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738 ODUHO ET AL. FIGURE 1 Chemical structure of menadione nicotinamide bisulfite. MATERIALS AND METHODS Animals and diets. All experiments were con ducted using male chicks from the cross of New Hampshire males and Columbian females (University of Illinois Poultry Farm). On d 8 posthatching, after food was withheld overnight, chicks were weighed and allotted to experimental groups so that each group had a similar average initial weight and weight distribution. Diets and water were freely available. Chicks were kept on a 24-h constant light schedule in heated, thermostatically controlled batteries with raised wired floors. All procedures were approved by the University of Illinois Committee on Laboratory Animal Care. Vitamin K activity. Assay 1 was conducted to determine the vitamin K (menadione) bioactivity of MNB (45.7% menadione, 32% nicotinamide, Vanetta SPA, Milano, Italy) when compared with MPB (45.5% menadione, Vanetta SPA). From hatching to d 7 posthatching, chicks were fed a vitamin K-deficient soybean concentrate diet (Table 1) estimated to contain no more than 30 /Â¿gvitamin K/kg. On d 8 posthatching, duplicate groups of four male chicks were assigned to the dietary treatments. Treatments consisted of graded levels (0, 5, 25, 50 and 400 /Â¿g/kg) menadione from MPB or MNB, which were added to the vitamin K-deficient diet (Table 1) at the expense of cornstarch. The NRC (1984) estimate of the dietary vitamin K requirement of young chicks is 500 Mg/kg. On d 22 posthatching, following the 14-d feeding period, chicks were weighed and their feed intake recorded, after which 1 mL of blood was drawn from each chick by heart puncture. Blood from all chicks in each pen was pooled and plasma was prepared. Plasma prothrombin time was determined by the method of Quick (1957), using sodium citrate as an anticoagulant and acetone-dehydrated chicken brain powder as a source of thromboplastin, prepared in our laboratory using methodology described by Griminger (1962 and 1965). Ingredient1 TABLE 1 Composition of vitamin K and niacin assay diets Cornstarch to Dextrose Corn Corn gluten meal (60% protein) Soybean protein concentrate (65% protein) Sucrose Solka floe Vitamin-K assay diet2 100.00 35.40 20.00 g/100 g 3.00 Niacin assay diet3 to 100.00 40.00 20.00 Vitamin-free casein (84% protein) 6.50 Corn oil 4.00 5.00 Mineral premix4 5.37 5.37 Vitamin premix (K or niacin-free)5 0.20 0.20 DL-choline chloride 0.20 0.20 all-rac-a-tocopheryl acetate (20 mg/kg) + + Ethoxyquin (125 mg/kg) + + Sulfathiazole 0.20 Amino acid premix6 1.60 DL-Methionine 0.30 1Cornstarch and dextrose obtained from A. E. Staley, Decatur, IL; corn gluten meal (60% protein) and soybean protein concentrate (65% protein) from Archer Daniel Midland, Decatur, IL; sucrose from Holly Sugar, Colorado Springs, CO; solka floe from James River, Berlin, NH; vitamin-free casein from U.S. Biochemicals, Cleveland, OH. Calculated analysis: 23% crude protein, 14.6 MJ/kg metabolizable energy and
niacin bioactivity of MNB when compared with nicotinamide (U.S. Biochemicals, Cleveland, OH). On d 8 posthatching, quadruplicate groups of four male chicks were assigned to the following dietary treat ments: 0, 2.5, 5.0 and 30 mg/kg nicotinamide (the latter a positive control) or 0, 2.5 and 5.0 mg/kg nicotinamide from MNB. The NRC (1984) estimate of the dietary nicotinic acid requirement of young chicks is 27 mg/kg. On d 22 posthatching, chicks and feed were weighed, and chick weight gain, feed intake and feed efficiency were determined. Chicks that had been fed 0 and 5.0 mg/kg nicotinamide (from both MNB and nicotinamide) were killed by cervical dislo cation at d 22 posthatching, and portions (0.25 g) of liver tissue were removed and pooled by replicate. Liver samples were homogenized and heated in a boiling water bath for 5 min. Hepatic pyridine nucleotides (NAD + NADH) were then determined by the enzyme-cycling (1969). procedure of Nisselbaum and Green Toxicity assays. Assay 3 was conducted to de termine the toxic effects of an acute oral (crop intu bation) dose of menadione from MPB (as a standard) or MNB. Preliminary work in our laboratory had es tablished that doses of MNB or MPB providing mena dione at 500 mg/kg body weight or less caused neither mortality nor morbidity. Assay 3 was therefore designed to provide logarithmic doses of menadione, starting at 100 mg/kg and working up to 1600 mg/kg body weight. Following dosing at d 8 posthatching, triplicate groups of four chicks at each dose level were fed a 23% protein corn soybean meal starter diet5 for a 14-d observation period. Assay 4 was a 14-d chronic toxicity study involving triplicate groups of four chicks at each menadione dose level (0, 30, 300, 3000 and 6000 mg/kg diet) from MPB or MNB. Additions were made to the cornsoybean meal starter diet. At termination of the assay on d 22 posthatching, blood was obtained by heart puncture after which 1-mL portions from each chick in a pen were pooled, resulting in three pooled samples for each of the nine treatments. Blood hemo globin was determined by the method of Crosby et al. (1954). Statistical analyses. Common-intercept multiple linear regression was conducted for data in Assays 1 and 2 using the General Linear Model procedure of SAS (1985). Data were first fitted to simple linear regression equations to establish that Y-intercepts were not different (P > 0.05) after which the multiplelinear regression model was fitted menadione biopotency assessment to the data. For in Assay 1, prothrombin time (s) was regressed on supplemental menadione intake (/Â¿g); for nicotinamide biopotency, weight gain (g) was regressed on supplemental nicotinamide intake (mg). The ratio of slopes (MNB: MPB) gave estimates of menadione or nicotinamide potency for MNB relative to MPB. MENADIONE NICOTINAMIDE BISULFITE 739 TABLE 2 Prothrombin time of chicks fed diets with graded levels of menadione from menadione dimethylpyrimidinol bisulfite (MPB) or menadione nicotinamide bisulfite (MNB) (Assay I)1 menadioneUS/kg052550400ProthrombinMPBs3528252519time3MNB3533272417 'Values are means of duplicate groups of four male chicks at d 22 posthatching; average initial weight was 76 g. The basal vitamin K assay diet (Table 1) was estimated to contain 0.05) and this indicated that MPB and MNB were equally efficacious in furnishing bioavailable menadione. Nicotinamide activity. Addition of graded doses of nicotinamide or MNB below the niacin requirement yielded linear (P < 0.01) responses in weight gain, feed 5Contained (g/100 g): corn, 52.8; dehulled soybean meal, 37.0; alfalfa meal, 1.0; menhaden fish meal, 2.0; corn oil, 4.0; dicalcium phosphate, 2.20; limestone, 1.00; DL-methionine, 0.20; NaCl, 0.40; trace mineral premix (Mn, FÃ©,Zn, Cu, I, Se), 0.15; flavomycin premix (4.4% flavomycin), 0.05; vitamin premix (retinyl acetate, cholecalciferol, all-rac-a-tocopheryl acetate, menadione sodium bi sulfite, vitamin B-12, niacin, D-Ca-pantothenate, riboflavin), 0.10; choline chloride (60% choline), 0.10. Downloaded from jn.nutrition.org by guest on April 2, 2013
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