Nephrocalcinosis is not uncommon in preterm infants, and elevated urinary oxalate excretions have been implicated as an important risk factor for its occurrence (1-3). Oxalate excretion, expressed as its molar creatinine ratio is higher in preterm than in term infants (4,5) and higher still in term infants than in older children or adults (6,7).
Renal stone formation may result from an imbalance of urinary lithogenic- and stone-inhibitory substance excretions (8). The excretion of either type of compound may be influenced by a variety of nutritional factors, dietary intake (9). For example, a higher urinary oxalate-creatinine ratio has been observed in formula-fed than in human milk-fed infants (4,5). It has been suggested but not proved that the oxalate content of formulas, compared with that of human milk influences this finding (4,5).
Therefore, we hypothesized that the oxalate content of several formula preparations was greater than in human milk. In addition to the measurement of oxalate in human or commercial milks, we measured the citrate and sulfate concentrations. Citrate is a potent stone-inhibitory substance (10), and urinary sulfate can reduce the urinary calcium oxalate saturation (11). We reasoned that the balance of stone-forming and stone-inhibitory substances in the dietary intake of infants also had relevance to the potential for nephrocalcinosis. To our surprise, milk oxalate concentrations did not differ, but the other anions were reduced in human milk compared with those in formula preparations.
PATIENTS AND METHODS
One human milk specimen was collected from each of 38 women after informed consent was obtained at the Children's Memorial Hospital in Chicago, Illinois, U.S.A., and the University Children's Hospital in Cologne, Germany. The study was approved by the ethical committees of the participating hospitals. Twenty-one milk specimens were collected during early lactogenesis (<8 days of lactation), and 17 samples were collected during established lactation (days 10-75), in which a steady state of milk anion concentration was assumed (12). Oxalate, citrate, and sulfate concentrations were analyzed in these human milk samples and in 16 commercial infant formula preparations. The formulas analyzed were those preparations most often used in the participating hospitals. Milk samples obtained in Cologne were frozen and shipped on dry ice to Chicago, where they all arrived frozen.
Formula preparations tested were freshly opened and prepared the same as the human milk samples: Before analysis, all milk samples were centrifuged at 1500g for 20 minutes at 4°C to separate the lipid layer from the aqueous milk content. To hinder oxalate neogenesis and to ensure accurate measurement of free inorganic sulfate, 20 µl 2 N hydrochloric acid was added to an aliquot of 1 ml of the aqueous portion (13,14). This aliquot was centrifuged again at 1500g for 20 minutes at 4°C until a clear, protein-depleted supernatant could be removed by needle aspiration. For measurement of oxalate, citrate, and sulfate concentrations, the supernatants were diluted with 0.3 mM H3BO3 (1:20 or 1:50) and injected automatically into an ion chromatography system (DX-500; Dionex, Sunnyvale, CA, U.S.A.), which was equipped with an analytical column (AS11) and a guard column (AG11) as the stationary phase. The mobile phase was NaOH; its 50% solution (J. T. Baker; Phillipsburg, NJ, U.S.A.) was diluted with water (>17.5 MΩ resistance) to obtain 5-mM and 100-mM concentrations. NaOH was continuously degassed with N2. Five millimolar NaOH was run for the first 10 minutes; thereafter, NaOH was run as a linear gradient from 5 mM to 52.5 mM for as long as 21 minutes (15). Eluent background conductivity was suppressed (>3 μ Siemens at the highest NaOH concentration) with an anion self-regenerating suppressor (ASRS-I; Dionex). Computer-based software (Peaknet; Dionex) was used to calculate the concentration of the measured anion peaks (15). Calibration standards (0.625-10 µM for oxalate, 10-100 µM for citrate, and 1.25-20 µM for sulfate) were run daily. Controls (2.5 and 7.5 µM for oxalate and sulfate, 25 and 75 µmol/l for citrate) were run before and after analysis of every six milk samples.
Oxalate and sulfate levels were expressed in micromoles per liter ± SE, and the citrate levels were expressed in millimoles per liter ± SE. The Mann-Whitney rank sum test and the Kruskal-Wallis one-way analysis of variance on ranks test were used for analyses of the three groups (human milk, early lactogenesis: HM-EL; human milk, established lactogenesis: HM; and formula preparations: F). P values less than 0.05 were considered to be significant.
The mean oxalate content of human milk increased about 27% from HM-EL to HM specimens (p < 0.05; Fig. 1), and was then no different than the mean oxalate concentration of the 16 formulas (Table 1). However, there was a wide range of measurements in HM samples (39.5-169.8 µmol/l) and a fourfold range of measured oxalate in F of differing nature (Table 2; Fig. 1).
The mean citrate content of HM increased only slightly (9.8%) from HM-EL, and each remained significantly lower than in F (Table 1). The mean sulfate concentration did not change with an increasing duration of lactation and was 13 times lower in HM than in F (p <0.0001; Table 1).
We examined the oxalate, citrate, and sulfate content of human milk during early lactogenesis and at established lactation compared with 16 commercial infant formula preparations to elucidate better the influence of nutritional intake of these substances on their urinary excretion in preterm and term infants. The mean oxalate content of human milk at established lactation and of the 16 formula preparations did not differ. There was, however, a wide range in measured single oxalate values in both groups. Our data suggest that it is not the dietary intake of oxalate that produces relative oxaluria in formula-fed infants (4-6). In contrast, both the citrate and sulfate contents of human milk were significantly lower than in formula preparations, which provides further evidence that they may be rate-limiting in relation to their urinary excretion in infants fed human milk.
We did not specifically ask the mothers to avoid oxalate-rich diets on the day the breast milk was collected, and therefore, the differences in the oxalate content of various samples may have been caused by differences in oxalate consumption in the maternal diet. Some human milk (n = 6) showed oxalate concentrations of more than 110 µmol/l, considerably higher than the mean oxalate level, but similar to levels in some of the infant formulas (Fig. 1). By design, we were unable to perform our analyses in individual women on multiple days of established lactation to exclude a variation in individual human milk oxalate levels linked to maternal oxalate consumption. Further studies will help to reveal such a possible influence.
What other factors may account for the variation in urinary oxalate excretion in infants fed different from milk sources but with similar mean oxalate levels? Although vitamin B6 levels represent a critical trigger for oxalate synthesis (16), an increase in endogenous oxalate production caused by pyrodoxine deficiency in most infants is unlikely. Adequate amounts of vitamin B6 are provided by formula (40-90 µg/100 ml) (17) and although pyridoxine concentration is lower in human milk (10 µg/100 ml), normal blood levels have been reported in human milk-fed infants (17,18). Alternatively, endogenous production of oxalate may be increased with higher levels of ascorbic acid (19). The concentration of ascorbic acid, an important precursor of oxalate (20), is higher in standard infant formulas (7.7-9 mg/100 ml) than in human milk (4.4 mg/100 ml) (17), but it remains unproven whether this difference induces an increase in oxalate excretion in formula-fed infants (19,21). In contrast, oxalate neogenesis is known to occur in biologic samples without proper preservation caused by nonezymatic oxidation of ascorbic acid to oxalate (13). This may cause an increase in oxalate concentration in formula fed hours after preparation and could therefore be a relevant factor in higher urinary oxalate excretions. Lower intestinal absorption of fat in preterm and formula-fed infants could account for their higher oxalate excretion (22,23). With relative fat malabsorption, a greater percentage of luminal oxalate remains soluble and will be absorbed, because calcium is bound to fatty acids in the lumen of the intestine instead of to oxalate (24).
Human milk levels of citrate and sulfate in the current study were comparable to those in previous observations (14,21,25,26), as was the noted increase in citrate concentration during established lactogenesis (12,21). Our data are unique, however, regarding sulfate and citrate levels in formula preparations.
Human milk citrate levels may be related to milk pH in an inverse manner, similar to urinary and plasma citrate levels (8,15). Thus, the increase in human milk and citrate after lactogenesis was inversely related to the decrease of colostrum pH (7.4) compared with that of milk (7.04) after the first 2 to 3 weeks of lactation (27,28). When the pH increased again at weaning from breastfeeding, milk citrate was reported to decrease again (28). Although citrate increased in human milk after lactogenesis, it nevertheless remained lower than in formula preparations. We noted also a peak in milk citrate concentrations between days 10 and 75 of lactation (27,28), which makes it even more obvious that the citrate level of human milk is considerably lower than in formula preparations.
What do our data on milk citrate levels mean? Because citrate is a potent stone-inhibitory substance, an increase in its excretion would help reduce urinary saturation (10). Most of the citrate ingested daily will be metabolized to bicarbonate. Therefore, a higher citrate intake with formula feeding may cause a tendency toward systemic alkalization in which the reabsorption of citrate is reduced in the proximal renal tubule and more citrate appears in the urine (29). This hypothesis remains untested at present. However, late incipient metabolic acidosis is a well-known complication in preterm infants fed formula preparations (30), and additional exogenous alkali administration is necessary to reduce the degree of acidosis (31). Such findings may contradict the idea that citrate content of formula influences systemic acid-base balance and later, its urinary excretion.
The primary source of urinary sulfate excretion is the metabolism of sulfur-containing amino acids. Increased urinary sulfate excretion was recently reported in preterm infants fed high concentrations of bovine milk proteins which caused an increased amount of urinary sulfate compared with that in infants fed human milk (32). Sulfate, when adequately excreted in the urine, may help to reduce urinary calcium oxalate saturation (11). In contrast, a higher urinary sulfate excretion may lead to an increase in renal tubular H+ secretion. Because the renal buffer capacity is weak in infants, urinary pH decreases under such circumstances and may thereby increase the risk of crystal aggregation (e.g., for calcium oxalate or uric acid). Therefore, the significantly higher sulfate concentration in formula preparations compared with that in human milk may be of clinical importance if translated directly into urinary anion excretion.
In conclusion, the higher oxalate excretion in formula-fed infants is not caused by its oxalate content. Urinary citrate and sulfate excretion may be influenced by a significantly higher concentration in formula preparations than in human milk. Changes in the urinary excretion of citrate and sulfate related to nutritional intake may be of clinical importance in populations at risk of development of nephrocalcinosis. Further studies beyond dietary intake must elucidate the exact mechanism that controls urinary anion excretions in infants.
Acknowledgment: This study was supported in part by a grant from the Deutsche Forschungsgemeinschaft, Bonn, Germany (Ho 1272/4-1) and by the Mineral Metabolism Research Fund at Children's Memorial Hospital, Chicago, Illinois, U.S.A.
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