Robert G. Jensen

Lipids in human milk.

I have reviewed recent (March 1995–December 1997) papers on human milk lipids including many on fatty acid (FA) composition. The effects of maternal diets on the profiles are apparent. However, more data on the composition of milk lipids are needed. It is noteworthy that so few papers on milk FA composition have reported analyses using high-resolution gas-liquid chromatography columns. Two of these were on milk from women in North America. The diets in North America are varied and the number of analyses few. We do not have a reliable data base showing the ranges of biologically important acids. Except for the gangliosides, few new data on the other lipids appeared during this period.

THE LIPIDS IN HUMAN MILK

The summary will be limited to the areas that should be intensively investigated. The first is: determination of fatty acid profiles using modern methods on a world wide basis. We have no more than five or six papers in which my criterion was applied, one from Canada and the remainder from Europe with some data from Africa. Obviously, milk cannot be used as the gold standard on this meager data base. The second area is analysis of TG structure. These analyses are difficult, but structure is one of the factors controlling digestion. Data on the effects of maternal diet on structure would be useful. The third area is the role of primary or derived milk lipids as microbicidal agents. The fourth area is examination of globule parameters, i.e. number, size, volume, surface, and how they are affected by diet. There are many others which may interest the reader.

Detection and determination of lipase (acylglycerol hydrolase) activity from various sources

Methods for the detection and determination of lipases (acylglycerol hydrolases) and preparation of assays are reviewed including substrates, conditions and screening. Some newer methods for the determination of lipase activity are discussed. Several of these are: (a) titrimetry, (b) colorimetry of Cu soaps of free fatty acids (FFA), (c) colorimetry of chromophores in the acyl chain of FFA or in glycerol, (d) radioassay, (e) gas liquid chromatography, (f) enzymatic treatment of FFA and measurment of the resulting products, and (g) direct immunological determination of the lipase. Examples and sensitivities are given and advantages and disadvantages are described.

Nutritional and biochemical properties of human milk: II. Lipids, micronutrients, and bioactive factors.

Human milk lipids contain preformed LCPUFA in considerable amounts, which serve as precursors for the formation of prostaglandins, prostacyclins, and other lipid mediators, as well as essential components in membrane-rich tissues (such as the brain and the retina), thus affecting functional outcomes. Besides a balanced nutrient composition and a number of conditionally essential nutrients, human milk provides different types and classes of bioactive factors, such as enzymes, hormones, and growth factors, many of which appear to have a role in supporting infantile growth and development. The bioactive agents include antimicrobial factors (e.g., secretory IgA, oligosaccharides, FA); anti-inflammatory agents; transporters (e.g., lactoferrin); and digestive enzymes (e.g., BSSL). Several nonpeptide hormones (thyroid hormones, cortisol, progesterone, pregnanediol, estrogens, and artificial contraceptive) and peptide hormones and growth factors (erythropoietin, hHG, gonadotropin-releasing hormone, epidermal growth factor insulin, insulin-like growth factor-I, nerve growth factor, transforming growth factor-alpha, gastrointestinal regulatory peptides and thyroid-parathyroid hormones) have been isolated and quantitated in human milk. Some of these components are also involved in the maturation of the gastrointestinal tract of the infant. In addition to the passive benefits provided by human milk, several data support the hypothesis that breastfeeding promotes the development of the infants own immune system, which might confer long-term benefits for the newborn infant. The risk of IDDM, Crohns disease, and atopic disease is lower in individuals who had been breastfed during infancy. Areas of major interest in human milk research include the study of human milk synthesis and the contributions of dietary composition and maternal metabolism to human milk composition, infantile utilization of human milk components, and the study of bioactive components, such as oligosaccharides, proteins and peptides, and lipids and their in vivo fate and biologic effects in the recipient infant.

Physiological aspects of human milk lipids

Human milk from healthy and well-nourished mothers is the preferred form of feeding for all healthy newborn infants. The nutrient supply with human milk supports normal growth and development of the infant. Here the general characteristics of human milk lipids and recent knowledge on lactational physiology, composition and functional aspects of human milk lipids are discussed. Lipids in human milk represent the main source of energy for the breastfed baby and supply essential nutrients such as fat-soluble vitamins and polyunsaturated fatty acids (PUFA). The essential fatty acids linoleic and alpha-linolenic acids (LA and ALA) are precursors of long-chain polyunsaturated fatty acids (LC-PUFA), including arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids (AA and DHA). LC-PUFA serve as indispensable structural components of cellular membranes and are deposited to a considerable extent in the growing brain and the retina during perinatal development. The supply of preformed LC-PUFA with human milk lipids has been related to functional outcomes of the recipient infants such as visual acuity and development of cognitive functions during the first year of life. Recent stable isotope studies indicate that the major portion of milk PUFA is not derived directly from the maternal diet, but stems from endogenous body stores. Thus, not only the womans current but also her long-term dietary intake is of marked relevance for milk fat composition.

Effect of fish oil on the fatty acid composition of human milk and maternal and infant erythrocytes.

To examine the effect of fish oil supplementation on the fatty acid (FA) composition of human milk and maternal and infant erythrocytes, five lactating women were supplemented with 6 g of fish oil daily for 21d. Usual maternal diets contained 1,147 mg of total n−3 FA, with 120 mg from very long-chain (>C18) n−3 FA. Supplementation increased dietary levels to 3,092 mg of total n−3 FA and 2,006 mg of very long-chain n−3 FA. Milk samples were collected daily, prior to fish oil ingestion, and at 4-h intervals on days 1, 7, 14 and 21. Milk n−3 FA content increased within 8 h and reached steady state levels within one week. The n−6 fatty acid content decreased. Erythrocyte eicosapentaenoic acid content increased from 0.24% to 1.4% (P<0.01) in mothers and from 0.11% to 0.70% (P<0.05) in infants. Docosapentaenoic acid increased from 1.4% to 2.2% (P<0.05) in mothers and from 0.30% to 0.78% (P<0.01) in infants. There was no significant change in docosahexaenoic acid or n−6 fatty acid content. Maternal platelet aggregation responses were variable. No differences in milk or plasma tocopherol levels were noted.

Rumenic acid: A proposed common name for the major conjugated linoleic acid isomer found in natural products

At the last American Oil Chemists’ Society meeting in Chicago, May 10–13, 1998, there was a formal discussion period after a day-long series of presentations on conjugated linoleic acid (CLA) attended by about 100 participants. One of the topics discussed was the possible naming of the major CLA isomer, cis-9, trans-11-octadecadienoic acid—found in milk, other dairy products, and meats of ruminant animals—as rumenic acid. The name rumenic acid has been proposed by Peter W. Parodi and is supported by a number of other scientists. There was extensive debate on this topic. CLA is a mixture of many positional and geometrical isomers of conjugated octadecadienoic acids both in natural products and in commercial preparations. In natural products, the predominant isomer (≥80% of total CLA) is cis-9, trans-11, whereas in commercial preparations the number and proportion of the isomers can vary widely depending on the conditions of preparation. The major arguments presented against naming any CLA isomer were: (i) the term CLA has been in common use for nearly two decades; (ii) cis-9, trans-11 appears also to be formed outside the rumen by desaturation of trans-11-18:1; (iii) the active isomer has yet to be identified; (iv) there appears to be evidence that cis-9, trans-11 isomer may not be the only active CLA isomer, therefore; how shall we name them? On the other hand, the major arguments for naming cis-9, trans-11-octadecadienoic acid rumenic acid were: (i) this is the major naturally occurring conjugated fatty acid in milk, other dairy products, and meats from ruminants; (ii) a major natural component can be named regardless of whether any biological activity has been ascribed to it; (iii) the name would avoid the misconception that it is a CLA having a methylene-interrupted double bond system, and (iv) it is an easy name, associated with the major place of origin, and may thus be less confusing. Additional names were suggested, such as bovinic acid. However, there was agreement that this name did not encompass the broad spectrum of natural products containing cis-9, trans-11-octadecadienoic acid, for example, from sheep and other ruminants. There was no complete consensus, but half the participants said they would use the new name in their future publications. We therefore recommend naming cis-9, trans-11-octadecadienoic acid as rumenic acid.

The lipids of human milk

The summary will be limited to the areas that should be intensively investigated. The first is: determination of fatty acid profiles using modern methods on a world wide basis. We have no more than five or six papers in which my criterion was applied, one from Canada and the remainder from Europe with some data from Africa. Obviously, milk cannot be used as the gold standard on this meager data base. The second area is analysis of TG structure. These analyses are difficult, but structure is one of the factors controlling digestion. Data on the effects of maternal diet on structure would be useful. The third area is the role of primary or derived milk lipids as microbicidal agents. The fourth area is examination of globule parameters, i.e. number, size, volume, surface, and how they are affected by diet. There are many others which may interest the reader.

Nutritional and Biochemical Properties of Human Milk, Part I

Human milk provided by healthy and well-nourished mothers is believed to cover the infants nutrient requirements during the first half year of life. It is composed of a mixture of nutritive components as well as other bioactive factors with relevant physiologic effects in the neonate infant. Human milk composition has a dynamic nature and varies with time postpartum, during a nursing, and with the mothers diet and certain diseases. The changes of human milk composition with time of lactation seem to match the changing needs of the growing infant over time. Human milk proteins are a source of peptides, amino acids, and nitrogen for the infant, but also in the protein fraction reside other properties of human milk that may benefit the breastfeeding infant. Specific whey proteins are involved in the development of the immune response (immunoglobulins), whereas others participate in the nonimmunologic defense (lactoferrin). In addition, human milk contains a complex mixture of oligosaccharides that are present only in minute amounts in other mammals milk. They may act as inhibitors of bacterial adhesion to epithelial surfaces, and thus play an important role in preventing infectious diseases in the newborn infant. Oligosaccharides may also promote the development of a so-called bifidus flora. In the next years, future research will lead to improved characterization of human milk components and elucidation of their individual mechanisms of action, which will increase our knowledge about the properties of human milk and the benefits of breastfeeding for the infant.

Determination of lipase specificity

Specificity of lipases is controlled by the molecular properties of the enzyme, structure of the substrate and factors affecting binding of the enzyme to the substrate. Types of specificity are as follows. I. Substrate: (a) different rates of lipolysis of TG, DG, and MG by the same enzyme; (b) separate enzymes from the same source for TG, DG and MG. II. Positional: (a) primary esters; (b) secondary esters; and (c) all three esters or nonspecific hydrolysis. III. Fatty acid, preference for similar fatty acids. IV. Stereospecificity: faster hydrolysis of one primarysn ester as compared to the other. V. Combinations of I–IV. Lipases with these specificities are: Ia, pancreatic; Ib, postheparin plasma. IIa, pancreatic; IIb,Geotrichum candidum, for fatty acids withcis-9-unsaturation, and IIc,Candida cylindracea. III,G. candidum for unsaturates. IV.sn-1, postheparin plasma andsn-3 human and rat lingual lipases. V. Rat lingual lipase. Methods for determination involve digestion of natural fats of known structure and synthetic acylglycerols followed by analysis of the lipolysis products. All of the types of specificity have been detected with use of synthetic acylglycerols. Detection of stereospecificity requires enantiomeric acylglycerols which are difficult to synthesize, so other methods have been developed. These involve the generation of 1,2-(2,3) DG and resolution of the enantiomers. Trioleoylglycerol or racemic TG can be used as substrates. If the lipase is stereospecific, then either thesn-1,2- or 2,3-enantiomer will predominate. The relative amounts of the enantiomers can be determined by measurement of specific rotation, and nuclear magnetic resonance spectra. The DG can also be separated by conversion to phospholipids and hydrolysis with phospholipases A-2 or C. Applications of these procedures are discussed and data on the specificity of various lipases presented.