Safety of milk from cows treated with bovine somatotrophin.
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Animals↗
Biomedical subjects
S M Donovan
Publications and source records attributed to S M Donovan.
Growth factors in milk as mediators of infant development.
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Animals↗
Insulin-like growth factors and insulin-like growth factor binding proteins in porcine serum and milk throughout lactation.
IGF-I, IGF-II, and IGF binding proteins (IGFBP) were characterized in porcine serum, colostrum, and milk on d 1-28 postpartum. IGF-I and -II were measured by heterologous RIA. Serum IGFBP were characterized by Western ligand blotting and milk IGF binding activity by [125I]-IGF binding assay. IGF-II accounted for 70-85% of serum IGF and rose 2-fold between d 1 and d 28. Serum IGF-I was unaffected by duration of lactation. Milk IGF-II concentrations were higher than IGF-I concentrations on d 1-7 postpartum. After d 10, milk IGF-I and IGF-II contents were not significantly different. Serum contained IGFBP with M(r) of 43, 39, 34, 28, and 24 kD. Over the course of lactation, the 43- and 39-kD bands increased, the 24-kD band decreased, and the 34- and 28-kD bands were unchanged. Milk IGF binding activity increased between d 1 (28%) and d 3 (44%), then declined until d 28 (7%). Serum and milk were separated by isoelectric focusing into 20 fractions, across a gradient from pH 3 to 10, that were screened for IGFBP by Western ligand blotting. Milk contained six IGFBP of similar M(r) as serum IGFBP; however, the relative amounts of the IGFBP and their apparent isoelectric points differed. In conclusion, porcine milk contains both IGF-I and -II, with IGF-II predominating. Several IGFBP with similar M(r) as those found in serum are present in milk. IGF peptide concentrations were highest in prepartum secretions and colostrum, whereas IGF binding activity peaked on d 4 of lactation.
Animals↗
Use of the piglet to study the role of growth factors in neonatal intestinal development.
Milk growth factors are thought to contribute to postnatal gastrointestinal growth. The roles of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) in the neonatal piglet intestine were investigated. In the first study, piglets were infected with rotavirus on d 4 postpartum and received formula containing 0, 500 or 1000 micrograms/l of EGF for 8 days. A non-infected control group received no EGF. Infected piglets developed severe diarrhea and gained 60% less weight than controls. Rotaviral infection caused a 37% decrease in villus height and 40% decreases in intestinal enzyme activities compared to control. Oral EGF increased villus height and lactase activity in a linear dose-response fashion. Our results suggest that supplementation of formulas with high physiological levels of EGF may aid in small intestinal recovery. A second study investigated absorption of orally administered IGF-I. Newborn piglets were fitted with catheters via the umbilical artery and vein. Piglets were given formula containing 25 microCi of [125I]-IGF-I and blood samples were drawn for 24O min. Total radioactivity, protein bound counts, and counts immunoprecipitable with an antibody to IGF-I were determined in plasma. Radioactivity was detected in portal and arterial plasma within 15 min and rose throughout the study, however, protein bound counts were stable at 20-30% of total counts between 30 and 180 min postgavage. Approximately 10% of the counts were immunoprecipitable by a polyclonal antibody to IGF-I, suggesting that up to 10% of orally administered IGF-I may be absorbed intact.
Animals↗
Regulation of insulin-like growth factor-binding protein expression by thyroid hormone in rat GH3 pituitary tumor cells.
Rat pituitary GH3 tumor cells express GH and insulin-like growth factor-I (IGF-I) mRNAs and possess specific IGF-I and IGF-II receptors which mediate the inhibitory effect of IGF on GH secretion. T3 increases the rate of cell replication and GH gene transcription, and causes an increase in IGF-I binding to cell membranes. Since the IGFs circulate in association with specific binding proteins (IGFBPs) that appear to modulate the access of IGFs to their receptors, we have investigated the effect of T3 treatment on the expression of IGFBPs in GH3 cells. Cells were grown in serum-free defined medium, and IGFBP secretion was determined by Western ligand blotting of conditioned medium after the addition of T3 and/or various peptides for 48-72 h. The conditioned medium of GH3 cells revealed a complex of bands migrating at 40-45 Mr, a pattern typical of rat (r) IGFBP-3. T3 treatment resulted in an increase in rIGFBP-3. IGF-I, IGF-II, and insulin did not alter rIGFBP-3 levels. After concentrating (10-fold) conditioned medium samples, two additional bands at 24K and 28K mol wt were also seen. These bands corresponded in size to rIGFBP-4 (24K) and its glycosylated form (28K). The mRNAs for both rIGFBP-3 and rIGFBP-4 were present in GH3 cells; T3 treatment increased steady state levels of rIGFBP-3 mRNA, but did not affect BP-4 mRNA levels. To learn whether the increased expression of IGFBPs could be responsible for the increased IGF-I binding seen after T3 treatment, [125I]IGF-I was cross-linked to GH3 membranes, and the proteins were separated on a 5-15% gradient sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. T3 treatment induced a large increase in the intensity of bands migrating at 135K and 280K, representing the alpha-subunit of the IGF-I receptor and an incompletely reduced alpha-alpha-dimer, respectively. No membrane-associated IGFBPs were detected. In conclusion, GH3 cells express two IGFBPs, rIGFBP-3 and rIGFBP-4, which are differentially regulated by T3. The increased binding of IGF-I to GH3 cell membranes after T3 treatment indicates that thyroid hormone induces an up-regulation of IGF-I receptors and that the increased IGF-I binding to GH3 membranes is not due to increased expression of membrane-associated IGFBPs.
Animals↗
Postprandial changes in the content and composition of nonprotein nitrogen in human milk.
The effect of a meal on human milk (HM) total nitrogen (TN) and nonprotein nitrogen (NPN) content and composition was examined. Two studies were performed in which milk and blood samples were collected 2-3 h after subjects consumed either a test breakfast or lunch. To monitor the rate of transfer of plasma urea into milk, two women were given [15N]2-urea with the meal. Milk TN concentrations were not significantly different from premeal values. However, concentrations of milk NPN, urea nitrogen, and alanine were increased by greater than or equal to 20% over premeal values. [15N]2-Urea appeared in plasma and milk within 15 min and reached maximum enrichments of 10% and 5.5% in plasma and milk, respectively. Several HM NPN components increase in concentration postprandially; however, these concentrations were not always correlated with changes in plasma concentrations, suggesting that milk NPN may also reflect metabolic activities within the mammary gland.
Adult↗
Insulin-like growth factors I and II and their binding proteins in human milk: effect of heat treatment on IGF and IGF binding protein stability.
Insulin-like growth factors (IGF-I and -II) are peptide growth factors that contribute to the growth-promoting properties of human milk. IGFs in extra cellular fluids are associated with high-affinity binding proteins (IGFBPs). In this study, IGF-I and -II, and IGFBPs in human milk were characterized, and the stability of the IGFs and IGFBPs to heat treatment was investigated. IGF-I and -II were quantified by radioimmunoassays of acid-chromatographed samples, and IGFBPs were characterized using Western ligand blotting. The concentration (mean +/- SD) of IGF-I in human milk was 1.5 +/- 0.5 micrograms/L, compared to 2.7 +/- 0.7 micrograms/L for IGF-II. Heat treatment did not significantly affect either IGF-I or -II content. Human milk contains a single, nonglycosylated, IGFBP, with an apparent Mr of 31 k, which was immunoprecititable by an antibody to IGFBP-2. Stability of the IGFBP to heat treatment was assessed and was not significantly affected by heat treatment. Therefore, both IGF-I and -II, and the IGFBP in human milk appear to be stable under heat treatment conditions routinely used for processing banked human milk.
Blotting, Western↗
Insulin-like growth factors I and II and their binding proteins in rat milk.
IGF-I and -II are peptide growth factors that may be important contributors to the growth-promoting properties associated with milk. IGF in extracellular fluids, including serum and milk, are carried by specific high-affinity binding proteins (IGFBP). In this study, the levels of IGF-I and -II in rat serum and milk were quantified by specific RIA, and the IGFBP were characterized using Western ligand blotting and autoradiography throughout lactation. The levels of IGF-I in both milk and maternal serum decreased during lactation. Serum IGF-I decreased from 743 +/- 187 micrograms/L on d 1 to 391 +/- 106 (mean +/- SD) on d 21 of lactation, and milk IGF-I levels fell from 30 +/- 10 to 13 +/- 8 micrograms/L. Levels of IGF-II in serum and milk were much lower than IGF-I, and were unaffected by lactation. In maternal serum, several IGFBP were identified: IGFBP-3, which migrates as four glycosylated bands with apparent Mr from 38 to 42 kD and one to two nonglycosylated bands with apparent Mr of 28 to 29 kD, and an IGFBP with an apparent Mr of 24 kD. In milk, IGFBP-3, the 24-kD binding protein, and a third IGFBP with an apparent Mr of 29 kD were identified. Treatment of milk and serum with Endoglycosidase F reduced the four glycosylated IGFBP-3 bands (38-42 kD) to two bands with apparent Mr of 35 and 32 kD. In rat milk, but not adult rat serum, the IGFBP with an apparent Mr of 29 kD was immunoprecipitable by an antibody that recognizes IGFBP-2.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Differential effects of insulin-like growth factor (IGF)-I and IGF-II on the expression of IGF binding proteins (IGFBPs) in a rat neuroblastoma cell line: isolation and characterization of two forms of IGFBP-4.
The isolation and hormonal regulation of two low molecular weight insulin-like growth factor binding proteins (IGFBPs) present in the conditioned medium (CM) of the rat neuroblastoma cell line B104 cells has been performed. IGFBPs were purified by ZnSO4 precipitation, insulin-like growth factor-I 1IGF-I) affinity chromatography, and reverse phase HPLC. Final isolation and N-terminal analysis was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting to polyvinylidene difluoride membranes, and sequencing of the bound proteins. Two IGFBPs, with apparent Mr of 28K and 24K were coisolated and sequenced. Both proteins had identical N-terminal sequences and appear to be two forms of IGFBP-4. Treatment of the IGFBPs with endoglycosidase-F caused a shift in the apparent Mr of the 28K IGFBP to 24K. However, there was no change in the apparent Mr of the 24K IGFBP. The data from this study suggest that the IGFBP-4 exists as both a glycosylated and nonglycosylated protein. Treatment of B104 cells with IGF-I increased the expression of both the 24K and 28K IGFBPs and also resulted in the appearance of IGFBP-3 and an unknown IGFBP at 29K. When added to subconfluent cells, IGF-I was also mitogenic in B104 cells. Similar to IGF-I, IGF-II treatment increased cell number and resulted in the appearance of IGFBP-3 and the 29K IGFBP. However, IGF-II treatment resulted in a significant decrease (approximately 50%) in the 24K IGFBP and also decreased the 28K IGFBP. This decrease in the expression of the 24K and 28K IGFBPs was dose-dependent and was blocked by addition of IGF-I to the cells. When an IGF-II receptor antibody was added to the cells it mimicked the effects of IGF-II on B104 cells, suggesting that the inhibitory effects of IGF-II are mediated through the type II IGF receptor. Although both IGF-I and IGF-II affected the amount of the 24K IGFBP in the CM, neither peptide affected the expression of the messenger RNA for the 24K IGFBP. In conclusion, we have isolated two IGFBPs from the CM of B104 cells. Both the 24K and 28K IGFBPs appear to be isoforms of the same protein, and sequence data suggest these proteins are two forms of IGFBP-4. IGF-I increases the expression of both of these IGFBPs, whereas IGF-II decreases their expression.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Differential regulation of the insulin-like growth factors (IGF-I and -II) and IGF binding proteins during malnutrition in the neonatal rat.
Insulin-like growth factor (IGF)-I and -II are known to play a major role in fetal and early postnatal growth. The IGF binding proteins (IGFBPs) are thought to be important in modulating the actions of the IGFs. In this paper, the effect of malnutrition in the neonatal rat on serum IGFs and IGFBPs and hepatic IGFBP messenger (m) RNA was examined. Control (C) dams (n = 9) were allowed ad libitum intake, whereas restricted (R) dams (n = 9) were limited to 50% of ad libitum intake throughout lactation, which results in decreased milk production and malnutrition of pups suckling on restricted dams. A subset of pups were cross-fostered from the R-dams to the C-dams from days 15-19 postpartum (PP) to investigate the effect of nutritional repletion (refed). Pups were killed on days 8, 12, 15, and 19 PP and liver and blood collected. Serum IGF-I and -II concentrations were measured by RIA after acid-chromatography to remove IGFBPs. Serum IGFBPs were characterized by Western ligand blot. Hepatic mRNA for IGFBP-1, -2, and -3 were determined by northern analysis. Body weight (BW) of R-pups was significantly less than C-pups by day 10 PP (P less than or equal to 0.05), and mean BW at day 19 was 56% of the C-pups. Refeeding from days 15-19 resulted in a significantly greater rate of growth vs. R-pups (3.2 vs. 0.9 g/day), and mean BW of refed pups at day 19 PP was 75% of C-pups. Malnutrition caused a significant reduction in both serum IGF-I and -II after day 12 PP, while causing an elevation in serum IGFBP-2. IGFBP-1 and IGFBP-2 mRNA expression were not significantly affected at days 8 and 12, but were elevated in livers of day 15 and 19 pups. Malnutrition caused a delay in the development shift from IGFBP-2 to IGFBP-3, which normally occurs between day 15 and 19 in the rat. Refeeding raised serum IGF-I and -II levels to those found in the C-pups and a trend toward normalization of IGFBP profiles. In conclusion, IGFs and IGFBPs are differentially regulated during neonatal malnutrition. The decrease in IGF peptide and induction of IGFBP-1 and -2 may provide protective mechanisms by inhibiting growth during malnutrition.
Animals↗
Maternal insulin-like growth factor-binding protein messenger ribonucleic acid during rat pregnancy.
Pregnancy is associated with rapid growth of maternal and fetal tissues. Insulin-like growth factors (IGF-I and -II) have roles in mediating both fetal and placental growth. In this study serum IGFs and IGF-binding proteins (IGFBPs) were characterized, IGFBP protease activity was quantified, and hepatic IGFBP-1, -2, -3, and -4 mRNA were investigated throughout rat pregnancy. IGF-I in maternal serum was elevated (P less than or equal to 0.001) on days 5 and 10 (d5 and d10) of gestation compared to levels in nonpregnant controls (NP), but was significantly decreased below NP levels (P less than or equal to 0.001) after d10 of pregnancy. Serum IGF-II levels were unaffected by pregnancy. Using Western ligand blotting (WLB), six IGFBP bands were visualized in NP, d5, and d10 pregnancy rat sera. At 15 and 20 days gestation, the IGFBP-3 bands were no longer detectable by WLB. Using an in vitro IGFBP protease assay, sera from rats at 15 and 20 days gestation proteolyzed 63 +/- 4% and 81 +/- 5% of recombinant human IGFBP-3, respectively. Regression analyses demonstrated that serum IGF-I was positively correlated with serum IGFBP-3 (r2 = 0.73; P = 0.001), whereas serum IGFBP-3 (r2 = -0.85; P = 0.001) and serum IGF-I (r2 = -0.78; P = 0.001) were negatively correlated with serum protease activity. In addition, no change was observed in liver IGFBP-3 mRNA during pregnancy, further suggesting that protease activity is primarily responsible for the decrease in serum IGFBP-3. However, IGFBP-1 and -4 mRNA levels were increased 3- to 11-fold after d5 of gestation. The hormonal and/or metabolic regulators of hepatic IGFBP-1 and -4 expression during rat pregnancy remain to be determined.
Animals↗
Insulinlike growth factor-binding proteins.
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Animals↗
Bioavailability of urea nitrogen for the low birthweight infant.
Previous studies have demonstrated increased retention (40%) of dietary urea nitrogen by term infants recovering from infection compared to healthy infants (13%), possibly due to a higher requirement for nitrogen. Since low birthweight infants also have a high requirement for nitrogen, the bioavailability of urea nitrogen was investigated in low birthweight infants using 15N,15N-urea. Four low birthweight infants (gestational age = 30 +/- 2.2 weeks [mean +/- SD], birthweight = 1.4 +/- 0.3 kg) were fed formula enriched with 15N,15N-urea. Metabolic balance studies (72 hours) were performed with urine and fecal collections. Nitrogen was quantitated by Kjeldahl analysis and 15N,15N-urea by gas chromatography/mass spectrometry. Mean nitrogen intake was 489 +/- 32 mg/kg/d, with 75% nitrogen absorption and 62% nitrogen retention. Maximum urinary enrichment was 8%. Cumulative 15N,15N-urea excretion was 72%, resulting in 28% retention. Thus the bioavailability of urea nitrogen for low birthweight infants appears to be intermediate between compromised and normal term infants.
Biological Availability↗
Partition of nitrogen intake and excretion in low-birth-weight infants.
Although nitrogen balance studies have been carried out in low-birth-weight infants, few have partitioned the nitrogen into its components. In this study, 72-hour balance studies were conducted in 24 low-birth-weight infants (gestational age, 30.7 +/- 1.6 weeks; birth weight 1.36 +/- 0.25 kg) fed their mothers' milk (preterm milk) or 50% preterm milk and 50% formula. Total nitrogen, nonprotein nitrogen, and whey protein intake and excretion were measured. Total nitrogen intake (preterm milk group, 452 +/- 138 mg/kg per day; preterm + formula group, 406 +/- 93 mg/kg per day), absorption (85%), and retention (71%) were not significantly different between groups. Intact and fragments of secretory IgA and lactoferrin were detected in soluble fecal extracts, and represented 25% and 9% of intake, respectively. Feeding preterm milk allows for nitrogen accretion similar to intrauterine growth rates for 5 weeks postnatally, and provides potentially functional proteins for the low-birth-weight infant.
Birth Weight↗
Isolation of the nonprotein nitrogen fraction from human milk by gel-filtration chromatography and its separation by fast protein liquid chromatography.
Human milk (HM) is unique compared with the milk of other species in that nonprotein nitrogen (NPN) constitutes 20-25% of the total N. The NPN fraction consists of a diverse group of compounds with molecular masses less than 10,000 Da (in the picogram to microgram per milliliter range), which have only been partially characterized. We developed a methodology to separate and concentrate the NPN fraction for further analysis. NPN was initially separated from other milk components by Sephadex G-25 gel filtration. Further isolation and separation was carried out by fast protein liquid chromatography gel filtration and ion-exchange chromatography. Molecular masses of unknown peaks were determined by using known molecular mass markers and standards. The methodologies developed lead to the discrete separation of NPN from other milk compounds and can be particularly valuable for isolating peptides in HM.
Chromatography, Gel↗
Development of a human milk protein standard.
Accurate quantitation of the protein content of human milk (HM) is clinically important for both determining protein and energy intakes of HM-fed infants. Protein can be determined by Kjeldahl analysis or colorimetric assays. Colorimetric assays are rapid and convenient, but usually overestimate the protein content when compared to Kjeldahl protein (KP). In this study a protein standard based on human milk protein (HMPS) was isolated by size exclusion column chromatography. Purity and composition of the standard was determined by gel electrophoresis, Kjeldahl and amino acid analyses. The protein content of 20 mature milk samples was determined by the Lowry, BCA and BioRad colorimetric assays, using bovine serum albumin, human serum albumin/IgG, and HMPS as standards. These results were compared with KP results using Student's t-test. All colorimetric assays overestimated milk protein content; however, the Lowry assay gave the lowest protein levels, with our protein standard yielding values closest to the KP value.
Amino Acids↗
Non-protein nitrogen and true protein in infant formulas.
Protein and nitrogen (N) requirements of infants and the optimum protein level of infant formulas are still under debate. Human milk (HM) N is considered adequate although 25% of the N is provided as non-protein nitrogen (NPN), half in the form of urea. Soluble N, insoluble N, NPN and urea N were determined in HM and cow's milk (CM), 5 CM-based infant formulas, 4 soy protein-based formulas (SF), and manufacturers' skim milk and whey protein sources. Total, peptide and free amino acids were also determined in all samples. Levels of NPN and urea N in formulas were highly dependent on the type of whey used, with ion-exchange whey being highest, followed by electrodialyzed and ultrafiltered whey, respectively. SF contained very little NPN with the exception of Soyalac. Consequently, true protein [(Total N-NPN) x 6.38] was lower than the reported levels in several formulas. Levels of peptide and free amino acids were also affected by the method of whey preparation. While it has been recognized that "casein-predominant" CM formulas will have different effects on metabolic indices than "whey-predominant" formulas, this study demonstrates that there are also pronounced differences among "whey-predominant" formulas.
Amino Acids↗
Ontogeny of serum insulin-like growth factor binding proteins in the rat.
Insulin-like growth factors (IGF-I and -II) are peptide growth factors that may be important for neonatal development. Specific high affinity IGF binding proteins (BPs) have been characterized in serum and extracellular fluids. The major serum binding complex in the adult has an apparent Mr of 150 K, while the predominant BP in the neonate is approximately 30 K. In the rat, the transition from the neonatal BP to the adult form occurs during the third postnatal week, concomitant with an increase in serum IGF-I and a decrease in serum IGF-II concentrations. Using specific RIAs and Western ligand blot analyses we have characterized the changes in serum IGF and IGF BPs, respectively, during the early postnatal period. Seven BPs were identified in serum with apparent Mr values of 42, 41, 40, 38, 28, 26, and 22 K. After deglycosylation, the 42, 41, 40, and 38 K BPs were reduced to two bands with apparent Mr values of 35 and 32 K, while the 28, 26, and 22 K BP were unchanged. In the neonate, the 28, 26, and 22 K BPs were present, with the 28 K BP in highest concentration. With increasing age, the 28 K BP decreased and the 42, 41, 40, and 38 K BPs appeared at approximately 19 days of age. Comparison of Western ligand blots of neonatal serum, BRL-3A conditioned media, rat amniotic fluid, and rat cerebrospinal fluid (CSF) demonstrated that all contained a prominent 28 K BP. A polyclonal antibody (alpha Hec 1) developed against the 31 K human IGF-BP (hBP-31) immunoprecipitated the 28 K BP from neonatal rat serum, BRL-3A media, rat amniotic fluid, and rat CSF, but did not react with adult rat serum. These findings suggest that, in the rat, the predominant neonatal serum BP is structurally and immunologically similar to the major BRL-3A, amniotic fluid, and CSF BPs, but distinct from the predominant adult serum BP.
Aging↗