Otakar Koldovsky

Hormones in milk

Abstract Hormones are present in milk of man and other mammals. Their concentration is influenced by various factors. Studies in rodents show that some hormones with large molecules retain physiological activity when administered perorally to suckling mammals.

Human Milk as a Potential Enteral Source of Erythropoietin

In addition to its content of traditional nutrients, milk is a rich source of hormones and peptides, which survive digestion in the neonatal gastrointestinal tract secondary to lower proteolytic activity and increased protein permeability. Previous studies have shown accelerated erythropoiesis or elevated serum erythropoietin (Epo) levels in neonatal (suckling) animals after maternal phlebotomy or maternal hypoxia exposure. We sought to determine whether significant quantities of Epo are present in human milk and whether Epo remains intact under physiologic digestion conditions. Immunoreactive Epo concentrations were determined in 409 human milk samples obtained from mothers of term and premature infants. Samples collected between birth and postpartum d 134 were divided into 11 postpartum day groups. Mean milk-borne Epo concentrations were within the normal range for plasma Epo concentrations and rose with postpartum day (F10,398 = 5.82, p < 0.0001). No differences were observed between milk collected from mothers of premature versus term infants. Estimated weekly human milk-borne Epo intakes approximated the lower range of published parenteral therapeutic doses. In simulated digestion at physiologic pH levels of 3.2, 5.8, and 7.4, milk-borne Epo resisted degradation at 1 and 2 h, compared with baseline. Therefore, we conclude that human milk contains considerable amounts of Epo which resist degradation after exposure to gastric juices at physiologic pH levels. These results support continued investigation into the fate and developmental roles of Epo in human milk.

Fate of Insulin-Like Growth Factors I and II Administered Orogastrically to Suckling Rats

ABSTRACT: Milk-borne insulin-like growth factors I and II (IGF-I and -II) may be of importance in the differentiation of the gastrointestinal tract of the suckling. To test this hypothesis, 10- to 11-d-old suckling rats were given via an orogastric tube 125I-IGF-I (n = 6) or 125I-IGF-II (n = 6) in rat milk and killed 30 min later. The results of this study demonstrated that approximately 40% of the radioactivity administered was detected in the gastrointestinal tract for both 125I-IGF-I and 125I-IGF-II experiments. Gel chromatography of acid extracts of homogenates of gastrointestinal tissues and luminal contents demonstrated that a significant fraction of recovered radioactivity eluted in a position identical to “native” IGF. These findings were confirmed by subjecting similarly treated samples to high performance liquid chromatography. In addition, radioactive material recovered from Mr 7,500 fractions bound specifically to crude membrane IGF-I and -II receptor preparations, further suggesting the preservation of biologic activity of the recovered peptides. Although skin homogenates contained large peptide fragments of 125I-IGF-I, no “intact” IGF was found in the blood or other tissues. These findings suggest that milk-borne IGFs are stable in the neonatal gastrointestinal tract and remain biologically active for as long as 30 min postingestion.

Gastric luminal digestion of lactoferrin and transferrin by preterm infants

Lactoferrin, a milk iron-binding protein, may play antimicrobial, iron-absorptive and growth-promoting roles in the developing gastrointestinal tract. To perform such functions, lactoferrin must survive digestive processes in the gut lumen in an active form. We investigated the gastric digestion of lactoferrin in addition to that of the other milk proteins, transferrin and casein, in preterm infants by measuring their degradation during incubation in vitro at 37 degrees C with gastric fluid at pH 1.8, 3.2 and 5.8. Fluid was obtained 1 h after a milk feeding, a time of maximum peptic activity, from 12 infants with a mean gestational age of 29.7 +/- 0.8 weeks at birth and a postnatal age of 24.7 +/- 3.2 days at sampling. Hydrolysis of all three proteins as indicated by generation of trichloroacetic acid soluble material from iodinated substrate was maximal at acid pH and declined by greater than 75% at pH 5.8, lactoferrin was less rapidly degraded than casein at low pH and transferrin breakdown was intermediate. Analysis of reaction mixtures by SDS-polyacrylamide gel electrophoresis showed degradation of lactoferrin and transferrin to low molecular weight products at pH 3.2 but minimal breakdown at pH 5.8. Several discrete fragments were generated at low pH, including species with molecular weights of 41,000-42,000 which may represent half-molecules. We conclude that dietary lactoferrin and transferrin may be degraded by preterm infant gastric fluid to discrete species, but that hydrolysis may be minimal at the prevailing postprandial pH. Consequently they may be rendered available for possible subsequent biological action within the infant.

Luminal hydrolysis of recombinant human epidermal growth factor in the rat gastrointestinal tract: Segmental and developmental differences

Epidermal growth factor (EGF), present in high concentrations in the milk of various species, is biologically active following oral administration to young animals. Although in vivo studies show gastrointestinal processing of dietary EGF during early postnatal development, the relative importance of luminal and mucosal digestion in such processing is undefined. To characterize the luminal metabolism of dietary EGF in the developing gastrointestinal tract, we incubated human recombinant 125I-EGF in vitro at 37 degrees with luminal fluid from the stomach and various segments of the small intestine of 12 day old suckling and 31 day old weanling rats and analyzed the resulting reaction products. The rate of EGF hydrolysis as determined by generation of acid soluble material was greater in weanling small intestine than in suckling, with maximal hydrolytic capacity observed in the mid-jejunum and ileum. Minimal hydrolysis was observed with stomach fluid from both age groups, and EGF retained its ability to elute as a single species on Sephadex G-25 columns and to bind to monospecific affinity columns and placental membrane receptors. Incubation with suckling small intestinal fluid produced little change in the chromatographic profile on Sephadex G-25, but a reduction in antibody and receptor binding was observed. In contrast, incubation with weanling small intestinal fluid yielded both a more pronounced loss of EGF-like material on G-25 columns and a greater reduction in receptor and antibody binding. We conclude that little luminal EGF degradation occurs in the rat stomach during the suckling and weanling periods, but that in the lumen of the small intestine breakdown increases during postnatal development.

Assessment of lactose absorption by measurement of urinary galactose

Individuals with sufficient intestinal lactase hydrolyze ingested lactose to galactose and glucose and these monosaccharides are absorbed. Lactose is not digested completely when intestinal lactase activity is low and the disaccharide is malabsorbed. Breath hydrogen excretion after lactose ingestion is used commonly to diagnose lactose malabsorption. However, no direct tests are currently used to assess lactose absorption. We tested a new method of assessing lactose absorption in 26 healthy individuals. Each subject ingested 50 g of lactose. Participants were evaluated for lactose malabsorption using a standard 3-h breath hydrogen test. In addition, the urinary excretions of galactose, lactose, and creatinine were quantitated for 3-5 h after lactose ingestion. On the basis of breath hydrogen analysis after lactose ingestion, 12 individuals were lactose malabsorbers (defined as a rise in the breath hydrogen concentration of greater than 20 parts per million above the baseline value). The 14 subjects who did not malabsorb lactose by breath hydrogen testing (defined as a rise in the breath hydrogen concentration of less than or equal to 20 parts per million above the baseline value), had significantly more galactose in their urine 1, 2, and 3 h after lactose ingestion than lactose malabsorbers. The ratio of excreted lactose to excreted galactose was significantly decreased in lactose absorbers compared with lactose malabsorbers (p less than 0.001). Determination of the ratio of urinary galactose to urinary creatinine separated lactose absorbers from lactose malabsorbers completely (p less than 0.001). We conclude from this study that the determination of urinary galactose, urinary lactose/galactose ratio, and urinary galactose/creatinine ratio may be used to assess lactose digestion and absorption in healthy adults.

Presence of multiple forms of peptidase inhibitors in rat milk.

Summary Peptides such as somatostatin (SS14), epidermal growth factor (EGF), transforming growth factor-α (TGFα), and insulin-like growth factors (IGF-I and IGF-II) are present in breast milk from various species, and their significance in the developing gastrointestinal tract has been suggested. Our recent studies have indicated that rat milk soluble fraction (RMSF) protects SS14 in the gastrointestinal lumen by inhibiting in vitro the luminal peptidolysis. In the present studies, we have shown that RMSF inhibited in vitro degradation by midjejunal luminal flushings of suckling rats of 125I-labeled somatostatin 14[Tyr11], EGF, TGFα, IGF-I and IGF-II, as well as trypsin activity in vitro against benzoyl-L-arginyl-p-nitroanilide. The inhibitory factors present in the RMSF were further fractionated by gel filtration on Sephadex G100, ion-exchange chromatography on DEAE-Sephadex, and fast protein liquid chromatography (FPLC). Gel filtration of Sephadex G100 separated RMSF into three peaks of proteins: G1, G2, and G3; peptidase inhibitor activities were present exclusively in G1. Ion-exchange chromatography on DEAE-Sephadex column resolved peptidase inhibitory activity (G1) into three different peaks, D1, D2, and D3, eluted at sodium chloride concentrations of 0.05 M, 0.1 M, and 0.2 M, respectively. Further purification of D2 by FPLC resulted in a fraction rich in peptidase inhibitory activity, which was essentially free of trypsin inhibitory activity. Results indicate the presence of at least three peptidase inhibitors in rat milk, which may play a role in the protection of milk-borne peptides in the gastrointestinal lumen.

Milk-borne epidermal growth factor modulates intestinal transforming growth factor-α levels in neonatal rats

Epidermal growth factor (EGF) is present in milk from various mammalian species, but its physiologic function in neonatal development remains unclear. Transforming growth factor-α (TGF-α) is a peptide structurally related to EGF, and its presence is detected in the developing small intestine of rats. The purpose of the present study was to examine the effect of milk-borne EGF on endogenous production of EGF and TGF-α in the small intestine of suckling rats. Neonatal rats were fed via gastrostomy either growth factor-free rat milk substitute (RMS) or RMS supplemented with EGF (100 ng/mL of RMS) from 8 to 12 d of age. Artificially reared rats were then compared with their dam-fed littermates. Animals fed the EGF-deficient diet RMS had markedly increased EGF and TGF-α mRNA levels in duodenum and ileum compared with dam-fed controls and significantly elevated total intestinal content of TGF-α peptide. Intestinal EGF content and EGF serum levels were significantly decreased in the RMS group compared with controls. The addition of EGF to the RMS diet normalized TGF-α mRNA levels in the duodenum and ileum, EGF mRNA levels in the ileum, and total intestinal TGF-α content and EGF serum levels to the levels measured in dam-fed littermates. Motility studies showed that enteral administration of EGF did not affect stomach emptying and intestinal transit. These studies indicate that exogenous milk-borne EGF modulates endogenous production of TGF-α in developing small intestine. It is likely that neither TGF-α nor EGF are solely responsible for small intestinal overgrowth of artificially reared neonatal rats.

Enteral absorption of erythropoietin in the suckling rat

Milk contains biologically relevant concentrations of erythropoietin (Epo), the primary hormone responsible for erythrocyte production. In animals, milk-borne Epo stimulates erythropoiesis. Epo receptors have been found in nonerythropoietic tissues including gastrointestinal tract. We hypothesized that milk-borne Epo is distributed to local gastrointestinal tissues, absorbed intact, and then distributed peripherally via the systemic circulation. Rat milk protected recombinant human Epo (rhEpo) from degradation in the suckling rat gastrointestinal tract. Simulated digestion of 125I-rhEpo in suckling rat gastrointestinal juices was performed. When measured by acid precipitation and immunoassay, rat milk protected rhEpo from gastrointestinal juices better than saline (p < 0.0001). The fate of enterally administered milk-borne 125I-rhEpo was examined in 10-d-old rats. RhEpo fed in rat milk was better protected from in vivo proteolytic degradation than rhEpo in saline (p < 0.05). After enteral 125I-rhEpo dosing, radiolabeled protein from gastric tissue comigrated on SDS-PAGE with intact rhEpo at 36.5 kD. To determine the local and systemic distribution of physiologic intakes of rhEpo, suckling rats were fed 125I-rhEpo in rat milk, and tissues were harvested 1, 2, and 4 h later. Intact 125I-rhEpo was found in gastric and small intestinal walls and lumens. Five percent of total administered dose was found intact in the plasma, whereas another 8 to 10% of total administered dose was localized to bone marrow, percentages comparable to those seen after parenteral administration. Radiolabel was also localized to liver and peripheral solid tissues. These patterns of localization and degradation of rhEpo after acute administration support both systemic absorption and gastrointestinal cellular processing.

Precocious increase of sucrase activity by carbohydrates in the small intestine of suckling rats. I: Significance of the stress effect of sugar-induced diarrhea

In this paper, we analyze the factors involved in the precocious increase of sucrase activity evoked by the early feeding of sucrose in suckling rats, and particularly, the role of diarrhea and stress in this phenomenon. Ten-day-old rats were removed from their mothers and gavage fed for 4 days at 3-h intervals either a basic low carbohydrate milk formula (10.8% fat, 8% protein, 1.4% carbohydrate; all by weight/volume) or basic low carbohydrate milk with: lactose (13%), fructose (13%), or Polycose (2%, 6%, or 13%); all formulas were isocaloric. Feeding the formula containing fructose or high (13%) Polycose led to diarrhea and evoked a concurrent increase of small intestinal sucrase activity. In further experiments, 11-day-old rats were fed the basic formula, the lactose (13%), the fructose (13%), and a sucrose (13%) formula for 8 h between 2 a.m. and 10 a.m. Also, 10-day-old rats were fed 0.5 ml of a solution of 5% mannitol in water while nursing with their mothers. The serum corticosterone levels were substantially increased within 8 h after the initiation of feedings with sucrose and fructose milks and the mannitol solution. The mannitol-fed rats also developed diarrhea within a day in association with a marked increase in sucrase activity. We conclude that a precocious increase of sucrase activity in the small intestine of suckling rats by dietary sugars is not caused by substrate induction, but is mainly due to the effect of stress. The stress is caused by diarrhea which is evoked by the feeding of indigestible and/or unabsorbable amounts of sugar.