Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland.

During lactation, branched-chain aminotransferase (BCAT) gene expression increases in the mammary gland. To determine the cell type and whether this induction is present only during lactation, female rats were randomly assigned to one of three experimental groups: pregnancy, lactation, or postweaning. Mammary gland BCAT activity during the first days of pregnancy was similar to that of virgin rats, increasing significantly from day 16 to the last day of pregnancy. Maximal BCAT activity occurred on day 12 of lactation. During postweaning, BCAT activity decreased rapidly to values close to those observed in virgin rats. Analyses by Western and Northern blot revealed that changes in enzyme activity were accompanied by parallel changes in the amount of enzyme and its mRNA. Immunohistochemical studies of the mammary gland showed a progressive increase in mitochondrial BCAT (mBCAT)-specific staining of the epithelial acinar cells during lactation, reaching high levels by day 12. Immunoreactivity decreased rapidly after weaning. There was a significant correlation between total BCAT activity and milk production. These results indicate that the pattern of mBCAT gene expression follows lactogenesis stages I and II and is restricted to the milk-producing epithelial acinar cells. Furthermore, BCAT activity is associated with milk production in the mammary gland during lactation.

Induction of expression of branched-chain aminotransferase and alpha-keto acid dehydrogenase in rat tissues during lactation.

This study was designed to determine the effect of lactation and weaning on the gene expression of branched-chain aminotransaminase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD) in different tissues of the lactating rat. BCAT activity increased in mammary tissue during lactation and was 6-fold higher than in virgin rats. This increase was associated with an increase in protein levels measured by immunoblot analysis, and with an increase in BCAT mitochondrial (BCATm) mRNA concentration. Twenty-four hours after weaning, BCAT activity, protein concentration, and mRNA levels in the dam decreased. BCAT activity, protein enzyme levels, and BCATm mRNA concentration in muscle were higher in weaning rats than in lactating rats. BCAT cytosolic (BCATc) mRNA was not expressed in mammary tissue, and there was no BCATc enzyme detected by Western blot in any physiological state. Mammary tissue BCKD activity increased and was active (dephosphorylated) during the lactation period. The level of enzyme also increased and the mRNA level for the E2 subunit in mammary tissue was 10-fold higher than the virgin values. Hepatic enzyme activity increased during weaning, and this was associated with the protein level and with the mRNA level of the E2 subunit. Muscle BCKD activity and protein content were the lowest of all tissues, and the E2 subunit mRNA level was barely detected by Northern blot analysis. The results suggest gene regulation of the two main catabolic enzymes of the branched-chain amino acid metabolism during lactation.

Amino acid intake during lactation and amino acids of plasma and human milk.

The aim of this study was to determine the free amino acid pool in plasma and milk in marginally nourished lactating women. Twenty-eight rural women (age, 23.9+/-5y; weight 50.2+/-4.9 kg; height, 148.2+/-4.8 cm) were studied under metabolic balance conditions. Subjects were divided into 6 groups (5-6 women in each), representing rural mothers postweaning and in the 15, 3rd, and 6th months of lactation; nonpregnant, nonlactating controls were from rural and urban areas. Amino acid analyses of diet and of plasma and milk samples were performed using a Beckman 6300 amino acid analyzer. Lysine intakes were lower than the recommended intake for lactating women (RDA). Plasma amino acid profiles differed between the lactating and weaned groups: aspartate and isoleucine increased at the 6th month (P < 0.05), while valine declined over weaning time (P < 0.05). In milk, valine and proline decreased at the 6th month (P < 0.05), while serine rose at the 3rd month. Free amino acid pools were 1- to 15-fold higher in plasma than in milk for branched-chain amino acids and basic, aromatic, and neutral amino acids. In mammary tissue these amino acids can be channeled to tissue and milk protein synthesis or to catabolic pathways. Glutamate was 40-fold higher in milk with respect to plasma content. This was the predominant amino acid in the free amino acid pool in milk. These results suggest selective amino acid transport in mammary tissue during lactation.

Negative balance of calcium during lactation in marginally nourished women.

Thirty-three rural Mexican women (age, 18-36y; weight, 50.3+/-3 kg; height, 148.3+/-2 cm) were studied under metabolic balance conditions. The objectives were to study the metabolic balances of calcium and phosphorus at the 1st, 3rd, and 6th months of lactation and postweaning and to determine the incorporation of calcium and phosphorus in milk. Subjects were divided into 5 groups of 5 to 10 each, representing: the 1st, 3rd, and 6th month of lactation, postweaning, and a control group of nonpregnant, nonlactating women. Metabolic balance was determined using identical diets and analysis of 24-hour urine (3 d), 72-hour feces, and 24-hour milk samples. Calcium content was determined by atomic absorption spectrophotometry and phosphorus by a colorimetric method. Calcium content in milk was similar at the 1st, 3rd, and 6th months. Positive calcium balances were observed in the control group, while balances were very negative in all lactation groups (-721.6+/-248 mg/d). Calcium urinary excretion was higher in the control and postweaning groups (P < 0.05), suggesting a regulatory mechanism to conserve calcium during lactation. No differences were observed in phosphorus content in milk at the 1st, 3rd, and 6th months. Positive balances were observed in the control and postweaning groups (331+/-139 and 87.1+/-130 mg/d, respectively, mean +/- SD), while the lactation groups presented more subjects (approximately 75%) in negative balance (mean +/- SD of -180.6+/-392 to -439+/-146 mg/d). High fecal calcium and phosphorus excretion (approximately 1,500 mg/d) likely contributed to the negative balance during lactation.

Characterization of methylaminoisobutyric acid transport by system A in rat mammary gland.

During lactation, the mammary gland has a large demand for amino acids for the synthesis of milk proteins and fatty acids. Arteriovenous differences in amino acids across the mammary gland show an elevated uptake of small neutral amino acids that are mainly transported via system A. The purpose of this study was to characterize the transport of methylaminoisobutyric acid (MeAIB), an amino acid analog used to model transport by system A in lactating rat mammary gland explants. MeAIB accumulation in mammary gland cells increased steadily, and after 3 hours of incubation, the intracellular concentration of the analog was 8-fold higher than the concentration in the medium. MeAIB transport into mammary gland explants showed a Km of 3.3 +/- 0.4 mmol/L and a maximal velocity (Vmax) of 555 +/- 23 pmol/microL intracellular fluid (ICF) x min, indicating a system with high capacity but low affinity for its substrate. MeAIB transport into mammary tissue depended highly on Na+, and the uptake was inhibited by addition of natural and analog small neutral amino acids. Cationic, anionic, and large neutral amino acids did not reduce MeAIB transport into mammary gland explants. Preincubation of mammary gland explants in an amino acid-free medium stimulated MeAIB transport, suggesting an adaptive regulation. The addition of an equimolar mixture of alanine, glycine, and serine to the preincubation medium inhibited stimulation of MeAIB transport. Furthermore, stimulation of MeAIB uptake by amino acid starvation was also prevented by the addition of actinomycin D, cycloheximide, tunicamycin, and colchicine. Dibutyryl cyclic adenosine monophosphate (cAMP) increased MeAIB uptake, whereas phorbol 12-myristate 13-acetate (PMA) did not stimulate MeAIB transport. During the first postweaning days, kinetic analyses showed a decrease of 27% in the Vmax. Injection of rat lactating mammary gland mRNA into Xenopus laevis oocytes induced expression of the MeAIB transport system; however, the induction was only 83% above background MeAIB uptake. The results of this study provide a partial explanation for the formation of the metabolic pool of small neutral amino acids in the lactating mammary gland.

[Calcium metabolism in lactating women]. / Metabolismo de calcio en la mujer durante la lactancia.

During lactation the main source of the breast-milk calcium seems to be maternal bone. The women who breast-fed, lost bone mineral content, which is recovered once breast-feeding ceased. Breast-milk calcium do not depend on an increase in calcium intake by the lactating mother. Calcium demand during lactation is associated with adjustments in the calcium metabolism, such as a decrease in urinary calcium excretion and mobilization of calcium from maternal bone. The classical calciotropic hormones concentrations (PTH and 1,25-dihydroxyvitamin D) are not associated with bone turnover markers or with changes in bone mineral content in the lactating women. However, serum estradiol is strongly associated with calcium balance, bone loss and calcium milk content.

[Calcium and phosphorus balance in rural lactating women in Mexico]. / Balance de calcio y fósforo en mujeres lactantes rurales de México.

OBJECTIVE: To compare the balance of calcium (Ca) and phosphorus (P) between lactation and weaning, and to determine the Ca and P milk production in Mexican rural lactating women. METHODS: Thirty-six women aged 18-36 y, weight 49 +/- 3 kg and height 148 +/- 2 cm, were divided in six groups: four groups of lactation (1st, 3rd, 6th and 12th month) one post-weaning group and one of non pregnant non lactating women. The balance studies were performed collecting duplicate diets, 24 h urine for 3 days, 72 h feces and 24 h milk samples for 2 days. The Ca content was determined by atomic absorption spectrophotometry and P by the molybdate method. RESULTS AND CONCLUSIONS: Ca content in milk was higher in the 3rd month of lactation. The Ca balances were negative in all lactation groups (789 +/- 165 mg/d). Ca urinary excretion was lower in the lactating group (p < 0.05) suggesting a regulatory mechanism to conserve Ca during lactation. No differences were observed in the P content in milk and positive balances of P were observed in the non lactating and the post-weaning groups, whereas they were negative in the lactation groups (115 to 475 mg/d). High fecal Ca and P excretion (approximately 1300 mg/d) was observed, which contributed to the negative condition of the balance during lactation. The production of Ca and P in the milk of these rural women was similar to the one seen in rural and urban groups in Africa, Asia, Europe and the U.S.

[Lactation performance in rural women with low bioavailability of nutrients from the regular diet]. / Lactancia en mujeres del área rural con baja biodisponibilidad de nutrimentos a partir de su dieta habitual.

Metabolizable energy (ME) and apparent nitrogen, fat and energy bioavailability from the habitual diet were measured in a group of lactating women from a Mexican rural community. Food intake was estimated in 40 women, age 24 +/- 3 y 3-5 m postpartum, body weight 48.7 +/- 5.4 kg and height 148 +/- 5 cm. Habitual diet was predominantly from vegetable sources based on maize (tortillas), beans, tomato, onion and chili. The daily intake was: 2224 +/- 336 kcal energy, 47.8 +/- 7.0 g protein, 44.7 +/- 11.3 fat and 408 +/- 70 g carbohydrates. Ten women were studied in balance conditions and consuming a controlled diet similar to their habitual diet. ME and apparent energy, nitrogen and fat availability were measured by metabolic balance. Milk output was measured by the test-weighing infant method and milk samples representative of 24-h were obtained for chemical analysis. Apparent digestibility of energy was 87 +/- 1.4%, from nitrogen 68.4 +/- 3.1% and 65.8 +/- 3.7 from fat. Measured ME was 1940 kcal/day. Metabolizable energy from the habitual diet by the marginally nourished lactating group was lower than the energy recommended allowance during lactation. Macronutrient's availability from the habitual diet was similar to that found in population with intake of predominantly vegetable diets and high dietary fiber content.

Regulation of branched-chain amino acid metabolism in the lactating rat.

There is evidence that during lactation, uptake of the essential branched-chain amino acids (BCAA) by mammary glands exceeds their output in milk protein. In this study, we have measured the potential of lactating rats to catabolize BCAA. The activity, relative protein and specific mRNA levels of the first two enzymes in the BCAA catabolic pathway, branched-chain aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD), were measured in mammary gland, liver and skeletal muscle obtained from rat dams at peak lactation (12 d), from rat dams 24 h after weaning at peak lactation and from age-matched virgin controls. Western analysis showed that the mitochondrial BCATm isoenzyme was found in mammary gland. Comparison of lactating and control rats revealed that tissue BCATm activity, protein and mRNA were at least 10-fold higher in mammary tissue during lactation. Values were 1.3- to 1. 9-fold higher after 24 h of weaning. In mammary gland of lactating rats, the BCKD complex was fully active. In virgin controls and weaning dams, only about 20% of the complex was in the active state. Hypertrophy of the liver and mammary gland during lactation resulted in a 73% increase in total oxidative capacity in lactating rats. The results are consistent with increased expression of the BCATm gene in the mammary gland during lactation, whereas oxidation appears to be regulated primarily by changes in activity state (phosphorylation state) of BCKD.

Leucine catabolism in mammary tissue, liver and skeletal muscle of dam rat during lactation and weaning.

BACKGROUND: This study was designed to determine the effect of lactation and weaning on the catabolism of branched-chain amino acids (BCAA). METHODS: Rates of transamination and oxidation of leucine and branched chain alpha-ketoacid dehydrogenase (BCKD) activity were measured in homogenates of mammary gland, skeletal muscle and liver on day 12 of lactation and 24 h after separation of dams from the litter (weaning). RESULTS: Lactating dams consumed 250% more protein than control rats, extra protein is required for protein synthesis by the mammary gland, the extent to which the excess of amino acids consumed during lactation is utilized or oxidized by different tissues is not known. The rate of transamination of [1-14C] leucine by mammary tissue of lactating dams was sixfold higher than in virgin rats. The rate of transamination remained elevated fourfold in postweaning dams. Rates of transamination were three times higher in mammary tissue than in muscle of lactating dams. Rate of oxidation [1-14C] leucine by lactating mammary tissue was tenfold higher than in control tissue. CONCLUSIONS: The capacity of mammary tissue for transamination and oxidation of leucine increased greatly during lactation, suggesting that the mammary gland may play an important role in the catabolism of BCAA during lactation.

[Free amino acids in plasma and milk of mexican rural lactating women]. / Aminoácidos libres en plasma y leche de mujeres lactantes rurales.

OBJECTIVE: To determine the free amino acid pool in plasma and milk in Mexican rural lactating women. METHODS: Twenty-eight women with an age 24 +/- 5.0 (+/- SD) years, weight 50 +/- 4.9 kg and height 148 +/- 4.8 cm were studied under metabolic balance conditions. Subjects were divided into five groups (three groups of lactation at 1st, 3rd and 6th month, one post-weaning group and a control group of non pregnant, non lactating women). Amino acid analyses of the diet and of plasma and milk samples were performed using an automated amino acid analyzer. RESULTS: Differences were observed between the lactation groups and the other groups: aspartate increased at the 6th month (p < 0.05) while leucine, valine and isoleucine declined in the 3rd month (p < 0.05). In milk, valine, proline and taurine decreased at 6 months (p < 0.05), while serine and threonine raised at 3 months. Plasma levels were > 4 fold greater than milk levels for branched chain amino acids and for the basic, aromatic and neutral amino acids. In contrast, glutamate was 40 fold higher in milk than plasma and it was the predominant amino acid in the free pool of milk. CONCLUSIONS: Our results suggest that the metabolic use of amino acids and the presence of specific amino acid transport systems during lactation, contribute to specific concentrations of free amino acids in milk that were not associated with the pool of free amino acids in plasma.

Significance of lipid consumption during lactation.

Human milk lipids are the main source of energy to support optimum growth of the breast-fed infant. The content and composition of milk lipids come from three main sources of fatty acids: the diet, mobilization of body fat stores and fatty acid synthesis de novo by the mammary gland. On account of these, the consumption and composition of the lipids from the diet and also the nutritional state, specifically the body fat percentage of the lactating woman, are elements that maintain a close relation with the content and composition of milk lipids which translates into the energy content given to the baby. The evidence suggests that the body fat stores significantly provide the demand imposed by lactation, and under suboptimal nutritional conditions where body fat stores are depleted, dietary lipid consumption is essential. It is necessary to elucidate the physiological regulatory mechanisms involved in the utilization of dietary lipids on milk synthesis. This information will be of great practical value, since it may allow the development of optimum diets for lactating women.

Alanine aminotransferase activity in mammary tissue, muscle and liver of dam rat during lactation and weaning.

Transamination reaction is the first step in the catabolism of most of the L-amino acids. Alanine is an important molecule in the inter-organ nitrogen transport, conveying them from muscle to the liver. Amino groups from this amino acid are generally first transferred to alpha-ketoglutarate in the cytosol of liver cells to form glutamate and leaving behind the corresponding alpha-keto acid analog. Measurements of the alanine aminotransferase (EC2.6.1.2.) activity were compared in liver, mammary gland and skeletal muscle in virgin, lactating and weaning dam rats. In this study liver was the principal tissue involved in alanine transamination, while muscle showed a reduction in the enzyme activity during lactation. Results indicate an increase in alanine amino-transferase activity in the mammary gland during lactation and weaning when compared with virgin rats. This suggests that mammary gland during lactation is an important extra-hepatic tissue involved in the metabolism of alanine and probably shunted into the pathways for amino group metabolism in terms of nitrogen economy.

Protein requirements of marginally nourished lactating women.

Few experimental data regarding protein requirements of lactating women are available. This study was designed to determine the protein requirement of seven healthy lactating women from a poor Mexican community who were 2-6 mo postpartum and had a mean body mass index of 21.8 +/- 2 kg/m2. Nitrogen balances were performed at intakes of 0.8, 1.0, and 1.2 g.kg body wt-1.d-1 of a mixed protein diet (70% derived from vegetable sources). A balance at 1.2 g.kg body wt-1.d-1 of a high-quality mixed protein (80% from animal sources) was the reference. The experimental diets were designed to resemble the habitual diet in terms of energy and macronutrients. The balance responses to nitrogen intake were -35.5 +/- 12.6, -10.5 +/- 14.8, and 7.8 +/- 19.2 mg N.kg body wt-1.d-1, respectively. Equilibrium balance was attained at 178.9 +/- 25.8 mg N (1.1 g protein.kg body wt-1.d-1), close to current recommended dietary allowances, albeit the dietary protein was mostly from vegetable sources.

Changes in the composition of mammary tissue, liver and muscle of rat dams during lactation and after weaning.

This investigation was conducted to evaluate the changes in the total content of protein and RNA in liver and muscles of rat dams before and after acute separation from their litters. Groups of 8-12 rats fed ad libitum were killed on the 12th (group L12) and 20th d (group L20) of lactation and on the 1st (group W1) and 7th d (group W7) after weaning. Nonpregnant, nonlactating rats paired for age served as controls. Dry matter, protein, DNA and RNA levels of the mammary gland, liver and muscles of the right hindlimb were determined. Wet weight and total organ or tissue protein, DNA and RNA were higher in mammary glands of L12 and L20 than in age-matched controls. These values were lower in groups W1 and W7 than in the lactating groups. No changes were noted in the total liver protein or DNA content, but total liver RNA was greater in groups L12 and L20 than in controls or group W1. Total muscle dry matter, DNA and RNA were significantly lower in groups L12 and L20 than in groups W1 and W7. Muscle protein content increased progressively from the 12th to 20th d of lactation to a peak in group W1, and it decreased to values found in age-matched controls in group W7. Although the muscle protein mass of the hindlimb during peak lactation (group L12) was only 63% of that in nonlactating control rats, within 1 d of weaning it was significantly higher than in nonlactating rats. Similar changes in RNA suggest that these changes in protein content are related to an adaptative mechanism designed to handle the surplus of plasma amino acids not used by the mammary gland after weaning.