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Home , Adenosine monophosphate, Cytidine diphosphate, Cytidine monophosphate, Guanosine, Guanosine monophosphate, Nucleoside

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provided by Elsevier - Publisher Connector Pediatrics and Neonatology (2011) 52,93e97

available at www.sciencedirect.com

journal homepage: http://www.pediatr-neonatol.com

ORIGINAL ARTICLE Profile of and in Taiwanese Human Milk

Kuo-Yu Liao a,b, Tzee-Chung Wu b,c,*, Ching-Feng Huang d, Chih-Chung Lin e, I-Fei Huang f, Lite Wu b

a Department of Pediatrics, Taoyuan Armed Forces General Hospital, Taoyuan, Taiwan b Children’s Medical Center, Taipei Veterans General Hospital, Taipei, Taiwan c National Yang Ming University School of Medicine, Taipei, Taiwan d Department of Pediatrics, Tri-Service General hospital and National Defense Medical Center, Taipei, Taiwan e Department of Pediatrics, Taichung Veterans General Hospital, Taichung, Taiwan f Department of Pediatrics, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

Received Mar 29, 2010; received in revised form Jun 18, 2010; accepted Jul 5, 2010

Key Words Background: Human milkeborne nucleotides and their related metabolic products have been human milk reported to have important physiological roles in breast-fed infants. The purpose of this study ; was to measure the concentrations of free nucleotides and nucleosides in human milk from Tai- ; wanese women. Taiwanese Methods: A total of 24 individual milk specimens were collected from women in Taipei and Kaohsiung, at four stages of lactation. Vegetarian or non-vegetarian dietary patterns were re- corded. The samples were analyzed for nucleotides and nucleosides by high-performance liquid chromatography. Results: Themean(Æstandard deviation) free nucleotide and nucleoside concentrations in Taiwanese human milk were 213.13 Æ 76.26 mmol/L and 16.38 Æ 7.11 mmol/L. The predominant nucleotide was diphosphate for almost all samples, regardless of the location, stage of lactation, or dietary status of the subjects. Overall, the mean concentrations of , , monophosphate, monophosphate, monophosphate, and monophosphate, in milk samples were 129.86 mmol/ L, 49.10 mmol/L, 5.60 mmol/L, 0.82 mmol/L, 2.96 mmol/L, and 25.25 mmol/L, respectively (equivalent to 61.0%, 23.1%, 2.6%, 0.4%, 1.0%, and 11.9% of free nucleotide composition). In free nucleosides, cytidine and uridine were predominant during all stages of lactation. The average concentrations of cytidine, uridine, adenosine, guanosine, and inosine, in milk samples were 9.25 mmol/L, 6.33 mmol/L, 0.18 mmol/L, 0.36 mmol/L, and 0.23 mmol/L,

* Corresponding author. Division of Gastroenterology and Nutrition, Children’s Medical Center, Taipei Veterans General Hospital, # 201, Shih-Pai Rd, Section 2, Taipei 11217, Taiwan. E-mail address: [email protected] (T.-C. Wu).

1875-9572/$36 Copyright ª 2011, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights reserved. doi:10.1016/j.pedneo.2011.02.012 94 K.-Y. Liao et al

respectively (equivalent to 56.5%, 38.7%, 1.1%, 2.2%, and 1.4% of free nucleoside composition). Comparing vegetarian and non-vegetarian statuses, it was found that the total free nucleotide concentration was high in the vegetarian group (p Z 0.037). Conclusion: Our data showed a wide range of concentrations of individual nucleotides and nucleosides in Taiwanese human milk. Unique dietary status could affect the nucleotide and nucleoside levels in human milk, especially the nucleotides in our study. However, the mech- anism of modulation of nucleotide and nucleoside levels in human milk is not clear. Copyright ª 2011, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. All rights reserved.

1. Introduction Kaohsiung. Eight subjects were vegetarians. The milk samples were categorized into one of four lactation stages: Approximately 15e30% of the total nitrogen in human milk Stage 1 of lactation was first week postpartum, Stage 2 was is nonprotein nitrogen and is made up of substances, first month postpartum, Stage 3 was second month post- including urea, free amino acids, nucleic acids, nucleo- partum, and Stage 4 was third to ninth month postpartum. tides, peptides, creatine, , , , Written informed consent was obtained from each subject and polyamines.1 At least 13 separate nucleotides have before enrollment. been found, accounting for 2e5% of the total nonprotein nitrogen.2 Nucleotides are made up of three components: 2.2. Sample preparation for high-performance (1) a nitrogenous heterocyclic base derivative of either liquid chromatography analysis a or ; (2) a ( or deoxyri- bose); and (3) one to three groups.3 Nucleotides The frozen milk specimens were thawed at room temper- and their related metabolic products play an essential role ature, and 5 mL of each specimen was mixed with 10 mL of in cell replication and . They store cellular 10% (w/v) trichloroacetic acid (TCA) solution. After allow- energy, mediate intracellular metabolic processes, and ing the mixture to stand on ice for 30 minutes, it was support metabolism and synthesis. Their functions centrifuged at 30,000 Â g for 15 minutes. The aqueous layer may be particularly important in rapidly growing infants.4 was recovered, and the residual cream and precipitate Nucleotide requirements are met by were washed twice with 5 mL of 7.5% (w/v) TCA solution. and by a salvage pathway that recovers metabolized The aqueous layers were combined, and TCA was removed nucleotides and nucleosides originating from dietary sour- by adding 20 mL of . The solution was then ces and intermediary metabolism.5 Polymeric forms of lyophilized and stored at À30C until analysis. Just before nucleotides (DNA and RNA) are generally the primary die- analysis, the lyophilized samples were dissolved in water tary sources of nucleotides. RNA and DNA are digested by and then brought to a volume of 1.5 mL with water. After ribonucleases and deoxyribonucleases to nucleotides and filtering through a 0.45-mm Chromatodisk (Kurabo, Osaka, then broken down by to nucleosides, the Japan), the nucleotides and nucleosides were analyzed by preferred form for absorption in the small intestine. high-performance liquid chromatography. It is estimated that an infant consuming human milk as a primary nutrition source would ingest 1.4e2.1 mg of 2.3. High-performance liquid chromatography 2 nucleotide nitrogen per day. Human milk is a rich source of analysis nucleotides for young infants, whereas cow’s milk lacks in 4 nucleotide content. Our study was designed to determine The nucleotides and nucleosides were analyzed using a the concentrations of free nucleotides and nucleosides in Hewlett Packard LC1050 system (Palo Alto, California, USA) Taiwanese human milk. We also evaluated the relationship equipped with a Capcellpak C18, type AG (Ø4.6 Â 500 mm; among diet, lactation stage or geographical area, and Shiseido, Tokyo, Japan) at 20C. Twenty-five millimolar nucleotide and nucleoside concentration. tetrabutylammonium hydrogen sulfated50 mM potassium phosphate (pH 3.5) was used as a solvent, and the flow rate 2. Methods was 0.75 mL/min. Nucleotides and nucleosides were detected by the absorbance at 254 nm. After each analysis, the column 2.1. Human milk specimens was washed with methanol for 15 minutes and equilibrated for 1 hour with initial conditions. Appropriate standards were The analysis of free nucleotides and nucleosides was done on used to establish the retention time of each nucleotide and 24 individual milk specimens. Each milk specimen containing nucleoside. 30 mL of was collected at home and was imme- diately put in the freezer, with temperature less than À4C. 2.4. Chemicals Then, the specimen was sent to our laboratory and put into another freezer at less than À70C. The milk specimens were Nucleotides and nucleosides for the standards were classified by the subject’s lactation stage, geographical area, purchased from Yamasa Co. (Chiba, Japan). All other chem- and whether they were vegetarian or not. Twelve specimens icals were purchased from Wako Pure Chemical Industries Ltd were collected from Taipei and another 12 specimens from (Osaka, Japan). Nucleotides and nucleosides in human milk 95

2.5. Statistical methods Table 2 Free nucleotide and nucleoside concentrations m Æ m Data were analyzed using SPSS 12.0 (SPSS Inc., Chicago, IL, Range ( mol/L) Mean SD ( mol/L) USA). Descriptive data were reported as mean Æ standard CMP 6.60e114.20 49.10 Æ 30.75 deviation. Comparison of parameters between the groups CDP 69.50e418.70 129.86 Æ 81.10 was performed with Mann-Whitney U test. A p value less UMP 1.00e26.80 5.60 Æ 5.75 than 0.05 was considered to be statistically significant. AMP 0.40e8.20 2.96 Æ 2.30 GMP 0e3.40 0.82 Æ 0.75 3. Results IMP 21.80e56.20 25.25 Æ 6.87 Total nucleotides 114.00e464.10 213.15 Æ 73.26 Cytidine 2.90e24.70 9.25 Æ 5.26 Twenty-four human milk samples were collected from women Uridine 2.20e15.10 6.33 Æ 3.74 in Taipei and Kaohsiung. Table 1 expresses the demographics Adenosine 0e1.20 0.18 Æ 0.28 of subjects who provided the milk samples. Table 2 summa- Guanosine 0.10e1.10 0.36 Æ 0.24 rizes all free nucleotide and nucleoside concentration data Inosine 0e0.90 0.23 Æ 0.23 showing the range, mean, and standard deviation. The Total nucleosides 0.65e3.05 16.38 Æ 7.11 predominant nucleotide was cytidine diphosphate (CDP) in almost all samples, regardless of the stage of lactation, AMP Z ; CDP Z cytidine diphos- inhabitation, or dietary status. Overall, the mean concen- phate; CMP Z cytidine monophosphate; GMP Z guanosine trations of CDP, cytidine monophosphate (CMP), ; IMP Z inosine monophosphate; SD Z standard monophosphate, , adenosine deviation; UMP Z uridine monophosphate. monophosphate, and inosine monophosphate (IMP) in the milk samples were 129.86 mmol/L, 49.10 mmol/L, 5.60 mmol/L, 0.82 mmol/L, 2.96 mmol/L, and 25.25 mmol/L, respectively (equivalent to 61.0%, 23.1%, 2.6%, 0.4%, 1.0%, and 11.9%, was no noticeable trend for free nucleoside concentrations at respectively, of free nucleotide). any stage of lactation. The total free nucleotide concentration in Stage 1 was Comparing vegetarian and non-vegetarian statuses, the 291.86 mmol/L; in Stage 2, 205.17 mmol/L; in Stage 3, total free nucleotide concentration was high in the vege- 200.39 mmol/L; and in Stage 4, 198.79 mmol/L. The average tarian group (p Z 0.037), but the total free nucleoside free nucleotide concentration was 213.15 Æ 76.26 mmol/L. concentration was not statistically different (p Z 0.076). The total free nucleotide concentrations were slightly higher However, it is not clear whether specific intake of the in Stage 1 than those in more mature milk samples, but there donors could cause differences in nucleotide and nucleo- was no noticeable trend over the course of lactation. side concentrations in milk because the number of samples For free nucleosides, cytidine and uridine were predomi- in this study was quite small. A similar result was found nant during all stages of lactation. Overall, the average when comparing free nucleotide and free nucleoside concentrations of cytidine, uridine, adenosine, guanosine, concentrations between Taipei and Kaohsiung (p Z 0.603 and inosine in milk samples were 9.25 mmol/L, 6.33 mmol/L, and 0.356). 0.18 mmol/L, 0.36 mmol/L, and 0.23 mmol/L, respectively (equivalent to 56.5%, 38.7%, 1.1%, 2.2%, and 1.4%, respec- 4. Discussion tively, of total free nucleoside). The total free nucleoside concentration in Stage 1 was In human milk, up to 30% of total nitrogen content is nonpro- m m 20.55 mol/L; in Stage 2, 16.36 mol/L; in Stage 3, tein nitrogen. Free nucleotides account for 0.4e0.6% m m 13.61 mol/L; and in Stage 4, 19.62 mol/L. The average free of nonprotein nitrogen and are present in a concentration of Æ m nucleoside concentration was 16.36 7.11 mol/L. There 50e150 mmol/L according to various reports.2 Our report of free nucleotides was 213.15 mmol/L(72.26mg/Lcorrectedfor recovery) and that of free nucleosides was 16.38 mmol/L Table 1 Demographics of subjects who provided milk (4.01 mg/L corrected for recovery). Leach et al6 and Tressler samples et al7 had reported the total potential available nucleoside Stage of Stage 1* Stage 2 Stage 3 Stage 4 concentrations in human milk from American, European, and lactation (N Z 3) (N Z 10) (N Z 8) (N Z 3) Southeast Asian women. Free nucleotides and nucleosides consisted of 36e39.9% and 6.6e8% of total potential available City nucleoside, respectively. The concentrations of free nucleo- Taipei 0 6 3 3 tides were 57.96 mmol/L, 68.04 mmol/L, and 80.99 mmol/L, Kaoshiung 3 4 5 0 and those of free nucleosides were 12.88 mmol/L, 15.12 mmol/ Vegetarian L, and 13.39 mmol/L, in human milk from American, European, 8 Yes 1 4 2 1 and Southeast Asian women (Figure 1). Sagawara et al No 2 6 6 2 analyzed by a method similar to ours, who reported the concentration of free nucleotides and nucleosides at different N Z number of cases. stages of lactation, in summer and winter, and in various areas, * Stage 1, first week postpartum; Stage 2, first month post- from human milk in Japanese women. They did not report the partum; Stage 3, second month postpartum; Stage 4, third to average data, but the concentration of total free nucleotides ninth month postpartum. and nucleosides were wide in the range of 8.4e115.0 mmol/L 96 K.-Y. Liao et al

(µmol/L) Free Nucleotides Taiwan and Japan was the IMP content. IMP constituted 250 only a small percentage of nucleotides in Japan; however, it constituted about 15% of nucleotides in Taiwan. In our 200 study, the range of IMP values was 21.8e56.2 mmol/L, with an average of 25.2 mmol/L. In another study, Janas and 150 Picciano2 measured IMP at various stages of lactation, and reported that the range of values was 1.5e18.4 mmol/L, 100 with an average of 6.5 mmol/L. Opposite results reported by Leach et al6 and Tressler et al7 revealed only low levels of 50 IMP in colostrums and were not able to detect it in more mature milk.6,7 0 The data reported by Leach et al6 showed that the Taiwan US Europe Southeast Asia concentrations of nucleosides in the colostrum stage were (µmol/L) Free Nucleosides slightly lower than those of other lactation stages, but our 25 data did not reveal this phenomenon. Cytidine was consis- tently the nucleoside in the greatest concentration in both 20 studies. However, the concentration of nucleosides tends to decrease gradually with advancing lactation according to 15 Schlimme et al.10 They also reported that the variations of cytidine and uridine in human milk were twofold and those 10 of adenosine and guanosine were up to fourfold greater than those in bovine milk. 5 According to the reports discussed earlier, this sample- to-sample variability appears to be a property of human 0 milk. We presume that the nucleotides and nucleosides in Taiwan US Europe Southeast Asia human milk are secreted in response to a nutritional Figure 1 Concentration of (A) free nucleotides and (B) free demand of the infant or result from the metabolic events nucleosides in human milk from Taiwan, the United States, within the mammary secretory cells. However, the mech- Europe, and Southeast Asia. anism of modulation of the nucleotide and nucleoside levels in human milk is still unknown. Because nucleotides can be synthesized endogenously and 21.0e66.0 mmol/L, respectively. The result reveals and there is no known deficiency syndrome, they are not geographical and seasonal variations in nucleotide and considered “essential” . However, dietary nucleo- nucleoside levels and suggests that highly variable dietary tides have been reported to be important in the growth and habits impact on the qualitative and quantitative expressions maturation of the developing gut and to play several roles in of nucleotides and nucleosides in human milk. immune function in animal models and human infants.11,12 The postpartum diets, including chicken soup, innards In bovine milk, nonprotein nitrogen only accounts for are usually encouraged for Taiwanese women, which are 2e5% of the total nitrogen, resulting in a nucleotide content rich in the components of nucleotides and nucleosides, in that is significantly lower than that in such as purine. The unique postpartum dietary supplement human milk. In addition, the nucleotide profile in bovine could play an important role in affecting the nucleotide and milk differs significantly; cytidine and adenosine derivatives nucleoside levels. Furthermore, there were 33% samples are present in relatively lower proportions.13 Supplemen- collected from vegetarians in our study. Many kinds of tation of infant formula with nucleotides was introduced in vegetarian food in Taiwan are made of beans, which is also Japan in 1965, in Europe in 1983, and in the United States in rich in the components of nucleotides and nucleosides. This 1989. The European Commission’s Scientific Committee for could be another important factor affecting the mean Food approved the supplementation of the five nucleotides nucleotide level in our study. to infant formula up to the following concentrations: The total amount of free nucleotides and nucleosides CMP, 2.5 mg/100 kcal; uridine monophosphate, 1.75 mg/ decreased gradually along with the lactation stages. The 100 kcal; adenosine monophosphate, 1.5 mg/100 kcal; data reported by Gil and Sanchez9 also revealed a similar guanosine monophosphate, 0.5 mg/100 kcal; and IMP, phenomenon; free nucleotide content is reduced to 75% of 1.0 mg/100 kcal.14 It has also recommended that the total that in human colostrums after 3 months of lactation. In our nucleotide concentration should be less than 5 mg/100 kcal. study, CDP was the predominant component during lacta- However, the recommendations from the United States are tion, which accounted for 48e77% of the total nucleotides for a maximum of 16 mg/100 kcal for term infant formula in our study. As a result, CDP could be the most important and 22 mg/100 kcal for low-birth-weight infant formula.15 content among the nucleotides. There are some differences Only nucleotides in the form of monophosphate salts are between our study and Sagawara et al’s study.8 They found used for supplementation.16 Infants fed with nucleotide- that CMP was the predominant nucleotide component, and supplemented formula compared with those fed with CDP was predominant only in the colostrum stage. nonsupplemented formula have a lower incidence of However, the sum of CMP and CDP consisted of more than diarrhea, higher titers after Haemophilius influ- 80% nucleotides in both studies in Taiwan and Japan. enza type b vaccination, and higher natural killer cell Another composition difference in nucleotides between activity.17 Studies of infants fed formula fortified with Nucleotides and nucleosides in human milk 97 nucleotides at concentrations equivalent to the free 5. Walker WA. Nucleotides and nutrition: role as dietary supple- nucleotide concentrations of human milk (10e29 mg/L) ment. J Nutr 1994;124:121Se3S. have reported beneficial effects on immune function and 6. Leach JL, Baxter JH, Molitor BE, et al. Total potentially on the incidence of diarrhea.18,19 Yau et al20 found that available nucleosides of human milk by stage of lactation. Am J e healthy term infants in Taiwan fed an infant formula forti- Clin Nutr 1995;61:1224 30. 7. Tressler RL, Ramstack MB, White NR, et al. Determination of fied with 72 mg/L of nucleotides for the first year of had total potentially available nucleosides in human milk from a significant reduction in the incidence of diarrhea from 8 Asian women. Nutrition 2003;19:16e20. weeks to 28 weeks of life and a trend of reduction in the 8. Sagawara M, Sato N, Nakano T, et al. Profile of nucleotides and incidence of diarrhea from 8 weeks to 48 weeks of life. The nucleosides of human milk. J Nutr Sci Vitaminol 1995;41: most widely hypothesized mechanism by which dietary 409e18. nucleotides might enhance immune maturation is that they 9. Gil A, Sanchez MF. Acid-soluble nucleotides of human milk at provide an increased supply of nucleotides at a time when different stages of lactation. J Dairy Res 1982;49:301e7. high metabolic demand (such as growth or clonal expansion 10. Schlimme E, Raezke KP, Ott FG, et al. as minor of immune cells) exceeds de novo synthetic capacity.21 milk constituents: dependence of nucleoside composition on There are several levels of nucleotide fortification avail- mammalian species and lactation stage. In: Gil A, Uauy R, editors. Nutritional and Biological Significance of Dietary able in commercial infant formulas. Further evaluation of Nucleotides and Nucleic Acids. Spain: Abbott laboratories; Taiwanese human milk nucleotide concentrations and the 1996. p. 70e86. most appropriate supplementation level are needed, 11. Duchen K, Thorell L. Nucleotide and polyamine levels in particularly in light of the beneficial role these molecules colostrums and mature milk in relation to maternal atopy and may play in the developing infant. atopic development in the children. Acta Paediatr 1999;88: In conclusion, our data showed a wide range of 1338e43. concentrations of individual nucleotides and nucleosides in 12. Uauy R, Stringel G, Thomas R, et al. Effect of dietary nucleo- human milk. Unique dietary status could affect the nucle- tides on growth and maturation of the developing gut in the e otide and nucleoside levels in human milk, especially the rat. J Pediatr Gastroenterol Nutr 1990;10:497 503. nucleotide levels in our study. However, the mechanism of 13. Yu VY. Scientific rationale and benefits of nucleotide supple- mentation of infant formula. J Paediatr Child Health 2002;38: modulation of nucleotide and nucleoside levels in human 543e9. milk is not clear. For improvement in the neonatal and 14. Scientific Committee for Food. Minutes of the 100th Plenary infant nutrition field, further randomized clinical trials are Session, March 7-8 1996, in Brussels. Directive 96/4/EC. encouraged to examine the benefits of dietary nucleotide Luxembourg: European Commission of the European Commu- supplements in infants, especially under specific clinical nities; 1996. p. 7. situations, such as extreme prematurity, being small for 15. Klein CJ. requirements for preterm infant formulas. gestational age, and poor enteral nutrient intake. J Nutr 2002;132:1395Se577S. 16. Cosgrove M. Perinatal and infant nutrition. Nucleotides. Nutrition 1998;14:748e51. References 17. Carver JD. Dietary nucleotides: effects on the immune and gastrointestinal systems. Acta Paediatr Suppl 1999;88:83e8. 1. Donovan SM, Lonnerdal B. Non-protein nitrogen and true protein 18. Brunser O, Espinoza J, Araya M, et al. Effect of dietary in infant formulas. Acta Paediatr Scand 1989;78:497e504. nucleotide supplementation on diarrhoeal disease in infants. 2. Janas LM, Picciano MF. The nucleotide profile of human milk. Acta Paediatr 1994;83:188e91. Pediatr Res 1982;16:659e62. 19. Carver JD, Pimentel B, Cox WI, et al. Dietary nucleotide effects 3. Molina JA, Romera JM, Gil A. Nucleotides and nucleic acids in upon immune function in infants. Pediatrics 1991;88:359e63. human milk. In: Gil A, Uauy R, editors. Nutritional and Bio- 20. Yau KI, Huang CB, Chen W, et al. Effect of nucleotides on logical Significance of Dietary Nucleotides and Nucleic Acids. diarrhea and immune responses in healthy term infants in Spain: Abbott laboratories; 1996. p. 63e7. Taiwan. J Pediatr Gastroenterol Nutr 2003;36:37e43. 4. Barness LA. Dietary sources of nucleotidesdfrom breast milk 21. Jyonouchi H. Nucleotide actions on humoral immune responses. to weaning. J Nutr 1994;124:128Se30S. J Nutr 1994;124:138Se43S.