Exploring Minor Proteins and Peptides in Human Milk: a Proteomic Analysis Across Lactation Stages

NCT06787963 | Recruiting DMC

• 1 other identifier: PROTEOM-MILK
• Study Type: observational
• Target: 150
• Locations: 1 country
• Sites: 2

Brief Summary

Human milk (HM) is the optimal food source for the nutrition, growth, and development of newborns. The protein fraction of HM plays a crucial role in the healthy development of infants. HM contains a wide variety of minor whey proteins and peptides with important bioactive functions, many of which are still unknown. Proteomics allows for the study of biological samples with inherently complex protein mixtures. Proteins are essential for the development of living organisms, both in quantitative and qualitative terms. The combination of proteomic techniques currently enables the study of protein variability and minor peptides in HM across different lactation stages and allows for differential quantification according to gestational age and birth weight. However, studies on the human milk serum proteome during these stages are limited. The aim is to explore the minor whey proteins and peptides in human milk through a longitudinal analysis of five groups of breastfeeding mothers (with 30 extremely low birth weight newborns, 30 very low birth weight newborns, 30 low birth weight newborns, 30 adequate birth weight newborns, and 30 high birth weight newborns). Gestational age will also be considered to ensure homogeneous group distribution according to this condition. HM samples will be collected from each mother during three lactation periods after birth: within the first 48 hours (colostrum), at 5-14 days (transitional milk), and at 100-120 days (mature milk) for the five birth weight groups. In these neonatal/infant groups, minor proteins from whey fraction and peptides will be separated, quantified, and identified using label-free proteomic techniques. This study aims to expand our understanding of the minor proteins and peptides in human milk and their bioactive roles in neonatal health. By examining these components across different birth weight groups and lactation stages, the research will offer insights into how protein and peptide profiles vary by gestational age and birth weight, potentially influencing neonatal development. The findings from this proteomic analysis could not only demonstrate the complexity of human milk composition but also contribute to targeted nutritional support for preterm or low-birth-weight infants, customizing protein supplementation in HM banks and therefore enhancing their growth and developmental outcomes.

Conditions

Low Birthweight Infant; Preterm; Very Low Birth Weight Preterms

Keywords

low birth weight; prematurity; gestational age; human milk; bioactive peptides; proteomics; lactation; whey protein

Outcome Measures

Primary Outcomes (7)

• Minor whey proteins: Lysozyme

  Concentration of lysozyme in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).

  Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)

• Minor whey proteins: Lactoferrin

  Concentration of lactoferrin in mg/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).

  Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)

• Minor whey proteins: Bile salt-dependent lipase (BSDL)

  Concentration of bile salt-dependent lipase (BSDL) in µg/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).

  Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)

• Minor whey proteins: Lactoperoxidase

  Concentration of lactoperoxidase in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).

  Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)

• Minor whey proteins: Alpha-1-antitrypsin

  Concentration of alpha-1-antitrypsin in ng/ml will be measured using immunoassays (Enzyme-Linked ImmunoSorbent Assay).

  Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)

• Proteome

  Protein signatures will be assessed through a proteomic analysis using nano-liquid chromatography (nESI-MS/MS). The analysis will be followed by bioinformatic analysis with dedicated software to analyze and achieve the relative quantification of differential proteins. A specific database representing the human proteome, updated in the UNIPROT repository, will be used. The results will be compared against the protein databases available in international platforms such as SWISS-Prot, NCBI, EBI, and TrEMBL. The results are typically expressed in units or formats that reflect the relative abundance or quantitative values of proteins or peptides, such as spectral counts, percentage and µg/ml.

  Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)

• Free peptides, primarily derived from β-casein

  Free peptides will be expressed in counts per peptide. Human milk samples will undergo trypsin digestion prior to analysis by liquid chromatography-mass spectrometry (LC-MS).

  Human Milk collection after birth - Time 1: at 48 hours (colostrum),Time 2: at 5-14 days (transitional milk), Time 3: at 100-120 days (mature milk)

Secondary Outcomes (2)

• Muscular ecography

  After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days

• Transfontanellar ecography

  After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days

Other Outcomes (3)

• General information - Mothers

  After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days (mothers will be asked if any changes have occurred)

• General information - Newborns

  After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days (mothers will be asked if any changes have occurred)

• Dietary intake and eating habits

  After birth - Time 1: at 48 hours,Time 2: at 5-14 days, Time 3: at 100-120 days

Study Arms (5)

30 extremely low birth weight

extremely low birth weight (\< 1000 g)

30 very low birth weight

very low birth weight (1000-1499g)

30 low birth weight

low birth weight (1500-2499g)

30 adequate birth weight

adequate birth weight (2500-3999g)

30 high birth weight

high birth weight (4000g or \>)

Eligibility Criteria

• Age: 0 Days - 120 Days
• Sex: all
• Healthy Volunteers: Yes
• Age Groups: Child (0-17)
• Sampling Method: Non-Probability Sample

Study Population

Five groups of breastfeeding mothers: Group 1 (n=30) - with neonates of extremely low birth weight; Group 2 (n=30) - with neonates of very low birth weight; Group 3 (n=30) - with neonates of low birth weight; Group 4 (n=30) - with neonates of adequate birth weight; Group 5 (n=30) - with neonates of high birth weight. Additionally, gestational age will be considered to achieve a homogeneous distribution of the groups according to this condition.

You may qualify if:

• Healthy mothers
• With monitored pregnancies within the care area of the Reina Sofía University Hospital in Córdoba
• With newborns expected to be exclusively breastfed until 4 months

You may not qualify if:

• Mothers whose newborns have any of the following conditions: congenital malformation, chromosomal abnormality, hypoxia-ischemia, gastroschisis, polycythemia, hypoglycemia, sepsis, blood incompatibility
• Pathological pregnancy, pregnancy by in vitro fertilization, or multiple pregnancies
• With no plan to exclusively breastfeed until 4 months
• Under medical treatment
• Have a drug addiction
• Refuse informed consent
• Have had previous breast surgery
• Live outside the metropolitan area

Sponsors & Collaborators

• Maimónides Biomedical Research Institute of Córdoba: lead
• Hospital Universitario Reina Sofia de Cordoba: collaborator
• Universidad de Granada: collaborator
• Universidad de Córdoba: collaborator

Study Sites (2)

Hospital Universitario Reina Sofía

Córdoba, Córdoba, 14004, Spain

RECRUITING

Maimonides Biomedical Research Institute of Cordoba (IMIBIC)

Córdoba, Córdoba, 14004, Spain

RECRUITING

Biospecimen

Retention: SAMPLES WITHOUT DNA

Human milk samples

MeSH Terms

Conditions

Premature Birth; Breast Feeding

Condition Hierarchy (Ancestors)

Obstetric Labor, Premature; Obstetric Labor Complications; Pregnancy Complications; Female Urogenital Diseases and Pregnancy Complications; Urogenital Diseases; Feeding Behavior; Behavior

Study Officials

• José Luis Gómez-Chaparro Moreno, MD, Ph.D

  Maimonides Biomedical Research Institute of Cordoba (IMIBIC)

  PRINCIPAL INVESTIGATOR

Central Study Contacts

José Luis Gómez-Chaparro Moreno, MD, Ph.D

CONTACT

+34957736467; drgomezchaparro@gmail.com

Ángel Gil, Professor

CONTACT

agil@ugr.es

Study Design

• Study Type: observational
• Observational Model: COHORT
• Time Perspective: PROSPECTIVE
• Sponsor Type: OTHER
• Responsible Party: SPONSOR

Study Record Dates

• First Submitted: January 8, 2025
• First Posted: January 22, 2025
• Study Start: December 2, 2024
• Primary Completion: January 1, 2026
• Study Completion (Estimated): December 1, 2027
• Last Updated: January 22, 2025

Record last verified: 2025-01

Data Sharing

• IPD Sharing: Will not share

Locations

ES (2)