Human breast milk is the preferred sole source of nutrition for infants through 6 months of age due to epidemiological evidence for reduced risk of disease1. Milk is a complete source of nutrition, containing macronutrients and micronutrients in addition to antibodies, growth factors, and bioactive components such as lactoferrin, oligosaccharides and phytochemicals including carotenoids (see Fig 1)2,3. While carotenoids are not currently considered essential nutrients besides the provitamin A activity of some, evidence suggests carotenoids may have particular roles in infant development and nutrition. Non-provitamin A carotenoids lutein and zeaxanthin protect against light stress and oxidation in the retinal pigment epithelium4, and increased risk of retinopathy of prematurity in preterm infants is associated with low serum lutein + zeaxanthin and non-detectable macular pigment optical density5. Provitamin A activity of β-carotene, α-carotene, β-cryptoxanthin, and perhaps α-cryptoxanthin may be particularly important for the mother and infant. Vitamin A is required for visual and immune development6, and evidence suggests provitamin A carotenoids are an important source of vitamin A in developing tissues7. Lycopene may also play a role in immune development8 and protection against inflammatory diseases9.

Milk composition fluctuates with lactation stage and is regulated by secretion and synthesis of components by mammary alveolar epithelial cells. At birth, colostrum is rich in lactoglobulins and other bioactive peptides. Mature milk composition is indicated around 5 days post parturition by changes such as increase in lactose and lipids, and is relatively stable through 6 months10. Milk production and intake averages 750–800 mL/day (30 g lipid/day) by 4 weeks, but variation between infants can range 400–1200 mL/day10. Lipid content of hind milk is greater than foremilk, and carotenoid concentration similarly increases as the breast is emptied11. Since breast fullness and completeness of milk expression influence lipid and carotenoid composition, variability of carotenoid composition is reduced when reported with respect to total lipid content12.

Carotenoids are transported to the epithelial surface via lipoproteins. Carotenes and lycopene associate mostly with VLDL/LDL, while lutein and zeaxanthin are equally distributed between LDL and HDL13,14. After release by lipoprotein lipase, carotenoids are likely transferred into mammary alveolar epithelial cells by fatty acid transporter and cluster determinant 36 (CD36). Lipids accumulate into droplets at the apical membrane surface, and are extruded into milk packaged within the milk fat globule membrane14.

The composition of human milk may be the best guide for development of dietary recommendations and phytochemical fortified infant formula due to lack of balance studies to determine optimal intake15. While milk macronutrient composition tends to be independent of maternal dietary intake10,15, carotenoid content of milk is associated with maternal diet16 and plasma carotenoid/ vitamin A status13,17. Since carotenoid content of breast milk varies by country due to carotenoids in the regional dietary pattern, reference values for individual countries are needed16. The aims of this study were to (1) expand on previous knowledge of carotenoid profiles by collecting longitudinal carotenoid profiles of human milk in select countries, and (2) develop associations of carotenoid transfer from maternal plasma to milk to the infant. We report here the carotenoid profiles of human milk from the cohorts in the Global Exploration of Human Milk study18.


Carotenoid profiles of human milk followed similar trends from donors in China, Mexico, and the USA, despite distinct dietary patterns. As lactation stage proceeded, total carotenoid content decreased, especially from week 2 to week 4. This is in agreement with previous studies that report carotenoid content per volume decreases from parturition to 4 weeks postpartum2,3,19 and stabilizes from 4–16 weeks postpartum1111,20. Total lipid content also decreased with lactation stage, which disagrees with previous reports that total lipid content increases steadily from parturition to 6 months postpartum13,21,22. Since both carotenoid content per volume and total lipid content decreased with lactation stage in the present study, total carotenoid content per lipid basis remained steady. Jewell, et al.19 reported carotenoid content per lipid basis was highest at parturition then dropped quickly, stabilizing by week 2 to week 4. Our previous study 2also observed a drop from week 1 to week 4 with carotenoids both per volume and per lipid basis. Since our sampling began at week 2, it may be that carotenoid content per lipid basis stabilized before longitudinal sampling began. In contrast to total and non-provitamin A carotenoids, provitamin A carotenoids were stable on a volume basis with lactation stage (increased per lipid basis). This physiological cause for this is unclear.

Cis-lutein isomers seem to be present in human milk at a roughly 1:3 ratio with all-trans-lutein. Presence of cis-lutein and zeaxanthin isomers have been identified before in human milk and plasma23 , as well as human and primate retina24,25. Some isomers may originate from dietary sources23 while some proportion may be isomerized from all-trans- species in vivo. Previous surveys of carotenoids across countries and lactation stages have not reported cis isomers, mostly likely due to lack of sufficient chromatographic separation of peaks. Depending on the chromatographic methodology cis isomers may be either uncounted or quantified with all-trans- isomers if the species were not resolved. Cis- isomers of lycopene were more prominent than all-trans-lycopene at a roughly 2:1 ratio in maternal plasma, neonatal plasma, and milk. This is consistent with previous observations that cis- isomers account for the majority of total lycopene in plasma and biological tissues but less than 10–25% in most foods such as unprocessed standard red tomatoes26.

While carotenoid content was expressed per volume and per lipid basis to facilitate comparisons to the literature, data from this study suggests that milk carotenoid concentration per mass lipid may best reflect transfer of carotenoids from mother to infant. Correlations of human milk with both maternal and neonatal plasma were stronger when milk carotenoid content was expressed in units of nmol/g lipid than in nmol/L. Previous studies indicate that carotenoid concentration follows the total lipid content of breast milk within a feeding as the breast is emptied, i.e. both increase from foremilk to hindmilk11,12. Even though carotenoids are associated with lipoproteins in circulation, once transferred to milk they are associated with the milkfat globule, and concentrations of carotenoids per total fat likely better reflect the secretion of carotenoids into the mammary gland. This would suggest that the total dietary intake of carotenoids by infants depends on the total intake of fat more than total volume of milk intake. However, since neonatal intake of human milk is often estimated on a volume basis, both measures may prove useful.

Neonatal plasma profile was similar to maternal plasma for xanthophylls (lutein, zeaxanthin, α-cryptoxanthin, and β-cryptoxanthin), but lower in the aliphatic carotenoids (α-carotene, β-carotene, and lycopene). Clinical, in vitro, and cell culture studies support the notion that polar xanthophylls are more bioavailable than carotenes, followed by lycopene27. Increased polarity may facilitate bioavailability by localizing xanthophylls at the surface of lipid structures, thereby enhancing transfer in and out of mixed micelles, lipoproteins, and milk fat globules. Carotenoid polarity is consistent with correlations between maternal plasma and milk carotenoid composition: the ratio of carotenoids in milk (nmol/L) to maternal plasma (nmol/L) was roughly 80% for lutein, 17% for cryptoxanthins, 7–13% for carotenes, and 4–5% for lycopene. This may also reflect a preference of mammary alveolar epithelial cells for HDL over LDL. However, this trend did not hold when comparing the ratio of neonatal plasma to human milk composition: 133% for lutein, 560–600% for cryptoxanthins, 270–300% for carotenes, and 480–650% for lycopene. Rubin, et al.28 similarly observed neonatal plasma levels in relation to carotenoid intake from human milk was greatest for lycopene, intermediate from β-carotene, and lowest for lutein. However, the seemingly low response of lutein in neonatal plasma from milk content may also reflect metabolism and tissue uptake (i.e. lutein and zeaxanthin deposition in retinal tissue).

Strengths of this study include the longitudinal sampling of the same donors to follow lactation stage. This design is more sensitive to trends in lactation stage than a larger and potentially more representative cross sectional sample of donors, each donating at only one lactation stage. All donors from each country lived within a reasonable distance of a single collection site for each country, which increased reliability of sample collection with the same protocol. Limitations of this study include a small sample size, with limited locations, and. Carotenoid profiles may reflect those of the particular location more than the country as a whole. The dietary pattern and resulting carotenoid profile may not necessarily reflect those in rural areas or geographically distinct cities within the same country. Plasma associations of carotenoid transfer were only derived from the Cincinnati, USA location at 4 weeks postpartum because the likelihood of exclusive breastfeeding was much greater than at 13 and 26 weeks. However, neonatalmaternal plasma associations may not reflect those of other locations and lactation stages.


This work deepens our understanding of neonatal exposure to carotenoids during development. The carotenoid content of human breast milk is highly variable between subjects, even within a given location and lactation stage, which makes it difficult to detect trends between countries and lactation stages unless those trends are very large. In general, carotenoid content tended to decrease with lactation stage as total lipid content decreased. Some particular carotenoids, such as lutein and lycopene, showed distinct differences between countries. We also report that the carotenoid content of maternal plasma is correlated to that of breast milk, which is again correlated to neonatal plasma content. Further work is needed in this area to understand the biological impact of carotenoid exposure to infants, and this study presents typical breast milk contents for such research. The breast milk carotenoid contents observed here across multiple nationalities and lactation stages may help guide dietary recommendations and the design of human milk mimetics.


1 Eidelman AI, Schanler RJ. Breastfeeding and the use of human milk. Pediatrics. 2012; 129(3):e827–41. Epub 2012/03/01. doi: 10.1542/peds.2011-3552 PMID: 22371471.
2 Song BJ, Jouni ZE, Ferruzzi MG. Assessment of phytochemical content in human milk during different stages of lactation. Nutrition. 2013; 29(1):195–202. Epub 2012/12/15. doi: 10.1016/j.nut.2012.07.015PMID: 23237648.
3 Ballard O, Morrow AL. Human milk composition: nutrients and bioactive factors. Pediatr Clin North Am. 2013; 60(1):49–74. Epub 2012/11/28. doi: 10.1016/j.pcl.2012.10.002 PMID: 23178060; PubMed CentralPMCID: PMC3586783.
4 Barker FM 2nd, Snodderly DM, Johnson EJ, Schalch W, Koepcke W, Gerss J, et al. Nutritional manipulation of primate retinas, V: effects of lutein, zeaxanthin, and n-3 fatty acids on retinal sensitivity to bluelight-induced damage. Invest Ophthalmol Vis Sci. 2011; 52(7):3934–42. Epub 2011/01/20. doi: 10.1167/iovs.10-5898 PMID: 21245404; PubMed Central PMCID: PMC3175953.
5 Bernstein PS, Sharifzadeh M, Liu A, Ermakov I, Nelson K, Sheng X, et al. Blue-light reflectance imaging of macular pigment in infants and children. Invest Ophthalmol Vis Sci. 2013; 54(6):4034–40. Epub2013/05/09. doi: 10.1167/iovs.13-11891 PMID: 23652486; PubMed Central PMCID: PMC3680006.
6 Bates CJ. Vitamin A. Lancet. 1995; 345(8941):31–5. Epub 1995/01/07. PMID: 7799706.
7 Kim YK, Wassef L, Chung S, Jiang H, Wyss A, Blaner WS, et al. beta-Carotene and its cleavage enzyme beta-carotene-15,15'-oxygenase (CMOI) affect retinoid metabolism in developing tissues. FASEB J. 2011; 25(5):1641–52. Epub 2011/02/03. doi: 10.1096/fj.10-175448 PMID: 21285397;PubMed Central PMCID: PMC3079298.
8 Ruhl R. Non-pro-vitamin A and pro-vitamin A carotenoids in atopy development. Int Arch Allergy Immunol. 2013; 161(2):99–115. Epub 2013/01/25. doi: 10.1159/000345958 PMID: 23343622.
9 Erdman JW Jr., Ford NA, Lindshield BL. Are the health attributes of lycopene related to its antioxidant function? Arch Biochem Biophys. 2009; 483(2):229–35. Epub 2008/11/06. doi: 10.1016/ 022 PMID: 18983972; PubMed Central PMCID: PMC2745920.
10 Kent J. How Breastfeeding Works. J Midwifery Womens Health. 2007; 52(6):564–70. doi: 10.1016/j.jmwh.2007.04.007 PMID: 17983993
11 Jackson JG, Lien EL, White SJ, Bruns NJ, Kuhlman CF. Major Carotenoids in Mature human Milk: Longitudinaland Diurnal Patterns. J Nutr Biochem. 1998; 9(1):2–7. doi: 10.1016/s0955-2863(97)00132-0
12 Giuliano AR, Neilson EM, Yap H-H, Baier M, Canfield LM. Quantitation of and inter/intraindividual variabilityin major carotenoids of mature human milk. J Nutr Biochem. 1994; 5:551–6.
13 Schweigert FJ, Bathe K, Chen F, Buscher U, Dudenhausen JW. Effect of the stage of lactation in humans on carotenoid levels in milk, blood plasma and plasma lipoprotein fractions. Eur J Nutr. 2004; 43(1):39–44. doi: 10.1007/s00394-004-0439-5 PMID: 14991268
14 Romanchik JE, Morel DW, Harrison EH. Distributions of carotenoids and alpha-tocopherol among lipoproteins do not change when human plasma is incubated in vitro. J Nutr. 1995; 125(10):2610–7. Epub1995/10/01. PMID: 7562097.
15 Stam J, Sauer PJ, Boehm G. Can we define an infant's need from the composition of human milk? AmJ Clin Nutr. 2013; 98(2):521S–8S. Epub 2013/07/12. doi: 10.3945/ajcn.112.044370 PMID: 23842459.
16 Canfield LM, Clandinin MT, Davies DP, Fernandez MC, Jackson J, Hawkes J, et al. Multinational study of major breast milk carotenoids of healthy mothers. Eur J Nutr. 2003; 42(3):133–41. Epub 2003/06/18.doi: 10.1007/s00394-003-0403-9 PMID: 12811470.
17 Azeredo VBd, Trugo NMF. Retinol, carotenoids, and tocopherols in the milk of lactating adolescents and relationships with plasma concentrations. Nutrition. 2008; 24(2):133–9. doi: 10.1016/j.nut.2007.10.011 PMID: 18053685
18 Woo JG, Guerrero ML, Ruiz-Palacios GM, Peng YM, Herbers PM, Yao W, et al. Specific infant feeding practices do not consistently explain variation in anthropometry at age 1 year in urban United States, Mexico, and China cohorts. J Nutr. 2013; 143(2):166–74. Epub 2012/12/14. doi: 10.3945/jn.112.163857 PMID: 23236024; PubMed Central PMCID: PMC3542908.
19 Jewell VC, Mayes CBD, Tubman TRJ, Northrop-Clewes CA, Thurnham DI. A comparison of lutein and zeaxanthin concentrations in formula and human milk samples from Northern Ireland mothers. Eur JClin Nutr. 2004; 58(1):90–7. doi: 10.1038/sj.ejcn.1601753 PMID: 14679372
20 Meneses F, Trugo N. Retinol,-carotene, and lutein + zeaxanthin in the milk of Brazilian nursing women: associations with plasma concentrations and influences of maternal characteristics. Nutr Res. 2005; 25(5):443–51. doi: 10.1016/j.nutres.2005.03.003
21 Bitman J, Freed LM, Neville MC, Wood DL, Hamosh P, Hamosh M. Lipid composition of prepartum human mammary secretion and postpartum milk. J Pediatr Gastroenterol Nutr. 1986; 5(4):608–15.Epub 1986/07/01. PMID: 3735011.
22 Jensen RG. Lipids in human milk. Lipids. 1999; 34(12):1243–71. Epub 2000/02/01. PMID: 10652985.
23 Khachik F, Spangler CJ, Smith JC Jr., Canfield LM, Steck A, Pfander H. Identification, quantification, and relative concentrations of carotenoids and their metabolites in human milk and serum. Anal Chem.1997; 69(10):1873–81. Epub 1997/05/15. PMID: 9164160.
24 Khachik F, Bernstein PS, Garland DL. Identification of lutein and zeaxanthin oxidation products in human and monkey retinas. Invest Ophthalmol Vis Sci. 1997; 38(9):1802–11. Epub 1997/08/01. PMID:9286269.
25 Johnson EJ, Neuringer M, Russell RM, Schalch W, Snodderly DM. Nutritional manipulation of primate retinas, III: Effects of lutein or zeaxanthin supplementation on adipose tissue and retina of xanthophyllfree monkeys. Invest Ophthalmol Vis Sci. 2005; 46(2):692–702. Epub 2005/01/27. doi: 10.1167/iovs.02-1192 PMID: 15671301.
26 Ferruzzi MG, Nguyen ML, Sander LC, Rock CL, Schwartz SJ. Analysis of lycopene geometrical isomers in biological microsamples by liquid chromatography with coulometric array detection. J ChromatogrB Biomed Sci Appl. 2001; 760(2):289–99. Epub 2001/09/04. PMID: 11530988.
27 During A, Harrison EH. Intestinal absorption and metabolism of carotenoids: insights from cell culture. Arch Biochem Biophys. 2004; 430(1):77–88. Epub 2004/08/25. doi: 10.1016/ PMID:15325914.
28 Rubin LP, Chan GM, Barrett-Reis BM, Fulton AB, Hansen RM, Ashmeade TL, et al. Effect of carotenoid supplementation on plasma carotenoids, inflammation and visual development in preterm infants. JPerinatol. 2012; 32(6):418–24. Epub 2011/07/16. doi: 10.1038/jp.2011.87 PMID: 21760585.