title: DETERMINATION OF CHOLESTEROL IN HUMAN MILK: AN ALTERNATIVE TO CHROMATOGRAPHIC METHODS.
abstract: BACKGROUND
  human milk (HM) is considered the best option for feeding healthy infants. Cholesterol (CHOL) is important for proper development of the nervous system, and for hormone and vitamin synthesis in growing infants. Breastfeeding and dietary CHOL intake during infancy have been suggested to affect blood lipid levels and the risk of cardiovascular disease in adulthood. Gas chromatography is the technique most widely used to determine CHOL in HM. Chromatographic methods are specific for the determination of CHOL and other sterols present in HM, but are extremely time consuming, and the costs and equipment requirements mean that they are not accessible to all laboratories.
  
  
  OBJECTIVE
  the present study describes the optimization and validation of an enzymatic-spectrophotometric method for CHOL determination in mature HM.
  
  
  METHODS
  determination of CHOL involves fat extraction with chloroform:methanol, hot saponification and extraction of the unsaponifiable fraction with diethyl ether. CHOL was determined by an enzymatic method in which the concentration of the lutidine dye formed is stoichiometric to the amount of CHOL, and is measured by the increase in light absorbance at 405 nm.
  
  
  RESULTS
  human milk fat (mg/mL) (27.5 ± 1.3) and CHOL (0.113 ± 0.004) in analyzed HM were within the ranges reported by others authors. Analytical parameters of the proposed method were assessed: The precision values (%) (expressed as the relative standard deviation (RSD)) were: 3.5 and 6.7 for intra- and inter-day, respectively. Accuracy, estimated by recovery assays, was 110 ± 1.6%.
  
  
  CONCLUSIONS
  the validated enzymatic-spectrophotometric method for determining CHOL in HM constitutes an alternative for fast and simple analysis of CHOL with equipment requirements accessible to any laboratory.
authors.affiliation: Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia (Valencia), Spain.. m.j.lagarda@uv.es.
authors.forename: Andrea
authors.initials: A
authors.lastname: Álvarez-Sala
authors.affiliation: Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia (Valencia), Spain.. m.j.lagarda@uv.es.
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Garcia-Llatas
authors.affiliation: Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia (Valencia), Spain.. m.j.lagarda@uv.es.
authors.forename: Reyes
authors.initials: R
authors.lastname: Barberá
authors.affiliation: Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia (Valencia), Spain.. m.j.lagarda@uv.es.
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Lagarda
mesh_terms: D000328:Adult; D002784:Cholesterol / Q000032:analysis*; D002789:Cholesterol Oxidase / Q000737:chemistry; D002849:Chromatography, Gas; D005260:Female; D006801:Humans; D007202:Indicators and Reagents; D008055:Lipids / Q000737:chemistry / Q000302:isolation & purification; D008895:Milk, Human / Q000737:chemistry*; D015203:Reproducibility of Results; D012997:Solvents; D013053:Spectrophotometry / Q000379:methods
chemical_list: D007202:Indicators and Reagents; D008055:Lipids; D012997:Solvents; D002784:Cholesterol; D002789:Cholesterol Oxidase