Folate transport proteins: Tissue distribution and effects of chronic ethanol intake in the micropig

Folate deficiency during chronic alcoholism may be mediated in part by changes in membrane composition and alterations in transport proteins responsible for translocation of monoglutamyl folates across intestinal, hepatic and renal membranes. To evaluate the role of folate binding protein (FBP) and reduced folate carrier (RFC) in folate homeostasis, we studied the biochemical and molecular properties of these proteins in Yucatan micropigs pair-fed diets containing 40% of daily Kcal as ethanol (n = 5) or corn starch control (n = 5) for 12 mos. Chronic ethanol feeding increased membrane fluidity, induced an accumulation of linoleic acid and decreased desaturation products of omega-6 and omega-3 fatty acids in liver plasma (LPM) and kidney brush border (KBB), whereas jejunal brush border (JBB) was unchanged. Folate binding of [ 3H]-folic acid was greater in LPM than KBB, while no activity was found in JBB. Binding of different [3H]-folate analogs by LPM showed higher affinity for 5-methyltetrahydrofolate, followed by folic acid and methotrexate. Northern blot analysis showed greater transcript intensity in liver than kidney, whereas FBP transcripts were absent in jejunum. FBP was identified by immunohistochemical staining in hepatocytes and renal tubular epithelial cells, whereas no staining was detected in JBB membranes. RFC transport was highest in LPM, 3-fold lower in KBB and 6-fold lower in JBB. Similarly, quantitative PCR showed greater RFC transcripts in liver, followed by kidney and jejunal mucosa, while transcript levels were lower in jejunal mucosa from ethanol-fed animals. Transport of [3H]-folic acid by JBB from ethanol treated animal showed decreased K_m and V_max, whereas K_m was decreased in KBB without changes in V_max. In summary, LPM and KBB membrane fluidity is regulated by ethanol induced fatty acid desaturation. FBP transcripts, immunoreactivity, and activity are expressed in pig liver and kidney and their relevant membranes, but no in the jejunum. RFC studies suggest that chronic ethanol exposure to micropigs decreases the intestinal absorption of monoglutamyl folates by altering the expression of RFC and its transport kinetics in JBB, whereas decreased RFC affinity in KBB may explain in part the abnormal urinary folate excretion during chronic ethanol exposure.