RESUMEN
The Wnt/beta-catenin/TCF4 pathway plays critical roles in the maintenance of small intestinal epithelium; however, downstream targets of the beta-catenin/TCF4 complex are not extensively characterized. We identified miR-30e as an immediate target activated by the beta-catenin/TCF4 complex. miR-30e was detected in the peri-nuclear region of the intestinal crypt IEC-6 cells. Bioinformatics analysis revealed clustered beta-catenin/TCF4 binding sites within the miR-30e promoter region. This promoter region was cloned into pGL3-control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV-SPORT6-beta-catenin expression vector dose-dependently increased luciferase activity, and co-transfection of pCMV-SPORT6-TCF4 expression vector further enhanced the promoter activity. Dexamethasone-induced IEC-6 cells differentiation caused a 2.5-fold increase in miR-30e expression, and upon beta-catenin siRNA transfection, miR-30e increased 1.3-fold. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding between beta-catenin/TCF4 complexes from IEC-6 nuclear extracts and the putative sequences in the miR-30e promoter. These results demonstrate that beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Diferenciación Celular/fisiología , Mucosa Intestinal/citología , MicroARNs/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , beta Catenina/metabolismo , Línea Celular , Inmunoprecipitación de Cromatina , Biología Computacional , Cartilla de ADN/genética , Dexametasona , Ensayo de Cambio de Movilidad Electroforética , Humanos , Immunoblotting , Mucosa Intestinal/fisiología , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Factor de Transcripción 4RESUMEN
Experimental studies indicate that zinc (Zn) and calcium (Ca) status, in addition to iron (Fe) status, affect gastrointestinal absorption of cadmium (Cd), an environmental pollutant that is toxic to kidneys, bone and endocrine systems. The aim of this study was to evaluate how various nutritional factors influence the uptake of Cd in women, particularly during pregnancy. The study was carried out in a rural area of Bangladesh, where malnutrition is prevalent and exposure to Cd via food appears elevated. The uptake of Cd was evaluated by associations between erythrocyte Cd concentrations (Ery-Cd), a marker of ongoing Cd exposure, and concentrations of nutritional markers. Blood samples, collected in early pregnancy and 6 months postpartum, were analyzed by inductively coupled plasma mass spectrometry (ICPMS). Ery-Cd varied considerably (range: 0.31-5.4microg/kg) with a median of 1.1microg/kg (approximately 0.5microg/L in whole blood) in early pregnancy. Ery-Cd was associated with erythrocyte manganese (Ery-Mn; positively), plasma ferritin (p-Ft; negatively), and erythrocyte Ca (Ery-Ca; negatively) in decreasing order, indicating common transporters for Cd, Fe and Mn. There was no evidence of Cd uptake via Zn transporters, but the association between Ery-Cd and p-Ft seemed to be dependent on adequate Zn status. On average, Ery-Cd increased significantly by 0.2microg/kg from early pregnancy to 6 months postpartum, apparently due to up-regulated divalent metal transporter 1 (DMT1). In conclusion, intestinal uptake of Cd appears to be influenced either directly or indirectly by several micronutrients, in particular Fe, Mn and Zn. The negative association with Ca may suggest that Cd inhibits the transport of Ca to blood.
Asunto(s)
Cadmio/farmacocinética , Absorción Intestinal/fisiología , Embarazo/sangre , Adulto , Bangladesh , Cadmio/sangre , Estudios de Cohortes , Cobre/sangre , Eritrocitos/metabolismo , Femenino , Ferritinas/sangre , Humanos , Absorción Intestinal/efectos de los fármacos , Hierro/sangre , Manganeso/sangre , Estudios Prospectivos , Distribución Aleatoria , Población Rural , Selenio/sangre , Estadísticas no Paramétricas , Adulto Joven , Zinc/sangreRESUMEN
This article presents the results of an expert consultation meeting aimed at evaluating the safety and public health implications of administering supplemental iron to infants and young children in malaria-endemic areas. Participants at this meeting that took place in Lyon, France on June 12-14, 2006 reached consensus on several important issues related to iron supplementation for infants and young children in malaria-endemic areas. The conclusions in this report apply specifically to regions where malaria is endemic.
Asunto(s)
Anemia Ferropénica/prevención & control , Suplementos Dietéticos , Enfermedades Endémicas , Hierro/uso terapéutico , Malaria/prevención & control , Anemia Ferropénica/epidemiología , Niño , Humanos , Lactante , Malaria/epidemiología , Organización Mundial de la SaludRESUMEN
Little is known about the acquisition of cobalamin by the mammary gland and its secretion into milk. Human milk and plasma contain at least two types of cobalamin binding proteins: transcobalamin II (TC) and haptocorrin (HC). In plasma, TC is responsible for the transport of cobalamin to tissues and cells; however, cobalamin in milk is present exclusively bound to HC. We show that human mammary epithelial cells (HMEC) exhibit high affinity for TC; Scatchard analysis revealed a single class of binding sites for the TC-[(57)Co]cyanocobalamin complex with a dissociation constant (K(d)) of 4.9 x 10(-11) M. Uptake of the TC-[(57)Co]cyanocobalamin complex at 37 degrees C was saturable by 24 h. Binding of free [(57)Co]cyanocobalamin to HMEC was not saturable and very limited binding of the HC-[(57)Co]cyanocobalamin complex was observed. Expression of the haptocorrin gene by HMEC was confirmed by Northern blot and PCR analysis. Thus, a specific cell surface receptor for the TC-cobalamin complex exists in the mammary gland and once cobalamin is internalized, it may be transferred to HC and subsequently secreted into milk as a HC-cobalamin complex.
Asunto(s)
Mama/metabolismo , Transcobalaminas/biosíntesis , Transcobalaminas/metabolismo , Vitamina B 12/metabolismo , Transporte Biológico , Células Cultivadas , Radioisótopos de Cobalto , Células Epiteliales/metabolismo , Humanos , Leche Humana/química , Leche Humana/metabolismo , ARN Mensajero/biosíntesis , Transcobalaminas/química , Transcobalaminas/genética , Vitamina B 12/químicaRESUMEN
The entire human beta-casein-encoding gene, Bca, was cloned and sequenced. The gene consists of eight exons ranging from 21 to 531 nucleotides (nt) in length and extending over 10,466 nt. Exon-2 contains the translational start, the entire signal sequence and the codons for the two first amino acids of the mature protein. This corresponds to the organization found in other species. The translational stop is localized to exon-7. Exon/intron boundaries are in accordance with the AG/GT rule and conform to suggested consensus sequences. Splice junctions are located between coding triplets. In all other species analyzed, Bca has been found to consist of nine exons; however, within intron-2 of the human gene, a sequence omitted from human mRNA, but corresponding to exon-3 of other known Bca genes, was revealed.
Asunto(s)
Caseínas/genética , Exones/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN Complementario/genética , Humanos , Intrones/genética , Datos de Secuencia Molecular , Plásmidos/genética , Conejos , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
A cDNA of 1065 bp encoding the human milk beta-casein was cloned and sequenced using a synthetic oligodeoxyribonucleotide probe and a human mammary gland library. The nucleotide (nt) sequence contained an open reading frame sufficient to encode the entire amino-acid (aa) sequence of a beta-casein precursor protein consisting of 210 aa and a signal peptide of 15 aa. The nt sequence shows 45-62% homology to those of bovine, ovine, rat, and mouse beta-caseins. The highly phosphorylated site, which is responsible for the calcium-binding capacity of beta-casein, the signal peptide, and a sequence encoding for an inhibitor to the angiotensin-converting enzyme seem highly conserved among the beta-caseins with known sequences.
Asunto(s)
Caseínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Fosfoproteínas/genéticaRESUMEN
Human milk has many unique properties that benefit the breast-fed infant. Several of these properties reside in the protein fraction of human milk; eg, host defense factors (such as immunoglobulins, lysozyme, and lactoferrin), digestive enzymes (lipases, proteases, amylase), specific binding proteins, and growth factors. Increased knowledge of human milk proteins and their biochemistry will aid our understanding of their physiological significance in the infant.
Asunto(s)
Proteínas de la Leche/fisiología , Leche Humana , Receptores de Superficie Celular , Proteínas Portadoras/análisis , Caseínas/análisis , Fenómenos Químicos , Química , Femenino , Receptores de Folato Anclados a GPI , Galactosiltransferasas/análisis , Glicoproteínas/análisis , Sustancias de Crecimiento/análisis , Humanos , Inmunoglobulinas/análisis , Lactalbúmina/análisis , Lactoferrina/análisis , Lactoperoxidasa/análisis , Lactosa/análisis , Lipasa/análisis , Proteínas de la Membrana/análisis , Proteínas de la Leche/análisis , Leche Humana/citología , Leche Humana/enzimología , Mucina-1 , Muramidasa/análisis , Oligosacáridos/análisis , Oxidorreductasas/análisis , Fragmentos de Péptidos/análisis , Péptido Hidrolasas/análisis , Inhibidores de Proteasas/análisis , Albúmina Sérica/análisis , Proteínas de Unión a Tiroxina/análisis , Transcobalaminas/análisis , Transcortina/análisis , alfa-Amilasas/análisisRESUMEN
Copper intakes of infants and adults are often much lower than current recommendations. Copper status, however, appears adequate in most populations. This suggests that copper requirements may be lower than believed earlier, except those for premature infants, who have high requirements as a result of low prenatal stores. Infants, in general, constitute a risk group because milk is low in copper. Bioavailability of copper from human milk is high, whereas it is lower from cow milk and infant formula. Protein source, amino acids, carbohydrates, and ascorbic acid can affect copper availability, whereas phytate, zinc, and iron appear to have little influence on copper absorption, at least physiologic intakes.
Asunto(s)
Cobre/farmacocinética , Adulto , Aminoácidos/farmacología , Animales , Ácido Ascórbico/farmacología , Disponibilidad Biológica , Cobre/análisis , Carbohidratos de la Dieta/farmacología , Fibras de la Dieta/administración & dosificación , Proteínas en la Dieta/farmacología , Humanos , Lactante , Alimentos Infantiles/análisis , Recién Nacido , Absorción Intestinal/efectos de los fármacos , Hierro/farmacología , Leche Humana/química , Estado Nutricional , Ácido Fítico/farmacología , Zinc/farmacologíaRESUMEN
Several human milk proteins have physiologic functions in infants. These proteins are involved in defense against infectious agents and in the optimization of nutrient uptake from milk. Therefore, interest in producing recombinant human milk proteins to use in infant formula has been growing. Microorganisms and transgenic animals can now be used for the production of bioactive proteins. However, the benefits of each protein must be evaluated in cells, animal models, and infants before claims can be made that adding them to formula improves the health or nutrition of infants. Once benefits are shown, proper manufacturing conditions must be developed for introducing the protein or proteins into formula. Processing conditions must be evaluated to ensure that biologic activity is maintained. Dry blending, aseptic processing, sterile filtration, and other techniques will likely be necessary for introducing proteins that require specific tertiary structure for activity. The importance of posttranslational modifications must also be considered: some proteins may require proper glycosylation or phosphorylation for physiologic activity.
Asunto(s)
Ingeniería Genética , Alimentos Infantiles , Proteínas de la Leche/genética , Proteínas de la Leche/normas , Leche Humana/química , Animales , Animales Modificados Genéticamente , Femenino , Humanos , Lactante , Recién Nacido , Proteínas de la Leche/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/normasRESUMEN
Full-term human infants are believed to possess adequate copper stores to last through weaning regardless of the copper content of the diet they are fed. This may not be generally true, however; a combination of low copper intake and low bioavailability from the diet may lead to copper deficiency. More information is needed on the bioavailability of copper from different infant diets, but it appears that copper is well absorbed from breast milk compared with infant formula. Several dietary factors that may affect copper absorption in infants, such as protein sources, amino acids, phytate, ascorbic acid, and other essential cations, need to be evaluated further. Studies in human infants evaluating these factors through use of stable isotope methods, as well as better indicators of copper status, are needed before the copper requirements of infants can be established. This is particularly important for premature infants who, born with inadequate copper stores, are prone to develop copper deficiency unless given higher provisions of copper. The possibility of copper excess also needs to be considered because there are limited opportunities to diagnose copper toxicity. Finally, the role of homeostatic regulation of copper metabolism in infants needs to be evaluated.
Asunto(s)
Cobre/farmacocinética , Alimentos Infantiles , Fenómenos Fisiológicos Nutricionales del Lactante , Animales , Disponibilidad Biológica , Cobre/administración & dosificación , Cobre/análisis , Humanos , Lactante , Alimentos Infantiles/análisis , Recién Nacido , Recien Nacido Prematuro , Leche/química , Estado NutricionalRESUMEN
Three methods for estimating the casein content of human milk were tested; isoelectric precipitation with washing and correction for co-precipitating proteins, sedimentation by ultracentrifugation, and indirect analysis (ie analyzing for the content of the major whey proteins and subtracting these from the total protein content). Gel electrophoresis and amino acid analysis were used to confirm some of the results. The casein content (mg/ml) of mature human milk (n = 9) was 2.33 +/- 1.69 by isoelectric precipitation, 1.80 +/- 0.48 by sedimentation and 2.96 +/- 1.08 by the indirect approach. A probable partition of nitrogen in breast milk would be casein N: whey protein N: non-protein N of 20:50:30; ie the correct ratio of casein nitrogen: whey nitrogen is approximately 20:80. Analysis of trace elements and minerals demonstrates that of total Ca 10%, Mg 5%, Zn 28%, Cu 17%, and Fe 27% is bound to casein when prepared by ultracentrifugation while isoelectric precipitation causes a redistribution of some of these elements. Since the protein ratio of human milk is considered a guideline when manufacturing infant formulas, these findings should be considered with regard to infant nutrition.
Asunto(s)
Caseínas/análisis , Leche Humana/análisis , Aminoácidos/análisis , Precipitación Química , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Punto Isoeléctrico , Proteínas de la Leche/análisis , Minerales/análisis , Nitrógeno/análisis , Espectrofotometría Atómica , Oligoelementos/análisis , UltracentrifugaciónRESUMEN
Mammalian milk iron (MFe) concentration decreases during lactation. In human attempts to increase MFe through supplementing the mother with iron during lactation have failed and no correlation between maternal iron status or intake and MFe has been determined, suggesting a regulatory mechanism. In contrast MFe in lactating rats can be affected by very high or low amounts of ingested iron. We previously described a transferrin receptor (TfR) mechanism that controls iron entry into rat mammary tissue. In this study lactating rats were used to determine effects of varying dietary iron during gestation and lactation on mammary TfR and MFe. Dams fed low-iron diets had a small increase in TfR, lower hematological indices (p less than 0.005), and lower MFe (p less than 0.005) than did controls or dams fed high iron. Dams fed the high-iron diet had a significant increase in TfR without a concomitant increase in MFe. There was no correlation between MFe and TfR. These findings suggest that control of MFe lies after iron entry into mammary tissue.
Asunto(s)
Hierro/administración & dosificación , Lactancia/efectos de los fármacos , Glándulas Mamarias Animales/efectos de los fármacos , Receptores de Transferrina/metabolismo , Animales , Dieta , Femenino , Deficiencias de Hierro , Masculino , Glándulas Mamarias Animales/metabolismo , Intercambio Materno-Fetal , Leche/metabolismo , Embarazo , Ratas , Ratas EndogámicasRESUMEN
Casein in human milk is believed to serve several biological functions in newborns. However, the content and subunit composition of human casein has so far received little attention. We recently developed a method to separate human-milk whey and casein by adjustment of whole human milk to pH 4.3 and addition of calcium followed by ultracentrifugation. In this study we analyzed and evaluated human casein prepared by different methods. We used fast protein liquid chromatography (FPLC) with an anion-exchange column (Mono-Q) and polyacrylamide gradient gel electrophoresis techniques to analyze the casein subunit composition. Total casein in human milk, as determined by the Kjeldahl method, varies during lactation; the casein content is approximately 20% of the total protein content in early lactation and 45% in late lactation. We found differences in both glycosylation and phosphorylation patterns of kappa-caseins and beta-caseins from premature and term milk samples.
Asunto(s)
Caseínas/aislamiento & purificación , Proteínas de la Leche/aislamiento & purificación , Leche Humana/análisis , Caseínas/clasificación , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Femenino , Liofilización , Humanos , Lactancia , Embarazo , Coloración y Etiquetado , UltracentrifugaciónRESUMEN
Human milk proteins are of nutritional and physiological significance to the newborn infant. To further study these proteins, a rapid procedure to separate and analyze human milk whey proteins was developed using fast protein liquid chromatography (FPLC). First, to separate whey proteins from casein, different variables such as low- or high-speed centrifugation at different temperatures with or without adjustment of pH to 4.6 or 4.3 and with or without addition of calcium to whole milk or skim milk were tested. Each variable was evaluated by gel filtration, anion-exchange chromatography, sodium dodecyl sulphate (SDS)-polyacrylamide gradient gel electrophoresis, immunoelectrophoresis, and immunodiffusion. The optimum method for a discrete separation of whey and casein is the adjustment of whole milk to pH 4.3 with addition of 60 mmol calcium/L, followed by ultracentrifugation. Rapid and sensitive separation and analysis of whey proteins was achieved by FPLC gel filtration and anion-exchange chromatography.
Asunto(s)
Proteínas de la Leche/aislamiento & purificación , Leche Humana/análisis , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Técnicas In Vitro , Proteínas de la Leche/análisis , Proteína de Suero de LecheRESUMEN
The appropriate amount of protein to use in infant formula is still under discussion. We found earlier that protein digestibility is higher from ultrahigh-temperature (UHT)-treated formula than from conventionally heat-treated formula. In this study, we evaluated the nutritional, hematologic, and biochemical effects of feeding infants whey-predominant UHT-treated formula with 13 (UHT-13) or 15 (UHT-15) g protein/L as compared with a conventional, powdered, whey-predominant formula (PF) with 13 g protein/L from 6 wk to 6 mo of age. Breast-fed infants served as control subjects. Growth was assessed at monthly intervals and venous blood samples were drawn at entry into the study and at 6 mo of age. At 6 mo, there were no significant differences in weight gain or linear growth, or hemoglobin, serum ferritin, zinc, or copper concentrations among the groups. Blood urea nitrogen concentrations were lowest for breast-fed infants; among the formula-fed groups the UHT-13 group had the lowest values. All formula-fed groups had higher plasma threonine concentrations than breast-fed infants. Infants fed the UHT-13 formula had threonine values closest to those of breast-fed infants. Concentrations of branched-chain amino acids were similar in breast-fed infants and those fed UHT-13 formula, whereas the other groups had higher values. Plasma tryptophan concentrations were significantly higher in the UHT-treated formula groups than in the other groups. Thus, infants fed UHT-13 formula had metabolic measures similar to those of breast-fed infants, possibly because of high protein digestibility, or a difference in the protein source used. Iron, zinc, and copper status was satisfactory in all groups. Selenium status, as indicated by serum glutathione peroxidase activity, varied with dietary selenium intake.
Asunto(s)
Aminoácidos/sangre , Lactancia Materna , Alimentos Infantiles , Proteínas de la Leche/análisis , Oligoelementos/sangre , Nitrógeno de la Urea Sanguínea , Ferritinas/sangre , Hemoglobinas/análisis , Humanos , Lactante , Polvos , Receptores de Transferrina/sangreRESUMEN
The effect of a low and a high protein diet (approximately 8 and 20% energy from protein, respectively) on the contents of different nitrogen-containing substances in breast milk was studied on three healthy Swedish mothers in full lactation. Each experimental diet was fed during a 4-day period and milk samples were collected during the last day. The milk volume was estimated by weighing the child before and after each feeding. The 24-hr outputs as well as concentrations of total nitrogen, true protein, and nonprotein nitrogen were significantly higher when the subjects consumed the high protein rather than the low protein diet. The higher content of nonprotein nitrogen was due to increased urea levels as well as to increased levels of free amino acids. Milk urea levels were closely correlated with plasma urea levels in samples obtained after overnight fasting when the subjects had consumed the experimental diets for 4 days. The 24-hr output of lactoferrin, alpha-lactalbumin, and serum albumin were higher when the subject consumed the high protein diet as compared to the low protein diet but the differences were not significant.
Asunto(s)
Proteínas en la Dieta/metabolismo , Proteínas de la Leche/análisis , Leche Humana/análisis , Nitrógeno/análisis , Adulto , Femenino , Humanos , Proteínas de la Leche/metabolismo , EmbarazoRESUMEN
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.
Asunto(s)
Proteínas de la Leche/análisis , Leche Humana/análisis , Nitrógeno/análisis , Cromatografía en Gel , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico , Diálisis , Liofilización , HumanosRESUMEN
BACKGROUND: Leptin, the product of the obese (ob) gene, is a regulator of food intake and energy metabolism. Immunoreactive leptin was detected recently in breast milk and it has been hypothesized that leptin may be absorbed and may contribute to differences in body composition between breast-fed and formula-fed infants. OBJECTIVE: The objective was to evaluate whether diet, adiposity, or sex affect plasma leptin in breast-fed and formula-fed infants. DESIGN: Venous blood samples were drawn from healthy, exclusively breast-fed or formula-fed Swedish infants at 1, 4, and 6 mo of age (n = 193) and from 12-mo-old Finnish infants (n = 79). Anthropometric measurements were made and plasma samples were analyzed for leptin, insulin, and glucose. RESULTS: There were no significant differences in plasma leptin between formula-fed and breast-fed infants at 1 and 4 mo of age, whereas formula-fed infants had significantly higher ( approximately 5%) leptin concentrations at 6 mo of age. Similar results were observed after correction for BMI. Plasma leptin was 15-25% higher in female than in male infants at 1, 4, and 12 mo of age (P < 0.05), also after correction for BMI. When all infants were analyzed together, a positive correlation (r = 0.34, P < 0.0001) was found between plasma leptin and BMI. Very low leptin concentrations were found in breast milk after centrifugation and the high concentrations reported previously were likely due to interference in the assay by milk fat. CONCLUSIONS: Plasma leptin concentrations are not higher in breast-fed than in formula-fed infants; however, sex and adiposity affect leptin concentrations even at this early age.
Asunto(s)
Lactancia Materna , Alimentos Infantiles , Fenómenos Fisiológicos Nutricionales del Lactante , Leptina/sangre , Obesidad/sangre , Glucemia/análisis , Índice de Masa Corporal , Estudios de Cohortes , Femenino , Humanos , Lactante , Recién Nacido , Insulina/sangre , Masculino , Obesidad/genética , Factores Sexuales , Suecia , Población Blanca/genéticaRESUMEN
BACKGROUND: Two heme-containing peroxidases, secretory lactoperoxidase and leukocyte-derived myeloperoxidase, which play host defense roles through antimicrobial activity, were previously identified in human colostrum. Within several days after the start of lactation, the relative contribution of myeloperoxidase to the peroxidase activity in milk was shown to decline as the number of milk leukocytes decreased. OBJECTIVE: Our knowledge of lactoperoxidase in human milk is still limited. The objective of this study was to use specific antibodies as a means of simplifying the purification and quantification of lactoperoxidase. DESIGN: Polyclonal antibodies were raised against recombinant human lactoperoxidase. Immunoglobulin G (IgG) was isolated by means of a protein A column and was characterized by immunoblotting. For the purification of lactoperoxidase from whey, a cation-exchange column and an immunoaffinity column with coupled IgG were used. The concentration of lactoperoxidase was determined by a sandwich enzyme-linked immunosorbent assay by using purified native lactoperoxidase as a standard. Native and biotinylated IgG were used as capture and detector antibodies, respectively. RESULTS: Two bands with molecular masses of approximately 80 and 100 kDa were detected in an immunoblot of human whey. Similar heterogeneity was observed in the sodium dodecyl sulfate-polyacrylamide gel electophoresis profile of purified lactoperoxidase. The mean (+/-SD) concentration of lactoperoxidase in 26 whey samples was estimated to be 0.77 +/- 0.38 mg/L. The concentrations were positively correlated with the peroxidase activity detected in these samples. CONCLUSION: Lactoperoxidase is commonly present in human milk throughout the lactation period and is likely to contribute to the protective effects of milk.
Asunto(s)
Lactoperoxidasa/análisis , Leche Humana/enzimología , Anticuerpos , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G , Técnicas de Inmunoadsorción , Lactancia , Lactoperoxidasa/inmunología , Lactoperoxidasa/aislamiento & purificación , Proteínas Recombinantes/inmunologíaRESUMEN
The contents of total nitrogen, nonprotein nitrogen, lactose, and individual milk proteins have been determined in human milk from well-nourished Swedish mothers. Breast milk samples from 50 mothers at different stages of lactation (up to 170 days) were collected. Furthermore, three mothers gave samples repeatedly throughout the whole lactation period. The protein content in mature milk was found to be 0.8 to 0.9% by amino acid analysis. The nitrogen content and the contents of the major human milk whey proteins, alpha-lactalbumin and lactoferrin, are very high for the first few days, then decrease rapidly and reach, thereafter, the more slowly declining level of mature milk. Nonprotein nitrogen and the nonspecific milk protein serum albumin are present in constant concentrations throughout lactation. The daily milk volumes were determined and found to be 500 to 600 ml in the very early part and 700 to 800 ml in the later part of the lactation period.