Cu,Zn- and Mn-Superoxide Dismutase Concentration in Human Colostrum and Mature Milk

Abstract

Superoxide dismutase (SOD, BC 1.15.1.1) catalyses the dismutation of the superoxide anion and is a metalloenzyme ubiquitous to living organisms. The enzyme is also contained in bovine milk and has been suggsted to diminish the oxidative problems associated with the superoxide anion. The present study is an attempt to determine the concentration of SOD in human colostrum and in mature milk to make a comparison with the values in bovine milk and human blood. Human colostrum and mature milk samples were respectively collected from 15 and 12 healthy lactating women at Machida Municipal Hospital and those living in Machida-shi. The samples immediately cooled in a refrigerator (at less than 5°C), skimmed by centrifugation at 1500 x 9 for 20 min and stored at - 80°C until being assayed. The total SOD activitiy of the milk was assayed in the absence of KCN, and the Mn-SOD activity in the presence of 1 mM KCN by the xanthine oxidase (XO)/nitrate method. CU,Zn-SOD activity is represented by subtracting the Mn-SOD activity from the total SOD activity. A standard curve was prepared from human SOD (Sigma Co.). One unit (NU) is defined as the amount that caused a 50% inhibition of blank autoxidation (ICso) in the XO/nitrate or cytochrome c assay. The ICsovalues for SOD by these assays were 0.1 and 0.19 j.tg/ml, and the specific activity was 3333 NU and 1754CU/mg of the protein, respectively. The concentration of SOD in human milk was obtained by measuring the volume of milk required to give IC 50 in this system. The SOD activity in milk during storage in the refrigerator (at less than 5°C) decreased rapidly with storaged time and fell to 40% of that of fresh skim milk after 5 days. Nagasawa et al. have reported that the disc gel electrophoretic patterns of human milk proteins changed during the storage of milk at about 5°C. The reduced SOD activity may have been casuded by the effect of other components or by the attack of original enzymes such as protease. In the milk kept in the freezer (-80°C) for 10 days, the SOD activity did not change in comparison with that of fresh skim milk. The concentrations of CU,Zn- and Mn-SOD in human milk are shown in Tables I and II. The average concentrations of total SOD in colostrum and mature milk were 1.25 (4.2NU, 2.2CU) and 1.42 (4.7NU, 2.5CU) j.tg/ml, respectively. Oyanagui has observed 22 NU of total SOD/ml in human blood plasma, the total SOD in human milk being approximately 5 times lower than that in human blood plasma. Holbrook and Hicks have reported that bovine milk contained 1.1 CU of SOD/ml by an XO/cytochrome c assay, which is approximately 100 times lower than that in bovine blood. It is apparent that the SOD concentration of human milk is 2.0 to 2.3 times higher than that of bovine milk. The average concentrations of CU,Zn- and Mn-SOD in colostrum were 0.14 and 1.11 j.tg/ml, and in mature milk 0.43 and 0.99 j.tg/ml, respectively. The percentages of Mn-SOD to total SOD in colostrum and mature milk were 88.8 and 69.7%, respectively. According to the report of Oyanagui, human blood plasma contained 12.3 NU (55.9% of total SOD) of CU,Zn­ SOD and 9.7NU (44.1%) of Mn-SOD/ml. Therefore, the proportion of Mn-SOD in human milk is higher than that in human blood. The concen­ tration of SOD in bovine milk has been reported by several investigators. Asada has suggested that cyanide-insensitive Fe- or Mn-SOD was not found in bovine skim milk. Korycka-Dahl et al. have reported that partially-purified SOD from bovine milk serum was identical with Cu,Zn­ SOD from bovine erythrocytes in their electrophoretic properties. According to their observations, no bands of Mn-SOD were observed on the polyacrylamide gels. However, the presence of trace amounts of Mn