Fatty Acids In Human Milk Research Articles

Effect of cold storage on vitamins C and E and fatty acids in human milk

Vitamins C and E and fatty acid levels in human milk were determined when fresh, after refrigeration at 4 °C for 96 h, and after freezing at −20 °C or −80 °C for 12 months. Total vitamin C content at 4 °C (6 h), −20 °C (8 months) and −80 °C (12 months) was significantly decreased. Vitamin E levels did not change at either refrigeration temperature (under 24 h) or at freezing or ultrafreezing temperatures. Our analysis revealed that fatty acids are not affected by cold storage. In conclusion, we recommend a change in milk storage practices; specifically, it should be stored up to 3 h in a refrigerator, up to 5 months in a freezer or up to 8 months in an ultrafreezer (−80 °C). Alternatively, vitamin C supplementation may be considered. In addition, we propose vitamin C as a marker for human milk stability.

Impact of iron and vitamin C-containing supplements on preterm human milk: In vitro

Stress due to reactive oxygen species (ROS) may lead to neonatal diseases, such as necrotizing enterocolitis and respiratory distress. Enteral supplements for premature infants (PREM) added to human milk (HM) to increase nutrient content may induce lipid oxidation due to free radical formation via Fenton chemistry. We hypothesized that ferrous iron and vitamin C-containing supplements added to HM in vitro cause oxidation of milk fats, affect intracellular redox balance, and induce DNA damage. Lipid peroxidation in HM was measured by FOX-2 and TBARS assays; fatty acid composition of supplemented HM was measured by gas chromatography. Two cell culture bioassays were used for assessing either intracellular oxidative stress or DNA damage: the former involved Caco-2BBe cells, a secondary differentiated cell line, and the latter utilized FHS-74 Int cells, a primary fetal small intestinal culture. Lipid oxidation products of HM increased after the addition of iron alone, iron and vitamin C, or iron and a vitamin C-containing supplement (Trivisol, TVS). A reduced content of mono and polyunsaturated fatty acids in HM was also observed. Iron, not iron+vitamin C, but iron+TVS induced significant intracellular oxidative stress in FHS-74 Int cells. In contrast, iron, either alone or in combination with TVS or vitamin C, increased DNA damage in Caco-2BBE cells. Iron supplementation may increase oxidative stress in PREM infants and should be given separately from vitamin C-containing supplements.

HAMLET, protein folding, and tumor cell death

HAMLET (Human a-lactalbumin made lethal to tumor cells) was discovered by serendipity. The first publication in 1995 described the discovery and the unusual properties of what later became the HAMLET complex [1]. A fraction of human milk was able to kill tumor cells by a mechanism resembling apoptosis, and many different types of tumor cells were susceptible to this effect while healthy differentiated cells were resistant. The activity was shown to reside in a complex between the human milk protein a-lactalbumin and oleic acid, which is the most abundant fatty acid in human milk [2]. Since then, the HAMLET complex has been characterized in detail in order to determine the structural basis and mechanism(s) of the tumoricidal activity, and HAMLET has been used to treat tumors in animals and patients [3,4]. In 2006, researchers working on HAMLET and related topics met in Lund in Sweden for the First International HAMLET Symposium. This review summarizes the current state of knowledge in this area based on the presentations made at the conference.

Trans Fatty Acids in Human Milk in Canada Declined with the Introduction of Trans Fat Food Labeling

Trans fatty acids in human milk have raised concerns because of possible adverse effects on infant growth and development. Analyses of human milk in the late 1990s in Canada showed high amounts of trans fatty acids from partially hydrogenated oils. Canada introduced labeling of trans fatty acids on retail foods in 2003. We analyzed trans and cis unsaturated and saturated fatty acids in human milk collected from 87 women in 2004–2006 and compared the levels to those in milk collected from 103 women in 1998 and analyzed using similar methods. The total trans fatty acids (mean ± SEM, g/100 g fatty acids) in human milk in Canada decreased significantly, from 7.1 ± 0.32 in 1998 to 6.2 ± 0.48, 5.3 ± 0.49, and 4.6 ± 0.32 over 3 consecutive 5-mo periods from November 2004 to January 2006. The milk total trans fatty acids were significantly and inversely related to 16:0, 18:2(n-6), 18:3(n-3), 20:4(n-6), 22:4(n-6), and 22:5(n-6) and positively related to 18:0 and conjugated linolenic acids (P < 0.05, n = 190). The estimated exposures of exclusively breast-fed infants to trans fatty acids decreased from a mean and 95th percentile intake of 2.0 and 4.4 g · infant−1 · d−1 in 1998 to 1.33 and 2.41 g · infant−1 · d−1, respectively, in late 2005. The estimated intake of the mothers was 4.0 (range 0.51–12.3) and 2.2 (0.56–7.65) g · person−1 · d−1 in 1998 and late 2005, respectively. Our studies show trans fatty acids have decreased in human milk in Canada, which suggests a concomitant decrease in trans fatty acid intake among lactating women and breast-fed infants.

Human milk fatty acid composition from nine countries varies most in DHA

Many published studies of breast milk FA composition are limited to populations from one or two countries. We aimed to examine the degree to which FA compositions vary across a number of diverse populations. Because diet and maternal adipose stores influence breast milk FA composition, differences in FA composition between groups most likely reflect habitual dietary differences. Approximately 50 breast milk samples (full breast expression) were collected from women in Australia, Canada, Chile, China, Japan, Mexico, Philippines, the United Kingdom, and the United States. The proportion of saturated FA was relatively constant among countries, with the exception of the Philippines, where levels of lauric and myristic acids were elevated (means greater than two times the mean of most other countries). Monounsaturated FA also varied little, with the exception of low levels of oleic acid in the Philippines and high levels of erucic acid in China. Although arachidonic acid (C20:4n-6) levels were similar among all countries (means ranging from 0.36 wt % to 0.49 wt %), mean DHA (C22:6n-3) levels ranged from 0.17 to 0.99 wt %, with the highest levels in Japanese milk and the lowest levels in Canadian and U.S. samples. The results of this study demonstrate that the proportion of saturated and monounsaturated FA are relatively constant across a large number of countries, whereas the level of some of the PUFA, especially DHA, are highly variable.

Impact of Enteral Supplementation in Human Milk on Lipid Oxidation and Redox Balance

Oxidative stress due to reactive oxygen species may be linked to the etiology of neonatal diseases, such as necrotizing enterocolitis and respiratory distress syndrome. Iron and vitamin C enteral supplements for premature infants (PT) added to human milk (HM) may induce lipid oxidation due to free radical formation. We hypothesized that these supplements added to HM causes oxidation of milk fats in vitro, and affects intracellular redox balance. We further hypothesized that these supplements induce oxidative DNA damage and apoptosis. Lipid peroxidation in HM was measured by Fox-2 and TBARS assays; fatty acid composition of supplemented HM was measured by gas chromatography. Two cell culture bioassays were used to determine the effects of iron and vitamin C supplementation; FHs-74 Int cells, a primary fetal intestinal culture, and Caco-2BBe intestinal cells, a secondary differentiated cell. Lipid oxidation products increased after the addition of iron in HM. This was accompanied by reduced content of mono and polyunsaturated fatty acids in HM. Vitamin C and iron admixtures in HM led to lower peroxides than iron alone in HM, contrary to expectations. The vitamins A and D (examined to study components of TRIVISOL, Mead Johnson, Evansville, USA) exhibited antioxidant activity in this study. Iron induced significant intracellular oxidative stress in FHs-74 Int cells and also increased DNA damage and apoptosis. We conclude that iron supplementation has the potential to cause formation of reactive oxygen species (ROS) both in HM and in cells exposed to iron supplemented milk. These effects in turn may increase oxidative stress in PT infants. Supported by Manitoba Institute of Child Health and Canadian Institutes of Health Research

High contents of both docosahexaenoic and arachidonic acids in milk of women consuming fish from lake Kitangiri (Tanzania): Targets for infant formulae close to our ancient diet?

Current recommendations for arachidonic (AA) and docosahexaenoic (DHA) acids in infant formulae are based on milk of Western mothers. Validity may be questioned in view of the profound dietary changes in the past 100 years, as opposed to our slowly adapting genome. Hominin evolution occurred in the proximity of East-African freshwater lakes and rivers and early homo sapiens had higher intakes of AA and DHA from a predominantly lacustrine-based diet. In search of milk AA and DHA contents of our African ancestors, we investigated the milk of 29 lactating women living in Doromoni near lake Kitangiri (Tanzania). They consumed sunflower oil-fried local fish as only animal lipid sources, maize and local vegetables. AA and DHA contents of Doromoni milk may be close to that of early homo sapiens, because of the similarity of their life-long consumption of East-African lacustrine-based foods. Human milk fatty acid relationships from our historical worldwide database and the literature revealed that disparities between the Doromoni diet and the presumed ancient diet (i.e. higher carbohydrate and linoleic acid intakes) are unlikely to affect milk AA and DHA contents. Doromoni milk had high contents of AA (median 0.70 mol%), DHA (0.75) and eicosapentaenoic acid (EPA, 0.17), and low AA/DHA ratios (median 0.91; 0.55–2.61). This tracks down to consumption of fish with high AA and DHA contents, and AA/EPA ratios. We conclude that the milk AA, DHA and EPA contents of Doromoni women might provide us with clues to optimize infant formulae and perhaps the milk of Western women.

Polyunsaturated Fatty Acids in Human Milk and Neurological Development in Breastfed Infants

Long-chain polyunsaturated fatty acids (LCPUFA) are bioactive compounds that include docosahexaenoic acid (DHA) and arachidonic acid (AA). DHA seems to be necessary to optimize growth and development of infant's neural functions. For preterm infants there is a wide agreement on LCPUFA benefits, while for term infants results are more controversial. There is presently a growing interest in supplementing pregnant and breastfeeding women with DHA. LCPUFA may act as coadjuvants in conditions of functional and organic brain impairments, as well as some inflammatory states, but caution is urged to prevent false expectations. Therefore, pediatricians should be aware of the present scientific evidence concerning dietary LCPUFA supplementations.

2 Polychlorinated Biphenyls and Levels of Polyunsaturated Fatty Acids in Human Milk

Background: Environmental contaminants such as persistent organic chlorines may contaminate human milk and negatively affect neural development. To what extent organic chlorines might influence levels of polyunsaturated fatty acids (PUFA) in milk has not been investigated yet. In this study we have investigated whether concentration of polychlorinated biphenyls (PCBs) may be associated with PUFA levels in human milk.Methods: Subjects: 25 healthy women (mean [SD, range] age 33 [6, 23–42] y., pre-pregnancy body mass index <25kg/m2, 88%, smoking during pregnancy, 24%, primiparous, 32%, who had been living in Milan or surrounding areas for at least 20 years and who delivered full term singleton infants participated in the study. Mothers exclusively breastfed for at least 4 months. Samples from colostrum, the first 2 days after delivery, and mature breast milk after 1 and 3 months were collected. The samples were analyzed for PCB 105, 118, 138, 153, 156 and 180, and for C18:2 n-6 (linoleic acid), C18:3 n-3 (alinolenic acid), C20:4 n-6 (arachidonic acid), C20:5 n-3 (eicosapentaenoic acid), C22:6 n-3 (docosahexaenoic acid) by means of gas-chromatographic techniques.Results: The concentrations of all examined contaminants were highest in colostral milk and then declined (P<0.001). PUFA levels did not show a definite smooth trend, except C20:4 n-6 and C22:6 n-3 whose concentrations declined (P<0.001). The concentrations of PCBs in milk were associated with mother's age, correlation coefficient (r) 0.57 < r <0.86, P <0.001. No significant association was found between levels of PUFAs and mother's age, P >0.14. No significant association was found of PCBs with PUFAs in human milk: colostrum, −0.16 < r < 0.25, P > 0.19; 1 month, −0.34 < r < 0.20, P0.10; 3 months, −0.27 < r <0.33, P0.11.Conclusion: Within the population of this study, no evident association was found between concentration of PCBs and PUFAs, in human milk.

Polyunsaturated fatty acids in human milk: an essential role in infant development.

The n-6 and n-3 fatty acids are essential dietary nutrients required for optimal growth and development, particularly of the brain and retina. Large amounts of the n-3 fatty acid docosahexaenoic acid (DHA) is accumulated in the brain grey matter and the visual elements of the retina during development, and reduced DHA in these tissues can result in decreased visual and psychomotor development. Although the possible importance of differences in n-6 and n-3 fatty acids, particularly DHA, between human milk and infant formulas has been the subject of intense clinical research, the variability in the essential fatty acid content of milk within and among different populations of women and implications of this to infant growth and development have received much less attention. Considerable research has shown that the DHA content of the maternal diet is the most important determinant of the amount of DHA secreted in milk, and thus the dietary intake of the breastfed infant. The DHA content of human milk varies over 10-fold, being lowest in women with no intake of DHA and highest in women with high intakes of DHA, which is found predominantly in fatty fish. The requirement for n-3 fatty acids, and the balance of n-6 and n-3 fatty acids for optimal growth and development of the brain and retina, and long-term minimization of risk of chronic disease remains as one of the most important questions in infant nutrition. Dietary recommendations to modifying dietary fat with the aim of reducing risk of chronic disease, including obesity and cardiovascular disease in adults, need to consider that when followed by pregnant women, these recommendations can have a marked effect on the amount and balance of n-6 and n-3 fatty acids secreted in milk.

Nutritional relevance of trans isomeric fatty acids in human milk

Trans isomeric fatty acids amount to about 2% of the fatty acid composition of human milk in Europe, whereas lower values were seen in Africa, and higher values were reported for North‐America. At least five human studies indicate that trans fatty acids may interfere with the metabolism of long‐chain polyunsaturated fatty acids. Conclusion: Trans isomeric fatty acids in human milk should be regarded as a potential confounding variable in studies investigating the role of long‐chain polyunsaturated fatty acids in the development of breastfed infants.

Trans fatty acids in human milk in Poland and their association with breastfeeding mothers’diets

Aim: To determine the content of trans fatty acids in human milk in relation to breastfeeding mothers’diet. Methods: Samples of milk were collected from 100 breastfeeding mothers and 7‐d dietary records and anthropometry from 69 mothers were obtained. Results: The following total trans fatty acids contents (median (lower‐upper quartile); % wt/wt) in milk samples were determined: 1) data for Spring: colostrum—1.37 (1.00–2.00), mature milk at 5–6 wk of lactation—2.59 (1.49–3.34) and at 9–10 wk of lactation—2.36 (1.55–3.92); 2) data for Autumn: colostrum—1.80 (1.42–2.48), mature milk at 5–6 wk of lactation—2.41 (1.79–4.31) and at 9–10wk of lactation—2.77 (1.53–4.18). The major sources of trans fatty acids in mothers’diets were bakery products, confectionery and snacks. Mothers who had high level of trans isomers in their milk consumed significantly higher amounts of these products.Conclusions: Bakery products, confectionery and snacks are a major source of trans fatty acids in maternal diet in Poland. The levels of trans fatty acids in human milk may reflect the current diet of the mother as well as the diet consumed early in pregnancy.

Infant intake of fatty acids from human milk over the first year of lactation.

Despite the importance of human milk fatty acids for infant growth and development, there are few reports describing infant intakes of individual fatty acids. We have measured volume, fat content and fatty acid composition of milk from each breast at each feed over a 24 h period to determine the mean daily amounts of each fatty acid delivered to the infant from breast milk at 1, 2, 4, 6, 9 and 12 months of lactation in five women. Daily (24 h) milk production was 336.60 (SEM 26.21) and 414.49 (SEM 28.39) ml and milk fat content was 36.06 (SEM 1.37) and 34.97 (SEM 1.50) g/l for left and right breasts respectively over the course of the first year of lactation. Fatty acid composition varied over the course of the day (mean CV 14.3 (SD 7.7) %), but did not follow a circadian rhythm. The proportions (g/100 g total fatty acids) of fatty acids differed significantly between mothers (P<0.05) and over the first year of lactation (P<0.05). However, amounts (g) of most fatty acids delivered to the infant over 24 h did not differ during the first year of lactation and only the amounts of 18:3n-3, 22:5n-3 and 22:6n-3 delivered differed between mothers (P<0.05). Mean amounts of 18:2n-6, 18:3n-3, 20:4n-6 and 22:6n-3 delivered to the infant per 24 h over the first year of lactation were 2.380 (SD 0.980), 0.194 (SD 0.074), 0.093 (SD 0.031) and 0.049 (SD 0.021) g respectively. These results suggest that variation in proportions of fatty acids may not translate to variation in the amount delivered and that milk production and fat content need to be considered.

Fatty acids in formulae for term infants: compliance of present recommendations with the actual human milk fatty acid composition of geographically different populations

Aim: Recommendations for formula fatty acids (FA) are largely based on the mature human milk FA composition. This study aimed to investigate whether current recommendations for formula FA for term infants comply with the actual breast‐milk FA composition of geographically distinct populations and to provide more realistic grounds for future recommendations. Methods: 455 mature breast‐milk samples were collected in different countries over 25 y. Recommendations of different organizations were projected on their FA data. FA interrelationships were calculated with Spearman's rank tests. FA compositions of 30 formulae were compared with those of breast milk. Results: Many samples from non‐Western communities did not meet the recommendations for formula 12:0, 14:0 and 18:2ω6, since these are mainly based on breast milk of mothers living in Western countries. Recommendations for 18:3ω3, 18:2ω6/18:3ω3, 20:4ω6 and 22:6ω3 were not met by many milk samples, which may point to the poorly developed recommendations for long‐chain polyunsaturated FA. Most of the investigated breast‐milk FA (12:0, 14:0, 16:0, 18:0, 18:3ω3, 22:6ω3, 18:2ω6, 20:4ω6, 18:lω9) were either positively or negatively interrelated. Many formulae had FA compositions that were not consistent with the physiological interrelationships of FA in breast milk. Conclusion: Future recommendations, if based on human milk, should derive from its FA balance, as indicated by the FA interrelationships. A “humanized” formula FA composition would in this sense be any composition that cannot be distinguished from that of breast milk by techniques such as principal component analysis.

Compliance of present recommendations of fatty acids in formulas for term infants with the actual human milk fatty acid composition in different populations

Fats are the only dietary agents possibly modulating the individual membrane composition, which otherwise is “genetically” determined. In infancy, human milk represents the first natural source of fats. Although the fat intake of breastfed infants is quantitatively and qualitatively unique, it is not known to what extent their varying requirements are met by the fat composition in the milk of their mothers. Conclusion: The fat composition of arbitrarily chosen human milk is not always the best standard for that in infant formula.

Trans fatty acids in human milk in Poland and their association with breastfeeding mothers' diets.

To determine the content of trans fatty acids in human milk in relation to breastfeeding mothers' diet. Samples of milk were collected from 100 breastfeeding mothers and 7-d dietary records and anthropometry from 69 mothers were obtained. The following total trans fatty acids contents (median (lower-upper quartile); % wt/wt) in milk samples were determined: 1) data for Spring: colostrum--1.37 (1.00-2.00), mature milk at 5-6 wk of lactation--2.59 (1.49-3.34) and at 9-10 wk of lactation--2.36 (1.55-3.92); 2) data for Autumn: colostrum--1.80 (1.42-2.48), mature milk at 5-6 wk of lactation--2.41 (1.79-4.31) and at 9-10 wk of lactation--2.77 (1.53-4.18). The major sources of trans fatty acids in mothers' diets were bakery products, confectionery and snacks. Mothers who had high level of trans isomers in their milk consumed significantly higher amounts of these products. Bakery products, confectionery and snacks are a major source of trans fatty acids in maternal diet in Poland. The levels of trans fatty acids in human milk may reflect the current diet of the mother as well as the diet consumed early in pregnancy.

Fatty acids in formulae for term infants: compliance of present recommendations with the actual human milk fatty acid composition of geographically different populations

Recommendations for formula fatty acids (FA) are largely based on the mature human milk FA composition. This study aimed to investigate whether current recommendations for formula FA for term infants comply with the actual breast-milk FA composition of geographically distinct populations and to provide more realistic grounds for future recommendations. 455 mature breast-milk samples were collected in different countries over 25 y. Recommendations of different organizations were projected on their FA data. FA interrelationships were calculated with Spearman's rank tests. FA compositions of 30 formulae were compared with those of breast milk. Many samples from non-Western communities did not meet the recommendations for formula 12:0, 14:0 and 18:2omega6, since these are mainly based on breast milk of mothers living in Western countries. Recommendations for 18:3omega3, 18:2omega6/18:3omega3, 20:4omega6 and 22:6omega3 were not met by many milk samples, which may point to the poorly developed recommendations for long-chain polyunsaturated FA. Most of the investigated breast-milk FA (12:0, 14:0, 16:0, 18:0, 18:3omega3, 22:6omega3, 18:2omega6, 20:4omega6, 18:1omega9) were either positively or negatively interrelated. Many formulae had FA compositions that were not consistent with the physiological interrelationships of FA in breast milk. Future recommendations, if based on human milk, should derive from its FA balance, as indicated by the FA interrelationships. A "humanized" formula FA composition would in this sense be any composition that cannot be distinguished from that of breast milk by techniques such as principal component analysis.

Trans fatty acids in subcutaneous fat of pregnant women and in human milk in the Czech Republic.

Using capillary gas chromatography, we determined total content of trans fatty acids (TFA) and C18:1 trans fatty acids in human milk and subcutaneous fat in 35 healthy Prague women. The average content of TFA in human milk fat was 4.22% (SD = 1.87%) of all fatty acids, and the value of trans C18:1 isomers was 3.63% (SD = 1.81%). The average concentration of total trans fatty acids in subcutaneous fat was 4.41% (SD = 0.79%) and the average content of C18:1 trans isomers was 2.81% (SD = 0.61%).

Estimated biological variation of the mature human milk fatty acid composition

We estimated the biological variation (CV biol) of 28 fatty acids (FA) in 465 mature human milk samples from The Netherlands, Caribbean, Jerusalem, Tanzania and Pakistan, by using data from the observed variation (CV obs) and analytical variation (CV anal). CV biol of the various regions was remarkably similar. The average CV biol of 455 samples, Pakistan excluded, ranged from 12.7% for 16:0 and 18.9% for 18:1ω9 to 68% for 22:6ω3 and about 100% for 20:5ω3. Those of 20:4ω6, 18:2ω6 and 18:3ω3 were 28.0, 33.0 and 37.3%, respectively. Because of the large CV biol and the many dietary changes in recent history, it seems impossible to consider the present human milk FA composition as the ‘gold standard’ for infant formula. Optimal human milk FA composition should rather derive from populations that consume traditional diets or from the scientific data that show the function of the individual FAs in neonatal development.

Comparison of two direct methods for the determination of fatty acids in human milk

We have validated and compared two direct methods for determining by capillary gas chromatography fatty acid composition in human colostrum and one method that involved lipid extraction and then methylation. Direct Method 1 (DM1) consisted of transesterification of fatty acids using acetyl chloride. Direct Method 2 (DM2) used boron trifluoride-methanol as esterification reagent. The two direct methods were compared with a traditional methylation method that involves prior lipid extraction. We found that there were no differences between the direct methods and the traditional method. We then validated both direct methods; recovery rates, inter-assay and intra-assay precisions were determined with satisfactory results. Both methods gave similar results, but DM2 has certain advantages over DM1, such as speed, safety and better recovery rates.