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Objective: To test whether breastfeeding's protection against anorectic responses to infection is mediated by n-3 fatty acids' attenuation of interleukin (IL)-1beta and tumor necrosis factor (TNF)alpha., Design: Experimental and observational studies., Setting: A hospital-based study was conducted., Subjects: Five groups of infants were followed; three in the experimental and two in the observational study., Methods: Breast-fed- (BF-1), DHA-supplemented formula- (SFF-1), and non-DHA-supplemented formula-fed (FF-1) infants were studied before and after immunization against diphtheria, tetanus, pertussis and haemophilus influenzae type b. Pre- and post-immunization energy intakes (EI) and serum IL-1beta and TNFalpha were measured. The two other groups, breast-fed (BF-2) and formula-fed (FF-2) infants with pneumonia were followed throughout hospitalization. EI, IL-1beta and TNFalpha were measured at admission and discharge. Baseline erythrocyte fatty acid contents were determined., Results: Both cytokines increased following immunization in all feeding groups. Post-immunization reductions in EI of SFF-1 infants (-11.8+/-5%, CI(95)=-23.3, 1.4%, P=0.07) were intermediate to those observed in BF-1 (-5.2+/-4.2%, CI(95)=-15.2, 5.9%, P=0.27) and FF-1 infants (-18+/-4.4%, CI(95)=-29%, -5.4%, P=0.02). In the observational study, TNFalpha (17.2+/-8.3 vs 3.4+/-3.0 ng/l, P=0.001) and decreases in EI (-31+/-43 vs -15+/-31%, CI(95)=-34%, 0.001%, P=0.056) were greater in FF-2 than in BF-2 infants at admission. Breastfeeding duration was associated positively with docosahexaenoic acid (DHA) erythrocyte contents, and negatively with admission TNFalpha. Decreases in EIs were associated with IL-1beta and TNFalpha concentrations., Conclusion: Reductions in EI following immunologic or infectious stimuli were associated with increases in IL-1beta and TNFalpha. Those reductions were attenuated by breastfeeding, and mediated in part by tissue DHA.

An attenuated severity of infections is among the well-documented benefits of breast-feeding. The degree to which this attenuated severity extends to the amelioration of anorexia is understood incompletely, and possible underlying mechanisms have received limited evaluation. This study was designed to test whether breast-feeding attenuates reductions in energy intake associated with a mild immunologic stimulus and to assess poststimulus relationships among putative reductions in energy intake and serum interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and leptin concentrations. A quasi-experimental, hospital-based study was conducted in 23 healthy fully breast- (BF) and formula-fed (FF) infants who received the quadruple diphtheria, pertussis, tetanus and hemophilus influenza (DPTH) immunization as an immunologic challenge. Only FF infants had decreased energy intakes (12 +/- 2%, P = 0.001) after immunization. Leptin concentrations increased after immunization only in FF infants (30 +/- 7%, P = 0.03). Correlations between postimmunization increases in IL-beta and reductions in energy intake were of borderline significance (r = -0.56, P = 0.08). These findings support the view that breast-feeding protects against anorectic responses to mild immunologic stimuli. Increases in leptin are associated with reductions in energy consumption in the postimmunization period in FF infants and postimmunization changes in IL-1beta concentrations likely are related to reductions in energy intake in response to immunologic stimuli.

The partitioning of dietary and endogenous nutrients during lactation is not well understood. To examine associations between plasma hormone and substrate profiles and indices of either maternal body protein metabolism or lactational performance, we measured plasma insulin, cortisol, prolactin, thyroxine, triiodothyronine, individual amino acid, blood urea nitrogen, and prealbumin concentrations in lactating and nulliparous women in the postabsorptive state. We related these measurements to the subjects' nitrogen balance, urinary 3-methylhistidine excretion, [1-13C]leucine metabolism and milk production. Insulin concentrations showed significant positive relationships with nitrogen balance and prealbumin concentrations; cortisol levels showed a significant negative relationship with nitrogen balance and a significant positive relationship with leucine incorporation into protein. Thyroid hormone concentrations showed significant positive relationships with urinary 3-methylhistidine excretion, leucine incorporation into protein, and milk production. Proline concentrations were associated positively with nitrogen balance and negatively with leucine incorporation into protein, whereas glutamate-glutamine concentrations showed positive associations with leucine oxidation and milk nitrogen concentrations. We propose that insulin and cortisol modulate the channeling of nutrients between anabolic and anti-anabolic aspects of maternal body protein metabolism, whereas thyroid hormones and cortisol modulate nutrient partitioning toward milk production and visceral protein synthesis. We suggest that some nonessential amino acids (proline, glutamate-glutamine) may become limiting during lactation because of their unique contributions to milk protein synthesis.

To determine whether growth faltering during early infancy was attributable to inadequate intake of human milk, the nutrient intakes and growth of 30 Otomi infants from Capulhuac, Mexico, were studied at 4 or 6 mo of age. Growth was monitored monthly from 1 through 6 mo of age. The 2H dose-to-the-mother method was used to measure human milk intake. Energy, protein, lactose, and fat concentrations in milk were analyzed by standard techniques. Mean (+/- SD) human milk intakes were 885 +/- 145 and 869 +/- 150 g/d at 4 and 6 mo, respectively. Protein and lactose concentrations in milk were normal but fat and consequently energy concentrations were abnormally low. Energy intakes averaged 81 +/- 14 kcal.kg-1.d-1 at 4 mo and 72 +/- 14 kcal.kg-1.d-1 at 6 mo. Growth faltering by 6 mo was evidenced by the significant decline in growth velocities and National Center for Health Statistics Z scores. Weight gain at 6 mo was 8.1 +/- 3.5 g/d and length gain was 1.0 +/- 0.34 cm/mo. Weight-for-age and length-for-age Z scores were -0.81 +/- 0.94 and -1.51 +/- 0.83, respectively. Growth velocities were not significantly correlated with nutrient intakes. Growth faltering among the Otomi infants despite energy intakes comparable to those of breast-fed infants in more protected environments may have resulted from an increase in the need for nutrients or from a growth-limiting nutrient, other than energy, in their diet.

We evaluated bone mineralization by single photon absorptiometry at 2 y in a cohort of preterm infants studied since birth. Infants were fed human milk fortified with Ca [to achieve 80 mg/dL (19.96 mmol/L)] and P [40 mg/dL (12.91 mmol/L)] from wk 2 through 8 after birth. After hospital discharge, infants were divided into two groups (HM and F) determined by the timing of the introduction of cow milk-based formula. Mid-radius bone mineral content (BMC) was assessed in 10 infants who were breast-fed (HM) for a minimum of 2 mo after hospital discharge and 11 who were bottle-fed (F). The mean duration of human milk-feeding differed by design between HM and F groups (31 +/- 15 versus 11 +/- 3 wk, respectively). Although we had observed previously that group F had significantly greater BMC values at 16, 25, and 52 wk compared with values in group HM, we found similarities in BMC values (180 +/- 30 mg/cm) between groups at 2 y. The 2-y cohort comprised healthy infants and the groups had similar birth weights, lengths of gestation, and values for weight (10.8 +/- 1.1 kg), length (82 +/- 2 cm), and bone width (7.8 +/- 1.1 mm). Follow-up outcomes at 2 y in preterm infants fed fortified human milk in hospital suggest that if they continue to receive human milk after hospital discharge, radius BMC will "catch-up" to that of similar infants given formula in the posthospitalization period.

Vitamin K was quantitated in the milk of four groups of 15 mothers from 1 d to 6 mo postpartum in a cross-sectional study. Concentrations were 7.52 +/- 5.90 and 6.36 +/- 5.32 nmol/L (3.39 +/- 2.66 and 2.87 +/- 2.40 micrograms/L) in colostrum and mature milk, respectively. Differences between colostrum and mature milk or among samples of mature milk collected at 1, 3, and 6 mo were not statistically significant. Because of significantly increased volumes of milk over the lactation period, approximately twice as much vitamin K was delivered in mature milk as in colostrum. Within normal ranges, concentrations of vitamin K in milk were not predicted by dietary intake of vegetables or fat. Vitamin K was correlated with fat in colostrum and was localized in the lipid core of the milk fat globule but was not associated with membranes. Vitamin K in human milk is insufficient to meet recommended intakes for infants aged less than 6 mo. Population and clinical studies are needed to assess the vitamin K status of exclusively breast-fed infants and to evaluate current recommendations.

The origin of intact (78-kD) lactoferrin found in the urine of human milk-fed preterm infants was investigated using human milk containing proteins enriched with [13C]leucine and [15N2]lysine or [2H4]lysine. Mothers of infants selected for the study were infused i.v. with [13C] leucine and [15N2]lysine or [2H4]lysine to label milk proteins. The labeled milk was collected from each mother, pooled, fortified with a lyophilized human milk fraction, and fed to her preterm infant by continuous orogastric infusion for a period of 48 h. Urine was collected from each infant for 96 h. Intact lactoferrin (78 kD) and DNA-binding lactoferrin fragments (51 and 39 kD) were purified from the urine by affinity chromatography on columns of immobilized single-stranded DNA-agarose. The concentration and isotopic enrichment of the intact lactoferrin and DNA-binding fragments were determined separately after their isolation by high-performance reverse-phase (phenyl) chromatography. Mass spectral analyses indicated that the isotopic enrichment of the purified urinary lactoferrin was 87 to 100% of that in the labeled human milk lactoferrin. Similar results were obtained for the isolated DNA-binding lactoferrin fragments. The ratios of isotopically labeled leucine to lysine in the purified milk lactoferrins and urinary lactoferrins were similar for each mother/infant pair. Isotopically labeled lysine, added to the milk as free amino acid, was not incorporated into the purified urinary lactoferrin. These results demonstrate that undegraded (78-kD) lactoferrin of maternal origin is absorbed by the gut and excreted intact in the urine of preterm infants; nearly all of the urinary lactoferrin was of maternal origin. The possible immunoregulatory functions of the absorbed intact, maternal lactoferrin are discussed.

A discrepancy between current recommendations for energy intakes and the reported intakes of human milk-fed infants has led to the suggestion that energy requirements of infants should be reevaluated. Gross energy intakes of 1-month-old, exclusively breast-fed infants are similar to recommended amounts of metabolizable energy; however, by 4 months of age, gross energy intakes of exclusively breast-fed infants are significantly less than current recommendations. Weight-for-age percentiles also fall during that period. Energy intakes (measured in kilocalories per kilogram) persist at less than recommended amounts, and weight-for-age percentiles continue to drop after solid foods are added to the diet. Energy intakes of formula-fed infants appear significantly higher than those of infants who are fed human milk. Growth rates, total daily energy expenditure, sleeping metabolic rates, minimal rates of energy expenditure, rectal temperature, and heart rates have been found to be lower in breast-fed infants. The maintenance of energy intakes and growth trajectories during the period of demand supplementation with solid foods suggests that intakes, growth rates, and rates of energy expenditure of human milk-fed infants are physiologic responses to an appropriate dietary intake.

The molecular forms of lactoferrin (LF) were examined in stools and urine collected at 2.5 or 5 wk of age from very low birth wt infants fed either a cow's milk formula or a fortified human milk preparation. LF was not found by Western blotting in excreta from infants fed cow's milk. In contrast, intact and fragmented forms of LF were detected in stools and concentrated urine of each infant who received human milk. Only intact LF was detected in the fortified human milk preparation, whereas many types of LF fragments were present in the stools and urine. The approximate molecular wt of the most prominent fragments were 44, 38, 34, and 32 kD. However, the stools also displayed lower molecular wt fragments that were not found in urines of those infants. The LF fragments in those excreta were similar in size to those produced in vitro by limited digestion of apo-LF with trypsin. Furthermore, fragments produced by in vitro proteolysis were immunoreactive in an ELISA for LF. Thus, the fragments of LF in stools of very low birth wt infants fed human milk appeared to be produced by in vivo proteolysis, and the close resemblance between the LF fragments in the stools and urine suggests that the urinary LF fragments originated in the gastrointestinal tract. It remains unclear, however, whether the whole LF molecules that were fragmented were derived solely from ingested LF in human milk or in part from LF produced by the infant in response to human milk feedings.

The macronutrient, trace mineral, and immunological contents of milk of five moderately controlled, insulin-dependent diabetic women were studied at 3 months postpartum. Concentrations of total nitrogen, lactose, fat, and energy were not distinguishable from those in milk of nondiabetic women. Glucose concentrations in milk from diabetic women were significantly higher (p less than 0.001) and more variable. The macro- and trace minerals (Ca, P, Mg, K, Zn, Cu, and Fe) were within normal ranges with the exception of Na which was higher in milk from the diabetic women (p less than 0.05). The concentrations of lactoferrin and secretory IgA were not statistically different from reference norms. Milk composition of five insulin-dependent diabetic women was not distinguishable from that of the reference population, with the exceptions of Na and glucose concentrations, which were slightly elevated.

The amounts of lactoferrin, lysozyme, SIgA and SIgA antibodies to E. coli somatic antigens in human milk ingested per day and per kg per day by breast-fed infants were determined during the first four months of life. A gradual decline in the amounts of lactoferrin, SIgA, and SIgA antibodies ingested per day or per kg per day was found, whereas the quantities of lysozyme ingested by the infants rose during that period. These data suggest that the production and secretion of these immunologic factors by the mammary gland may be linked to the ontogeny of the production or catabolism of those components at mucosal tissues of the recipient infant.

The relationships between lactation performance and maternal diet and body protein metabolism were determined at 1, 5, and 12 months postpartum in lactating women who consumed a controlled diet of measured protein and energy. Milk production was measured by the 24-h test weighing procedure. Maternal body protein metabolism was evaluated by nitrogen balance and a primed, constant infusion of [1-13C]leucine and [alpha-15N]lysine. Milk production was associated positively with lysine flux (P less than 0.05, r = 0.59), leucine incorporation into body protein (P less than 0.05, r = 0.58), nitrogen intakes (P less than 0.05, r = 0.56), and energy intakes (P less than 0.01, r = 0.69). When adjusted for postpartum time, significant associations between total nitrogen concentrations in milk and nitrogen balance also were present (P less than 0.05, r = 0.77). These observations document associations among lactation performance, maternal diet, and the metabolic responses of body protein stores in well-nourished women and suggest strategies for the improvement of milk production in settings where nutrient insufficiency and malnutrition prevail.

A longitudinal study of the effect of prematurity on the development of several components of the immunologic system in human milk was performed. Milk was obtained during the second through the twelfth week after parturition. The mean concentrations of lactoferrin and lysozyme were greater in preterm than in term milk during each interval of lactation. The patterns of change in these components were similar for term and preterm milk. Secretory IgA was the predominant form of IgA in preterm milk. The mean concentrations of IgA were greater in preterm milk throughout the study period. Furthermore, total and secretory IgA levels in preterm milk rose linearly during the sixth through the twelfth week, whereas the concentrations of IgA did not change in term milk during that period. In most preterm mothers, secretory IgA antibodies to Escherichia coli somatic antigens increased as lactation proceeded. These increments in specific antibodies usually did not correlate with changes in total secretory IgA. In addition, leukocyte counts in preterm milk were usually lower at two weeks and higher at 12 weeks than in term milk. Thus the concentrations of certain components of the immunologic system in human milk are altered by premature delivery. A decrease in milk volume may account for some changes, whereas certain alterations may be the result of other consequences of premature delivery or less stimulation by the premature infant.

Human milk is a highly complex fluid with a nutrient balance and an array of functional properties that may promote a level of metabolic efficiency that is not attainable when a cow milk-based formula is fed. This is not a novel idea. Mitchell in 1933 proposed that the level of efficiency of energy use is determined by the nutrient "balance" in the diet. Nonetheless, difficulties remain in the attempt to reconcile the low levels of intake with established estimates of energy needs. If the amount of energy that appears necessary for growth and maintenance of a 4-month-old infant is compared with that from his or her intake of an exclusive human milk diet, the infant should have little or no energy left for activity. Do metabolic economies contribute to more efficient uses of energy for growth and maintenance in breastfed infants? Are there differences in body composition? Does more efficient use of energy occur as a result of a decrease in clinical and subclinical infections? Is efficient energy utilization accomplished by significant curtailment in activity? If an excess level of energy is consumed by bottlefed infants, what are the positive or negative short-term or long-term consequences? These questions are the focus of research in numerous laboratories. Field and clinical studies of breastfed infants and in vitro studies of human milk offer unique opportunities to understand basic mechanisms of human adaptations to nutrient intake and environmental challenges.

Cortisol (F), Na, and K were measured in human milk samples taken every 4 h during 28 24-h cycles in 19 subjects. F in milk, measured by radioimmunoassay of unchromatographed extracts, demonstrated a circadian variation similar to that of plasma F and was dexamethasone-suppressible. Values ranged from 220 +/- 41 (SE) ng/dl at 2300 h to 1,214 +/- 210 at 0700 h. When milk extracts were chromatographed, a similar circadian variation was seen, and F ranged from 87 +/- 27 (SE) ng/dl at 2300 h to 784 +/- 164 at 700 h. The morning values of F exceeded previous estimates of corticosteroid binding capacity in milk, indicating the possibility of significant levels of unbound F. The subjects were divided into three groups according to time postpartum: I, 3.5-6 wk; II, 8-18 wk, and III, 20-32 wk. In milk, normalized values of F (unchromatographed) and electrolytes (x/means) were compared, with x the value at time t and means the mean value for a 24-h cycle. A significant circadian variation was seen in milk for (x/means)Na, (x/means)K, (x/means)Na/K, and x/means)F. Correlation of (x/means)F with (x/means)Na 4 h later was negative in all three groups (r = -0.36 to -0.56, P less than 0.10-0.001) as was (x/means)F vs. (x/means)Na/K (r = -0.33 to -0.61, P less than 0.10-0.001); (x/means)F vs. (x/means)K 4 h later was positive (r = 0.38 to 0.52, P less than 0.05-0.001). Dexamethasone administration caused a significant decrease in milk means Na and an increase in meansK in all five subjects (P less than 0.01). Thus, milk Na and K concentrations appeared regulated by adrenal corticosteroids.

Mineral homeostasis often is disrupted in the very low birth weight (VLBW) infant fed either human milk or commercial formula that contains insufficient quantities of available calcium (Ca) and phosphorus (P). Alterations in mineral homeostasis include abnormal patterns of serum (Ca and P concentrations and alkaline phosphatase activity) and urine (Ca and P) biochemical markers, low net Ca and P retentions in comparison with intrauterine estimates of mineral accretion, and decreased bone mineral content. A two-phase study was conducted in our laboratory to test for these alterations in mineral homeostasis. In phase 1, VLBW infants fed a preparation of fortified human milk (either human milk-derived fortifier I or II or cow milk-derived fortifier) or cow milk-based formula specially designed for VLBW infants were evaluated during their hospitalization. In phase 2, after hospitalization, these infants were evaluated during the first 6 months of life when fed either their mother's milk or routine formula exclusively. The bioavailability of Ca and P from the tested preparations varied widely. Although the fortification of human milk resulted in both an improved biochemical pattern and net retention of Ca and P, optimal intrauterine mineral accretion was not achieved in any group tested. Longitudinal assessments of bone mineralization, by single photon absorptiometry, demonstrated that human milk-fed former VLBW infants had reduced bone mineral content. These investigations suggest that former VLBW infants fed human milk exclusively may be at risk for Ca and P deficiencies.

Fatty acid metabolism and the contribution of dietary fatty acids to milk cholesteryl ester (CE) and phospholipid (PL) were investigated in normal lactating mothers. The approach used was to feed mixtures of triglycerides containing deuterium-labeled palmitic acid (16:0-2H2), oleic acid (18:1-2H6), and linoleic acid (18:2-2H4). Milk and plasma samples were collected for 72 hr. Triglyceride (TG), CE, and PL fractions from milk, plasma, and lipoprotein were isolated and analyzed by gas-liquid chromatography and mass spectrometry. Data for the milk CE and PL fractions showed a definite selectivity for incorporation of 16:0-2H2 and 18:1-2H6 relative to 18:2-2H4. Based on the ratios of the deuterated fatty acids incorporated into the milk CE and PL samples, their incorporation times and isotopic enrichment data, it appears that these fatty acids are supplied mainly by the TG derived from chylomicrons and very low density lipoproteins. Plasma and lipoprotein CE data showed a progressive increase in 18:2-2H4 content, with 16:0-2H2 and 18:1-2H4 remaining relatively constant over the collection period. Plasma and lipoprotein PL data showed a higher rate for incorporation of 18:2-2H4 than 16:0-2H2 and 18:1-2H6 over the course of the sampling period. Comparison to previous data from adult males indicates lactation does not have a major effect on the general metabolism of these fatty acids. An increase with time in the isotopic enrichment of 18:2-2H4 in the plasma and lipoprotein CE and PL samples was observed which is consistent with in vitro selectivities reported for lecithin:cholesterol acyltransferase and phosphatidylcholine acyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of fortified human milk feedings on the urinary excretion of lactoferrin, lysozyme, secretory component, IgA, and secretory IgA antibodies to Escherichia coli O antigens were investigated in very low birth wt infants. Infants were maintained on either a human milk or a cow's milk preparation. The amounts of each immune factor that were ingested and excreted were quantified during balance studies conducted at 2.5 and 5 wk of age. Serum levels of these immune factors were similar in both feeding groups. The urinary excretion of all factors except lysozyme was 7- to 150-fold greater in infants fed human milk than in those fed cow's milk formula. IgA was the only factor for which the amount of the factor excreted correlated with the amount ingested. Fragments as well as whole molecules of lactoferrin were found in the urine of the infants fed human milk, but the molecular sizes of the excreted proteins exceeded those normally filtered by the kidneys. Therefore, the genesis of the enhanced levels of host defense factors in the urine of infants fed human milk is not clear. Gastrointestinal absorption and subsequent renal excretion as well as enhanced production of immune factors in the infant's urinary tract are possible explanations.

Ten lactating mothers (five of preterm and five of term infants) 9-17 d postpartum consumed a 5% fat, 15% protein, and 80% carbohydrate diet for 5 d. Their milk was analyzed for fatty acid composition by gas chromatography. Significant increases in the sum of the absolute and relative concentrations of C10:0, C12:0, and C14:0 fatty acids and significant decreases in the absolute and relative concentrations of C18:0, C18:1, and C18:2 fatty acids were detected on day 4 in both groups (p less than 0.01). Women who delivered prematurely or at term responded similarly in early lactation to a low-fat, high-carbohydrate diet with an increase in the concentration of fatty acids less than 16 carbons in length. The magnitude of this response is highly variable and may be controlled by total energy balance as well as by individual endocrine responses.

The effects of the duration of lactation upon lactoferrin, lysozyme, total IgA, SIgA, SIgA antibodies to Escherichia coli somatic antigens and leukocytes in human milk were investigated. Longitudinal and cross-sectional studies were performed with milk collected from women 20 to 35 years of age during te first year of lactation. Collection and storage conditions and immunologic analyses were controlled to minimize confounding variables. The concentrations of lactoferrin, total IgA, and leukocytes and the uptake of 3H-thymidine by phytohemagglutinin-stimulated lymphocytes fell during the first several weeks of lactation; afterward, the levels of lactoferrin and IgA stabilized. Approximately 90% of total IgA in human milk during the year was SIgA. Secretory IgA antibody titers to E. coli increased in some individuals studied longitudinally suggesting that the enteromammary gland pathway of SIgA antibody production was active after several weeks of lactation. Moreover, the concentrations of lysozyme, after falling to a nadir of 20 to 30 micrograms/ml at 2 to 4 weeks, rose to 200 to 300 micrograms/ml by six months and remained elevated. The immunologic system in human milk undergoes remarkable changes which may represent adaptations for the recipient infant.

The amounts of lactoferrin, lysozyme, total IgA, secretory IgA (SIgA), and specific SIgA antibodies to a pool of Escherichia coli O antigens were measured in 96-h collections of feces obtained from 28 very low birth weight infants, 28-30 wk of gestation, studied at 2.5 and 6 wk of age. Eighteen of these infants were fed their mothers' milk fortified with fractions of skim and cream derived from pasteurized, lyophilized, mature human milk (FM) and 10 infants were fed commercial cow's milk-based formula. The concentrations of these selected immune factors in the FM and formula also were measured. Specific SIgA antibodies to E. coli O antigens were detected in the feces of 90% of the FM-fed infants, but in none of the feces of the formula-fed infants. The feces obtained from FM-fed infants had markedly greater quantities of lactoferrin (p less than 0.001), lysozyme (p = 0.006), and IgA (p less than 0.001) than those of cow's milk formula-fed infants. The concentrations of total and secretory IgA were correlated significantly (r = 0.88, p less than 0.001) and 95% of total IgA was SIgA. The fecal concentration of specific SIgA antibodies to E. coli O antigens in FM-fed infants correlated with the concentration of these antibodies in their milk (p less than 0.001). However, there were no direct relationships between the milk concentrations or the infant's intakes of the other selected immune factors and the excretion of these factors in the feces.(ABSTRACT TRUNCATED AT 250 WORDS)

Mothers' milk, fortified daily with skim and cream components derived from mature donor human milk, was fed fresh during the first 2 postnatal months to 18 (group FMM) healthy, very low birth weight (VLBW) infants. Following the same feeding protocol, 16 additional VLBW infants were fed commercial formula that had a similar range of energy and nitrogen. Infants were evaluated for growth, metabolizable energy, balances of nitrogen and fat, and biochemical markers of nutritional status. Growth measurements from birth to 1800 gm were similar in the two groups. Nutritional balance was performed at approximately 2.5 (first study period) and 6 (second study period) weeks. Metabolizable energy (average 109 kcal/kg/day) was similar in both groups and met theoretical needs of the growing VLBW infant. Apparent nitrogen retention was similar and equivalent to estimates of intrauterine nitrogen accretion (approximately 325 mg/kg/day). Fat absorption increased from the first (70% of intake) to the second study (80% of intake) and was similar in both groups. Serum values for albumin, total protein, prealbumin, hemoglobin, and creatinine were also similar, and declined with increases in postnatal age. Significant differences were observed between feeding groups for serum phosphorus and calcium. These results indicate that healthy VLBW infants maintain adequate growth and macronutrient balance for the first 2 months postnatally when fed mothers' milk fortified with additional skim and cream components.

Human milk sodium ([Nal]) and potassium ([K)] concentrations were measured every 4 h for 24 h in 28 subjects 3.5 to 32 wk postpartum. A diurnal variation in milk Na was seen, which was reciprocal to K. Significant negative correlations between Na and K were seen in these periods: 3.5 to 6; 8 to 18, 20 to 32 wk postpartum (p less than 0.01 for each). The mean 24 h milk sodium concentrations (x(Na)) decreased between 3.5 and 18 wk postpartum (p less than 0.005 by paired t test). Changes in mean potassium (-XK) were not statistically significant. Diet apparently does not affect milk Na. Administration of a low Na diet: 10.8 +/- .9 (SD) mEq Na/day and 60 to 100 mEq K/day for 2 days did not change x(Na). But urinary Na decreased 7-fold as aldosterone increased 5-fold. No significant correlation was seen between 24 h Na excretion in urine and x(Na) in milk (n = 51). A significant positive correlation was seen between urinary K and -XK in milk (r = 0.36), p less than 0.001).

A comparison was made between the dose-to-the-mother deuterium-dilution method and the conventional test-weighing technique for determining human-milk intake in five exclusively breast-fed infants and in four breast-fed infants who received supplemental foods. After administration of 2H to the mothers human milk and infant urine were sampled over 14 d and analyzed for 2H:1H ratios by gas-isotope-ratio mass spectrometry. Infant total body water was determined by 18O dilution. The test-weighing procedure was conducted for 5 d consecutively. The intake of human milk (mean +/- SD) estimated by 2H dilution was 648 +/- 63 g/d and estimated by test-weighing was 636 +/- 84 g/d. The mean difference between the two methods was not significantly different from 0. The 2H-dilution and test-weighing techniques provide similar estimates of human-milk intake.

A stable isotope tracer method was utilized to measure quantitatively the secretion of diet-derived fatty acids (FA) into human milk. A mixture of [2H6]tripalmitin, [2H18]-triolein, and [2H12]trilinolein was administered to three healthy, lactating women 22 to 30 years of age. Milk and blood samples were collected sequentially for 72 hr. The FA composition and concentration of total plasma, lipoprotein, and milk triglycerides were determined by gas-liquid chromatography (GLC) and the isotopic enrichment was determined by gas-liquid chromatography-mass spectrometry (GLC-MS). There were no statistically significant differences in mammary secretion of the individual fats, either by a single individual or between subjects. The mean secretion of fat by one breast was 5.11 +/- 1.26% of the dose (CV = 25%). There was a significant 6.0-hr delay between peak occurrence of the tracer in plasma and its occurrence in milk. The lipids are transported to the mammary gland primarily by the chylomicron and very low density lipoprotein triglycerides.

The concentrations of protein nitrogen (PN), non-protein nitrogen (NPN), energy, fat, sodium (Na), calcium (Ca), phosphorus (P), magnesium (Mg), and zinc (Zn) were determined in human milk from mothers giving birth to full-term (n = 13) and preterm infants (n = 8). Milk samples were collected under controlled conditions at two-week intervals for 12 weeks postpartum. Statistically significant differences in PN, Ca, and P concentrations were detected between the milk from mothers of preterm and term infants. The mean PN concentration in the preterm milk was statistically higher than that of term milk (198 vs. 164 mg N/dl), in contrast to the lower mean Ca (220 vs. 261 mg/l) and P (125 vs. 153 mg/l) concentrations detected in the preterm milk. No other differences in mean nutrient concentration were observed between the two groups. Concentrations of PN, NPN, Na, P, and Zn decreased over time. The concentration of Mg increased slightly. The content of fat, energy, and Ca did not change.

The concentration of gross energy in 24-h pooled samples of milk is compared with that of single samples obtained during the same period. Samples were collected from mothers exclusively breastfeeding their infants on demand. The objective of the comparison was to evaluate simplified sampling schemes for approximating the concentration of energy of milk produced over 24 h. The lowest concentration of energy of single samples occurred in milk collected between 12 midnight and 6 a.m. The concentration of energy of milk was related negatively to the volume expressed and the time interval preceding each expression. The interval preceding each expression was correlated positively to the volume expressed. These results indicate that the pattern of milk expression is important in the design of sampling schemes. Therefore, sampling schemes may not be transferable between populations with distinct feeding patterns. The highest correlation between single samples and the 24-h value was obtained from samples collected between 12 midnight and 6 a.m. (r = 0.84, p less than 0.001). The widths of the 95% confidence and prediction intervals for the equation relating values from single samples to the 24-h pools were +/- 2.7% and +/- 10.5%, respectively, of the mean 24-h pool sample value when three single samples (collected between 12 midnight and 6 a.m., 6 a.m. and 12 noon, and 6 p.m. and 12 midnight) were included in the predictive equation.

The bone mineral status of former very low birth weight infants previously fed fortified human milk was evaluated during the posthospitalization period. Anthropometric measurements, bone mineral content, bone width, serum calcium, phosphorus, and albumin concentrations, and alkaline phosphatase activity were evaluated at 10, 16, and 25 postnatal weeks. Infants were fed either commercial formula or unfortified human milk after their hospital discharge. At 16 and 25 weeks postnatally, human milk-fed infants (group HM) had lower bone mineral content (P less than 0.01), bone mineral content/bone width ratio (p less than 0.01), serum phosphorus concentration (p less than 0.03), and higher alkaline phosphatase activity (p less than 0.01) than commercial formula-fed infants (group CM). Growth was similar in both groups. Bone mineral content was correlated positively to serum phosphorus (r = 0.52, p less than 0.05) and inversely to alkaline phosphatase activity (r = -0.63, p less than 0.01) at 25 weeks postnatally. The use of serum biochemical markers, however, could account for only 44% of the variability in bone mineral content. The exclusive feeding of human milk to very low birth weight infants after hospital discharge may place them at risk for mineral deficiencies.

Intakes of calcium, phosphorus, magnesium, zinc, sodium, potassium, iron, and copper of 45 exclusively breast-fed infants were determined during the first 4 mo of life. Direct 24-h measurements of milk intake and mineral contents of human milk were used to estimate mineral intakes. Daily intakes of Ca, P, Zn, K, Na, Fe, and Cu decreased significantly over the study period while the intake of Mg increased. With the exception of Mg, mineral intakes on a weight basis displayed significant quadratic trends over the 4 mo. In spite of seemingly low-mineral intakes, growth progressed satisfactorily.

Volume, fat and energy contents were measured in human milk samples collected over 2 24-h periods with either an electric or hand-operated breast pump. The energy content was significantly higher and the variance for this measurement was significantly lower in samples obtained by the electric pump. The type of pump used is an important consideration when the content of energy and other nutrients associated with the fat compartment in human milk is to be evaluated. This observation is especially pertinent to cross-study comparisons, eg studies of well- and malnourished populations.

Enhanced calcium and phosphorus retention was achieved in 16 very low birth weight infants (birth weight 1117 +/- 42 g, gestation 29 +/- 0.2 weeks) fed a preparation of fortified human milk augmented with calcium lactate and monobasic and dibasic phosphate salts. Measurements of growth and macronutrient utilization were similar to those obtained in a previous study of infants fed a preparation of fortified human milk that contained lower levels of calcium and phosphorus. However, unlike the relative hypophosphatemia, hypophosphaturia, and hypercalciuria noted in the infants in our earlier study, normal serum and urine phosphorus and urine calcium values were observed in this study. Postnatal calcium and phosphorus retentions correlated significantly with respective intakes, the absorption of fat, and the retention of nitrogen. The relationships among calcium and phosphorus intake and retention predict that 160 mg/kg/d and 94 mg/kg/d, respectively, must be fed to achieve retention equivalent to intrauterine estimates. Although postnatal retention of calcium and phosphorus may be increased to levels accumulated by the fetus, technical considerations for the preparation of a formula with sufficiently high levels of calcium and phosphorus must be resolved.

The mineral adequacy of fortified mothers' milk for very low birth weight (VLBW) infants was tested during their first two postnatal months. Metabolic balance and serum Ca, P, Mg, Na, and K values were evaluated at 2.5 and 6 weeks of life in 32 VLBW infants (less than 1.3 kg). Infants were fed either their mothers' milk fortified with skim and cream components derived from heat-treated, lyophilized mature human milk (FMM) or commercial cow milk-derived formulas. Despite Ca and P concentrations 50% to 100% higher in the fortified human milk than is usual in unfortified human milk, group FMM's Ca and P intakes remained significantly below those fed formula (P less than 0.001). Serum calcium levels and alkaline phosphatase activity were higher and serum phosphorus lower (P less than 0.002) in group FMM, whereas serum levels of magnesium, sodium, and potassium were similar in both groups. Ninety-six-hour urinary excretion of Ca was greater and 96-hour urinary excretion of P was less in group FMM (P less than 0.02). Retention of Ca and P in both groups was significantly below estimates of intrauterine accretion. Mg retention was significantly higher in group FMM (P less than 0.002) despite intakes of Mg significantly below those in infants fed formula. Although intakes of Na were below recommended levels in both groups, by the second balance period all infants demonstrated Na retention that was greater than expected from the sum of estimates of intrauterine accretion and dermal losses. Similar findings were noted for K. Biochemical and balance data indicate that relative to the needs of the VLBW infant, fortified mothers' milk was deficient in Ca and P, but adequate in Mg, Na, and K.

Six lactating women were recruited at 5 to 7 months postpartum for a study of milk composition during gradual weaning. At recruitment all subjects were exclusively breast-feeding their infants. The subjects were asked to reduce the frequency or duration of breast-feeding episodes by approximately one-third each month for a period of 3 months. Milk was collected before weaning began and at 2-wk intervals for 12 wk. The concentrations of protein and Na were 142 and 220% of base-line values, respectively, at the end of this period. Changes in fat composition were difficult to interpret. A linear trend was observed through wk 10, but values for wk 12 were similar to those obtained before weaning was initiated. The concentration of Fe at wk 12 was 172% of the base-line value. The concentration of Ca did not change and that of Zn fell to 58% of the base-line value at wk 12. The significance of the changes in nutrient concentrations, their relationship to the involution of the mammary gland, and the suitability of milk from this stage of lactation for milk banking are evaluated.

Issues regarding the efficacy of feeding human milk to premature infants include the development of optimal protocols for collecting, storing, and processing human milk. Studies of the nutritional and immunologic composition of milk produced by women who delivered term or premature infants and who weaned their infants gradually from human milk have been studied to identify optimal donors. The effects of specific collection, storage, and processing conditions on the composition of mature human milk also have been evaluated. Collection, storage, and processing conditions have distinct effects on specific functional components. The caloric content of milk, the content of nutrients carried in the lipid portion of milk, and selected enzymatic activities depend on the completeness with which the breast is emptied. Storing milk in polyethylene, polypropylene, and pyrex containers influences key immunologic components in human milk as do storage temperatures. None of the nutrient compositions of milks studied matched current estimates of the nutritional needs of premature infants. Importantly, both the concentrations and the pattern of change in nutrient and immunologic contents are distinct in milks of women delivering infants at term or prematurely. Further changes are seen during the process of gradual weaning.

Milk intake and growth in 45 exclusively breast-fed infants were documented during the first 4 months of life. Energy and protein intakes were substantially less than current nutrient allowances. Energy intake declined significantly from 110 +/- 24 kcal/kg/day at 1 month to 71 +/- 17 kcal/kg/day at 4 months. Protein intake decreased from 1.6 +/- 0.3 gm/kg/day at 1 month to 0.9 +/- 0.2 gm/kg/day at 4 months. Infant growth progressed satisfactorily, compared with National Center for Health Statistics standards. A reevaluation of energy and protein intakes and allowances during infancy is merited.

The incubation of milk, at 4 degrees C, from mothers of infants with breast milk jaundice (BMJ) is reported to result in significantly higher levels of free fatty acids (FFA) compared with milk from controls. Single milk samples collected under standard conditions were obtained from four mothers of infants with BMJ and 14 control donors matched for stage of lactation. Milk samples were analyzed for the concentrations of FFA, using thin-layer gas chromatographic techniques. In addition, serum total fatty acids were measured in mothers and infants. The concentrations of FFA increased after storage of the milk from both the jaundiced and control groups. No differences were observed in the composition of milk FFA before or after incubation, when respective values were compared between these two groups. Similarly, no differences were detected in serum total fatty acids in either infants or mothers. The observation that increased levels of FFA in milk are associated with BMJ was not confirmed.

Because of the importance of breast-feeding to child health, research in the formation, composition, and biological effects of human milk should be strongly encouraged. Future exploration should include the processes that are responsible for the synthesis and secretion of human milk; the nature and function of the physical compartments in human milk; the structure and function of certain proteins and other components in human milk; and the in vivo fate and effect of a host of constituents including nutrients, growth factors, hormones, inducers, immunological factors, and antiinflammatory agents. Because of the potential public health benefits, these studies should be a high priority in pediatric research for the next decade.

Variability observed in the energy, total nitrogen, lactose and fat concentrations of human milk was partitioned among subjects, days and sample duplicates. Highly significant differences were detected between individuals and between days within the individual for all four nutrients (P less than 0.001). Subjects, days and sample duplicate determinations contributed 89, 9 and 2 per cent, respectively, to the variance for energy; 87, 12 and 1 per cent for nitrogen; 75, 21 and 4 per cent for lactose; and 92, 8 and less than 1 per cent for fat. The reliability of mean estimates for a group or for an individual was derived for various combinations of subjects, days and duplicates to provide a tool for planning future studies.

The effects of collection technique, storage container, and the duration and temperature of storage on selected nutrient concentrations in mature human milk were evaluated. Milk samples were collected during the fourth week of lactation from women 20-35 years of age by hand expression or suction. Greater volumes and fat concentrations were observed in milks collected by suction. Vitamin A, zinc, iron, copper, sodium, and protein nitrogen concentrations were not affected by storage of milk in either pyrex or polypropylene containers for up to 24 h. The storage temperature had a significant effect on protein nitrogen and ascorbic acid concentrations. These findings indicate that collection methods and storage procedures used for comparatively brief periods will affect the concentrations of selected nutrients of mature human milk. Specific recommendations are made for the collection and storage of milk.

Midmorning plasma amino acid levels were measured in 31 healthy, very low birth weight infants (mean age 16 days, mean birth weight 1180 g, gestation 29 wk) during 96-h balance studies. All infants received continuous enteral infusion of isonitrogenous, isocaloric preparations of either human milk fortified with pasteurized, lyophilized fractions of mature human milk (n = 18) or whey-dominant cow milk-based formula (n = 13). Weight gain (15 g/kg/day), nitrogen retention (303 mg/kg/day), and metabolizable energy (104 kcal/kg/day) were similar between groups. Plasma levels of threonine, valine, and the sum of essential amino acids were significantly greater in the whey-dominant formula-fed infants (p less than 0.01). Taurine and cystine were measured in significantly greater concentrations in the fortified human milk and threonine, valine, methionine, and lysine in the whey-dominant cow milk formula (p less than 0.01). Relationships between plasma amino acid levels and indices of nitrogen utilization differed between groups. These differences suggest that further modifications of whey-dominant formulas may be indicated.

Growth, 96-hour balance of nutrients (nitrogen, fat, calcium (Ca), phosphorus (P), and magnesium), metabolizable energy, and serum biochemical markers of mineral status (Ca and P concentrations and alkaline phosphatase activity) were measured in 22 very low birth weight infants to investigate the bioavailability of minerals from specialized formula and from human milk fortifiers. The intakes of Ca and P were similar between group FORM ("Preemie" SMA) and group CMF (1:1 wt/wt, human milk and Similac Natural Care or Similac Special Care). The intakes of nitrogen, energy, fat, and magnesium differed between groups. Group CMF had significantly greater fecal losses and significantly lower absorption and retention of Ca and P in comparison with those of group FORM. Retention of Ca and P in both groups, however, was greater than 25% below intrauterine estimates of accretion. Retention rates of Ca, P, and magnesium were not correlated with their respective intakes. Weight gain during the balance study and during the entire study interval was significantly less in group CMF. The ratio of Ca retention to either weight gained or nitrogen retained was lower in group CMF, which suggested that the low retention of Ca was related less to the slower rate of growth in these infants than to their greater fecal losses of Ca. Although the cause of the greater fecal losses of Ca and P in this group is unclear, the data suggest an insolubility of the mineral sources. Our results indicate that sole reliance on the absolute mineral concentrations of the milk selected for very low birth weight infants may be unrealistic; the bioavailability of Ca and P from particular mineral sources should be evaluated.