Main

Recently there has been an increasing interest in the nutritional aspects of dietary nucleotides. Possible biologic effects such as enhancement of immune function, increase in iron bioavailability, modification of intestinal microflora, changes in plasma lipids, and promotion of gut growth and maturation have been reviewed(1–4). In general, dietary nucleotides are considered mainly to be products of nucleic acid degradation. Fig. 1 depicts the main catabolic pathways of dietary RNA in human adults. The enzymes necessary for this degradation originate essentially in exocrine pancreatic secretion and from small intestinal mucosa(5).

Figure 1
figure 1

The main catabolic pathways of RNA in the human. Modified fromreferences 5–7. The enzymes are: 1, ribonucleases; 2, phosphodiesterases; 3, AMP deaminase, 4, 5, 6, 15, and 16, 5′-nucleotidase/alkaline phosphatase; 7, adenosine deaminase,8, nucleosidase; 9 and 10, purine nucleoside phosphorylase/nucleosidase; 11, adenine deaminase; 12 and14, xanthine oxidase; 13, guanine deaminase; 17, cytidine deaminase; 18, nucleosidase; 19, uridine phosphorylase/nucleosidase; 20, cytosine deaminase; 21, dihydrouracil dehydrogenase; 22, dihydropyrimidinase; 23, 3-ureidopropionase.

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Human milk is assumed to be of optimal composition for the infant and serves as a reference in development of infant formulas. Recently, much interest has therefore focused on the fact that human milk contains considerable amounts of nucleotides(4, 8–11). The content in bovine milk is less and also of different composition(4, 11, 12). Because bovine milk is the main ingredient for most infant formulas, the question of nucleotide supplementation has been raised, and infant formulas supplemented with monophosphate nucleotides are already on both the American and European markets(4).

Nucleosides are the preferred form for absorption by the intestinal mucosa(13–16). Apart from nucleotides, nucleic acids, originating from cells in the milk, may be considered a potential source that could be used for nucleoside formation. However, previous studies on nucleic acids in human milk report widely different levels(17, 18). Recently Leach et al.(19) investigated total potentially available ribonucleosides in human milk. They found, in pooled milk collected from 100 European mothers at four stages of lactation, that 48 ± 8% of the total potentially available ribonucleosides were present as polymeric nucleotides(RNA), 36 ± 10% as nucleotides, 8 ± 6% as nucleosides, and 9± 4% as nucleotide adducts.

However, there is still a substantial lack of knowledge considering the origin of the nucleotide-related compounds in human milk. For instance, RNA and nucleotide catabolic enzymes have been identified in human milk(20, 21), but their possible influence on the nucleotide profile has not been evaluated. Furthermore, there is a lack of knowledge about whether the nucleotides in milk are actively secreted in response to the nutritional demand of the infant, or are the result of other, in this respect, nonspecific events within the mammary gland.

The capacity of the newborn infant to digest and absorb nucleotide-related compounds from intestinal contents has not been established. In fact, several enzymes involved in nutrient digestion have been shown to be not fully developed at birth and therefore limiting for efficient nutrient utilization(22). With respect to supplementation of infant formulas, the chemical form in which nucleotides preferentially should be added (as nucleic acids, nucleotides, or nucleosides) is still unsolved.

The aim of this study was to use newly developed HPLC analytical techniques to determine the content of nucleic acid and ribonucleotide metabolites in human milk and to evaluate the catabolic fate of these substances when exposed to a homogenate containing luminal and mucosal enzymes from human fetal small intestine.

METHODS

Chemicals. Nucleotides, nucleosides, nucleobases, uric and orotic acids, as well as RNA from baker's yeast were from Sigma Chemical Co.(St. Louis, MO). All other chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany).

Human milk and infant formula. Breast milk samples were collected via a breast pump from 14 Swedish mothers at 3-24 wk of lactation. Generally the milk was collected after a meal by completely emptying the breast from which the infant had not been fed, or when fed from both breasts, the breast from which it was last fed. The samples were immediately frozen and kept at -80 °C until used, except for three of the samples of which an aliquot was first used fresh in incubation experiments as described below, whereas the remaining part was immediately frozen and kept at -80 °C for later use.

A whey predominant cow's milk-based infant formula, Baby Semp 2 (Semper AB Sweden), was used together with an experimental batch of this formula supplemented with monophosphate nucleotides.

Fetal small intestinal mucosa homogenate. The fetal small intestine used was from a 22-wk-old fetus. The material was purchased from the International Institute for the Advancement of Medicine (Exton, PA) and stored at -80 °C until used.

After careful thawing, a 25-mm long segment of the fetal small intestine was put onto a Petri dish kept on ice and placed under a stereomicroscope. The segment was opened, and the mucosa was carefully scraped off and collected together with luminal material. The homogenization was carried out in 0.15 M NaCl in an ice bath(23) by use of the small Dounce tissue grinder (Kontes Glass Co., Vineland, NJ) with the tight-fitting (type B) pestle. The homogenate thus obtained was either frozen at -80 °C or kept on ice if used immediately.

Incubations. The homogenate from fetal small intestine was characterized by incubation with the following solutions: RNA (67.0 mg/L); nucleotide mixture (5′-CMP 25.0 μmol/L, 5′-UMP 25.0 μmol/L, 5′-IMP 12.6 μmol/L, 5′-GMP 9.0 μmol/L, 5′-AMP 16.6 μmol/L); nucleoside mixture (cytidine 35.0μmol/L, uridine 35.5 μmol/L, inosine 18.3 μmol/L, guanosine 15.2μmol/L, adenosine 16.0 μmol/L); nucleobase mixture (cytosine 9.0 μmol/L, uracil 8.9 μmol/L, guanine 13.2 μmol/L, adenine 18.5μmol/L).

All substances were dissolved in 10 mM sodium acetate and 0.12 M NaCl, pH 6.50. The homogenate, originating from fetal small intestine, was also incubated with samples of human milk, unsupplemented and nucleotide-supplemented infant formula. Details of the various incubations are given in respective figure and table legends.

Preparation of samples. Preparation of milk and formula samples for HPLC was carried out on ice, by mixing one volume of milk or formula(prepared according to the manufacturer's recommendation) with one volume of 1.0 M perchloric acid. After standing for 10 min, the procedure was followed by centrifugation at 13,400 × g for 10 min at 4 °C. Perchloric acid was extracted from the supernatant by mixing (Vortex) with an equal volume of 0.5 M trioctylamine in 1,1,2-trichlorotrifluoroethane (Freon)(24).

Immediately after incubation, mixtures of intestinal homogenate and substrate were chilled on ice and subsequently centrifuged at 13,400 ×g for 2 min at 4 °C. The incubation mixtures containing human milk or infant formula were chilled and centrifuged in a Microcon 30 concentrator (Amicon Inc., Beverly, MA) at 12,000 × g for 10 min at 4 °C, and the permeate was collected. All samples were filtered through a 0.45-μm filter before HPLC analysis.

HPLC analysis of nucleic acid metabolites. Samples (20 μL) were analyzed on a μBondapak C18 reversed phase column (3.9 mm inside diameter × 300 mm, Waters, Millipore Corp., Milford, MA), connected to a Perkin-Elmer LC-235 diode array detector (Perkin-Elmer Corp., Norwalk CT). The spectral information was used for peak identification and evaluation of peak purity by the Omega-2 and Omega-235 software from Perkin-Elmer.

The three separation systems used are shown in Figure 2. System A was used for the examination of the fetal small intestine, whereas system B was used for the examination of human milk, infant formula, and incubation mixtures of fetal small intestinal homogenate with human milk and infant formula. System C was used for samples containing orotic acid because this compound elutes together with 5′-CMP in system A and B.

Figure 2
figure 2

HPLC elution profiles of standard mixtures for the three separation systems used in this investigation. System A, solvent A = 0.1 M KH2PO4, pH 4.00; solvent B = 50% methanol in water. A linear gradient (0-50% solvent B) started after 10 min of isocratic elution and was maintained for 20 min. Flow rate 1 mL/min, detection 254 nm.System B, solvent A = 0.15 M KH2PO4, pH 4.00; solvent B = 25% methanol in solvent A. A linear gradient (0-100% solvent B) started after 2 min of isocratic elution and was maintained for 28 min. Flow rate 1 mL/min, detection 254 nm. System C, mobile phase = 0.1 M trisodium citrate, pH 6.50. Flow rate 1 mL/min, detection 280 nm. The peaks are:1, 5′-CMP; 2, 5′-UMP; 3, uracil;4, cytidine; 5, 5′-GMP; 6, 5′-IMP;7, uric acid; 8, guanine; 9, hypoxanthine;10, uridine; 11, xanthine; 12, 5′-AMP;13, inosine; 14, guanosine; 15, adenosine;16, orotic acid.

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Determination of nucleic acid. Samples of human milk and infant formulas were hydrolyzed for 1 h at 90 °C in 1 M perchloric acid to quantitatively liberate the purine bases adenine and guanine. To determine guanine and adenine from nucleotides, nucleosides, and bases, one part of a chilled sample was mixed with one part of ice-cold 2 M perchloric acid. After standing for 10 min on ice, the procedure was followed by centrifugation at 13,400 × g for 10 min at 4 °C. The supernatant was thereafter centrifuged in a Microcon 30 concentrator at 12,000 ×g for 10 min. The permeate was collected and hydrolyzed as described above. After hydrolysis, perchloric acid was removed by extraction of 1 volume of the hydrolyzed sample with 2 volumes of trioctylamine/Freon. For the separation a 10-μm LiChrospher column (4 mm inside diameter × 250 mm, Merck, Darmstadt, Germany) was used. The mobile phase was 0.20 M KH2PO4, pH 3.95, the flow rate was 1 mL/min, and 20 μL were injected. Guanine and adenine were detected at 254 nm and quantified by use of external standards. The levels of these two bases, after correction for the guanine and adenine contribution from acid soluble nucleotides, nucleosides, and bases, were used to calculate the total amount of nucleic acid, based on the assumption of molar equality between purine and pyrimidine bases. Recovery of RNA from baker's yeast was 97.5 ± 2.2%.

RESULTS

Human milk composition. In Table 1 the average milk composition of nucleotide-related compounds is shown. The total amount of nucleic acid, expressed as nucleotide equivalents, was 68 ± 55 μmol/L. Of the nucleotides 5′-CMP and 5′-UMP were found at higher levels than 5′-GMP and 5′-AMP, whereas 5′-IMP was not detected. Of the nucleosides, only cytidine and uridine were found. Guanine was detected at a low level in only four of the samples, whereas uric acid, the end product of purine catabolism in humans, was found at a relatively high level in all samples. Orotic acid was not detected. The unsupplemented formula was devoid of nucleic acid and 5′-ribonucleotides, but contained ribonucleosides and uric and orotic acids at similar levels to the supplemented formula. The supplemented formula also contained the five 5′-ribonucleotides at expected levels.

Table 1 Levels of nucleic acid and metabolites in human milk, at 3-24 wk of lactation, and in unsupplemented and ribonucleotide supplemented formula
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To elucidate if the specific nucleotide profile of human milk, to some extent, might result from enzymatic digestion, incubation of fresh and frozen samples was performed. The results from these incubations are shown inTable 2. In all three samples 5′-CMP and 5′-UMP were partly transformed to cytidine and uridine, whereas 5′-GMP and 5′-AMP were partly transformed to guanine and uric acid. This indicates that the milk contains the complete sequence of enzymes(Fig. 1) necessary to convert purine nucleotides all the way to uric acid. As pyrimidine nucleosides accumulated during incubation, enzymes responsible for their further degradation were less active or absent. The changes during incubation were similar, independent of whether the milk was used fresh or was frozen and thawed before incubation, indicating no significant further contribution of enzymes from any cells ruptured by freezing and thawing.

Table 2 Changes in levels of nucleotides, nucleosides, nucleobases, and uric acid during incubation of human milk for 24 h at 23°C
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Capacity of fetal small intestine to catabolize RNA and its metabolites. To evaluate the endogenous catabolic capacity of the fetal small intestine, different mixtures of nucleotides, nucleosides, or 0.15 M NaCl as a control were incubated with fetal intestinal mucosa homogenate. The HPLC elution profiles after incubation are shown in Fig. 3. The control mixture (no exogenous substrate) shows small peaks of uracil, cytidine, uric acid, uridine, xanthine, inosine, and guanosine after incubation for 1 h (Fig. 3A). Most likely the main parts of these peaks were generated from small amounts of RNA in the homogenate. During incubation of the nucleotides (compare Fig. 3,B and C) 5′-CMP and 5′-UMP were hydrolyzed to cytidine and uridine, respectively. The nucleotides 5′-GMP and 5′-IMP were hydrolyzed to guanosine and inosine, respectively, which in turn were further catabolized to hypoxanthine, xanthine, and uric acid according to the pathways illustrated in Fig. 1. The purine nucleotide 5′-AMP was catabolized to inosine, most likely via adenosine, and further on to hypoxanthine, xanthine, and uric acid. When the nucleosides were incubated with homogenate (compare Fig. 3, D and E), cytidine and uridine were only slightly affected, whereas the purine nucleosides were catabolized to hypoxanthine, xanthine, and uric acid in the same way as when the corresponding 5′ nucleotides were incubated. Because there was no adenosine after incubation(Fig. 3,C andE), this indicates a relatively high adenosine deaminase activity.

Figure 3
figure 3

HPLC elution profiles of different mixtures after incubation for 1 h at 37 °C with the small intestinal homogenate.(A) 125 μL of homogenate + 375 μL of buffer (control);(B) 125 μL of 0.15 M NaCl + 375 μL of 5′-nucleotides(control); (C) 125 μL of homogenate + 375 μL of 5′-nucleotides; (D) 125 μL of 0.15 M NaCl + 375 μL of nucleosides (control); (E) 125 μL of homogenate + 375 μL of nucleosides. Separation system A (Fig. 2A) was used. The peaks are: 1, 5′-CMP; 2, 5′-UMP; 3, uracil; 4, cytidine; 5, 5′-GMP; 6, 5′-IMP; 7, uric acid; 8, hypoxanthine; 9, uridine; 10, xanthine; 11, 5′-AMP; 12, inosine; 13, guanosine; 14, adenosine.

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The homogenate was also incubated with the four bases cytosine, uracil, guanine, and adenine. Table 3 shows the recovery after incubation. Guanine was catabolized to xanthine and uric acid (data not shown). The high recovery of cytosine, uracil, and adenine indicates very low activities of cytosine deaminase, dihydrouracil dehydrogenase, and adenine deaminase.

Table 3 Recovery of pyrimidine and purine bases after incubation with homogenate from fetal small intestine for 1 h at 37°C
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RNA was also incubated with the homogenate at 37 °C. During this incubation, cytidine and uridine accumulated, whereas 2′3′-CMP and 5′-CMP were found as intermediates during degradation of RNA(Fig. 4). The identification of 2′3′-CMP and 5′-CMP as intermediates supports the presence of ribonuclease and phosphodiesterase. During degradation of purines (Fig. 4), hypoxanthine and xanthine accumulated, whereas the concentrations of inosine and guanosine suggested them to be intermediates. As in the other incubation experiments, there was no adenosine, presumably because this nucleoside was rapidly converted to inosine.

Figure 4
figure 4

Formation of pyrimidines and purines during incubation of RNA with homogenate of fetal small intestine for 4 h at 37 °C. The following incubations were performed: 1) 375 μL of homogenate + 1125 μL of RNA, 2) 375 μL of homogenate + 1125 μL of buffer(control); 3) 375 μL of 0.15 M NaCl + 1125 μL of RNA(control).

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Availability of nucleic acids in human milk and infant formula. To get a better understanding of the availability of nucleic acid in milk and infant formulas, further incubations with homogenate from fetal small intestine were carried out.

When human milk and homogenate were incubated at 37 °C for 4 h (see column A in Table 4), the levels of cytidine, uridine, xanthine, and uric acid increased. This is in agreement with the results obtained from incubations of homogenate with nucleotides, nucleosides, and RNA as described above. The incubation of human milk and homogenate generated a net increase in pyrimidines and purines of 24 μmol/L(Table 4, column A). This increase most likely results from degradation of human milk nucleic acids and nucleotide adducts. When human milk, to which exogenous RNA had been added, was incubated with homogenate, the net increase in pyrimidine and purine concentration was about twice as high, 56 μmol/L (Table 4, column B), compared with the results of incubation with human milk alone.

Table 4 Changes in levels of nucleotides, nucleosides, nucleobases, and uric acid after 4 h of incubation at 37 °C of fetal small intestinal homogenate with human milk, with and without added RNA, and infant formula with and without added RNA
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The incubation of infant formula and homogenate generated only a small net increase of pyrimidines and purines (Table 4, column C), compared with the incubation with human milk (Table 4, column A). This probably reflects the low level of nucleic acid in the formula(Table 1). When infant formula with added RNA was incubated (Table 4, column D), the net increase of pyrimidines and purines was almost equal to that found after incubation of human milk with additional RNA (Table 4, column B).

The nucleotide supplemented formula was also incubated with the homogenate(data not shown). The pyrimidine nucleotides, 5′-CMP and 5′-UMP, just as in previous incubations accumulated as cytidine and uridine, whereas the purine nucleotides were converted to hypoxanthine, xanthine, and uric acid. The net change of pyrimidine and purine levels was this time 0,1μmol/L, again indicating no significant contribution from endogenous nucleic acid in the formula in agreement with the low levels detected(Table 1).

DISCUSSION

In the present study the total nucleic acid content of human milk was determined. Expressed as nucleotide equivalents, the mean level was 68± 55 μmol/L. Leach et al.(19) determined polymeric nucleotides (RNA) in pooled human milk samples from 100 European women and found a mean level of 91 μmol/L expressed as total potentially available nucleosides.

This value is somewhat higher than what we found, although within the same range despite the fact that these authors did not determine DNA. However, it is likely that DNA constituted only a minor portion of the total nucleic acid content; as in all cells, the concentration of RNA is considerably higher than that of DNA, the former being relatively constant, whereas the latter varies with the stage of the cell cycle. Sanguansermsri et al.(18) found for milk of European mothers the DNA to constitute 5-10% of the total nucleic acid level. However, the analytical methods used by these authors have been criticized for being unspecific, prone to overestimation in complex sample matrixes(19), and showing poor reproducibility(4).

We also analyzed ribonucleotides and ribonucleosides and found mean levels for these groups to be 84 ± 25 μmol/L and 10 ± 2 μmol/L, respectively, which is similar to those reported by Leach et al.(19), i.e. 68 and 15 μmol/L, respectively. In addition, they also determined nucleotide adducts and reported a mean level of 17 μmol/L. Nucleotide adducts, as well as nucleic acids, may be considered a potential source of nucleosides.

Whether the nucleotides and nucleosides in human milk are actively secreted into the milk in response to a nutritional demand of the infant, or indirectly result from other metabolic events within the mammary secretory cell(i.e. synthesis of lactose, protein, and fat), is still not known. However, cellular metabolites are thought to enter milk by their secretion within Golgi vesicles(25, 26), together with cytoplasmic fragments liberated during fat secretion(27) and by diffusion across the apical membrane(26, 28). For instance, during synthesis of lactose and sialyllactose, which occurs within Golgi vesicles, 5′-UMP and 5′-CMP are formed(29, 30) and may therefore be released into the alveolar lumen during exocytosis.

We found the specific profile of nucleotides and nucleosides to have a predominance for pyrimidines, whereas purines were found mainly as uric acid(Table 1). Because uric acid is the end product of purine catabolism in man, this suggests the possibility that the nucleotide profile, at least to some extent, might be the result of postsecretory changes within the milk during storage in the breast. Our data from the in vitro incubation of human milk (Table 2) support the presence of enzymes necessary to catabolize nucleotides to cytidine, uridine, and uric acid. The catabolic enzymes, as well as nucleic acids, may originate from the various types of cells identified in human milk throughout lactation(31). Ho et al.(31) found during the 2nd week of lactation that epithelial cells started to increase, accompanied by numerous cell fragments, probably derived from the process of apocrine secretion. However, it is obvious that there is a lack of information on how levels and composition of nucleotides and related compounds in the milk are regulated.

The results from the characterization of the human fetal small intestinal homogenate are well in agreement with what other investigators have found when various nucleotides and nucleosides were either exposed to rat or hamster small intestinal mucosa in an everted sac model(13, 14), or perfused through the lumen of isolated loops of rat jejunum(15, 16). In vivo, luminal digestive enzymes originate from different sources, e.g. brush border epithelium(32, 33), pancreatic juice(34), and bile(35). The luminal digestive process may also be influenced by the high turnover of mucosal enterocytes, resulting in leakage of intracellular enzymes from rejected cells. In our incubations with homogenate from intestinal mucosa, the luminal content was included. It is therefore possible that the results also reflect the influence of basal pancreatic secretion. The formation of 2′3′-CMP and 5′-CMP during incubation with RNA(Fig. 4) clearly indicates the presence of ribonuclease and phosphodiesterase. Although it is possible that the concentration of intracellular enzymes, originating from ruptured enterocytes in the homogenate, may be higher than under in vivo conditions, the overall composition of catabolic enzymes is likely to reflect the physiologic situation, on a qualitative basis.

The incubations of human milk with homogenate indicated that RNA present in human milk can be used for pyrimidine and purine formation. However, further studies are needed to clarify whether the digestive process has sufficient capacity, or if nucleotides and nucleosides in the milk compensate for a low endogenous digestive capacity. Recently, Witte et al.(36) demonstrated, in situ, an increase of purine catabolic mucosal enzymes during postnatal maturation of the gastrointestinal tract in the mouse.

In conclusion, the nucleotide/nucleoside profile of human milk could, at least to some extent, result from limited catalysis during storage of the milk in the breast. Furthermore, the results from this investigation, taken together with what was previously known about pyrimidine and purine digestion, indicate that it is likely that even the preterm infant, on a qualitative basis, can digest RNA as well as nucleotides and nucleosides using normal pathways, and that supplementation of infant formulas with either RNA, nucleotides, and nucleosides or a combination of these would be relevant from at least the compositional point of view.