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Concentrations of zinc in human milk are notable for the extent of the decline that occurs through the course of lactation and for the relatively low levels in mature milk that result from this decline. Although poor maternal zinc status may affect the rate of this decline, such an effect is at most quite small, and the decline is primarily physiologic(1, 2). Although zinc deficiency has been well documented in a few term, otherwise healthy, breast-fed infants(3, 4) and the zinc derived from human milk alone may not be adequate with more prolonged lactation(5), the needs for dietary zinc appear to be met quite adequately for fully breast-fed infants in the first few months of life. To accomplish this, bioavailability of zinc from human milk must be favorable, and homeostatic mechanisms in the fully breast-fed infant must approach maximal efficiency to absorb and retain sufficient zinc to meet the growth requirements of young infants.

Although studies in animal models(6) and in normal adults(7) have indicated favorable absorption of zinc from human milk, studies of zinc homeostasis directly in the normal breast-fed infant have not been reported. The objective of this study was to determine fractional and total absorption of zinc and fecal excretion of endogenous zinc in normal fully breast-fed infants by application of stable isotope techniques.

METHODS

Study design. Healthy exclusively breast-fed infants were investigated in a cross sectional study. Oral administration of extrinsic zinc stable isotope label was combined with metabolic collection techniques that were developed specifically for this study. Fractional absorption of zinc was determined by fecal monitoring; an isotope dilution technique was used to calculate fecal excretion of endogenous zinc; and net absorption of zinc was calculated. The study was conducted in the subjects' homes after careful training of the parents. One of the investigators (C.J.R.) was present for all isotopically labeled feeds and visited the homes frequently throughout the study.

Subjects. Nine male infants were studied between 2 and 5 mo of age. All infants had been born at term and were healthy. None had received any formula or solids before or during the study, and none was on medications or vitamin-mineral supplements. The infants were recruited from advertisements in clinics and hospitals and by word of mouth. The study was approved by the Human Subjects Committee of the University of Colorado Health Sciences Center. After explanation of the purpose and requirements of the study, signed consent was obtained from the parents of all subjects in the study.

Isotope preparation and administration. 70Zn-enriched zinc oxide powder (99.72 atom%) was obtained from Oak Ridge National Laboratories (Oak Ridge, TN). Accurately weighed quantities of the isotope were dissolved in 1 N H2SO4, diluted in triply deionized water, and titrated to pH 5 with metal-free ammonium hydroxide. The concentration of zinc in the isotope preparation was determined in triplicate by atomic absorption spectrophotometry, with correction made for the higher atomic weight of the enriched zinc. Expressed milk from the infants' own mothers was extrinsically labeled with an accurately weighed dose of the 70Zn solution. The quantity of label added to the milk was that calculated to be the minimum necessary to ensure detectable enrichment for sufficient duration to measure endogenous zinc in the feces. Aliquots of unlabeled and labeled milk were reserved for later isotopic and total zinc analyses.

The labeled milk was allowed to equilibrate for 4 h and was then frozen. On d 1 of the study, the milk was thawed and divided into six small aliquots, approximately 5 mL each, and administered to the infants orally via small plastic syringes immediately before routine feeds over 24 h. Each syringe was weighed before and after the dose was given. Any drooling or regurgitation was collected on ashless filter papers and saved for determination of total zinc and isotopic enrichment. The final actual dose of 70Zn was determined by subtracting the amount collected on the filter paper from the total weighed amount fed to the infant.

Fecal and urine collections. All stools were collected from the time of the first isotopically labeled feed on d 1 through at least 8 d. A baseline fecal specimen was obtained before administration of label. Feces were collected primarily by use of a portable collection seat with a removable zinc-free plastic liner bag. When the infant was out of the seat, such as during feeds, stools were collected on ashless filter papers placed in zinc-free plastic-lined diapers. Any “losses” of feces were dabbed up on ashless filter papers for analysis with the remainder of the sample. Samples were stored in individual plastic bags in small freezers provided to each subject for the study. Actual losses, which were rare, were quantified by the mothers, all of whom had been trained before the beginning of the study. All stools and notations regarding the collections were recorded on a daily log sheet which was reviewed with the investigators. A 3-d metabolic period (d 4-6) was demarcated by brilliant blue fecal markers; stool collections continued until the second marker was passed.

Urine was collected during the metabolic period in a resealable zinc-free plastic bag which was attached to the scrotum with an adherent patch(Stomahesive Wafer, Squibb, Inc., New Brunswick, NJ). The bags were drained frequently with a zinc-free syringe, and the urine was added to a sterile zinc-free plastic specimen cup. Collections were made twice daily (AM and PM) for approximately 4-6-h time blocks. The actual collection interval was dependent on the time required to obtain a minimum of 100 mL for each collection period. The starting and ending times for each period were noted on the specimen cup and the log sheets. Fecal and urine specimens were frozen until analysis.

Milk sampling and test weighing. Milk samples of 5 to 10 mL were hand expressed for each feed during the metabolic period. The nipple and areola were cleaned before each collection with deionized water. The mothers of the subjects were given instructions for collection procedures to avoid zinc contamination of the milk samples; zinc free vials were provided. Test weighing procedures, which have been described in detail previously(8), were used to measure infant milk intakes for the 3 consecutive days of the metabolic period. Electronic digital balances(Sartorius Corp, Bohemia, NY), which integrate 100 rapid serial measurements to provide a mean weight to the nearest 1 g, were set up in the subject's home for the study period. Naked infant weights were obtained at approximately the same time every day during the study. Average rate of weight gain was calculated from a linear regression on day versus weight for the 8 d of the study.

Laboratory analyses. Individual fecal samples were ashed at 425°C. The ashed samples were quantitatively dissolved in 6 N HCl and total zinc was determined on a diluted aliquot with an atomic absorption spectrophotometer fitted with a deuterium arc background correction lamp(Perkin-Elmer, Norwalk, CT). Other inorganic elements were removed from reconstituted ashed samples by ion exchange chromatography with AG 1 ion exchange resin (Bio-Rad Laboratories, Richmond, CA). Isotopic enrichment was determined by measurement of isotope ratios by fast atom bombardment-induced secondary ion mass spectrometry on a double-focusing mass spectrometer (model VG 7070E HF; Fisons-VG Analytical, Manchester, UK) equipped with an Ion Tech(London, UK) atom gun. The mass spectrometer was operated at low resolution, and ion counting detection and peak switching were used to measure70 Zn/66Zn ratios. Fecal and urine samples containing 1 to 2μg of zinc were analyzed; the precision of the measured ratios was 0.5 to 1.0% relative SD. Enrichment, defined as all zinc in sample from isotopically enriched source divided by total zinc in the sample, is calculated from the measured ratios by use of a standard curve(9).

Preparation of individual urine samples included wet digestion with concentrated nitric acid and H2O2(10). Dried samples were then ashed in muffle furnace as described above for fecal samples. Reconstituted ash was placed on chelating resin column to remove major minerals. The resulting eluents were then placed on ion exchange resin columns and isotopic enrichment determined as described above for the fecal specimens. Zinc concentrations in milk samples were determined by previously described methods(1).

Calculations. Total dietary zinc intake was determined by multiplying the weight of each feed by the zinc concentration in the sample from that feed. The total zinc intake over the metabolic period was divided by the exact number of hours between administration of the nonabsorbable markers and multiplied by 24 to calculate an average daily zinc intake. The quantity of isotope tracer (as a fraction of administered dose) present in each fecal sample was calculated by multiplying the total zinc in the sample by its enrichment and dividing by the dose of isotope administered. The cumulative values of these data were plotted against time from isotope tracer administration to show cumulative fecal excretion of isotope for each subject. Correction for absorbed isotope that was subsequently secreted into the intestinal lumen and excreted in the feces was made by extrapolating to time zero a linear regression line through the final points of the cumulative fecal excretion plot (after excretion of unabsorbed isotope was apparently complete)(11). The corrected cumulative fractional fecal excretion was subtracted from one to determine fractional absorption of zinc. Multiplication of fractional absorption by total daily dietary zinc determined total absorbed zinc. Endogenous fecal zinc was calculated by a modification of the technique described by Jackson(12), according to the equation Σ(F × f)/(u × 3), where F is total fecal zinc, f is isotope enrichment for each stool sample during the metabolic period (divided by 3 for days of metabolic period), and u is average urine enrichment over the metabolic period. The validity of substituting urine for plasma enrichment and using an orally, instead of i.v., administered isotope has been examined in adult studies(11). The latter substitution is dependent on completion of elimination of unabsorbed isotope before the “metabolic collection period.” Net absorption of zinc was determined by subtracting endogenous fecal zinc from total absorbed zinc.

Data analysis. Summarizing of data and statistics were performed with the Statistix 3.5 program (Analytical Software, St. Paul, MN). Data are presented as mean (±SD) and statistical significance was considered at the p ≤ 0.05.

RESULTS

The mean age of the infants was 3.4 mo, with a mean weight of 6.12 ± 0.95 kg (Table 1). Mean weight change during the study period was 20 ± 7.9 g/d. There was a trend for a positive correlation between age and fractional absorption (r = 0.48, p = 0.19) but no suggestion of a correlation between other parameters and age. Age was, therefore, disregarded in subsequent analyses.

Table 1 Descriptive data and results for individual subjects
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Zinc intake from human milk ranged from 9.7 to 32.3 μmol/d (0.63 to 2.1 mg/d). The dose of 70Zn given ranged from 2.34 to 3.26 μmol (153 to 213 μg) and accounted for 15.9 ± 5.8% of the daily zinc intake. A representative plot of cumulative excretion of isotope depicting the correction for isotope that was absorbed, secreted into the intestine and excreted in the feces is shown in Figure 1. Mean fractional absorption was 0.54 ± 0.075. This resulted in a total absorbed zinc of 9.5 ± 3.5 μmol/d (0.62 ± 0.23 mg/d). Fractional absorption was not correlated with absolute quantity of isotope dose or dose as a percentage of total zinc intake.

Figure 1
figure 1

Plot from representative subject (no. 1) of cumulative fecal excretion of isotope, depicting correction for isotope that was absorbed, secreted back into intestine and excreted in feces.

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The mean (n = 7) excretion of endogenous fecal zinc was 4.7± 2.3 μmol/d (0.31 ± 0.15 mg/d), and 0.8 ± 0.3μmol/kg/d (0.05 ± 0.02 mg/kg/d). Calculation of endogenous fecal zinc was not obtained in 2 of the subjects because of apparent prolonged fecal excretion of unabsorbed isotope, indicated by the lack of an inflection point in the cumulative fecal enrichment curve and by isotopic enrichment in the final fecal samples that exceeded enrichment in corresponding urine samples. The calculated net absorption ranged from 2.4 to 11.8 μmol/d (0.15 to 0.77 mg/d). Means for total absorbed zinc, endogenous fecal zinc and net absorbed zinc for these seven infants are depicted in Figure 2.

Figure 2
figure 2

Mean total absorbed zinc, endogenous fecal zinc, and net absorbed zinc for seven subjects for whom endogenous fecal zinc could be determined.

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The correlation coefficient between total absorbed and fecal excretion of endogenous zinc was 0.66 (p = 0.15).

DISCUSSION

Because of the changes in human milk zinc concentrations as lactation progresses, with progressive narrowing of the gap between ingested zinc and requirements for infant growth(5), age-related changes in some of the parameters included in this study were considered. Apart from a trend for an increase in fractional absorption, possibly related to the decrease in milk zinc concentrations, no age-related changes were observed. Not included in the results of this report were longitudinal data for four fully breast-fed infants studied at both 2 and 4 mo of age. The results of these studies were in accord with those reported here. It is plausible that age-related changes would have been observed if studies were undertaken over a wider age range, especially starting at a younger age.

Zinc homeostasis is regulated principally in the small intestine where both fractional absorption of exogenous zinc and net secretion of endogenous zinc are modulated in response to dietary zinc intake and zinc nutritional status. Fractional absorption when zinc is administered with water averages approximately 60%. Fractional absorption of zinc from individual foods or composite meals is quite variable but typically is much less than absorption from water(13). It is notable, therefore, that the mean fractional absorption for the breast-fed infants in this study approached the average figure for zinc absorption from water. This figure is higher than fractional absorption reported when human milk was administered to adult subjects(7). It is also greater than the fractional absorption of zinc from a low zinc cow milk based infant formula in a study of normal infants(14). The mean daily zinc intake of the formula-fed infants was, however, approximately 25% higher than that of the infants in the present study and the age span was greater. Without additional experimental data it is not entirely clear to what extent the relatively high absorption of zinc from human milk is an adaptation to the relatively low dietary zinc intake rather than or in addition to a reflection of the favorable bioavailability attributable to the composition of human milk. The latter explanation is suggested by the association of development of severe zinc deficiency at the time of weaning in breast-fed infants with acrodermatitis enteropathica and the therapeutic response of human milk feeding to formula fed infants with this disorder(15). Indirect support for favorable bioavailability of zinc in human milk also is provided by recent observations in animals(16) and humans(17) that modulation of fractional absorption may not be the major homeostatic response to chronic low zinc intake.

The study design did not include i.v. administration of zinc stable isotopes and did not include plasma sampling. To determine the excretion of endogenous zinc in the feces by isotope dilution, we used, therefore, a modification of the technique described by Jackson et al.(12). The latter was a modification of the technique originally developed and validated in animals by Weigand and Kirchgessner(18), who also validated substitution of urine isotopic enrichment for enrichment in plasma and selected solid tissues. Preliminary data in adults are in accord with this finding and have also demonstrated that an orally administered isotope can be substituted for an isotope administered i.v.(11). Use of an orally administered isotope depends on continuing fecal collections beyond the excretion of unabsorbed isotope, which occurred by 3 d for most of the infants. Fecal enrichment after that is due primarily to absorbed isotope being secreted into the gastrointestinal tract and subsequently being excreted. These modifications of the isotope dilution technique allowed the determination of fecal excretion of endogenous zinc in infants without relying on traditional balance data.

Although fractional absorption was relatively favorable, it was not sufficiently high, given the relatively low dietary zinc intake, to provide much margin in net absorption for the accommodation of losses of endogenous zinc. It therefore seems likely that these infants had generally adapted nearly maximally to achieve the greatest possible conservation of endogenous zinc and that the endogenous fecal zinc excretion was approaching obligatory losses. On a body weight basis, endogenous zinc excretion was higher than the 0.52 ± 0.24 μmol/kg/d reported for infants fed a low zinc formula(14). This difference may be attributable to methodology. Using the same methodology as Ziegler et al.(14), combining fractional absorption with balance data to calculate endogenous fecal zinc, the calculated mean for the breast-fed infants in this study was 0.50 ± 0.28 μmol/kg/d. Net (apparent) absorption of zinc could also be calculated from the metabolic balance data. The wide range of results obtained from this approach, including negative values, was very similar to that reported by other investigators(19). This range, which was approximately twice that for net absorption calculated from the isotope label studies, likely reflects the recognized difficulties of achieving accurate balance data.

Mean net absorption of zinc appeared marginal to meet calculated requirements for growth and to replace the minor losses by routes other than the intestine(5). Several possible reasons merit consideration. First, requirements may be less than previously estimated. Urine and integumental losses were not measured in this study, and it is quite feasible that these losses are lower than calculated previously(5), which would give a modest decrease in calculated requirements for net absorption. Second, the composition of new growth was not determined, and if this had included a relatively high proportion of adipose tissue, growth requirements would be less than average. Thirdly, young infants appear to have a modest store of hepatic zinc(20). Although data on the utilization of these stores are limited, it appears unlikely that this would be an important factor after 2 mo of age. Finally, despite maximal adaptation, these infants may not have been absorbing sufficient zinc to support continued optimal growth. It is possible that zinc may become a limiting nutrient for growth in breast-fed infants(21). However, the positive correlation between total absorbed zinc and endogenous fecal zinc, which has also been observed for a group including both formula-fed and breast-fed infants(22), suggests that endogenous losses may have been fine tuned to regulate retention, depending on the amount of zinc absorbed. If this conclusion is correct, it implies that not all of these infants were conserving endogenous zinc maximally and were, therefore, unlikely to be deficient. More data at low levels of total absorbed zinc would be necessary to definitively assess this possibility.

The results of this study indicate that fully breast-fed infants maintain zinc homeostasis by a combination of relatively high fractional absorption and conservation of endogenous zinc by the intestine. The favorable fractional absorption is likely to be due to the favorable composition of human milk for zinc bioavailability as well as to the low zinc intake. The conservation of endogenous zinc plays a crucial role in achieving zinc retention adequate to meet the demands of growth in the face of modest intake.