Article pubs.acs.org/JAFC
Qualitative and Quantitative Analyses of Glycogen in Human Milk Hiroko Matsui-Yatsuhashi,*,† Takashi Furuyashiki,† Hiroki Takata,† Miyuki Ishida,† Hiroko Takumi,† Ryo Kakutani,† Hiroshi Kamasaka,† Saeko Nagao,‡ Junko Hirose,§ and Takashi Kuriki† †
Institute of Health Sciences, Ezaki Glico Company, Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan Nagao Maternity Hospital, Terado-cho, Muko-shi, Kyoto 617-0002, Japan § Department of Food Science and Nutrition, School of Human Cultures, University of Shiga Prefecture, 2500 Hassaka-cho, Hikone-shi, Shiga 522-8533, Japan ‡
ABSTRACT: Identification as well as a detailed analysis of glycogen in human milk has not been shown yet. The present study confirmed that glycogen is contained in human milk by qualitative and quantitative analyses. High-performance anion exchange chromatography (HPAEC) and high-performance size exclusion chromatography with a multiangle laser light scattering detector (HPSEC-MALLS) were used for qualitative analysis of glycogen in human milk. Quantitative analysis was carried out by using samples obtained from the individual milks. The result revealed that the concentration of human milk glycogen varied depending on the mother’s conditionsuch as the period postpartum and inflammation. The amounts of glycogen in human milk collected at 0 and 1−2 months postpartum were higher than in milk collected at 3−14 months postpartum. In the milk from mothers with severe mastitis, the concentration of glycogen was about 40 times higher than that in normal milk. KEYWORDS: glycogen, human milk, period postpartum, inflammation, enzymatic treatment
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INTRODUCTION
Recently it has been reported that glycogen extracted from scallops and oysters shows immunomodulating activities.20 Kakutani et al. showed that orally administered glycogen enhances the antitumor as well as natural killer activity in mice.21 It has been suggested that the effects of the immunomodulatory activities of glycogen might be dependent on its molecular size.21,22 Furthermore, it has been reported that glycogen activates immunocytes such as macrophages through Toll-like receptor 2.23 The presence of glycogen in bovine milk has been reported; however, it remains unclear whether glycogen is contained in human milk or not. In 1965, Cecil et al. found that glycogen was involved in bovine milk and that mastitis resulted in a significant increase in glycogen in milk.24 Naidu et al. have also reported that leukocytes extracted from bovine milk contain glycogen.25 It has been suggested that glucose supplied from glycogen in the white blood cells is used as an energy source to prevent bacterial infection.25 Arthur et al. has suggested that glucose does not limit the rate of lactose synthesis in women during lactation.26 If human milk contains glycogen, there would be important implications, especially for breast-fed children. Newborns and infants could acquire immune activities by taking glycogenas well as other immunity-related molecules contained in human milkfrom birth until the end of the lactation period. In this study, we identified glycogen in human milk by three qualitative methods. In addition, we quantified glycogen and showed that the glycogen content is changed by the condition of the milk.
Human milk produced in the mammary gland is the first and best food for infants from birth throughout the defined breastfeeding period. Human milk contains a wide range of nutrients such as proteins, lipids, and carbohydrates. These nutrients are generally known to promote growth and develop the infant immune system.1,2 Human milk involves a high abundance of carbohydrates approximately 7%. Lactose, the major component of carbohydrates, comprises approximately 6% of human milk, and the remainder is considered to be various types of milk oligosaccharides.3−7 Lactose, the source of galactose and glucose, is known as an important nutrient for the growth of infantsthe osmotic agent that also induces innate immunity.8 More than 100 oligosaccharides have been identified and characterized from human milk to date. Some of them have important functions such as antipathogenic, immunomodulatory, and prebiotic activities.9−11 Other oligosaccharides have been reported to prevent the invasion of pathogens in the developing intestinal tract of newborns.12,13 They also reinforce the mucosal immune system by strengthening a tight junction and by activating lymphoid elements.14 Glycogen, consisting of glycosidic linkages of glucose with high molecular weight, is the primary energy storage of polysaccharides in animals.15,16 Most glycogens in mammals are stored in the muscles and liver, and some are ubiquitously stored in various organs such as the brain, skin, kidney, heart, and blood leukocytes.17,18 Glycogen in muscles is hydrolyzed to form glucose, which provides energy for muscle contraction. Glycogen in the liver acts to release glucose to the bloodstream for regulation of the blood sugar level.19 In addition, glycogen is also known to be present in human dietary foods such as oysters, scallops, sea mussels, and sweet corn. © 2017 American Chemical Society
Received: Revised: Accepted: Published: 1314
August 16, 2016 February 1, 2017 February 3, 2017 February 3, 2017 DOI: 10.1021/acs.jafc.6b03644 J. Agric. Food Chem. 2017, 65, 1314−1319
Article
Journal of Agricultural and Food Chemistry
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Quantitative Analysis of Glycogen. Quantitative analysis was carried out for the milk samples obtained from 63 mothers: normal (n = 42) and mastitis milk (n = 21). On the other hand, we collected milk from six mothers whose one breast was normal but the other side was infected to compare the normal and mastitis milk of the same participants. Each sample was obtained by the procedure described under Extraction of Glycogen from Human Milk. The milk sample was treated with α-amylase and isoamylase at 37 °C for 2 h, followed by αglucoamylase for a further 18 h as described above, and then the concentration of glycogen was determined as the glucose equivalent using a glucose measurement kit (Glucose CII Test Wako, Wako). We confirmed that the data presented in this study were on the linear region of our calibration curve and were beyond the limit of quantitation (1 μg/mL). Statistical Analysis. All experimental data are presented as the mean ± SD. Statistical analysis was carried out using the appropriate tests, such as the unpaired or paired t test (Bonferroni correction).36 A difference between groups of P < 0.05 was considered significant.
MATERIALS AND METHODS
Materials. Bovine liver glycogen and mussel glycogen were purchased from Sigma (Osaka, Japan). Enzymatically synthesized glycogen (ESG) produced from starch according to the method reported previously27−30 was also used as a standard. The properties of ESG are identical to those of natural glycogen as reported previously.29,30 α-Amylase from porcine pancreas (type 1A) was purchased from Sigma. Isoamylase and pullulanase were obtained from Hayashibara Co., Ltd., Okayama, Japan. Glucoamylase was from Toyobo Co., Ltd., Osaka, Japan. All other chemicals used in this study were of analytical grade and obtained from Wako Pure Chemical Industries (Osaka, Japan). Human Milk. Milk was collected by using breast massage treatments of the participants who came to Nagao Clinic in Kansai region, Japan, between July 2013 and February 2014, 0−25 months after their deliveries. All participants were given informed consents for this study. It is important to consider the influence of glucose involved in daily food to the concentration of glycogen in human milk. However, it was difficult for the midwives to restrict food intake of the participants in order not to give any stress. All of the participants were advised to take a well-balanced diet in their daily lives. This study was approved by the Ethics Committee of the University of Shiga Prefecture (No. 210-3) and followed the Declaration of Helsinki. Informed consent was obtained from all milk donors. Extraction of Glycogen from Human Milk. The expressed milk from the participants was immediately heated at 100 °C for 10 min to prevent degradation of glycogen. Then the milk was rapidly frozen and kept at −20 °C until the analyses. Because of the extremely low concentration of glycogen compared to that of lactose in human milk, we carefully collected glycogen in human milk by using the modified extraction method of Carroll et al.31 and Futagawa et al.32 Milk was defrosted and centrifuged for 10 min at 10000g. The upper cream layer of the milk was excluded. To remove proteins from the milk, a sulfosalicylic acid solution was added to the milk at a final concentration of 5%, and the solution was then centrifuged for 10 min at 10000g again. After the centrifugation, the supernatant was collected. The glycogen was then precipitated by adding ethanol to the supernatant at a final concentration of 50%, and the components were well mixed and centrifuged twice at 7500g for 15 min to obtain glycogen. Through these steps, glycogen could be separated from other low molecular weight sugars such as lactose, glucose, and oligosaccharides. The precipitate was dissolved in distilled water for the following analyses. Qualitative Analysis of Glycogen. Qualitative analysis of glycogen was carried out for 600 mL of milk collected from 150 participants. The milk was randomly selected within 25 months of parturition. The milk was concentrated by lyophilization before the analysis. Bovine liver glycogen, mussel glycogen, and ESG were used as standards for the qualitative analysis. High-performance size exclusion chromatography (HPSEC) with a multiangle laser light scattering detector (MALLS, DAWN HELEOS, Wyatt Technology Corp., Santa Barbara, CA, USA), and a differential refractive index detector (RI)28,30 was used to identify glycogen. High-performance anion exchange chromatography (HPAEC, DIONEX ICS-300, Thermo Fisher Scientific K.K.) was also used to identify the chain length of glycogen. The procedure of the qualitative analysis of glycogen is described briefly, as follows. The sample was analyzed using HPSECMALLS-RI after reaction of the sample with or without treatment of enzymesα-amylase (18 U/mL) and isoamylase (74 U/mL)at 37 °C for 2 h, followed by α-glucoamylase (50 U/mL) for 18 h. The reaction was carried out in 50 mM acetate buffer at pH 5.4. Enzyme treatment of glycogen was carried out as described by Takata et al. and Rani et al.33,34 The sample was then eluted through the column (Shodex OH-pak SB-806 M HQ, Shoko Co., Ltd., Tokyo, Japan) at 40 °C with 0.1 M NaNO3 at a flow rate of 1 mL/min. HPAEC was carried out by using a modified method of Takata et al. after treatment of the sample with isoamylase and pullulanase (2 U/ mL) for 18 h at 37 °C.33 These enzymes hydrolyze the α-1,6-linkage of glycogen selectively.35
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RESULTS Qualitative Analysis of Glycogen in Milk. HPSECMALLS-RI Analysis. The sample was extracted from 600 mL of human milk as described above and concentrated by lyophilization. Milk was obtained from total of 150 participants randomly selected within 25 months of their parturition. A portion of the samples was subjected to HPSEC-MALLS-RI analysis. This analysis showed that the sample contained macromolecules. To compare the elution time of macromolecules in the analysis, glycogen aqueous solutions [0.01% (w/v) or 0.1% (w/v)] extracted from bovine liver, mussel, or aqueous solutions of ESG [0.1% (w/v)] were used as standards. The HPSEC-MALLS chromatograms show glycogen obtained from bovine liver (Figure 1A), mussel (Figure 1B), and
Figure 1. HPSEC-MALLS chromatograms of bovine liver glycogen (A), mussel glycogen (B), ESG (C), and the sample obtained from human milk (D). Aqueous glycogen solutions of (A) [0.01% (w/v)], (B) [0.1% (w/v)], and (C) [0.1% (w/v)] were eluted at 8.0−9.5 min.
ESG (Figure 1C). All glycogen was eluted for 8.0−9.5 min regardless of its origins. The average molecular weights of bovine liver glycogen, mussel glycogen, and ESG were 2.69 × 106, 1.28 × 106, 3.79 × 106, respectively (Figure 1A−C). In Figure 1D, the chromatogram of the sample obtained from human milk shows an elution time of around 8.0−9.5 min, which is the same as that in the case of other glycogens. The average molecular weight of the sample was estimated to be 3.91 × 106. These results clearly indicated that human milk contains macromolecules with molecular sizes similar to those of bovine liver glycogen, mussel glycogen, and ESG. 1315
DOI: 10.1021/acs.jafc.6b03644 J. Agric. Food Chem. 2017, 65, 1314−1319
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Journal of Agricultural and Food Chemistry Enzymatic Treatment. To determine the nature of the macromolecule obtained from human milk, we next carried out enzyme treatment followed by an HPSEC-MALLS-RI analysis. Carbohydrates absorb neither UV nor visible light, so the response of RI is proportional to the concentration. The concentration of macromolecule before and after the reaction with α-amylase, isoamylase, and α-glucoamylase was measured using an RI detector. These enzymes act on the specific linkages for α-1,4 and α-1,6 bonds so that the reaction could result in the degradation from α-D-glucan to glucoses.37,38 The result of the analysis revealed that the sample without enzyme treatment was eluted markedly for 8.0−9.2 min, and the concentration was 49.5 μg/mL (Figure 2a). In contrast, the
of bovine liver glycogen (Figure 3a). On the other hand, the unit-chain peaks of α-1,4-linkages without the treatment of isoamylase and pullulanase were not found in either bovine liver glycogen or the sample obtained from human milk (data not shown). These results confirmed that human milk contains glycogen. Quantitative Analysis of Glycogen in Milk. Glycogen Concentration in Normal Human Milk. The glycogen concentration in milk from each mother was determined as described under Materials and Methods. Milk obtained from participants with no clinical sign of mastitis was divided into three groups, depending on the period postpartum. Because the difference in the constituents between colostrum and mature milk is well-known, according to the period of postpartum, we divided the milk samples into three groups: (I) within 1 month postpartum; (II) between 1 and 2 months postpartum; (III) between 3 and 14 months postpartrum. The results showed that the glycogen concentrations in groups I and II were 3.86 ± 2.86 μg/mL milk (n = 15) and 3.75 ± 3.51 μg/mL milk (n = 15), respectively. However, the concentration of glycogen in group III was significantly decreased to 1.72 ± 0.71 μg/mL milk (n = 12) (Figure 4). The amount of glycogen was quite
Figure 2. HPSEC-MALLS-RI chromatogram of the sample obtained from human milk before and after enzymatic treatment. The peak of human milk eluted in 8.0−9.2 min (a) disappeared after treatment with enzymes that specifically break the linkages of α-1,4 and α-1,6 bonds (b).
peak of the sample disappeared after enzyme treatment (Figure 2b). Similar results were obtained from bovine liver glycogen, mussel glycogen, and ESG (data not shown). From these results, it is suggested that the macromolecule obtained from human milk is α-D-glucan having α-1,4- or α-1,6-linkages. HPAEC Analysis. Next, we analyzed the chain length distribution of the sample obtained from human milk after debranching with isoamylase and pullulanase using the HPAEC method. Isoamylase and pullulanase specifically hydrolyzed α-1, 6 glucosidic linkages to liberate α-1,4-linked glucose chains from glycogen, and the HPAEC system can detect α-1,4-linked chains having 1−40 glucose units. That of glycogen extracted from bovine liver was also detected as a standard. Figure 3 shows the chromatogram of unit-chain distributions of bovine liver glycogen and the sample obtained from human milk after the treatment with the enzymes. The distribution of the sample obtained from human milk (Figure 3b) was very similar to that
Figure 4. Concentration of glycogen in normal milk obtained from healthy mothers. Normal human milk was divided into three groups depending on the postpartum period: (I) within 1 month; (II) between 1 and 2 months; and (III) between 3 and 14 months after childbirth. All values are means ± SDs, n = 12−15. A significant difference between groups I and III was determined by the unpaired t test (Bonferroni correction).
small, but detected in all samples obtained from normal milk. These results showed that the glycogen concentration is higher in the early period of lactation than in the late period of lactation. Glycogen Concentration in Inflammatory Human Milk. Next, we investigated the concentration of glycogen in human inflammatory milk. It is known that the concentration of many componentssuch as proteinsin human inflammatory milk is extremely different from that in human normal milk. The inflammatory milk was divided into two groups depending on the degree of inflammation. Level I was the milk extracted from participants with stiff, painful, or swollen breasts who did not have an attack of fever. Level II was the milk extracted from participants with a fever of >38 °C for whom clinically serious mastitis was suspected. Normal milk obtained from healthy mothers without inflammation was used as a control. The results showed that the mean concentration of glycogen in normal milk was 3.21 ± 2.84 μg/mL milk (n = 42), whereas those of levels I and II inflammatory groups were found to be 14.0 ± 24.2 μg/mL (n = 17) and 127 ± 109 μg/mL (n = 4), respectively (Figure 5a). These data indicate that the glycogen concentration in human milk is increased by inflammation.
Figure 3. HPAEC chromatograms of bovine liver glycogen (a) and the sample obtained from human milk (b). The numbers above peaks represent the number of glucose units in α-1,4-linked chains liberated by hydrolyses of the α-1,6-linkages by treatment with specific enzymes. 1316
DOI: 10.1021/acs.jafc.6b03644 J. Agric. Food Chem. 2017, 65, 1314−1319
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Journal of Agricultural and Food Chemistry
milk.42 Because the inflammation is strongly related to leukocytes, glycogen and leukocytes could increase in parallel when the participants have clinical signs such as mastitis. Thus, we consider that the glycogen in human milk could originate mainly from leukocytes. It has been reported that there is no significant relationship between the number of leukocytes in bovine milk and that in the blood.25 The observation of glycogen particles in human mammary glands has previously been reported.43 Therefore, it is suggested that the portion of glycogen produced in the mammary glands transferred directly to milk when the glycogen originates from leukocytes. In fact, factors such as transforming growth factor beta (TGF-β) which is present mainly at the early stages of lactation of human milk and shows immune activityhave been reported to move directly from the mammary gland to human milk.44 However, it remains uncertain whether glycogen in human milk is originated from leukocyte or not. A difference has been reported in the enterobacterial fIora between breast- and formula-milk fed infants during the first year of life. 45 It is considered that >100 kinds of oligosaccharides, comprising about 1% of human milk, have some immune activity in human milk. It has also been reported that the biological activity of structurally larger glycans is clinically significant.46,47 In addition, the indigestible oligosaccharides have been shown to exhibit prebiotic activity.48 In the present study, we found that the concentration of glycogen within 1 month postpartum in normal milk was 3.86 μg/mL. Although the concentration appears to be lower than that of other sugars in human milk, we could say that a portion of the glycogen may reach the intestine or colon because the molecular weight of glycogen is too high to completely be digested. Recently, we reported that glycogen is harder to digest by digestive enzyme than starch and works as a prebiotic.33,49 The digestive enzymes of babies and infantssuch as αamylaseare not yet developed, so a higher level of glycogen particles is expected to reach the intestine of infants more than that of adults. Thus, it could be suggested that glycogen is capable of showing some effect on the intestinal tracts of infants.50 Kakutani et al. have reported that glycogen not only conducts signal transduction in the epithelial cells in the gut but also affects the immune activities in the intestines.21,23 Other studies have shown that glycogen-administered mice have significantly stronger immune activity than those to whom water was administered.21,51,52 Therefore, glycogen in milk may possibly confer effects such as immune function and prebiotics for infants, although the amount of glycogen in milk is very small. Further studies should clarify the role of glycogen in milk, such as that in the intestinal tracts of infants. Thereafter, it would be worthwhile to assess the significance of the levels of glycogen in human milk. In conclusion, our results demonstrate the existence of glycogen in human milk by chromatographic analyses. The concentration of glycogen in normal human milk was 3.21 ± 2.84 μg/mL milk. We also confirmed that the concentration of glycogen was higher in the earlier lactation period between 0 and 2 months compared to that in the later lactation period of between 3 and 14 months. Furthermore, a positive correlation between the concentration of glycogen and the level of inflammation was indicated in mastitis milk. It is therefore critically important to study the role of glycogenfor instance, in the immune activities of infantsin human milk.
Figure 5. (a) Concentration of glycogen obtained from normal or mastitis human milk. All values are means ± SDs, n = 4−42. (b) The relative value of glycogen concentration in normal and mastitis milk was obtained from the same participants. All values are means ± SDs, n = 6.
We next performed further analysis to clarify the positive correlation between glycogen concentration and inflammation. Participants with clinical signs in only one breast were selected. Milk samples were expressed from both breasts of the participants. Their inflammatory level was I or II. We analyzed the glycogen concentrations in the milk obtained from the normal breast and that from the inflammed breast. The results showed that the concentration of glycogen in the normal side was 1.55 ± 0.96 μg/mL milk, whereas that of the inflammed side was 28.6 ± 41.3 μg/mL (n = 6). This revealed that the relative value of the glycogen concentration of the inflammed breast was increased to 12.3 times that of the normal breast, regardless of individual differences (Figure 5b). These results suggested that there is a positive correlation between glycogen and inflammation in human milk.
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DISCUSSION We identified glycogen itself in human milk for the first time to our best knowledge. Furthermore, we measured the concentration of glycogen in human milk by the hydrolyzation of it to glucose with specific enzymes. The average concentration of glycogen throughout lactation in normal human milk was 3.21 μg/mL milk. The quantitative analysis of glycogen in bovine milk has been studied by the Anthrone reagent method.24,39−41 The concentration of glycogen in normal bovine milk has been determined to be 13 μg/mL by the Anthrone reaction.24 The difference may arise not only from the difference in nutritional composition derived from species (such as the different ratios of carbohydrate) but also from the difference in extraction and quantitative analysis methods. The glycogen concentration of bovine milk exhibiting clinical symptoms has been reported to be 125 μg/mL,24 which is about 9.6 times higher than that in normal bovine milk. Our results showed that the concentration of glycogen in human mastitis milk was approximately 4−40 times higher than that in normal milk. Thus, it is suggested that the concentration of glycogen is strongly correlated with the degree of mastitis. In these results, the mean concentration of glycogen in mastitis milk was obviously high; however, we could not detect any significant differences in statistics between groups. We consider that the lack of significant differences is due to the large variation and the small number of samples of mastitis milk. When mothers suffer mastitis, the number of leukocytes is known to increase in the blood, mammary glands, and human 1317
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AUTHOR INFORMATION
Corresponding Author
*(H.M.-Y.) Phone: +81-6-6477-8425. Fax: +81-6-6477-8362. E-mail:
[email protected]. ORCID
Hiroko Matsui-Yatsuhashi: 0000-0002-5231-8171 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to the mothers who voluntarily participated in our study and provided samples of their milk.
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ABBREVIATIONS USED ESG, enzymatically synthesized glycogen; HPSEC, highperformance size exclusion chromatography; MALLS, multiangle laser light scattering detector; RI, differential refractive index detector; HPAEC, high-performance anion exchange chromatography
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REFERENCES
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DOI: 10.1021/acs.jafc.6b03644 J. Agric. Food Chem. 2017, 65, 1314−1319
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DOI: 10.1021/acs.jafc.6b03644 J. Agric. Food Chem. 2017, 65, 1314−1319
