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Eighty female Sprague-Dawley rats were fed a semisynthetic diet during gravidity which was supplemented with 5 mg vitamin B6 per kg diet. The daily food intake was 14 g. During the following lactation the rats were assigned to one of 10 vitamin B6 treatment groups (0, 3, 6, 9, 12, 15, 18, 36, 360 and 3,600 mg per kg diet). The feed was given ad libitum. At day 14 of lactation the rats were decapitated. Parameters for determination of the vitamin B6 status were concentration of pyridoxine, pyridoxal and pyridoxamine in liver and body analyzed by using HPLC. Body was defined without the gastroenteral tract that was divided into carcass (extrahepatic compartments without liver) and total body (extrahepatic compartments plus liver). The mean weight of liver was 13 g with a dry mass of 33%; there was no difference between the treatment groups. The vitamin B6 concentration was lowest in rats fed 0 mg vitamin B6/kg diet (5 micrograms/g fresh matter, FM) and highest in the rats fed 3600 mg vitamin B6/kg diet (10.9 micrograms/g FM). The total vitamin B6 consisted on the average of 38% pyridoxal and 62% pyridoxamine. This was only changed significantly at the highest supplementation level, where 20% pyridoxine were detected instead of pyridoxamine. The mean weight of carcass averaged 212 g at a dry matter content of 31%. The vitamin B6 concentration ranged in the treatment groups from 0 mg to 360 mg vitamin B6/kg diet between 2.1 micrograms/g FM and 2.8 micrograms/g FM. It was highest in the 3600 mg vitamin B6 treatment group at 7.5 micrograms/g FM. The total vitamin B6 consisted of 63% pyridoxal and 37% pyridoxamine. It was only significantly affected in the 3600 mg vitamin B6 treatment group, where also pyridoxine could be found in the amount of 56%. The results indicate that alimentary vitamin B6 supply had more influence on liver vitamin B6 concentration than on carcass concentration. Total body concentration is very similar carcass concentration, as 95% of vitamin B6 is located there. The suitability of the parameters by the evaluation of the vitamin B6 requirement was confirmed the comparison of two statistical methods. It is concluded that a vitamin B6 supply of 5 to 6 mg/kg diet is necessary to meet the requirements during lactation.

Simple methods of determining the water-soluble vitamins B1, B2, B6 and B12 in milk by HPLC are described. Compared to existing procedures, the following improvements can be realized. The oxidation of vitamin B1 to thiochrome is stopped by the addition of sodium sulphite. This step significantly increases repeatability. Thiochrome is then extracted with butan-1-ol, which results in fewer co-extracts and greater selectivity. After the hydrolysis of the 5-phosphates of the vitamin B6 (pyridoxine, pyridoxal and pyridoxamine), these three vitamers are determined by isocratic HPLC as DDS-ion-pairs and with fluorimetric detection. As only microbiological methods have so far been used for the determination of minute quantities of vitamin B12 in milk, a new HPLC procedure is proposed with a detection limit of 0.2 micrograms vitamin B12/L milk.