Determination of hyaluronan molecular mass distribution in human breast milk

Han Yuan, Ripal Amin, Xin Ye, Carol A. De La Motte, Mary K. Cowman

Research output: Contribution to journalArticlepeer-review


Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M ≤ 20 kDa, M ∼ 20 to 60 kDa, and M ∼ 60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M ≥ 110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.

Original languageEnglish (US)
Pages (from-to)78-88
Number of pages11
JournalAnalytical Biochemistry
StatePublished - Apr 1 2015


  • Electrophoresis
  • Hyaluronan
  • Ion exchange
  • Milk
  • Molecular mass
  • Quantification

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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