134 S. Ezaki et al. present study was conducted to elucidate the significance of breastfeeding with a view toward preventing oxidative stress. Thus far there have been no reports on long-term total antioxidant capacity (TAC) in breast milk. A new method to measure TAC was used to compare the AC in formula milk and breast milk and to clarify the relationship between AC in breast milk and the postnatal age in days when collected. Materials and Methods At our facility, we put breast milk provided by mothers for their babies into breast milk packs and freeze it at −20°C. Later, when an opportunity to give it to the babies comes, we slowly thaw the breast milk at 37°C and give it to the babies as nourishment. During the current study, we used thawed breast milk instead of fresh breast milk to measure the degree of AC supplied to babies. Since breast milk is an important source of neonatal nutrition, we used the excess portion of breast milk in the packs to ensure they have the necessary amount of milk, milk that would normally be destroyed. We obtained consent from the parents before thawing the milk, and took measurements. All samples were taken from breast milk that was thawed within 48 hours after being frozen following collection. Extraction of defatted milk Before taking measurements, we prepared defatted milk through a double centrifugal process. The breast milk was centrifuged at low speed (500 g for 25 min) to settle out the suspended cellular components. Next, the cellular compo- nents were removed and the decelled milk was centrifuged at high speed (5000 g for 30 min) to separate it into defatted milk and fat content. The defatted milk was used for taking the measurements. The same method was used to prepare defatted milk using two types of formula milk having a composition similar to the breast milk of Japanese women [18–20], and the measurements taken (Table 1). Mesurements of TAC TAC levels were determined using the biological anti- oxidant potential (BAP) test, which is based on the ability of a colored solution, containing a source of ferric (Fe3+) ions adequately bound to a special chromogenic substrate, to decolour when Fe3+ ions are reduced to ferrous ions (Fe2+), as it occurs by adding a reducing/antioxidant system [21]. Practically, 50 µ L of ferric chloride reagent are transferred into the cuvette containing the thiocyanate derivative reagent. The resulting colored solution, after gently mixing by inversion, undergoes 550 nm photometric reading. Then, 10 µ L of sample milk are added in the same cuvette and the obtained solution is gently mixed and incubated into the thermostatic block for 5 min at 37°C. After incubation, the sample can be read for absorbance of 550 nm. BAP test, by exploiting the same chemical principle of the well-known ferric reducing ability of plasma (FRAP) test—i. e. the reduction of ferric to ferrous ions—provides a reliable measure of biological antioxidant potential of blood plasma and food [22, 23]. Statistical analysis An unpaired t test was used for comparison of numerical data between the two groups. All p values were based on two-sided tests. Statistics were compiled using SPSS10.0J (SPSS Inc, Chicago, IL) and Graph Pad Prism 4 (Graph Pad Software, Inc., San Diego, CA). Simple linear regression analysis was used to determine the correlation between different postnatal ages in days and TAC. Results The study was performed on 56 samples of breast milk collected from mothers of premature infants born with a mean ± SD gestational age of 33 ± 4 weeks (range: 24–37 weeks) that was collected at postnatal age 39 ± 43 days (range: age 4–145 days). The compositions of formula milk A and formula milk B are shown in Table 1. No significant difference was observed (p = 0.51) between the TAC in formula milk A Table 1. Exogenous antioxidants in breast and formula milk in Japan Type of milk Day after delivery (days) Cu (µ g/dl) Zn (µ g/dl) Vitamin A (µ g/100 ml) β-carotene (µ g/100 ml) α-tocopherol (µ g/100 ml) Vitamin C (mg/100 ml) 3–5 45.1 487 192 23 1.2 7.4 6–10 42 411 145 17 0.9 6.3 44.5 352 109 6 0.5 7.5 Breast milk 38.3 280 77 4 0.4 7.1 11–15 16–30 31–60 61–120 121–240 35.4 207 76 3 0.4 6.7 28.6 125 62 4 0.4 6 25.3 80.9 64 5 0.4 5.7 Formula milk A 43 410 53 9.5 0.84 6.8 B 41 340 59 5.2 0.51 6.2 J. Clin. Biochem. Nutr.