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American Journal of Clinical Nutrition, Vol. 78, No. 4, 782-789, October 2003
© 2003 American Society for Clinical Nutrition

Original Research Communication

Milk folate but not milk iron concentrations may be inadequate for some infants in a rural farming community in San Mateo, Capulhuac, Mexico1,2,3

Salvador Villalpando, Marie E Latulippe, Gorethy Rosas, Maria Jose Irurita, Mary Frances Picciano and Deborah L O’Connor

1 From the Basic Nutrition Division, Instituto Nacional de Salud Publica, Cuernavaca, Mexico (SV, GR, and MJI); the Nutrition Department, Pennsylvania State University, University Park (MEL and MFP); and The Hospital for Sick Children and University of Toronto, Toronto (DLO).

2 Supported by Nacional de Ciencia y Tecnologia, Mexico.

3 Address reprint requests to DL O’Connor, Department of Nutritional Sciences, University of Toronto, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. E-mail: deborah_l.o\|[rsquo ]\|connor{at}sickkids.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: We were interested in identifying possible nutritional reasons for growth faltering among breastfed infants in the rural farming community of San Mateo, Capulhuac, Mexico (2800 m above sea level).

Objective: We examined the prevalence of inadequate iron and folate status among lactating Otomi women and determined to what extent their iron and folate nutriture influenced the milk concentrations of these nutrients.

Design: Lactating women (n = 71) provided blood and milk samples and dietary information at a mean (± SD) of 22 ± 13 d postpartum. Blood indexes included hemoglobin, hematocrit, serum iron, total-iron-binding capacity, ferritin, transferrin receptor, mean cell volume, plasma folate, and erythrocyte folate.

Results: Approximately 62% and 58% of the women had nutritional anemia defined as a hemoglobin concentration <= 133 g/L and a hematocrit value of 41.0%, respectively. With the use of a 3-index iron assessment model, 2 of the 66 women whose iron status was assessed (3%) had iron-deficient erythropoiesis, and 24 (36%) had iron deficiency anemia. Among the 67 women whose folate status was assessed, 29 (43%) had a low plasma folate concentration, and 13 (19%) had a low blood folate concentration in conjunction with a low hemoglobin concentration. Milk iron content was unrelated to maternal iron status, and the milk provided more than adequate amounts of iron to the infants. In contrast, the infants’ predicted folate intake was {approx}45 µg/d, or 70% of the current recommended intake.

Conclusion: Milk folate concentrations in Otomi women are low and may not support optimal folate status in all breastfed infants.

Key Words: Iron status • folate status • lactation • milk folate • milk iron • iron-folate interaction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the predicted prevalence of iron deficiency with or without anemia varies widely, it is clear that a disproportionate number of reproducing women throughout the world show evidence of iron deficiency ({approx}50–60% compared with 20–30% of nonpregnant, nonlactating women) (1–3). Similarly, up to one-third of reproducing women are estimated to have some degree of folate undernutrition (4). Even well-nourished women develop a certain degree of folate depletion (eg, an increase in serum homocysteine concentration) during lactation (5, 6). Nonetheless, milk folate concentrations are conserved at the expense of maternal body reserves (5, 7). As such, it is generally accepted that during maternal folate deficiency, folate intakes in breastfed infants are protected until the introduction of solid foods (8). Results from animal studies in our laboratories suggest, however, that maternal iron deficiency may be detrimental to both maternal and infant folate nutriture. We reported that milk collected from iron-deficient rats and pigs contains up to {approx}50% less folate than that collected from iron-sufficient animals (9–12). This reduction in milk folate content was sufficient to impair folate status and growth in nursing rats and pigs. Matoth et al (13) showed that folic acid supplementation of apparently healthy infants who were fed a low-folate boiled cow milk diet had better growth than did infants fed the same diet without supplemental folic acid.

Milk iron content is unaffected by iron intake in well-nourished women; however, no information is available to confirm that this relation holds true in undernourished women (14, 15). To our knowledge, the possible consequences of maternal iron deficiency alone, or in combination with folate deficiency, on milk folate and iron concentrations in humans has not been studied.

We were interested in determining whether maternal iron or folate deficiency, or an interaction between the 2, could be contributing to the disproportionate decline in growth velocity and National Center for Health Statistics z scores among healthy Otomi breastfed infants (4–6 mo of age) from San Mateo, Capulhuac, Mexico, relative to their US counterparts (16, 17). Preliminary data collected from blood samples of lactating women in this community provide biochemical evidence of iron and folate deficiency (18).

Although variations in the energy density of milk are present, Otomi infants meet their energy demands by increasing their milk intake, so that corresponding infant intakes of energy, protein, and carbohydrates from milk are comparable with those of US breastfed infants having normal growth (19). Similarly, mean calcium, magnesium, phosphorous, copper, and zinc concentrations in milk produced by Otomi women at 4 and 6 mo postpartum have been shown to be comparable with those in milk secreted by healthy American women (20). Except for zinc, estimated intakes of each nutrient would meet or exceed adequate daily intakes for term infants up to 6 mo of age (19–22). The purpose of the present study was to use sensitive and specific indexes of iron and folate status to assess the extent to which iron status and folate status affect milk folate and iron content in Otomi women.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Lactating women (n = 71) residing in the rural, farming, Indian community of San Mateo, Capulhuac, Mexico, which is located 2800 m above sea level, furnished blood and milk samples, as well as dietary and demographic information, at 22 ± 13 ( ± SD) d postpartum. A detailed description of this community and the dietary habits of its residents was published previously (19). Briefly, Otomi women subsist on a maize-predominant diet, and the proportion of meat included in the diet is limited. Protein from animal sources (mostly eggs) makes up only 30% of all protein consumed. Dark leafy vegetables and legumes are frequently eaten after prolonged boiling, and hence the folate content of these usually good sources of folate is low (23). The calculated dietary intake of phytic acid is very high because of the consumption of corn (65% of energy intake) and other phytate-containing vegetables. In most cases, exclusive breastfeeding extends for >= 4 mo. In the few cases in which infants are not exclusively breastfed at 4 mo, foods other than human milk constitute only 2% of total energy intake (16). The total duration of breastfeeding averages 18 mo.

Women who were willing to exclusively breastfeed their offspring were recruited from the local medical clinic and enrolled in the study if they met the eligibility requirements. The eligibility requirements included the following: 1) having an age between 17 and 37 y, 2) being free of chronic diseases, 3) not currently taking any medication, 4) not consuming alcoholic beverages, 5) being free of pregnancy complications, 6) having given birth to a term infant of appropriate length and weight, and 7) being willing to comply with the experimental protocol. The participation of human subjects was approved by the Institutional Review Boards of both the Instituto Mexicano del Seguro Social, Mexico City, and the Pennsylvania State University, State College.

Anthropometric measurements, dietary intakes, and blood sample collection
Maternal weight and height and infant weight and length were measured in the clinic with the use of standardized procedures (24). Weights were measured by using electronic scales with precisions of ± 0.1 g for infants (Sartorious, Gotttingen, Germany) and ± 50 g for women (Tanita W8; Tanita Inc, Tokyo, Japan). Heights and lengths (± 1 mm) were measured with the use of a clinical stadiometer and an infant measuring board (Holtain Ltd, Croswell, Crymych, United Kingdom), respectively. Head circumferences (± 1 mm) were measured with the use of a nonstretchable tape measure (InserTape; Abbott Laboratories, Columbus, OH). For each infant, z scores were calculated for weight-for-age, length-for-age, weight-for-length, and head circumference–for–age by using the year 2000 growth charts from the Centers for Disease Control and Prevention and supporting software (25).

Usual maternal dietary intakes of energy, protein, fat, carbohydrates, iron, folate, and vitamin B-12 were estimated by using 3 d of dietary data collected according to a detailed standardized protocol. Briefly, community workers trained by a nutritionist recorded the dietary intakes of the study participants in their homes for 2 consecutive days. Dietary intakes were assessed during one weekend day by using an interactive 24-h dietary recall method that included information previously collected on standard serving sizes. Nutrient intakes were tabulated by using Mexican food composition tables (26).

After the subjects fasted overnight, blood samples were collected either into tubes containing EDTA or into tubes that were free of trace elements and did not contain an anticoagulant (Vacutainer; Becton Dickinson, Franklin Lakes, NJ). A portion (100 µL) of whole blood was diluted in 10 volumes of a 0.1-mol potassium phosphate/L buffer containing 0.05 mol sodium ascorbate/L. Milk was collected by using an electric breast pump (Egnel, Libertyville, Illinois) to completely express the milk from one breast >= 2 h after the previous feeding. For folate analyses, 1% sodium ascorbate was dissolved into milk and plasma samples, and all milk and blood samples were frozen at -70 °C. Milk and serum samples for determination of milk and blood indexes of iron status were immediately frozen at -20 °C until analyzed.

Biochemical analyses
A complete blood count analysis, including measurements of hematocrit, hemoglobin concentration, and mean cell volume (MCV), was performed by using fresh blood samples and an electronic particle counter analyzer (ACT8 Coulter Counter; Beckman Coulter, Miami). C-reactive protein concentrations were measured by immunoprecipitation (SANOFI; Pasteur Diagnostic, Mexico City).

To assess folate status, erythrocyte and plasma folate were determined by microbiological assay with cryoprotected Lactobacillus casei (ATCC #7469; American Type Tissue Culture Collection, Manassas, VA) as the test organism (27). Before the microbiological assay, whole blood lysates were incubated for 25 min at 37 °C to convert folates to their microbiologically assayable forms (eg, short-chain folylpolyglutamates). Erythrocyte folate content was determined by using the analyzed whole blood folate concentration minus the plasma folate content corrected for hematocrit. Milk samples were processed with a trienzyme digestion procedure before microbiological assay as described in detail by Mackey and Picciano (6). The accuracy and reproducibility of these assays were assessed by using infant formula with a certified mean (± SD) value of 1.29 ± 0.28 mg folic acid/kg (standard reference material no. 1846; National Institute of Standards and Technology, Gaithersburg, MD). Analysis in our laboratory yielded a folate content of 1.33 ± 0.05 mg folate/kg.

Serum iron and total-iron-binding capacity were determined by using the colorimetric method of Fielding (28) modified for determination in microliter sample volumes. Fetal bovine serum with a certified value of 32 ± 2 µmol Fe/L (176 ± 10 µg Fe/dL) (lot #7000C; Atlanta Biologicals, Norcross, GA) was used to verify the accuracy and reproducibility of this method. Analysis in our laboratory yielded an iron content of 30 ± 0.7 µmol/L.

For milk iron measurement, quartz vials containing thawed milk were dried to a constant weight. Samples were heated for 12 h in a muffle furnace at 500 °C, and then the ashes were cooled in a dessicator. The ashes were then dissolved in a 2-mol HNO3/L solution containing 2% thioglycolic acid for iron reduction. An aliquot of 50 µL was placed in a 96-well plate, and 100 µL chromogen (as for serum iron analysis) was added. Infant formula with a certified value of 11.4 ± 0.7 mmol Fe/kg (63.1 ± 4.0 mg Fe/kg) (standard reference material no. 1846) was used to assess the accuracy and precision of the milk iron assay. Analysis in our laboratory yielded an iron content of 10.7 ± 0.5 mmol/kg (58.9 ± 3.0 mg/kg).

Plasma ferritin concentrations were measured by radioimmunoassay (Diagnostic Products, Los Angeles) and verified with the use of a ferritin standard (19 ± 1.5 µg/L). Results from our laboratory yielded a plasma ferritin concentration of 20 ± 1.1 µg/L. Transferrin receptor concentrations were measured by enzyme immunoassay (Ramco Laboratories Inc, Houston), for which high and low quality-control serum samples were provided.

Maternal iron and folate status assessment models
A 3-index iron assessment model and normative cutoff values for nonpregrant women, including ferritin (<= 12 µg/L), transferrin saturation (TS; <= 16%), and MCV (<= 80 fL), which were described by Cook et al (29), were used in the present study. With the use of receiver operating characteristic curve analysis and blood samples from lactating women in this community, a sample-specific hemoglobin cutoff of 133 g/L was derived and reported (30, 31). This hemoglobin cutoff was used in the present study and is in agreement with the World Health Organization (32) recommended value, which was adjusted for altitude (33–35). According to the 3-index model, subjects who were positive for >= 2 of these iron deficiency measures (ferritin, TS, and MCV) were considered iron deficient. On the basis of this 3-index model and hemoglobin concentrations, the subjects were classified as follows: 1) iron sufficient: adequate iron and adequate hemoglobin, 2) iron-deficient erythropoietic: low iron and adequate hemoglobin, 3) iron-deficient anemic: low iron with low hemoglobin, and 4) indeterminate iron status: one index of iron status (ferritin, TS, or MCV) indicative of iron depletion with or without low hemoglobin.

The following criteria were applied in the assessment of folate status. 1) Folate-sufficient subjects had an erythrocyte folate (EF) concentration > 360 nmol/L (24), a plasma folate (PF) concentration > 10 nmol/L (36), and an MCV < 94 fL (29) with or without adequate hemoglobin. This plasma folate cutoff is based on the use of plasma homocysteine as an indicator of folate undernutrition (36). 2) Folate-deficient subjects had an EF or PF concentration below the respective cutoff value but adequate MCV and hemoglobin. 3) Subjects with a low EF or PF concentration in addition to an elevated MCV but with adequate hemoglobin were classified separately. 4) Subjects with a low EF or PF concentration, an MCV < 94 fL, and low hemoglobin were classifed as folate deficient with a hemoglobin concentration <= 133 g/L. 5) Finally, subjects with a low EF or PF concentration, an elevated MCV, and low hemoglobin were considered folate-deficient anemic.

Statistical design and analyses
All statistical analyses were performed with SAS for WINDOWS, release 8.1 (SAS Institute Inc, Cary, NC), and unless indicated otherwise, statistical significance was set at P < 0.05. All values in the text are expressed as means ± SDs unless noted otherwise. All variables that were not normally distributed were transformed by using either the natural log or square root. We were unable to produce normal distributions for the following study variables: dietary folate intake, dietary vitamin B-12 intake, and milk iron concentration, and, as appropriate, nonparametric statistics were applied as described below.

Pearson correlation coefficients were computed to assess whether and to what extent relations existed between normally distributed or normalized infant anthropometric, blood iron, and blood folate measures. Dietary intakes of energy, iron, folate, and vitamin B-12 were divided into quartiles. Milk and blood iron and folate measures were compared across these energy and nutrient quartiles by using either analysis of covariance controlled for days postpartum and energy or Kruskal-Wallis tests of statistical significance. Likewise, either analysis of covariance or Kruskal-Wallis statistics were used to compare milk folate and milk iron concentrations between the women categorized as iron sufficient, iron deficient, or of indeterminate iron status. Pairwise comparisons of milk and blood iron and folate measures across quartiles of dietary nutrient intake or categories of maternal iron status (eg, iron sufficient, iron deficient, indeterminate iron status) were performed by using least significant difference tests if a P value for analysis of covariance statistics was < 0.05. No P values < 0.05 were generated for Kruskal-Wallis statistics, and hence pairwise comparisons were not performed. A Bonferroni-adjusted {alpha}-value (0.05/6, or 0.0083) was assigned as the level of statistical significance for these post hoc pairwise comparisons (37).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subject characteristics
Seventy-one women aged 24 ± 5 y (range: 17–37 y) were recruited. All had exclusively breastfed their infants from birth. A complete set of biochemical data for determination of iron status and folate status was available for 66 and 67 women, respectively. The average weight and height of the women in the sample was 53 ± 7.6 kg (range: 38–77 kg) and 1.5 ± 0.1 m (range: 1.3–1.7 m), respectively. All but 9 of the subjects were stunted (height < 152.7 cm), but none were wasted [body mass index (in kg/m2) < 17.8] (25). Twelve percent of the subjects were overweight (body mass index > 26.5) (25). The average parity was 3, and 17 (24%) of the women were primiparous. Almost 90% of the women had < 3 y of education. Evidence of infection or inflammation was apparent in 6 subjects on the basis of a C-reactive protein concentration > 11 mg/L. The average weight and length of study infants at the postpartum clinic visit was 3.5 ± 0.6 kg (range: 2.3–5.4 kg) and 51 ± 2.4 cm (range: 47–58 cm), respectively. As shown in Table 1Go, the infants were small in comparison with the 2000 reference population from the Centers for Disease Control and Prevention (25).


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  		TABLE 1 . Distributions of weight, length, and head circumference measurements among Otomi infants at a mean (±SD) of 22 ± 13 d postpartum1
 
Dietary assessment
Calculated dietary intakes of energy, iron, folate, and vitamin B-12 are summarized in Table 2Go. Median energy intakes were {approx}53% of the estimated energy requirement for an active person (38). As a percentage of total energy consumed, carbohydrate intake in our study population was higher than that in lactating women in the US, but intakes of fat and protein were lower in our study population (40, 41). Dietary macronutrient intakes, as a percentage of total energy consumed, were consistent with current recommendations regarding adequate macronutrient distribution ranges (38).


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  		TABLE 2 . Comparison of tabulated energy and nutrient intakes in lactating Otomi women at a mean (±SD) of 22 ± 13 d postpartum with dietary reference intakes (DRIs) and reported intakes in US lactating women1
 
The mean dietary iron intake in our study population was considerably higher than the currently recommended intake (21). In contrast, the median dietary folate intake (86 µg/d) was {approx}17% of that recommended (500 µg/d) and <= 29% of that in US lactating women (299 or 411 µg/d) (40, 41). Likewise, the median vitamin B-12 intake among Otomi women was {approx}50% of that recommended and considerably less than actual intakes among US women (39, 40, 41).

Evaluation of blood indexes of iron and folate status
Approximately 62% and 58% of the women had signs of nutritional anemia on the exclusive basis of either a hemoglobin concentration <= 133 g/L (31, 33, 34) or a hematocrit < 41.0% (31, 35) (Tables 3Go and 4Go). The prevalence of low serum iron (<= 11 µmol/L), TS (<= 16%), and ferritin (<= 12 µg/L) each approached 50%. Using the 3-index iron assessment model with and without hemoglobin, 2 women (3%) were classified as having iron deficient erythropoiesis, 24 (36%) had iron deficiency anemia, and 19 (29%) had indeterminate iron status. The latter category included women who had at least one of the following in the range indicative of iron deficiency: ferritin, TS, or MCV, with or without a low hemoglobin concentration. Exclusion of women who had an elevated C-reactive protein concentration did not appreciably alter the aforementioned results.


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  		TABLE 3 . Iron and folate measures in blood and milk samples collected from Otomi women at a mean (±SD) of 22 ± 13 d postpartum1
 

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  		TABLE 4 . Biochemical iron and folate status of lactating Otomi women at a mean (±SD) of 22 ± 13 d postpartum1
 
Twenty-nine women (43%) had a low plasma folate concentration. Thirteen subjects (19%) had a low blood folate concentration in conjunction with a hemoglobin concentration <= 133 mg/L. Three additional subjects ({approx}5%) had a low blood folate concentration in conjunction with both an elevated MCV (> 94 fL) and a low hemoglobin concentration. Thirteen subjects (20%) showed signs of both iron and folate deficiencies. Four subjects (6%) had anemia of unknown origin.

Maternal plasma folate concentrations were associated with the length-for-age z scores of the infants at 22 d postpartum (r = 0.34, P = 0.0007), which suggests a relation between maternal folate status and infant birth size. This observation is consistent with the results of several reports in the literature that link maternal folate status in both developed and developing countries with infant birth size (42). Maternal folate status was not related to the infants’ weight-for-age, weight-for-length, or head circumference–for–age. Likewise, no significant associations were found between maternal indexes of iron status and infant size.

Correlation of iron and folate status measures
The measures of biochemical iron status (ie, hemoglobin, hematocrit, MCV, serum iron, TS, and ferritin) were strongly correlated (r: 0.51–0.96, P < 0.0001). As anticipated, these iron indexes were inversely correlated with transferrin receptor concentrations (hemoglobin: r = -0.60, P < 0.0001; hematocrit: r = -0.57, P < 0.0001; MCV: r = -0.39, P = 0.0009; serum iron: r = -0.22, P = 0.0659; TS: r = -0.27, P = 0.03; ferritin: r = -0.49, P < 0.0001). The expression of this receptor is known to increase with diminution of iron status. Total-iron-binding capacity did not correlate with other measures of iron status and decreased with the number of days postpartum, regardless of iron status. Plasma and erythrocyte folate concentrations were positively correlated (r = 0.524, P < 0.0001).

Neither plasma nor erythrocyte folate concentrations were correlated with any biochemical index of iron status when subjects with a C-reactive protein concentration > 11 mg/L were included in the analysis. Once these latter 6 subjects were removed, inverse correlations of erthyrocyte folate concentrations with serum iron and TS were found (r = -0.27 and -0.25, respectively, P < 0.05).

Dietary intakes, blood indexes of iron and folate status, and their effect on the iron and folate content of milk
Biochemical blood indexes of iron and folate status were compared across quartiles of dietary intakes of energy, iron, folate, and vitamin B-12. As appropriate, the analyses were controlled for days postpartum. There were no differences in any biochemical index of iron or folate status across quartiles of dietary intakes of energy, iron, folate, and vitamin B-12. Likewise, there was no relation of dietary intakes of energy, iron, folate, and vitamin B-12 with milk iron concentrations. As summarized in Table 5Go, milk folate concentrations were found to be related to dietary iron intake after women with a C-reactive protein concentration > 11 mg/L were removed from the analysis (P = 0.02). In this latter model, both energy intake (P = 0.0101) and the number of days postpartum (P = 0.01) were also predictive of milk folate concentration. The women in the lowest quartile of dietary iron intake had significantly lower milk folate concentrations than did those in the second and third quartiles, at an {alpha}-level < 0.05. The milk folate concentrations in the women in the highest quartile of dietary iron intake did not differ significantly from those in the women in the other quartiles. Only the difference between the lowest quartile and the third quartile remained significant after the use of a Bonferroni-adjusted {alpha}-value (0.05/6, or 0.0083) to address the issue of multiple comparisons (37).


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  		TABLE 5 . Concentrations of folate and iron in the milk of Otomi women at a mean (±SD) of 22 ± 13 d postpartum across quartiles of intakes of dietary iron, folate, and vitamin B-121
 
Except for hematocrit and hemoglobin concentration, blood biochemical indexes of iron and folate status were unrelated to milk folate concentrations. Significant inverse associations were found between hematocrit and milk folate concentrations and between hemoglobin and milk folate concentrations (r = -0.39 and -0.36, respectively, P < 0.005). These associations remained significant even after energy intake and the number of days postpartum were controlled for and subjects with an elevated C-reactive protein concentration were included in the analysis. Because milk iron concentrations were not normally distributed and could not be normalized, statistical comparisons of blood biochemical indexes of iron and folate status with milk iron concentrations were not performed.

Comparison of mean milk folate concentrations between women who were categorized as iron sufficient, iron deficient, or iron indeterminate with the use of the 3-index model did not show any significant differences even after the number of days postpartum and energy intake were controlled for. Likewise, no significant differences in mean milk iron concentration were detected between women classified as iron sufficient, iron deficient, or iron indeterminate.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A significant proportion of lactating Otomi women in this study had compromised biochemical indexes of iron status. On the basis of a 3-index iron assessment model, 26 women (39%) had either iron-deficient erythropoiesis (n = 2) or iron deficiency anemia (n = 24). Fifty-four percent of the women had low iron stores (ferritin concentration <= 12 µg/L). The women’s absolute dietary iron intakes exceeded the 2001 recommended dietary allowance for this nutrient during lactation (9 mg/d) (21). The disjuncture between adequate iron intakes and the high prevalence of iron deficiency probably reflects the low bioavailability of iron in the diets consumed and the net maternal iron deficit accrued during pregnancy (2001 recommended dietary allowance for pregnancy = 27 mg/d). Indeed, it is well documented that the typical Mexican rural diet contains predominately nonheme sources of iron rather than the more bioavailable heme iron, is high in phytate and oxalate (iron-absorption inhibitors), and is low in ascorbic acid (iron-absorption enhancer) (43).

In addition to the high prevalence of iron deficiency, 43% of the women showed evidence of folate depletion. Dietary intakes of folate were extremely low: the median intake was 86 µg/d (first and third quartiles: 40, 137 µg/d), compared with the currently recommended intake during lactation of 500 µg/d (39). These intakes may, in fact, overestimate actual folate intakes because vegetables in this community are typically boiled for long periods, a practice that probably destroys heat-labile folates (23). Twenty-nine women (43%) had a low plasma folate concentration. Thirteen subjects (19%) had a low blood folate concentration in conjunction with a hemoglobin concentration <= 133 mg/L.

The iron concentrations in the milk produced by the women in this study were well within the range of those reported for milk from well-nourished women in the United States (21), and this milk would provide {approx}0.37 g highly available iron/d to an infant consuming 0.85 L human milk/d. The latter milk volume was previously shown to be the mean quantity of milk consumed by infants in this community (21). The current adequate intake of iron for breastfed infants aged 0–6 mo is 0.27 g/d. In contrast with milk iron, the folate content of the milk collected from the Otomi women was considerably lower than that reported for milk from American women (40, 41). The mean folate concentration in the milk collected from the Otomi women (120 ± 129 nmol/L) was {approx}60% of the 192 nmol/L that we reported for well-nourished American women by using optimized assay conditions (44–46). The latter value of 192 nmol/L was used to calculate the adequate intake of folate for infants aged 0–6 mo in the dietary reference intakes for folate (39). With the assumption of an average milk intake of 0.85 L/d for Otomi infants aged 0–5 mo (16), the mean folate intake of the infants in this community is {approx}45 µg/d, or 70% of the currently recommended intake (39). Consequently, some infants in this community may be ingesting insufficient folate to maintain appropriate tissue concentrations.

The mechanisms for the low milk folate concentrations observed in the present study are not clear. In well-nourished women, milk folate concentrations are believed to be fairly well preserved across a range of maternal folate intakes and blood folate concentrations (6, 7, 47). In the case of severe maternal folate deficiency, as evidenced by megaloblastic anemia, milk folate concentrations decrease precipitously (7). The prevalence of folate depletion was widespread in this sample but was infrequently associated with megaloblastic anemia, although megaloblastic anemia may have been masked in many instances by a concomitant iron deficiency. The fact that milk folate concentrations were associated neither with dietary folate intake nor with plasma or erythrocyte indexes of folate status makes it difficult to absolutely conclude that inadequate maternal dietary folate intakes per se were responsible for the lower-than-expected milk folate concentrations reported in the present study.

In the present study, there was evidence of an interaction between iron and folate metabolism, although this relation was much more complex than previously observed in our animal studies (9–12). Specifically, maternal hematocrit values and hemoglobin concentrations were inversely related to milk folate concentrations (r = -0.39 and -0.36, respectively, P < 0.005), even after energy intake and the number of days postpartum were controlled for. In addition, after subjects with evidence of infection were removed from the statistical analysis, an inverse correlation of erthyrocyte folate concentrations with serum iron and TS was found (r = -0.27 and -0.25, respectively, P < 0.05).

In contrast with our experiments using iron-deficient animal models, in which an ample supply of dietary folate was typically provided, the mothers in the present study had both poor iron status and poor folate status. As we reviewed in detail elsewhere, observations both in humans and in animal models suggest that iron and folate deficiencies can develop independently of each other, secondary to poor dietary sources of each, or that iron deficiency, particularly during reproduction, can negatively alter folate metabolism (48). Yet, several circumstances have been described in which correction of a primary iron deficiency either causes folate deficiency or exacerbates a mild form of this disorder (48–50). We suggest that in the present study, the inverse relation of milk and erythrocyte folate concentrations with some biochemical indexes of iron status reflects the latter situation. In the present study, a significant percentage of the women showed evidence of iron deficiency, folate deficiency, or both. Among the iron-depleted women, a sufficient supply of folate appeared to be available to support the reduced level of erythropoiesis. In contrast, the negative interaction between some biochemical indexes of iron status and erythocyte folate status suggests that when iron is no longer limiting, there is a greater demand for folate, which reflects a lower erythrocyte folate concentration and reduced secretion of folate into milk.

The possibility that poor maternal vitamin B-12 status is responsible, at least in part, for the low milk folate concentrations reported in the present study cannot be discounted. Although the amount of vitamin B-12 consumed by the Otomi women in this study was not related to milk folate content, the median vitamin B-12 intake of the lactating Otomi women was {approx}50% of that recommended and was considerably less than that of women in the US (40, 41). The low dietary vitamin B-12 intakes reported in the present study are consistent with those previously reported among rural Mexican and periurban Guatemalan lactating women (51, 52). Vitamin B-12 serves as a cofactor for the conversion of methyltetrahydrofolate to tetrahydrofolate. The latter is believed to be a preferred substrate for folylpolyglutamate synthetase, an enzyme responsible for attaching glutamate residues onto folate, which is a critical step in maintaining and concentrating this vitamin within mammary epithelial cells (48).

We acknowledge that the previously reported growth faltering of otherwise healthy breastfed infants in this rural farming community between 4 and 6 mo of age is probably due to many factors, including both nutritional and nonnutritional factors (eg, altitude, infection, etc) (16, 17). As previously shown, milk concentrations of energy, protein, carbohydrates, calcium, magnesium, phosphorus, and copper are probably not responsible for growth faltering because intakes appear to be comparable with those of healthy, breastfed American infants and exceed adequate intakes (16, 20–22). Although the zinc concentration in the milk of Otomi women at 4 and 6 mo postpartum is comparable with that in the milk of American women (20), there is controversy in the literature about whether either is sufficient to fully meet the growth requirements of rapidly growing infants after 4 mo of age (21). On the basis of unpublished data collected from 40 lactating Otomi women, we suspect that zinc intakes are low ( ± SEM = 2.6 ± 2.5 mg/d) relative to the recommended intake of 12 mg/d (21). National data from Mexico suggest that the median zinc intake of females aged 12–49 y is 6 mg/d (first, third quartile: 4.2, 8.2 mg/d) (43). Thus, Otomi infants may be born with low zinc stores and, as a consequence, may be disproportionately vulnerable to low milk zinc concentrations.

The relatively high altitude of Capulhuac (2800 m) may also influence the physical growth of Otomi infants. Birth weight and postnatal body size are lower at altitudes > 1500 m than at lower altitudes (53, 54). Although there appears to be some controversy as to whether postnatal weights are affected once they are adjusted for birth weight, there does appear to be a greater effect on linear growth after birth length is controlled for.

In conclusion, results from the present study suggest a high prevalence of suboptimal iron status, folate status, or both among lactating women in the rural farming community of San Mateo, Capulhuac, Mexico. In addition, median vitamin B-12 intakes were markedly lower than the intakes recommended during lactation. Despite poor maternal iron status, the milk iron concentrations reported in the present study would provide more than adequate amounts of bioavailable iron for breastfed infants. In contrast, milk folate concentrations may be limiting for some infants. Whether the milk folate concentrations reported in the present study are indeed sufficient to alter the folate metabolism and growth of exclusively breastfed infants in this community is worthy of further investigation.


   ACKNOWLEDGMENTS
 
SV and MFP designed the study. MEL, GR, and MJI collected the data and performed biochemical analyses. MEL and DLO performed data analysis and wrote the article. SV and MFP contributed significantly to the execution of the study and provided editorial assistance in writing the manuscript. None of the authors had any personal or financial conflicts of interest with the sponsoring institution.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication January 10, 2003. Accepted for publication April 29, 2003.




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