Hirai Y, Kawakata N, Satoh K, Ikeda Y, Hisayasu S, Orimo H, Yoshino Y
Department of Biochemistry, Nippon Medical School, Tokyo, Japan.
J Nutr Sci Vitaminol (Tokyo). 1990 Dec;36(6):531-44. doi: 10.3177/jnsv.36.531.
Lactoferrin (LF) was isolated from human milk by serial procedures of 45% ammonium sulfate precipitation, CM Sephadex C-50 ion-exchange chromatography, Sephadex G-100 gel filtration, and Cu-affinity chromatography, in which 59Fe lactoferrin was used as the tracer. The recovery of LF from human milk was 3.3%. LF from human milk was a single component having a molecular weight of 78k on SDS-PAGE, and showed pI 8.02 by isoelectric focusing on slab gel. The LF concentration was measured by rocket immunoelectrophoretic assay using anti-human LF antiserum in human colostrum and milk, from 1 to 60 days after parturition (125 samples). The LF concentrations in colostrum (1-3 days of puerperium, n = 35), the transitional milk (4-7 days, n = 60), and mature milk (20-60 days, n = 30) were 6.7 +/- 0.7, 3.7 +/- 0.1, and 2.6 +/- 0.4 (mean +/- SEM) g/liter, respectively. Both the LF and total protein (TP) concentrations showed significantly inverse correlations with the days after parturition (p less than 0.001). The lactoferrin/total protein ratio (LF/TP) in the mature milk (16.1 +/- 1.4%) was significantly less than that in the colostrum (20.4 +/- 1.2%, p less than 0.05) and the transitional milk (21.4 +/- 0.9%, p less than 0.05). Furthermore, iron concentration (Fe) in human milk was also measured by the internal standard technique of the spiked method on atomic absorption, and the lactoferrin iron saturation (LS%) was calculated. Neither Fe nor LS% had significant difference among these three stages of the lactation. The means (n = 125) of Fe and LS% were 60.6 +/- 5.4 micrograms/100 ml and 11.8 +/- 1.1%, respectively. However, significant correlation was observed between LF and Fe (p less than 0.005) or between LF/TP and both of Fe (p less than 0.05) and TP (p less than 0.001) in the mature milk. These results suggest that the mechanism stimulating the synthesis and secretion of LF is different from those of other proteins and LF can play variable roles in iron nutrition of babies at the different stages of lactation.
通过45%硫酸铵沉淀、CM Sephadex C - 50离子交换色谱、Sephadex G - 100凝胶过滤和铜亲和色谱等一系列步骤，从人乳中分离出乳铁蛋白（LF），其中使用59Fe乳铁蛋白作为示踪剂。人乳中LF的回收率为3.3%。人乳中的LF在SDS - PAGE上是分子量为78k的单一成分，通过平板凝胶等电聚焦显示其pI为8.02。使用抗人LF抗血清，通过火箭免疫电泳法测定产后1至60天（125个样本）人初乳和乳汁中的LF浓度。初乳（产后1 - 3天，n = 35）、过渡乳（4 - 7天，n = 60）和成熟乳（20 - 60天，n = 30）中的LF浓度分别为6.7±0.7、3.7±0.1和2.6±0.4（平均值±标准误）g/升。LF和总蛋白（TP）浓度均与产后天数呈显著负相关（p＜0.001）。成熟乳中的乳铁蛋白/总蛋白比值（LF/TP）（16.1±1.4%）显著低于初乳（20.4±1.2%，p＜0.05）和过渡乳（21.4±0.9%，p＜0.05）。此外，采用原子吸收加标法的内标技术测定人乳中的铁浓度（Fe），并计算乳铁蛋白铁饱和度（LS%）。在哺乳期的这三个阶段，Fe和LS%均无显著差异。Fe和LS%的平均值（n = 125）分别为60.6±5.4微克/100毫升和11.8±1.1%。然而，在成熟乳中，观察到LF与Fe之间存在显著相关性（p＜0.005），LF/TP与Fe（p＜0.05）和TP（p＜0.001）之间也存在显著相关性。这些结果表明，刺激LF合成和分泌的机制与其他蛋白质不同，并且LF在哺乳期不同阶段对婴儿的铁营养可发挥不同作用。
