Chetta Katherine E, Newton Danforth A, Wagner Carol L, Baatz John E
Division of Neonatology, Department of Pediatrics, Darby Children's Research Institute, Medical University of South Carolina, Charleston, SC, United States.
Front Nutr. 2022 Jul 5;9:918872. doi: 10.3389/fnut.2022.918872. eCollection 2022.
Human milk, the best enteral selection for a preterm infant, becomes altered during freezing and soluble free fatty acid is generated over time. Free fatty acids may form complexes, such as the oleic acid-bound protein called HAMLET (human α-lactalbumin made lethal to tumor cells). We determined the biological activity of preterm human milk protein-oleic complexes (HAMLET-like complexes) and tested the hypothesis that laboratory-synthesized HAMLET exhibits cytotoxicity in human immature epithelial intestinal cell culture. Thirty-four milk samples from 15 mothers of hospitalized preterm infants were donated over time. Milk fractions were tested repeatedly for FHs 74 Int and HIEC-6 fetal cell cytotoxicity, using a sensitive viability assay. Protein and fatty acid identities were confirmed by Western blot, high performance liquid chromatography, and mass spectrometry. Cytotoxicity of intestinal cells exposed to milk increased respective to milk storage time ( < 0.001) and was associated with free oleic acid ( = 0.009). Synthesized HAMLET was cytotoxic in cultures of both lines. Preterm milk samples killed most cells in culture after an average 54 days in frozen storage (95% C.I. 34-72 days). After prolonged storage time, preterm milk and HAMLET showed a degree of cytotoxicity to immature intestinal cells in culture.
母乳是早产儿最佳的肠内营养选择，但在冷冻过程中其成分会发生变化，随着时间推移会产生可溶性游离脂肪酸。游离脂肪酸可能会形成复合物，比如与油酸结合的蛋白质，即人α-乳白蛋白制成的肿瘤细胞致死因子（HAMLET）。我们测定了早产母乳蛋白 - 油酸复合物（类HAMLET复合物）的生物活性，并验证了实验室合成的HAMLET在人未成熟肠上皮细胞培养中具有细胞毒性这一假设。随着时间推移，我们从15位住院早产儿母亲那里收集了34份母乳样本。使用灵敏的活力测定法，对母乳组分反复进行FHs 74 Int和HIEC - 6胎儿细胞的细胞毒性测试。通过蛋白质印迹法、高效液相色谱法和质谱法确认蛋白质和脂肪酸的特性。暴露于母乳的肠细胞的细胞毒性随着母乳储存时间的增加而增加（<0.001），并且与游离油酸有关（=0.009）。合成的HAMLET在两种细胞系的培养中均具有细胞毒性。早产母乳样本在冷冻储存平均54天后（95%置信区间34 - 72天）杀死了培养中的大多数细胞。在延长储存时间后，早产母乳和HAMLET对培养中的未成熟肠细胞均表现出一定程度的细胞毒性。
