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Aim To determine the concentration of vitamin K including K1 (phylloquinone) and K2 (menaquinone-n; MK-n) in the maternal milk of Japanese women. Methods We collected human milk samples from more than 4000 mothers living throughout Japan from December 1998 to September 1999, and analysed the contents of vitamin K1 and K2 in 834 of the samples. We defined as group A the 555 samples among them that met the following conditions: breast milk of mothers who were under 40 y old, not in the habit of smoking and/or using vitamin supplements, and whose babies showed no symptoms of atopy and whose birthweights were 2.5 kg or more. Vitamins extracted from the enzymatic hydrolysates of the human milk were purified with a Sep-Pak Plus silica cartridge, and then measured by a method based on high-performance liquid chromatography (HPLC) combined with coulometric reduction and fluorometric detection. Results The mean concentration of vitamin K (K1 + K2) of mothers of group A and all groups were 0.434 +/- 0.293 and 0.517 +/- 1.521 microg/100 ml (average +/- SD), respectively, and menaquinones containing 4, 6 and 7 isoprenoid residues could be detected in the milk samples. Vitamins K1 and MK-4 were found to be predominant in the milk samples, and the concentration of MK-4 in colostrum was higher than that of MK-4 in mature milk. We also found that the MK-7 concentration in the milk of mothers living in eastern Japan was higher than that of mothers living in western Japan. Conclusion The different features of vitamin K1 and MK-7 concentrations in the milk of Japanese women may be due to differences in dietary foods.

Colostrum, mature milk, and paired plasma samples were obtained from 10 postpartum women who had not been previously studied. The geometric mean concentration of IgG4 in colostrum (3.3 micrograms/ml) was similar to the mean concentration in mature milk (3.0 micrograms/ml). The arithmetic mean for the percent of IgG = IgG4 was 10.3 +/- 3.3% for colostrum, 10.3 +/- 3.1% for mature milk, 2.6 +/- 0.3% for early plasma, and 1.7 +/- 0.3% for later plasma. Local mammary production of immunoglobulin was determined by subtracting the estimated serum contribution from the measured concentration in colostrum or milk. Evidence for local mammary production of IgG4 was found in 5 of 10 colostrum samples and 8 of 10 mature milk samples. These observations indicate that the previously observed selective enrichment of IgG4 in colostrum is also true for mature milk. These are the first studies suggesting continued local production of any immunoglobulin other than IgA in mature human breast milk.

Selenium in human milk sampled at different intervals after parturition was analyzed on two occasions five years apart. The selenium content of colostrum and transitional milk was higher than that of mature milk. Mature milk sampled in 1983 contained more selenium than mature milk sampled in 1978 (11.9 (0.8) vs. 9.4 (0.5) micrograms/l, mean (SD), p less than 0.01). The selenium and protein content in milk were positively correlated, which may be explained by the occurrence of seleno-amino acids in proteins. The selenium content was not correlated to that of tocopherol or of highly unsaturated fatty acids in mature milk. The values for the selenium content in human milk found here are lower than those previously reported from other countries.

The concentration of epidermal growth factor (EGF) was determined by radioimmunoassay in colostral and mature human milk. The content of EGF in colostral milk was 25-38 ng/ml compared to 5.2-11.5 ng/ml in samples of mature milk. The radioimmunoassayable EGF content in mature milk was not influenced by pasteurization and EGF was detected in a preparation of human milk protein at a concentration similar to that found in mature milk. The growth promoting activity in human milk measured by 3H-thymidin incorporation in human fibroblasts was higher in colostral milk (stimulated incorporation 8.7.fold) than in mature milk (stimulated incorporation about 7-fold).

Breast milk samples obtained from 74 women, at different stages of lactation, were analysed for leukocyte concentration and their bactericidal activity. The total leukocyte count in colostrum was 5 000/mm3 a concentration which is similar to that in circulation. As lactation became established, there was a drop in leukocyte concentration and the average count in mature milk was around 2 000/mm3 after the first 3 months. Polymorphs and mononuclear leukocytes together constituted 90--95% of the cells and the rest were lymphocytes. Bactericidal activity of milk leukocytes was similar to that of circulating leukocytes, irrespective of the stage of lactation. These results indicate that breast milk is rich in living leukocytes and that these cells may have a protective role against infection in the infant. The total leukocyte concentration as well as their bactericidal capacity were similar in well nourished and undernourished women suggesting that the protective factors in milk are not influenced by nutritional status of the mother.

The concentration of tri-iodothyronine (T3) and thyroxine (T4) in human milk was determined by radioimmunoassay (RIA). The analysis of T3 was performed on unextracted milk and on ethanol extracts of defatted milk. Analysis of unextracted milk was complicated by artifacts. Reliable and reproducible results were achieved only with the milk extracts. In 10 colostral milk samples the mean T3 levels +/- SD were 0.80 +/- 0.52 nmol/l before feeding (early milk) and 0.93 +/- 0.62 after feeding (hind milk). The T3 concentration in colostrum did not change significantly during the feeding to the infant. In 12 mature milk samples collected between infant feedings, the mean T3 +/- SD was 1.19 +/- 0.42 nmol/l. T4 was not detected in any of the samples analysed (detection limit 3 nmol/l).