early step in the development of folate deficiency in chronic alcoholics.     

Haem and folate transport by proton-coupled folate transporter/haem carrier protein 1 (SLC46A1)

Haem carrier protein 1 (HCP1) was originally identified and characterised as a mammalian haem transporter. However, recent evidence has shown that it is also a proton-coupled folate transporter (PCFT) and mutations in the gene cause hereditary folate deficiency in humans. We therefore investigated haem and folate transport characteristics of PCFT/HCP1 both in vivo and in vitro in CD-1 mice and in the presence or absence of a blocking antibody for PCFT/HCP1, and also in cultured cells (which express PCFT/HCP1 endogenously) to elucidate the specificity and selectivity of PCFT/HCP1. The in vivo study showed that the addition of folic acid inhibited 59Fe-labelled haem transport in hypoxic mice but had no effect in normal mice. Using in vitro methods, the results showed increased [3H]folate uptake into everted duodenum from hypoxic mice but uptake was reduced by the addition of haem or PCFT/HCP1 antibodies to the medium. Caco-2 cells transiently transfected with small interfering RNA (siRNA) PCFT/HCP1 duplex oligos resulted in a 69 % reduction in PCFT/HCP1 mRNA when compared with the control siRNA. Both haem and folate uptake were significantly (P < 0.05) reduced in cells transfected with PCFT/HCP1 siRNA; however, the magnitude of reduction with folic acid uptake was greater (48 %) than that of haem (22.5 %). Overall the data support PCFT/HCP1 as a primary folate transporter with a lower affinity for haem. PCFT/HCP1 could therefore play a physiological role in Fe nutrition and the data highlight the potential for the interaction of folate and haem at the level of intestinal absorption.     

Point mutations alter the cellular distribution of the human folate receptor in cultured Chinese hamster ovary cells

Diminished cellular need for folate results in decreased function of the human folate receptor (FR) but increased expression of this protein in cells grown at different rates. Much of this FR is intracellular and not available for vitamin transport, raising the following question: what is the function of this excess receptor? In this study, we characterized the effects of three point mutations on FR regulation in Chinese hamster ovary cells stably transfected to express either wild-type receptor or FR containing mutations at positions 67(S-->P),144(E-->D), and/or 201(N-->D). The 201(N-->D) FR responded functionally like the wild-type receptor but was localized predominantly at the cell surface (>90% vs. <40% for wild-type). This mutation disrupted a N-linked glycosylation site and generated a partially deglycosylated receptor. The 67(S-->P) mutation also shifted the cellular distribution such that more FR was surface accessible ( approximately 80%) but did not affect glycosylation. Because previous results showed that these mutations influence the conformation of FR, our findings suggest that structural changes in the receptor facilitate its trafficking to the cell surface. FR containing the 67(S-->P) mutation with either a 144(E-->D) or 201(N-->D) change was not processed from the high-mannose to complex glycoform but was still transported to the cell surface and able to transport folates. Thus, conformational changes introduced by specific point mutations can influence FR processing and/or trafficking to the cell surface. Furthermore, the fact that mutated FR can be trafficked to the cell surface more efficiently suggests that the native receptor may be retained intracellularly to perform some function there.     

Folate absorption in alcoholic pigs: in vitro hydrolysis and transport at the intestinal brush border membrane

We used the miniature pig to evaluate the effect of ethanol ingestion on the hydrolysis of pteroylpolyglutamate and on the uptake of pteroylmonoglutamate (PteGlu) by the intestinal brush border membrane, processes that are required for folate absorption. After feeding ethanol or sucrose at 60% of calories for 11 mo, the uptake of PteGlu by jejunal brush-border-membrane vesicles was similar in both groups of animals. Jejunal brush border pteroylpolyglutamate hydrolase was decreased by one-half in the ethanol-fed group. Jejunal brush-border-membrane fluidity, measured by fluorescence polarization, was similar in both groups. Acute exposure of the jejunal vesicles to ethanol increased membrane fluidity and decreased hydrolase activity but had no effect on PteGlu transport. Inhibition of jejunal folate hydrolase by chronic exposure to ethanol may be an early effect in the pathogenesis of folate malabsorption and deficiency in chronic alcoholism.     

Maternal-fetal folate transfer: effect of ethanol and dietary folate deficiency

The effects of acute ethanol treatment and dietary folate deficiency on maternal-fetal folate transfer were studied to asses the hypothesis that the potentiation of ethanol's toxic effect on the fetus during ongoing folate deficiency was due to the impairment of folate transfer. Sprague-Dawley rats were fed either a folate-deficient diet (0.2 mg/kg) or a folate-sufficient diet (2 mg/kg) for an average of 11 weeks before pregnancy and continued until gestation day 11 when they were sacrificed. On gestation day 11, pregnant rats were treated with either ethanol (2.5 g/kg body weight) or isocaloric sucrose (control) followed by an intravenous administration of 3H-folate (2 muCi/100 g body weight) at 120 minutes. At 210 minutes, maternal blood and feto-placental tissues were removed for radioactivity measurement. Folate status and ethanol treatment had no effect on the distribution of 3H-folate in maternal circulation. However, contrary to the hypothesis, the uptake of 3H-folate by feto-placental tissues was increased in folate deficiency and by ethanol treatment, indicating that folate transfer was not impaired by the ethanol treatment. Other possibilities are discussed.     

Decreased axonal transport in rat nerve following acute and chronic ethanol exposure

The axonal transport system, which supplies essential proteins and other cellular components to the distal portions of peripheral nerve axons, has been postulated to be the primary site of vulnerability inducing the peripheral neuropathies associated with neurotoxin exposure. Axonal transport was examined in normal rat dorsal root ganglia-sciatic nerve preparations incubated 28 hours in vitro in the presence of 79, 198 or 395 mg% ethanol. Exposure of the nerves to 395 mg% ethanol significantly reduced the accumulation of radiolabeled protein by 70%. Also, groups of rats were pair-fed an ethanol or isocaloric control diet for 9, 16 or 28 weeks. In vitro axonal transport was found to be unchanged in nerves of rats fed the ethanol diet for 9 weeks, but was significantly reduced 44% after 16 weeks and 47% after 28 weeks of ethanol feeding. These results suggest that interference with the axonal transport machinery by ethanol or perhaps acetaldehyde, its primary metabolite, may lead to the development of alcoholic peripheral neuropathy.     

Effect of ethanol administration on Mg2+ transport across liver plasma membrane.

Acute and chronic ethanol administration results in a decrease in cellular Mg2+ content and an alteration of Mg2+ transport in liver cells. In this study we investigated the extent to which ethanol affects the Mg2+ transport mechanisms in the liver cell membrane. The functionality of these transport mechanisms was assessed in plasma membrane vesicles purified from livers acutely perfused with varying concentrations of alcohol, livers of animals fed with 6% ethanol for 3 weeks, and the respective controls. Acute alcohol administration had little or no effect on the Mg2+ extrusion mechanisms present in the apical and basolateral domains of the hepatocyte but completely impaired the Mg2+ entry mechanism present in the basolateral side of the cell. This effect was already evident at a dose of alcohol as small as 0.01% (approximately 1.5 mM). The chronic administration of ethanol, instead, impaired all the Mg2+ transport mechanisms irrespective of the location and directionality in a time-dependent manner. Taken together, these data indicate a selective sensitivity of the Mg2+ entry mechanism to acute alcohol administration, whereas the Mg2+ extrusion mechanisms are affected only after prolonged exposure to alcohol. These results suggest that the defect in hormone-activated Mg(2+) transport observed in the chronic EtOH model [Young, A., Cefaratti, C., & Romani, A. (2003). Chronic EtOH administration alters liver Mg2+ homeostasis. Am J Physiol 284, G57-G67] depends not only on a reduced cellular Mg2+ content but also on the impaired Mg2+ transport mechanisms present in the hepatocyte plasma membrane, in particular the Mg2+ entry pathway, which prevents the liver cell from restoring cellular Mg2+ homeostasis.     

Effect of cow milk on food folate bioavailability in young women

BACKGROUND: The findings of animal and human studies suggest that a certain component in milk enhances food folate bioavailability. OBJECTIVE: The objective was to evaluate whether cow milk enhances the bioavailability of food folate in humans. DESIGN: Thirty-one young women were fed low-folate diets on a 4-d rotation with (n=17) or without (n=14) cow milk for 8 wk. Plasma and erythrocyte folate and plasma total homocysteine (tHcy) concentrations were monitored. RESULTS: Plasma folate concentrations steadily declined in both the milk and the nonmilk groups; however, the declines were not significantly different between groups (P=0.09). In contrast, erythrocyte folate concentrations declined only in the nonmilk group and remained unchanged in the milk group; the difference between the 2 groups was significant (P=0.01). Plasma tHcy concentrations significantly increased in the nonmilk group and remained unchanged in the milk group; the difference between the 2 groups was significant (P=0.02). CONCLUSION: The inclusion of cow milk in the diet enhanced the bioavailability of food folate as assessed by the changes in erythrocyte folate and plasma tHcy concentrations but not in plasma folate concentrations. The mechanisms of action by the components of cow milk remain to be investigated.     

Ethanol-induced alterations in exocrine pancreatic amino acid transport and secretion

The effects of ethanol on exocrine pancreatic amino acid transport and secretion were investigated during perfusion of the isolated rat pancreas with ethanol concentrations ranging from 0.06% to 4.1%. Amino acid transport was quantitated using a rapid dual isotope dilution technique in which unidirectional substrate uptake (15-20 sec) is assessed relative to an extracellular tracer. Pancreatic secretion evoked by 0.3 microM carbachol was abolished during perfusion with 0.32% ethanol. Influx of L-lysine, L-serine and methylaminoisobutyric acid (MeAIB) was marginally increased by 0.32% ethanol but significantly inhibited during subsequent perfusion with 1.28-4.1% ethanol. Pancreatic oxygen consumption and effluent PCO2 levels decreased with increasing ethanol concentration, and the control venous pH (7.21 +/- 0.01, n = 8) gradually approached arterial pH values (7.46 +/- 0.02, n = 9). These results indicate that low concentrations of ethanol readily inhibit secretagogue-induced pancreatic secretion. Amino acid transport at the basolateral membrane of the exocrine pancreatic epithelium appears only to be inhibited after acute exposure to high ethanol concentrations.     

MODULATION OF CANCER GROWTH BY VITAMIN E AND ALCOHOL

Seventy-five percent of esophageal cancers are alcohol related,yet alcohol is not a carcinogen. Ethanol may promote carcinogenesisvia increased free radical products during its metabolism, asindicated by data from this and other studies. Ethanol is oxidizedto acetaldehyde by alcohol dehydrogenase, catalase and the microsomalethanol oxidizing system (MEOS). Free radicals (FR) are releasedduring the oxidation of ethanol by the MEOS. An increased formationof FR in tissues would increase their oxidative stress and mayincrease their susceptibility for developing chemically inducedcancers. FR and some FR products can rapidly react with biologicalmaterials, i.e. lipids, proteins and nucleic acids, formingtoxic products. This study focuses on the effects of FR and/orFR products on cancer promotion during alcohol metabolism. Eightgroups of mice were fed nutritionally adequate diets supplementedwith vitamin E and/or ethanol. Some groups of mice were alsoorally gavaged with N-nitrosomethylbenzylamine (NMBzA), an esophagealcarcinogen. Following the feeding of the various diets for 22weeks, livers and esophagi were removed and the FR burden inthe liver measured by the presence of lipid peroxide productsand the number of tumors in each esophagus determined. Thesestudies indicate that a linear relationship exists between theincreasing number of esophageal tumors and increasing levelsof lipid peroxide products that are formed during FR activity.These results show that FR and/or FR products are the cancerpromoters during ethanol metabolism, since diets supplementedwith high levels of vitamin E, which inhibits ethanol-inducedFR activity and the formation of FR products, suppress the promotionof cancer by ethanol Therefore, we conclude that FRs producedduring ethanol metabolism and/or the products of FR activityare the cancer promoters attributed to ethanol.     

Aging: effect on hepatic metabolism and transport of folate in the rat

Effects of aging on hepatic folate metabolism and transport were assessed in male Fisher 344 rats. Total serum and hepatic folate levels were measured. Hepatic folates were measured by high-performance liquid chromatography and by Lactobacillus casei assay. Transport of 5-methyltetrahydrofolate (5-CH3-H4PteGlu) was measured in isolated hepatocytes. Serum folate declined with aging; however, neither the total folate level nor the distribution of hepatic folate coenzymes was affected by the aging process. The level of liver folate monoglutamates was not significantly different in any group. The initial rate of uptake of 5-CH3-H4PteGlu was significantly decreased in hepatocytes from the 24-mo-old rats, as was the ability to concentrate this folate from the medium. Aged rats maintain apparently normal levels of hepatic folates despite decreased serum levels and decreased ability to take up folates, suggesting that membrane transport of folates may not be a limiting factor in hepatic folate assimilation.     

Chronic ethanol feeding and acute ethanol exposure in vitro: effect on intestinal transport of biotin

This study examined the effects of chronic ethanol feeding and acute ethanol exposure in vitro on biotin transport in rat intestine. Transport studies were performed with intestinal everted sacs. Ethanol was fed to rats for 6-7 wk. Compared with pair-fed controls, ethanol feeding significantly decreased plasma biotin concentrations and transport at physiological concentrations (0.01, 0.1, and 0.3 mumol/L) but not at pharmacological concentration (100 mumol/L). When added to the incubation medium of everted jejunal sacs from dry-food-fed rats, ethanol (2% vol:vol) significantly inhibited the transport of biotin at a physiological concentration (0.1 mumol/L) but not at a pharmacological concentration (100 mumol/L). The inhibitory effect of ethanol on the transport of 0.1 mumol biotin/L increased with increasing concentration of ethanol in the incubation medium [0.5% to 5% (vol:vol)]. Acetaldehyde, the major ethanol metabolite, also significantly inhibited biotin transport at 0.2% (vol:vol). These data demonstrate that chronic ethanol feeding and acute ethanol exposure in vitro inhibit the intestinal transport of biotin by the carrier-mediated process. Chronic ethanol feeding is also associated with a significant decrease in plasma biotin concentrations. The ethanol-induced inhibition in intestinal transport of biotin may be a contributing factor in reducing plasma biotin concentrations.     

Food restriction beneficially affects renal transport and cortical membrane lipid content in rats.

Food restriction (FR) exerts a variety of beneficial effects and may prolong life in both humans and animals. However, studies of its effects on the cortical brush border membrane (BBM) and basolateral membrane (BLM) lipid concentration, which may be pertinent to renal function, have not been reported in detail. We hypothesized that FR would decrease renal work and lower renal membrane lipid concentration. The changes in lipid concentration would be most dramatic in BBM because this membrane is the entry site for the recovery of filtered ions and nutrients. Young male Fischer 344 x Brown-Norway F1 rats consumed food ad libitum (AL) or were food-restricted (FR, 60% of AL consumption) for 6 wk. AL rats had higher fractional excretions of Na(+), K(+), and Cl(-) than did the FR group (P < 0.001). Renal Na,K-ATPase activity in AL rats was 100% higher than in FR rats (P < 0.001), reflecting greater renal work. The work required for renal proton secretion was lower in FR than in the AL rats. In FR rats, all BBM phospholipid concentrations (phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) were approximately 50% lower than in the AL rats (P < 0.001). In the BLM, food restriction resulted only in lower phosphatidylcholine concentration, while the other phospholipids were unaffected. Plasma and renal membrane (BBM and BLM) cholesterol concentrations were significantly lower in FR than in AL rats. These results show that a nutritionally complete, but energy restricted, diet improves renal function. It also prevents renal membrane lipid deposition and decreases plasma cholesterol. Prolonged food restriction might attenuate the renal injury that occurs in obese humans as a consequence of insulin resistance and atherosclerosis.     

Relative folate bioavailability from diets containing human, bovine and goat milk

The present study was designed to determine the relative folate bioavailability from diets containing human, bovine or goat milk and the relative sensitivity of various response criteria used in assessing folate bioavailability. Following a 12-wk depletion period, 16 groups of male rats (n = 5/group) were fed experimental diets with or without 20% milk solids and graded levels of folic acid for 4 wk. Total folates were measured in plasma, erythrocytes, liver and kidney. Bioavailability of dietary folate was determined using slope-ratio statistics. Plasma response was found to be the most sensitive indicator of folate bioavailability based on steepness of slope, goodness of fit (r = 0.96, P less than 0.01) and linearity of response over the entire range of folate intakes. Kidney folate concentration also showed a significant linear relationship to total folate intake (r = 0.69, P less than 0.01). Liver and erythrocyte folate concentrations were not correlated with folate intake (r = 0.33 and r = 0.22, respectively). Using plasma folate as the response criterion, dietary incorporation of human milk significantly enhanced folate bioavailability by 75% (P less than 0.01). With kidney as the response tissue, folate bioavailability from diets containing human and bovine milk was significantly enhanced over milk-free diets. These results show that incorporation of human or bovine milk into diets enhances folate bioavailability and that plasma and kidney folate concentrations are sensitive and specific indicators of folate bioavailability.