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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.

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.

The energy intake, expenditure, and deposition of 40 breast-fed and formula-fed infants were investigated at 1 and 4 mo of age to explore possible differences in energy utilization between feeding groups. Energy intake was calculated from 5-d test-weighing records or pre- and postweighing of formula bottles, in combination with bomb calorimetry of the milks. Total daily energy expenditure (TDEE) was determined by the doubly labeled water method. Sleeping metabolic rate (SMR) and minimal observable energy expenditure were measured by indirect calorimetry. Activity was estimated as the difference between TDEE and SMR. Energy deposition was estimated from dietary intake and TDEE. Energy intakes were significantly higher for the formula-fed than breast-fed infants at 1 mo (118 +/- 17 versus 101 +/- 16 kcal/kg/d) and 4 mo (87 +/- 11 versus 72 +/- 9 kcal/kg/d) (p less than 0.001). TDEE averaged 67 +/- 8 and 64 +/- 7 kcal/kg/d at 1 mo and 73 +/- 9 and 64 +/- 8 kcal/kg/d at 4 mo for the formula-fed and breast-fed infants, respectively, and differed between feeding groups (p less than 0.04). SMR and minimal observable energy expenditure (kcal/min) were higher among the formula-fed infants at 1 and 4 mo (p less than 0.005). The energy available for activity and the thermic effect of feeding did not differ between feeding groups. Rates of weight gain (g/d) and energy deposition (kcal/kg/d) tended to be greater among the formula-fed infants at 1 and 4 mo (p less than 0.006).(ABSTRACT TRUNCATED AT 250 WORDS)

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 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.

We studied the effects of a diet that was low in fat, high in carbohydrate (CHO) on milk lipid composition and de novo endogenous fatty acid synthesis by the mammary gland in five lactating women. The women consumed either a low fat (LF) (5% fat, 80% CHO) diet or a high fat (HF) (40% fat, 45% CHO) diet. Fat synthesis was determined after an oral dose of 500 mg/kg D2O by measuring the incorporation of deuterium into C10:0 to C18:0 saturated fatty acids of milk fat and plasma triglycerides by gas chromatography-mass spectrometry. Synthesis of plasma C16:0 and C18:0 triglycerides was barely detectable while women consumed the HF diet, but was increased 6-fold during the LF diet. Medium chain fatty acids secreted by the mammary gland increased from 12.8% (HF diet) to 16.3% (LF diet) in milk fat from four of five subjects (p = 0.027). Medium chain fatty acid secretion, however, increased from 13.9% (HF diet) to 29.9% (LF diet) in one subject. The primary fatty acids synthesized during lactation were C10:0, C12:0, and C14:0 in the majority of women studied. The LF diet significantly increased the apparent synthesis of C14:0 (p = 0.05), whereas no changes were observed in C12:0, C16:0, or C18:0. One subject had highly enriched C16:0 and C18:0 fatty acids in her milk on the LF diet, which could have been the result of mammary synthesis or of transport and secretion of hepatically synthesized lipids.

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)

We measured bone mineral content (BMC) in 18 neonatal miniature piglets by single photon absorptiometry, total body calcium (TBC) by total body neutron activation analysis, growth, and serum indices of mineral status (calcium, phosphorus, alkaline phosphatase activity). Measurements were begun on day 6, when the piglets were weaned, and were continued to day 19. After weaning, the piglets were assigned randomly to receive one of three diets which differed only in their concentrations of calcium and phosphorus: 100% of the recommended level (diet A), 60% (diet B), and 20% (diet C). No differences were observed among groups during the 19-day study, either in weight gain (48 +/- 2 g/day) or increment in crown-rump length (2.4 +/- 0.2 cm/wk). BMC correlated significantly (p less than 0.001) with TBC at 6 (r = 0.83), 13 (r = 0.77), and 19 (r = 0.93) days. BMC correlated significantly (p less than 0.001) with the ash weight (r = 0.87) and calcium content (r = 0.90) of the corresponding tibial bone segment. Anthropometric parameters and serum indices of mineral status did not predict TBC as accurately as did BMC measurements. We observed a range in BMC measurements in this study that was similar to the range reported for infants in the 1st yr of life. The high correlation between BMC and TBC suggested that BMC is useful in the assessment of mineral status in infants.

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.

An acoustic plethysmograph designed to measure body volumes of infants is described. This method uses the principle of the Helmholtz resonator in which the resonant frequency of a chamber is inversely proportional to the square root of the volume of air inside the chamber. After an object is placed inside the chamber, the change in resonant frequency is used to measure the volume of the object. The coefficient of variation of repeated measures of body volumes of 13 miniature piglets (5 to 18 days old, body weights 1253 to 2631 g) ranged from 0.3 to 3.2%. Body volumes measured by the acoustic method agree closely with those calculated from carcass analysis in which body volume is the sum of the volumes of total body water, fat, protein, and minerals. Fat-free mass computed from densitometry using a four-compartment model and body volume measurements from the acoustic method agrees with fat-free mass obtained from carcass analysis. The mean difference between methods was not significant: 1 ml for body volume and 3 g for fat-free mass. The limits of agreement between methods was +/- 75 ml for body volume and +/- 214 g for fat-free mass. Our results indicate that the acoustic method can measure a change in body volume of 75 ml and a change in fat-free mass of 214 g in a 2000 g infant.

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.