Nutrition for Health Folate Requirement of Growing Kittens to Prevent Elevated Formiminoglutamic Acid Excretion Following Histidine Loading 1 , 2

Folate is a generic descriptor referring to a group of related compounds of which folic acid is the simplest form. Folic acid is readily assimilated and converted to the active cofactors necessary for single-carbon metabolism and DNA synthesis. Folate has been shown to be an essential dietary component for growing kittens (Thenen and Rasmussen 1978). Because the folate requirement of growing kittens had not been experimentally determined, the National Research Council (1986) suggested a tentative requirement of 0.8 mg/kg diet based on data from swine. In this study, we determined the minimal folate requirement of growing kittens based on hematological parameters and formiminoglutamic acid excretion after a histidine load. Materials and methods. The experimental protocols adhered NIH guidelines (NRC 1985) and were approved by the Animal Use and Care Administrative Advisory Committee of the University of California, Davis. Animals and housing. Specific-pathogen–free domestic short-haired kittens (initial mean body weight 1.33 6 0.03 kg) from the Feline Nutrition and Pet Care Center of the University of California, Davis were used. Kittens were housed individually in stainless steel metabolism cages (60 3 60 3 60 cm) in rooms with a controlled temperature (21 6 2°C) and light cycle (lights on: 0600–2000 h). They had free access to the experimental diets and tap water. Diets. A gelatin-based purified diet supplemented with amino acids was used as a basal diet, the diet contained all nutrients in sufficient amounts to meet the requirements of growing kittens except for folate. Experimental diets were prepared by adding various amounts of folic acid (pteroylglutamic acid, Sigma Chemical, St. Louis, MO) to the basal diet at the expense of cornstarch. The folic acid concentrations of the experimental diets were 0.1, 0.15, 0.2, 0.3, 0.6, 0.9 and 1.2 mg/kg diet, which were confirmed by a Lactobacillus casei microbiological assay (Martin et al. 1990). Experimental design. Kittens were weaned at 8 wk of age and given the basal diet and tap water until 10 wk of age (wk 0 of the experiment) when they were randomly allocated into seven groups of eight kittens (4 males and 4 females) in each group. Each group was randomized to one of the seven experimental diets and maintained on the diet for 20 wk. Food intake was measured daily and body weight weekly. Blood samples were taken at wk 0 and 20 from the jugular vein of unanesthetized kittens with potassium EDTA as an anticoagulant. Plasma was prepared from the blood samples by centrifugation at 1100 3 g for 20 min. For the measurement of formiminoglutamic acid (Figlu), urine was collected for a period of 48 h from kittens given a histidine load (0.22 g L-histidine/kg body weight) administered by gastric tube at wk 0 and 20. Urine was collected into a plastic bottle containing 1 mL of 18 mol/L sulfuric acid (Fisher Scientific, Fair Lawn, NJ). Food was withheld overnight before the oral histidine load. A broken-line method (Robbins 1986, Robbins et al. 1979) was employed to estimate the minimum folate requirement of growing kittens. Sample analysis. Hematological parameters were measured using a blood cell counter (Mascot, CDC Technologies, Oxford, CT). A microbiological assay described by Tamura (1990) with L. casei (7469, American Type Culture Collection, Rockville, MD) and the Bio-Rad Model 2550 EIA 1 Presented as part of the Waltham International Symposium on Pet Nutrition and Health in the 21st Century, Orlando, FL, May 26–29, 1997. Guest editors for the symposium publication were Ivan Burger, Waltham Centre for Pet Nutrition, Leicestershire, UK and D’Ann Finley, University of California, Davis. 2 Supported by Waltham Centre for Pet Nutrition, Leicestershire, U.K. and a grant from the Center for Companion Animal Health, School of Veterinary Medicine, University of California, Davis. 3 To whom correspondence should be addressed. 4 Composition of the basal diet (g/kg): gelatin (150 Bloom Type A, Grayslake Gelatin, Grayslake, IL), 380; amino acid mixture, 40; sucrose, 100; animal fat (Florin Tallow, Dixon, CA), 300; starch 93 (Melojel, Bridgewater, NJ); cellulose, 20; mineral mixture (Williams et al. 1987), 50; vitamin mixture, 10; choline chloride, 4.7 and taurine, 2.5. The amino acid mixture (Ajinomoto U.S.A., Raleigh, NC) was composed of (g/kg diet): L-isoleucine, 4.64; L-leucine, 7.32; L-methionine, 7.04; L-phenylalanine, 3.12; L-threonine, 3.72; L-tryptophan, 2.32; L-valine, 4.24; L-histidine, 3.20 and L-lysine z HCl, 4.40. The vitamin mixture contained (mg/kg diet) retinyl palmitate, 40; cholecalciferol, 5; dl-a-tocopheryl acetate, 320; menadione, 15; thiamin mononitrate, 25; riboflavin, 10; pyridoxine z HCl, 10; nicotinic acid, 100; calcium pantothenate, 20; myo-inositol, 200; cyanocobalamin in mannitol, 0.05; D-biotin, 1; ascorbic acid, 400 (as a preservative). 5 Abbreviations used: Figlu, formiminoglutamic acid; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume.