Repletion of Folate-Depleted Rats with an Amino Acid—Based Diet Supplemented with Folic Acid

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Abstract
Folate depletion and repletion protocols are not well standardized. Weanling rats were moderately depleted of folate in 28 d with a folate-free purified diet based on 17% amino acids as the nitrogen source. They were then folate repleted for 23 d with the amino acid diet supplemented with either 125, 250, 500, 1000 or 2000 µg folic acid/kg. Hematology, growth and tissue folate levels were measured in subsets of the rats when they were 24 (baseline), 52 (depleted) and 75 d old (repleted). The same measurements were made in control rats that had been fed 2 mg folic acid/kg of the amino acid diet for the same period of time. Our findings show that with repletion, growth of previously depleted rats is in direct proportion with the level of supplementation up to 1000 µg folic acid/kg diet. Serum folate levels of repleted rats also increased in proportion to supplementation between 500 to 2000 µg/kg diet, and liver folate levels increased proportionally with the level of supplement within the range of 125 to 2000 µg/kg diet. The 2000 µg/kg supplement was sufficient to restore liver folate levels equivalent to that of controls, but body weight and serum folate levels failed to catch up with that of controls in the 23-d repletion period. There was a nonlinear relationship between serum and liver folate levels: serum folate remained constant at about 6 µg/l as liver folate increased to about 7 µg/g, then serum folate diverged by increasing to 120 µg/l with only minor increases in liver folate. Because of the nonlinear relationship between serum and liver folate levels and the greater expense and complexity of measuring tissue folate, growth may be the most reliable as well as the simplest measurement criteria. Data suggest that a rat assay system using a folate-free diet, based on 17% amino acids as the nitrogen source, is a reliable system for assessing relative bioavailability of food folates. Under the standardized conditions reported, growth is a sensitive, reliable and practical response indicator for measuring depletion and repletion with folate.

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