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Free fatty acid and α‐lactalbumin‐oleic acid complexes in preterm human milk are cytotoxic to fetal intestinal cells in vitro

ABSTRACT: 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 in vitro 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 (p<0.001) and was associated with free oleic acid (p=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. Protein was reduced 221 in 1 mM dithiothreitol and alkylated in 5.5 mM

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Perinatal Necrotizing Enterocolitis

SUBMITTER: Jennifer Bethard

LAB HEAD: Katherine Chetta

PROVIDER: PXD034652 | Pride | 2022-08-12

REPOSITORIES: Pride

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Publications

Free Fatty Acid and α-Lactalbumin-Oleic Acid Complexes in Preterm Human Milk Are Cytotoxic to Fetal Intestinal Cells in vitro.

Chetta Katherine E KE Newton Danforth A DA Wagner Carol L CL Baatz John E JE

Frontiers in nutrition 20220705

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 in vitro biological activity of preterm human milk protein-oleic complexes (HAMLET-like complexes) and tested the hypothesis that laboratory-synthesized HAMLET exhibits cytotoxicity in human i ...[more]

PMID: 35866080

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Project description:The GOLDN study was initiated to assess how genetic factors interact with environmental (diet and drug) interventions to influence blood levels of triglycerides and other atherogenic lipid species and inflammation markers (registered at <a href="http://clinicaltrials.gov/ct2/show/NCT00083369">clinicaltrails.gov</a>, number NCT00083369). The study recruited Caucasian participants primarily from three-generational pedigrees from two NHLBI Family Heart Study (FHS) field centers (Minneapolis, MN and Salt Lake City, UT). Only families with at least two siblings were recruited and only participants who did not take lipid-lowering agents (pharmaceuticals or nutraceuticals) for at least 4 weeks prior to the initial visit were included. A total of 1048 GOLDN participants were included in the diet intervention. The diet intervention followed the protocol of Patsch et al. (<a href="http://www.ncbi.nlm.nih.gov/pubmed/1420093">1992</a>). The whipping cream (83% fat) meal had 700 Calories/m2 body surface area (2.93 MJ/m2 body surface area): 3% of calories were derived from protein (instant nonfat dry milk) and 14% from carbohydrate (sugar). The ratio of polyunsaturated to saturated fat was 0.06 and the cholesterol content of the average meal was 240 mg. The mixture was blended with ice and flavorings. Blood samples were drawn immediately before (fasting) and at 3.5 and 6 hours after consuming the high-fat meal. For the GOLDN lipidomics study, sterols and fatty acids were measured from stored plasma (-80 degrees Celsius) collected at fasting and 3.5 hours after the diet intervention using TrueMass Panels from Lipomics (West Sacramento, CA). A total of 11 sterols were quantified in nmols/gram of sample including total cholesterol, 7-dehydrocholesterol, desmosterol, lanosterol, lathasterol, cholestanol, coprostanol, beta-sitosterol, campesterol, stigmasterol, and 7alpha-hydroxycholesterol. A total of 35 fatty acids were quantified in nmols/gram of sample inlcuding myristic acid (14:0); pentadecanoic acid (15:0); palmitic acid (16:0); stearic acid (18:0); arachidic acid (20:0); behenic acid (22:0); lignoceric acid (24:0); myristoleic acid (14:1n5); palmitoleic acid (16:1n7); palmitelaidic acid (t16:1n7); oleic acid (18:1n9); elaidic acid (t18:1n9); vaccenic acid (18:1n7); linoleic acid (18:2n6); gamma-linolenic acid (18:3n6); alpha-linolenic acid (18:3n3); stearidonic acid (18:4n3); eicosenoic acid (20:1n9); eicosadienoic acid (20:2n6); mead acid (20:3n9); di-homo-gamma-linolenic acid (20:3n6); arachidonic acid (20:4n6); eicsoatetraenoic acid (20:4n3); eicosapentaenoic acid (20:5n3); erucic acid (22:1n9); docosadienoic acid (22:2n6); adrenic acid (22:4n6); docosapentaenoic acid (22:5n6); docosapentaenoic acid (22:5n3); docosahexaenoic acid (22:6n3); nervonic acid (24:1n9); and plasmalogen derivatives of 16:0, 18:0, 18:1n9, and 18:1n7.

Dataset Information

Free fatty acid and α‐lactalbumin‐oleic acid complexes in preterm human milk are cytotoxic to fetal intestinal cells in vitro

Publications

Free Fatty Acid and α-Lactalbumin-Oleic Acid Complexes in Preterm Human Milk Are Cytotoxic to Fetal Intestinal Cells <i>in vitro</i>.

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