INTRODUCTION
Breast milk has been considered the standard for optimal nutrition of term newborn infants. For this reason, there have been innumerable studies that have analyzed the composition of human breast milk. These studies have centered upon the amino acid and protein content (1-4 5-8 9-11 12-16 17-19
There has been far less attention paid to the free sugars and sugar alcohols in breast milk (17 alcohols has increased in recent years. Many of these sugars and polyols are being offered commercially as supplements to one's diet with the purported purpose of stimulating the production of glycans and improving the immune response. In addition, there is clear evidence of a therapeutic effect of mannose supplementation in patients with the disease carbohydrate-deficient glycoprotein syndrome (20-22
The present study was stimulated by recent studies in our laboratory that have shown that the concentrations of the sugars other than glucose and of the sugar alcohols are much higher during fetal development compared with maternal concentrations. This has been true in both ovine pregnancy (23 24,25
Therefore, the goal of the present study was to determine whether there are significant concentrations of these same sugars and sugar alcohols in human breast milk. For purposes of comparison, similar measurements were made from samples of term formulas, preterm formulas, and cow's milk.
MATERIALS AND METHODS
Breast milk samples were obtained from 16 lactating women who had delivered term infants after uncomplicated pregnancies and from 17 lactating women who had delivered preterm. Tables 1 and 2 present the clinical information about these patients. The milk samples were collected over a range from 3 days to 29 days after delivery and over a gestational age range from 29 to 41 weeks. All samples were immediately frozen at −70°C until analyzed.
TABLE 1: Term breast milk
TABLE 2: Preterm breast milk
Milk Carbohydrate Analysis
A breast milk sample of 0.1 mL was analyzed as follows: 0.1 mL of 0.3 N zinc sulfate containing 30 mg% xylitol as internal standard was added to 0.1 mL sample. The mixture was mixed well and another 0.1 mL of 0.3 N barium hydroxide was added. The mixture was centrifuged at 14,000 × g for 10 minutes, and the supernatant was filtered through a 0.45 μm filter before loading on a refrigerated autosampler for high-performance liquid chromatography (HPLC) analysis. For lactose, which is at much higher concentrations than the other sugars and sugar alcohols , the sample was diluted before rerunning on the HPLC system.
A Dionex HPLC analyzer equipped with a CarboPac MA1 anion-exchange column was used for the separation of the hexose, polyols, and lactose (Dionex, Sunnyvale, CA). The analysis was run isocratically with 500 mmol/L sodium hydroxide for 25 minutes, followed by a step change to 400 mmol/L sodium hydroxide for 20 minutes at ambient temperature. The flow rate was 0.4 mL per hour. Figure 1 presents the chromatogram (upper plot) of sugar standards for the CarboPac MA1 column.
FIG. 1: Chromatograms of standard sugars and polyols for each of the two columns presented. Upper chromatogram depicts peak for xylitol, the internal standard we have used.
A separate aliquot of the supernate was analyzed on a CarboPac PA 10 column for galactosamine and glucosamine concentrations. The system was run isocratically with 18 mmol/L sodium hydroxide at ambient temperature. The flow rate was 0.6 mL per hour. The sodium hydroxide solution was prepared with degassed, deionizer water. All the peaks were quantified by using a pulse amperometric detector (Dionex ED40 Electrochemical Detector) with a gold electrode. The Dionex PeakNet software was used for instrument operation and data analysis. Figure 1 presents the chromatogram (lower plot) of sugar standards for the CarboPac PA10 column.
For purposes of comparison, samples were obtained from four milk formulas used for feeding term infants and from eight milk formulas prepared for preterm infants. A commercially available 1% cow's milk was also analyzed. Differences between groups for breast milk and formula concentrations were tested using an unpaired t -test where appropriate after testing for normality or by a Mann-Whitney U test.
RESULTS
The primary purpose of this study was to determine the concentrations of the free sugars and sugar alcohols in breast milk. It quickly became apparent that there were two distinctly different patterns to the HPLC chromatograms. These are shown in Figure 2 . The more common pattern found in 14 breast milk samples is shown in the lower chromatogram of Figure 2 . The sugars and sugar alcohols are shown with retention times for each compound identical with the standard mix. The data for all sugars and polyols were included in the normal breast milk data. A second pattern is shown in the upper chromatogram of Figure 2 . In these samples, there were other unidentified compounds that separated with retention times very close to those of erythritol, arabitol, sorbitol, mannitol, and mannose, making it impossible to determine their concentrations in these samples. Only the data for the other carbohydrates were included in the calculation of normal breast milk values from these samples. The concentrations of all other sugars and sugar alcohols did not differ between those samples with extra peaks in the chromatograms and those without such peaks. Thus, the concentrations from both sets of breast milk samples have been pooled for those sugars.
FIG. 2: Two patterns encountered in breast milk samples, both term and preterm. Note that in upper chromatogram (marked with asterisk), there are large peaks with same elution times as erythritol, arabitol, sorbitol, and mannitol. These compounds are not present in lower chromatogram.
We also tested whether there were any significant differences between breast milk collected from women who had delivered at 36 weeks gestation or alter (Table 1 ) and those that had delivered preterm (Table 2 ). No differences were found for any sugar or alcohol except lactose. Lactose concentration in term pregnancies was 161.6 ± 4.7 mmol/L and in PRETERM pregnancies was 149.2 ± 3.0, with a P < 0.04. Thus, in Table 3 , the concentrations of the sugars and sugar alcohols are presented separately for each group and as a pooled mean for all sugars except lactose.
TABLE 3: Breast milk
Figure 3 presents a comparison of the mean ± SE of the sugar and polyol concentrations, which are relatively high in breast milk, term formula milk, preterm formula milk, and 1% cow's milk. Glycerol concentrations are higher in breast milk compared with any of the other milks. Myoinositol concentrations are also higher in breast milk compared with term formula milk or to cow's milk. Some preterm formula milk has been supplemented with inositol so that its concentration exceeds that of breast milk. Figure 4 presents the same comparisons of breast milk versus formula for those carbohydrates at relatively low concentrations in all milks.
FIG. 3: Breast milk concentrations are compared with those of term formula, preterm formula, and cow's milk. Preterm formula was subdivided on the basis of whether it had been supplemented with myoinositol. Breast milk data represents combined data of both term and preterm samples. Concentration of glucose in preterm inositol-supplemented formula milk should be multiplied 5 times; lactose in all breast milk and formula should be multiplied 100 times. The data for myoinositol, galactose, and glucose concentrations are presented for comparison with maternal and fetal plasma as well as with term and inositol-fortified preterm formulas in
Figure 5 .
FIG. 4: Same comparison as in Figure 3 is made for those sugars and polyols at relatively low concentrations.
FIG. 5: Maternal and fetal plasma concentrations taken from a previous study (
21 ) compared with breast milk and term formula milk, as well as preterm inositol-supplemented formula milk, for myoinositol, galactose, and glucose. Glucose concentration in preterm formula should be multiplied five times.
The high concentrations of inositol in breast milk and preterm formula milk is evident. Note that galactose and glucose concentrations are quite high in formula milk, whether preterm or term formulas.
DISCUSSION
It is appropriate that the composition of human breast milk has been used as a gold standard for comparison with milk formulas prepared for term infants. It is not the purpose of this report to review all of the previous work directed at the protein and lipid composition of the milk. However, carbohydrates have their own special role to play in nutrition. Lactose is the principal sugar in milk. When it is hydrolyzed, it yields glucose and galactose in equimolar amounts. Glucose intake and metabolism have been studied in some detail because of the frequency of hypoglycemia in newborns. Galactose has also been studied, although not in as much detail. Other free carbohydrates have received little or no attention. Specifically, mannose intake and metabolism have not been the focus of any clinical studies. Recently, we reported that there was a significant umbilical uptake of mannose into the fetal circulation in term normal pregnancies (23,24 24 26
As to the sugar alcohols , myoinositol has received attention largely from studies suggesting a role of inositol in the developing lung (27,28 29 alcohols , including arabitol, sorbitol, ribitol, and mannitol, are present in breast milk as well as in both term and preterm formulas.
The presence of free glucose and galactose in formulas, which are absent in breast milk, probably reflects some lactose hydrolysis during formulation. Given their low concentrations relative to lactose, their presence should have little clinical significance.
The present study clearly demonstrates that many of the nonglucose carbohydrates including the sugar alcohols are present in breast milk. Given that these compounds are also found at increased concentration in human fetal blood compared with maternal, it is not unreasonable to attribute some nutritional significance to their presence. Further studies of their metabolism and absorption are needed to better define their potential importance.
Finally, a comment needs to be made on the extra peaks appearing in some of the breast milk samples. Thus far, we have been unable to identify these compounds. However, they have been found in both term and preterm breast milk. They may be a function of diet, although we could not detect any pattern related to the time of day the milk was collected. But this study was not directed at this issue. It was a rather surprising finding for us. There have been recent reports (16,30
CONCLUSION
This study has analyzed breast milk samples from 33 lactating women with a postnatal age range of 3 to 29 days and a gestational age range from 29 to 41 weeks gestation. The free sugar and sugar alcohol concentrations were determined. No differences were found between term and preterm milk samples. Concentrations of some sugars and alcohols were significantly higher in breast milk than in formula milk (e.g., myoinositol). Some preterm formulas had quite high concentrations of free glucose and galactose. A surprising finding was the presence of additional carbohydrate compounds, as yet unidentified, in approximately 30% of the samples.
REFERENCES
1. Ferreira IM. Quantification of non-protein nitrogen components of infant formulae and follow-up milks: comparison with cows' and human milk.
Br J Nutr 2003;90:127-33.
2. Groneberg DA, Doring F, Theis S, et al. Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein.
Am J Physiol Endocrinol Metab 2002;282:E1172-9.
3. Lonnerdal B. Nutritional and physiologic significance of human milk proteins.
Am J Clin Nutr 2003;77:1537S-43S.
4. Ramirez I, DeSantiago S, Tovar AR, et al. Amino acid intake during lactation and amino acids of plasma and human milk.
Adv Exp Med Biol 2001;501:421.
5. Fewtrell MS, Abbott RA, Kennedy K, et al. Randomized, double-blind trial of long-chain polyunsaturated fatty acid supplementation with fish oil and borage oil in preterm infants.
J Pediatr 2004;144:471-9.
6. Innis SM. Perinatal biochemistry and physiology of long-chain polyunsaturated fatty acids.
J Pediatr 2003;143:S1-8.
7. Mitoulas LR, Gurrin LC, Doherty DA, et al. Infant intake of fatty acids from human milk over the first year of lactation.
Br J Nutr 2003;90:979-86.
8. Morera S, Castellote AI, Jauregui O, et al. Triacylglycerol markers of mature human milk.
Eur J Clin Nutr 2003;57:1621-6.
9. Huisman M, van Beusekom CM, Lanting CI, et al. Triglycerides, fatty acids, sterols, mono- and disaccharides and sugar
alcohols in human milk and current types of infant formula milk.
Eur J Clin Nutr 1996;50:255-60.
10. Mitoulas LR, Kent JC, Cox DB, et al. Variation in fat, lactose and protein in human milk over 24 h and throughout the first year of lactation.
Br J Nutr 2002;88:29-37.
11. Paul VK, Singh M, Srivastava LM, et al. Macronutrient and energy content of breast milk of mothers delivering prematurely.
Indian J Pediatr 1997;64:379-82.
12. Clemens K and Silvia R. Physiology of oligosaccharides in lactating women and breast fed infants.
Adv Exp Med Biol 2000;478:241-50.
13. Coppa GV, Pierani P, Zampini L, et al. Characterization of oligosaccharides in milk and feces of breast-fed infants by high-performance anion-exchange chromatography.
Adv Exp Med Biol 2001;501:307-14.
14. Erney R, Hilty M, Pickering L, et al. Human milk oligosaccharides: a novel method provides insight into human genetics.
Adv Exp Med Biol 2001;501:285-97.
15. La Ferla B, Prosperi D, Lay L, et al. Synthesis of building blocks of human milk oligosaccharides. Fucosylated derivatives of the lacto- and neolacto-series.
Carbohydr Res 2002;337:1333-42.
16. Stahl B, Thurl S, Henker J, et al. Detection of four human milk groups with respect to Lewis-blood-group-dependent oligosaccharides by serologic and chromatographic analysis.
Adv Exp Med Biol 2001;501:299-301.
17. Indyk HE and Woollard DC. Determination of free myo-inositol in milk and infant formula high-performance liquid chromatography.
Analyst 1994;119:397-402.
18. Boehm G, Lidestri M, Casetta P, et al. Supplementation of a bovine milk formula with an oligosaccharide mixture increases counts of faecal bifidobacteria in preterm infants.
Dis Child Fetal Neonatal Arch Ed 2002;86:3-F178.
19. Moro G, Mosca F, Miniello V, et al. Effects of a new mixture of prebiotics on faecal flora and stools in term infants.
Acta Paediatr Suppl 2003;441:77-9.
20. Alton G, Kjaergaard S, Etchison JR, et al. Oral ingestion of mannose elevates blood mannose levels: a first step toward a potential therapy for carbohydrate-deficient glycoprotein syndrome Type I.
Biochem Mol Med 1987;60:127-33.
21. Niehues R, Hasilik M, Alton G. Carbohydrate-deficient glycoprotein syndrome type Ib. Phosphomannose isomerase deficiency and mannose therapy.
J Clin Invest 1998;101:1414-20.
22. Panneerselvam K, Etchison JR, Skovby F, et al. Abnormal metabolism of mannose in families with carbohydrate-deficient glycoprotein syndrome type I.
Biochem Mol Med 1997;61:161-7.
23. Teng C, Tjoa S, Fennessey P, et al. Transplacental carbohydrate and sugar alcohol concentrations and their uptakes in ovine pregnancy.
Exp Biol Med 2002;227:189-95.
24. Brusati V, Jozwik M, Jozwik M, et al. Fetal and maternal non-glucose carbohydrates and polyols in normal human pregnancies at term.
Pediatr Res 2005. In Press.
25. Brusati V, Paolini C, Marconi AM, et al. Non glucose carbohydrates and polyols concentration in normal singleton (AGA) and twin (T) human pregnancies.
J Soc Gynecol Invest 2002;9(Suppl 1).
26. Panneerselvam K, Freeze HH. Mannose enters mammalian cells using a specific transporter that is insensitive to glucose.
J Biol Chem 1996;271:9417-21.
27. Hallman M, Pohjavuori M, Bry K. Inositol supplementation in respiratory distress syndrome.
J Pediatr 1987;110:604.
28. Hallman M, Bry K, Hoppu K, Hoppu K, et al. Inositol supplementation in premature infants with respiratory distress syndrome.
N Engl J Med 1992;326:1285-7.
29. Groenen P, Peer P, Wevers R, et al. Maternal myo-inositol, glucose, and zinc status is associated with the risk of offspring with spina bifida.
Am J Obstet Gynecol 2003;189:1713-9.
30. Erney R, Hilty M, Pickering L, et al. Human milk oligosaccharides: a novel method provides insight into human genetics.
Adv Exp Med Biol 2001;501:285-97.
