Orientador: Prof. Dr. Jesuí Vergílio Visentainer

Data da Defesa: 29/05/2020



INTRODUCTION. Human milk (HM) is the most adequate and complete nutritional source for neonates, in addition to having a protective role against infectious diseases and ear infections, especially in the first six months of life. The protective effects of HM are related to the duration of breastfeeding, suggesting a potential cumulative effect, due to “metabolic programming” preventing obesity, type 2 diabetes and leukemia in adulthood. Consistent evidence indicates that children who received HM also perform better on intelligence tests and have a higher level of education when compared to infants who were not breastfed. Until the 7th day after delivery, HM is considered colostrum, between the 8th and 14th day postpartum it is considered transitional milk and after the 15th day postpartum it is considered mature milk. HM consists of more than 150 different components such as water, vitamins, minerals, immunocompetent substances, hormones, trophic factors or growth modulators, in addition to carbohydrates, proteins and lipids. Lipids are a wide and complex group of compounds soluble in organic solvents and insoluble in water, and can be divided into different lipid classes based on their chemical structures. Among the main lipids in HM are triacylglycerols (TAGs), diacylglycerols (DAGs) and monoacylglycerols (MAGs). TAGs mainly represent the lipid composition of HM, ranging from 97.6% in colostrum to 99% in mature milk, being the main source of energy for the neonate and also of polyunsaturated, monounsaturated and saturated fatty acids (FAs). Other lipid components such as phospholipids and cholesterol are present in smaller proportions as part of the HM fat globule, representing a maximum of 2% of the total lipid, despite having important functions. Among HM phospholipids, sphingomyelin is present exclusively in the fat globule membrane and is capable of modulating the activity of intestinal lipase, contributing positively to the development of the infant intestinal microbiota. Cholesterol is also one of the components of cell membranes and its intake by the neonate has been suggested as a beneficial factor, as it is involved in the synthesis of myelin present in neurons, contributing to the development of the central nervous system. The determination of lipids in HM requires the use of methods that consider the variations of these compounds, since such variations can influence the results. Therefore, it is necessary to know the characteristics of the compounds of interest (analytes) contained in the HM (matrix) in order to choose the ideal method to be employed. In addition, for the proper application of each method, it is usually necessary that the matrix be subjected to an extraction and / or concentration process, so that it is possible to obtain and detect the analytes using the method employed. Chemical analysis of a sample includes the application of analytical methods, qualitative or quantitative, in order to identify and quantify, respectively, its constituents. Such analytical methods have stood out in recent years, due to their technological advances and for contributing to the determination of a wide range of lipid substances.
AIMS. Due to the lipid importance of HM for the development of the neonate, its complete constitution has been continuously studied. For this purpose, different analytical methods have been applied and improved over the years, involving advanced analytical techniques, such as gas chromatography (GC), high performance liquid chromatography (High Performance Liquid Chromatography - HPLC) and mass spectrometry (Mass Spectrometry - MS). Therefore, the objective of this work is to present a bibliographic review on the main analytical methods described in the literature, currently used to carry out the determination of several classes of HM lipids.
MATERIAL AND METHODS. The study design was based on a bibliographic review with experimental design, carried out in an electronic database, looking for scientific articles on the subject. The languages used in the research were Portuguese and English, the terms being delimited in: 1 - Human Milk; 2 - Human Milk Lipid Composition; 3 - Fatty Acids in Human Milk; 4 - Human Milk Lipids; 5 - Methods; 6 - Analytical Methods; 7 - Lipid Determination. Relevant studies on analytical methods used in the extraction and determination of HM lipids were included. The research was carried out involving books in the area of Chemistry, Analytical Chemistry and scientific articles indexed in the electronic databases PubMed (US National Library of Medicine), Scientific Electronic Library Online Brazil (SciELO), Web of Science and Portal Capes. Were selected 81 articles that addressed the theme of this paper.
RESULTS AND DISCUSSION. Regarding the dosage of the total lipid content, lipid extraction can be performed through liquid-liquid extraction, using solvents that are capable of solubilizing all lipid compounds, so that the result found is as correct as possible . When these solvents are added to the HM, two phases are formed, one aqueous and the other organic. Lipids are dissolved in the organic phase, which is the lower phase, while carbohydrates, salts and water are solubilized in the upper, aqueous phase. In this way, it is possible to collect the extracted lipids effectively and dose them using gravimetric methods. In these methods, the total evaporation of the solvent is carried out and the lipid dosage is quantified by the difference in the weight of the empty glassware when starting the analysis and glassware with the lipid phase, after lipid extraction. Among the methodologies widely used for lipid extraction, the procedure proposed by Soxhlet (1879), Folch et al. (1957) and Bligh and Dyer (1959). Lucas et al. (1978), developed the method called Creamatocrit, which separates the portion of cream by centrifuging a sample of HM. This lipid portion, or “cream”, is routinely used to calculate the fat percentage of milk samples in brazilian Human Milk Banks. Solid phase extraction (SPE) can also be used in the extraction of different classes of lipids, using cartridges containing a solid phase for the extraction of lipids, which are then eluted with suitable organic solvents, while the interferents remain retained in the phase solid, promoting the separation of the phases of interest. Due to the complexity, a complete analysis of all the lipids present in the HM sample requires more than one instrument. The GC coupled to the flame ionization detector (Flame Ionization Detector - FID) is the most widely used method of separation and determination because it is cheaper and widely used to determine the composition in FAs. With the advancement of technology, other techniques such as LC, especially HPLC (High Performance Liquid Chromatography), UHPLC (Ultra High Performance Liquid Chromatography) and MS (Mass Spectrometry) systems have also started to be used in the analysis of other HM lipid classes.
CONCLUSIONS. Until now, there have been few technological advances in the development of lipid extraction methods, since most studies still use the old methods, which require large volumes of toxic solvents. The analytical methods available have been able to assist in a greater understanding of the characteristics of HM lipids. Chromatographic methods (GC, HPLC and UHPLC) and ESI-MS are currently the most used methods for the identification, characterization and quantification of different lipid classes. Among the chromatographic methods, GC-FID can be considered as an established method in the determination of FAs, as it presents satisfactory precision, in addition to the possibility of using several types of available columns. In addition, the results obtained by this method can be easily reproducible, thanks to the use of empirical and theoretical correction factors, which provide reliability and allow comparison between results of different studies. The technological advances developed to date are capable of providing an amplitude in the knowledge of lipid characteristics of HM, mainly in the area of mass spectrometry. It is noteworthy that continuous scientific advances are of great relevance to the area, in order to increasingly improve the analytical methods in relation to the time of analysis, costs and technological quality, providing, mainly, the evolution of knowledge about the characteristics of the lipid species, nutrients so important in the health of the infant.
Key words: Human milk; Lipid composition; Identification; Quantification; Analytical methods.


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