lipids and human milk

Lipid Technology May 2012, Vol. 24, No. 5 103

Feature

Lipids and human milk

Jongsoo Kim and James Friel

J.F. is Professor in Human Nutritional Sciences at the University of Manitoba, Winnipeg, Manitoba, Canada.E-mail: [email protected]. is Research Associate in the same department.

Summary

To support the growth and development of the breast-fed infant, human milk provides the dietary essential fatty acids, linoleic acid (LA;18:2n-6), a-linolenic acid (ALA, 18:3n-3), as well as longer-chain polyunsaturated fatty acids including arachidonic acid (20:4n-6) anddocosahexanoic (DHA 22:6n-3). The linoleic acid, alpha-linolenic acid, DHA and arachidonic acid concentration of pasteurized andunpasteurized human milk remains stable during the first month of storage at – 208C and –808C. However after the first month, a slowdecrease in concentration progresses until the end of 6 months of storage at both temperatures. The levels of n-6 and n-3 fatty acids,particularly linoleic acid, alpha-linolenic acid and DHA, in human milk vary widely within and among different populations, and arereadily changed by maternal dietary intake of the respective fatty acid. The present paper reviews recent understanding from keyresearchers of maternal diet and human milk fat composition and form our work the effect of milk fat composition on storage conditions. Itis important to understand that maternal diet can affect human milk fat composition and subsequently infant development and growth.

Introduction

Human milk is a synergism of essential nutrients and bioactivecomponents. Milk is a very complex fluid with carbohydratesand salts in solution, caseins in colloidal dispersion, cells andcellular debris, and lipids mostly in emulsified globules [1]. Themain milk lipids are esterified triacylglycerols (TG, 98%), phos-pholipids (PL, 0.8%), cholesterol (C, 0.5%), and others. Lipids (3–5%) occur as globules emulsified in the aqueous phase (87%) ofmilk. A small portion of fatty acids is esterified in the form ofphospholipids, included in the membrane that surrounds andstabilizes the lipidic core of the milk fat globule. The globulesare covered with bipolar materials (phospholipids, proteins,mucopolysaccharides, cholesterol, enzymes), organized into aloose layer called the milk lipid globule membrane (MLGM). TheMLGM performs as an emulsion stabilizer and represents themembrane of the secretory mammary gland cell. The diameterof globules range in size from 1 to 10 lM. While most of the glo-bules are less than 1 lM, those around 4 lM accounts for most ofthe weight. The globules present a large surface area (4.6 m2/dL)to lipolytic enzymes and other adhesive components. The sys-tem in milk delivers energy, nutrients, protective components,and metabolic messages to the infant.

Milk fat synthesis

Milk fat consists predominantly of polyunsaturated fatty acids(PUFA, about 98 g/100 g milk total fat). PUFA are synthesizedfrom glycerol and non-esterified fatty acids in the alveolar cellsof the mammary gland [1]. Fatty acids of C-16 are taken up fromplasma chylomicrons and very long chain-polyunsaturated fattyacids through the action of the mammary lipoprotein lipase.The fatty acids in human milk originate from recent dietaryfatty acid intake, fatty acids released from maternal adipose tis-sue, de novo synthesis, and from metabolism of dietary fattyacids in the maternal liver. Fatty acid synthesis in the mammarygland proceeds via the fatty acid synthetase enzyme complex,

starting with acetyl-CoA, followed by sequential addition of C-2units as malonyl-CoA, which is similar to other cells. In tissuesother than the mammary gland, long-chain acyl thioesterase ispart of the fatty acid synthetase complex and cleaves the fattyacyl chain at C-16 to give rise to the palmitic acid (16: 0). In themammary gland, the cytosolic medium-chain acyl thioesterasecleaves the fatty acyl chain at C-14 to generate myristic acid(14:0) due to the presence of medium-chain fatty acids in humanmilk and their increase during high carbohydrate diets. Conver-sion of acetyl-CoA to malonyl-CoA catalyzed by acetyl-CoA car-boxylase is the rate-limiting step in fatty acid synthesis, and it isregulated by insulin in a similar process to that in other tissues.

Several important differences in mammary gland fatty acidsynthesis and in the effect of dietary lipids on milk fatty acidsoccur between ruminants and non-ruminants [1]. In ruminantsthe primary sources of cholesterol for mammary-gland fattyacid synthesis are acetate and b-hydroxybutyrate, and as a resultof metabolism by the rumen bacteria, dietary fatty acids have lit-tle influence on the composition of cow's milk fatty acids. Cow'smilk typically contains a2 g linoleic acid and alpha-linolenicacid/100 g total fatty acids and has negligible amounts of EPAand DHA. In humans, glucose-derived acetyl-CoA is the majorsource of C-2 units for fatty acid synthesis. Maternal dietaryintake of PUFA affects the quantity of n-6 and n-3 fatty acidssecreted in milk. The long-chain saturated fatty acids, cis- mono-unsaturated fatty acids, trans-fatty acids and n-6 and n-3 PUFAare taken up by the mammary gland and then acylated intoPUFA for secretion into milk. Mammary gland-specific acyl trans-ferases preferentially esterify 16:0 and 18:0 at the centre sn-2position of human-milk PUFA, rather than at the sn-1 or sn-3positions seen in other tissues. The products of hydrolysis ofhuman-milk PUFA by gastric and pancreatic lipases are non-essential fatty acids, released from the PUFA sn-1 and sn-3 posi-tions and sn-2 monoacylglycerols enriched in 16:0 and 18:0. Thisunusual structure of human-milk PUFA is important becausenon-esterified 16:0 and 18:0 have melting points (about 638Cand 698C respectively). These are considerably above body tem-

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DOI 10.1002/lite.201200190

104 May 2012, Vol. 24, No. 5 Lipid Technology

peratures and readily form insoluble soaps with Ca and otherdivalent cations at the pH of the intestine, giving rise to fecalloss of both fat and important minerals. Whether in breast-fedinfants the distribution of fatty acids in the plasma chylomicronPUFA with 16:0 and 18:0 in the centre position, influences tissuen-6 and n-3 fatty acid delivery is not known. After absorption, sn-2 monoacylglycerols are re-esterfied mainly via the 2-monoacyl-glycerol pathway, maintaining the unusual position of fattyacids in human milk fat.

Linoleic acid and alpha-linolenic acid cannot be formed bymammalian cells, and consequently all the derivative and n-6and n-3 fatty acids secreted in milk must come from the mater-nal diet, either directly, after storage or after further metabo-lism in maternal tissues. Linoleic acid and alpha-linolenic acidare present in the diet in greatest amounts from unsaturatedplant, seed and nut oils, although different oils vary consider-ably in their linoleic acid and alpha-linolenic acid content. Onceobtained from the diet, linoleic acid and alpha-linolenic acidcan be further metabolized by D6-desaturation, elongation andD5-desaturation to form arachidonic acid and EPA respectively.This involves two successive elongations of EPA to form 24:5n-3,then desaturation at position 6 to yield 24:6n-3. Subsequentlytranslocation to the peroxisomes generates 22:6n-3 by a singlecycle of b-oxidation. Further metabolism of n-6 fatty acids toform docosapentaenoic acid (22:5n-6) occurs through a similarpathway. A characteristic feature of animals fed an n-3 fattyacid-deficient n-6 fatty acid-sufficient diet is a decrease in tissuelevels of DHA and an increase in n-6 docosapentaenoic acid.Essential fatty acid deficiency involving an inadequate intake ofboth n-6 and n-3 fatty acids, on the other hand, results in adecrease in arachidonic acid and DHA and increased desatura-tion and elongation of the n-9 fatty acid oleic acid (18:1n-9) togive increased levels of 20:3n-9 and 22:3n-9.

The synthesis of arachidonic acid, EPA, DHA and their inter-mediates is limited to animal cells, and they are found in thediet only as part of animal tissue lipids. In general, the richestdietary source of EPA and DHA is fish andmost dietary arachido-nic acid originates from poultry and other meats. As arachidonicacid, EPA and DHA secreted into milk, they may originate eitherfrom the maternal diet or as a result of synthesis from their lino-leic acid and alpha-linolenic acid precursors respectively inmaternal tissues. In vitro, the D6-desaturase that catalyses thefirst step in fatty acid desaturation shows substrate preferencein the following order: alpha-linolenic acid > linoleic acid >oleicacid. However, in vivo a considerable proportion of alpha-linole-nic acid undergoes b-oxidation for energy, rather than under-going desaturation and elongation. As a result, only smallamounts of alpha-linolenic acid are converted to DHA.

Lipids and storage

The emulsion in the milk is thermodynamically unstable, but itmaintains its original compartmentalization during the nur-sing process for a short period of time. Thereafter, some lipidconstituents in milk may be altered, but most of the originalcontent of milk lipids apparently is not changed. Off-flavoursmay develop to the extent that the infant will not consumestored milk after exposure to various storage conditions.

In a study from our group, the 4 fatty acids (linoleic acid,alpha-linolenic acid, DHA and arachidonic acid) maintained gen-eral stability after refrigeration, frozen storage and Holder pas-

teurization [4]. During storage for 8 days at 48C, there was no dif-ference in the linoleic acid, alpha-linolenic acid, DHA and ara-chidonic acid contents of unpasteurized human milk. The samefatty acids both pasteurized and unpasteurized remained stableduring 4 weeks of storage at –208C and at –808C. However afterthe first month, a slow decrease in content is possible until 6months (Table 1).

Diet effect on milk fat composition

Over 200 fatty acids of different chain length and unsaturationhave been identified in human milk. The levels of n-6 and n-3fatty acids, particularly linoleic acid, alpha-linolenic acid andDHA, in human milk vary widely within and among differentpopulations, and they are strongly affected by the maternal diet-ary intake of the respective fatty acid.

The content of 12:0 and 14:0 is high in milk from Nigerianwomen, consuming a high-carbohydrate diet deficient in 18:3n-3, the precursor of 22:5-6. Palm oil was the major dietary fat.This oil contains about 40% each of 16:0 and 18:1 and virtuallyno 12:0, 14:0, or 18:3n-3 [3]. The quantities of 22:6n-3 in milkfrom Sudanese women were low, 0.07 € 0.03 wt%. The majordietary fat was unhydrogenated cottonseed oil. Regional dietsare diversified and the fatty acid profiles of milk from thoseregions typify the diets, i. e., high carbohydrate-low fat, and thesources of particular fatty acids. The 22:6n-3 contents of themilk from women in the marine region of China is extraordina-rily high (2.78%) due to high consumption of seafood . Fatty acidprofiles from various population groups including Spanish,Dutch, full-term and preterm confirm dietary effects on milkcomposition. In very preterm milks (a31 wk), the 22:6n-3 and20:4n-6 content decreases as lactation progresses. In pretermmilk (31 to 36 wk), 22:6n-3 did not change and 20:4n-6 decreased[2].

Milk fat from women consuming vegan diets, which lackDHA, contains a0.1 g DHA/100 g total fatty acids, whereas muchhigher amounts, often >1 g DHA/100 g total fatty acids, arefound in the milk of women consuming diets high in fish andother marine animals [1].

Milk fat and infant development

All human milks meets and exceeds the dietary requirementsfor n-6 fatty acids with an intake of only 1–2% energy from lino-leic acid. The intake of linoleic acid by the breast-fed infant issimilar to that of adults in the same population. Similar to lino-leic acid, the levels of DHA in human milk also vary widelywithin and among populations, which again results in differ-ences in the plasma and cell membrane levels of DHA amongbreast-fed infants.

High concentrations of DHA are present in phosphatidylser-ine and the ethanolamine phosphoacylglycerols (ethanolamineplasmalogen and phosphatidylethanolamine (PE)) of brain greymatter and the outer segments of rod and cone photoreceptorsin the retina.

DHA plays a role in photoreceptor signal transduction byinfluencing the ability of photons to transform rhodopsin to theactivated meta-rhodopsin ll state and the efficiency of G-protein-coupled receptor signalling. There are specific binding sites forDHA in the rhodopsin molecules. In the brain, DHA is enriched


Lipid Technology May 2012, Vol. 24, No. 5 105

in synaptic terminal membranes and has diverse roles in braingrowth and function and in protecting against oxidative stress.Early DHA deficiency has the potential to result in long-termeffects on cognitive and behavioural functions. Growth of mem-brane structures, cell division, myelination and such processesas neurite extension and dendritic arborisation, requires thesynthesis of large amounts of new membrane material, whichresults in a high demand for DHA. The rate of brain growth is atpeak velocity during the last 3 months of gestation and the firstfew months after birth (1), leading to the general concept thatDHA requirements in the human infant may be particularlyhigh during the later stage of gestation and in early infancy. Aswith linoleic acid, the mean level of DHA in milk among groupsof women presents wide variability among individuals.

An increase in DHA intake among lactating women throughsupplementation with fish, fish oils, single-cell PUFA, DHA-enriched eggs or other sources of DHA increases the secretion ofDHA in milk, and as a result, increases infant development.Increased intakes of alpha-linolenic acid, however, reportedfrom an intervention study using supplements of alpha-linole-nic acid and supplements of DHA had little or no effect inincreasing the secretion of DHA in human milk. The mostimportant factor determining the secretion of DHA in humanmilk is the mother’s intake of DHA.

The inclusion of DHA in infant formulas increases DHA in adose-dependent manner in the blood lipids of infants fed for-mula. In addition, the addition of DHA to infant formulasincreases visual and neural system maturation in term-gestationinfants who are fed formula from birth or after initial breast-feed-ing. The circulating blood levels of DHA in breast-fed infantsdepend on the concentration of DHA in the mother’s milk, whichin turn depends on the amount of DHA in the mother’s diet.

References

[1] Innis S. M., Proceedings of the Nutrition Society 2007, 66,397–404.

[2] Jensen R.G., Lipids 1999, 34, 1243–1271.

[3] Sauenvald T.U., Demmelmair H., Fidler N. & Koletzko B.,Polyunsaturated fatty acid supply with human milk. Phy-siological aspects and in vivo studies of metabolism. In:Short and Long Term Effects of Breast Feeding on ChildHealth. Eds. Koletzko et al., Kluwer Academic/PlenumPublisher 2000, 261–270.

[4] Abramovich M., Human Milk Storage Conditions inregard to safety and optimal preservation of nutritionalproperties, A Thesis for the degree of Master of Science, Uni-versity of Manitoba, 2011.


Table 1. The stability of linoleic acid, alpha-linolenic acid, DHA and arachidonic acid after refrigeration, frozen storage and Holder pasteurization.

Weeks of storage Temperature Fatty acid Stability Treatment

8 days 48C Linoleic acid, alpha-linolenicacid, DHA and arachidonic

Stable Unpasteurized

4 weeks –208C and at –808C Linoleic acid, alpha-linolenicacid, DHA and arachidonic

Stable Unpasteurized andpasteurized milk

1 month –208C and at –808C Linoleic acid, alpha-linolenicacid, DHA and arachidonic

Stable Unpasteurized andpasteurized milk

6 months – Linoleic acid, alpha-linolenicacid, DHA and arachidonic

Slow decrease is possible,after first month

Unpasteurized andpasteurized milk

lipids and human milk

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