RESUMEN
We report the presence of bile salt-stimulated lipase in milk collected from dog and cat. This enzyme has previously been found only in the milk of human and gorilla. Bile salt-stimulated lipase activity in individual dog milk specimens (range: 4.8-107.4 U/ml; 1 U = 1 mumol [3H]oleic acid released/min) was similar, while that in cat milk specimens (range: 2.2-16.9 U/ml) was lower than in human milk (range: 10-80 U/ml). Longitudinal patterns for bile salt-stimulated lipase activity differed depending upon the enzyme source: in dog milk, lipase activity was lowest in colostrum, while in cat milk, lipase activity was highest in colostrum and decreased at mid-lactation. In human milk, bile salt-stimulated lipase activity levels remain fairly constant throughout the first 3 months of lactation. Dog, cat and human milk bile salt-stimulated lipase activity had a neutral-to-alkaline pH optimum of 7.3-8.5, was stable at low pH (above 3.0 for at least 1 h), and was inhibited 95-100% by eserine (at concentrations greater than 0.6 mM). The lipase in the milk of the three species studied had an absolute requirement for primary bile salts (tauro- and glycocholate), and was inhibited by secondary bile salts (tauro- and glycodeoxycholate). These data are the first to report bile salt-stimulated lipase activity in milk from mammals other than the highest primates. Presence of this lipase in non-primate milk will permit the study of the factors that regulate the ontogeny, synthesis and secretion of the enzyme during pregnancy and lactation as well as its function in neonatal fat digestion.
Asunto(s)
Ácidos y Sales Biliares/farmacología , Lipasa/análisis , Leche/enzimología , Animales , Gatos , Perros , Femenino , Concentración de Iones de Hidrógeno , Lactancia , Lipoproteína Lipasa/análisis , Estudios Longitudinales , Fisostigmina/farmacología , Embarazo , Especificidad de la EspecieRESUMEN
We examined effects of acute unilateral enucleation on incorporation from blood of intravenously injected unsaturated [1-14C]arachidonic acid ([14C]AA) and [1-14C]docosahexaenoic acid ([14C]DHA), and of saturated [9,10-3H]palmitic acid ([3H]PA), into visual and nonvisual brain areas of awake adult Long-Evans hooded rats. Regional cerebral metabolic rate for glucose (rCMRglc) values also were assessed with 2-deoxy-D-[1-14C]glucose ([14C]DG). One day after unilateral enucleation, an awake rat was placed in a brightly lit visual stimulation box with black and white striped walls, and a radiolabeled fatty acid was infused for 5 min or [14C]DG was injected as a bolus. [14C]DG also was injected in a group of rats kept in the dark for 4 h. Fifteen minutes after starting an infusion of a radiolabeled fatty acid, or 45 min after injecting [14C]DG, the rat was killed and the brain was prepared for quantitative autoradiography. Incorporation coefficients k* of fatty acids, or rCMRglc values, were calculated in homologous brain regions contralateral and ipsilateral to enucleation. As compared with ipsilateral regions, rCMRglc was reduced significantly (by as much as -39%) in contralateral visual areas, including the superior colliculus, lateral geniculate body, and layers I, IV, and V of the primary (striate) and secondary (association, extrastriate) visual cortices. Enucleation did not affect incorporation of [3H]PA into contralateral visual regions, but reduced incorporation of [14C]AA and of [14C]DHA by -18.5 to -2.1%. Percent reductions were correlated with percent reductions in rCMRglc in most but not all regions. No effects were noted at any of nine non-visual structures that were examined. These results indicate that enucleation acutely reduces neuronal activity in contralateral visual areas of the awake rat and that the reductions are coupled to reduced incorporation of unsaturated fatty acids into sn-2 regions of phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Reduced fatty acid incorporation likely reflects reduced activity of phospholipases A2 and/or phospholipase C.
Asunto(s)
Encéfalo/metabolismo , Enucleación del Ojo , Ácidos Grasos/metabolismo , Animales , Ácido Araquidónico/metabolismo , Autorradiografía , Ácidos Docosahexaenoicos/metabolismo , Glucosa/metabolismo , Procesamiento de Imagen Asistido por Computador , Masculino , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Ratas , Ratas Endogámicas , Análisis de RegresiónRESUMEN
Milk lipids from six mothers with cystic fibrosis were compared with milk lipids from six mothers without cystic fibrosis. Mean neutral lipids (in g/dL) were colostrum, 2.4; transitional, 2.9; mature milk, 2.9, suggesting that milk fat content was sufficient to support energy needs of the infant. Lipid class composition was normal, 98% being present as triglyceride. Cholesterol and cholesteryl ester concentrations were similar in the milk of both groups of lactating women. Phospholipid class distribution of cystic fibrosis milk was different from that in control milk. Approximately 85% of the lipids were present in six major fatty acids from 12:0 to 18:2 in both groups, indicating that the profile of most of the lipid was similar. The linoleic acid content of cystic fibrosis milk was decreased significantly to 75% of normal values. Other polyunsaturated fatty acids were elevated in cystic fibrosis milk. Concentrations of 16:2, 18:3, and several longer chain polyunsaturated fatty acids were twice that in the milk of the control women, suggesting increased delta 6-desaturation. Elevated proportions of several other polyunsaturated fatty acid metabolites suggested increased chain elongation to C20 and C22 polyunsaturated fatty acids. These differences in fatty acid patterns in cystic fibrosis milk were similar to differences found in blood lipid patterns of children with cystic fibrosis. These results suggest abnormal polyunsaturated fatty acid nutrition or metabolism in cystic fibrosis.
Asunto(s)
Fibrosis Quística , Lípidos/análisis , Leche Humana/análisis , Colesterol/análisis , Ésteres del Colesterol/análisis , Fibrosis Quística/metabolismo , Ácidos Grasos/análisis , Femenino , Humanos , Fosfolípidos/análisis , Triglicéridos/análisisRESUMEN
The purpose of the present study was to determine the effect of inhibiting the mitochondrial beta-oxidation of free fatty acids on the incorporation of radiolabeled free fatty acids into brain lipids. To this end, methyl 2-tetradecylglycidate (MEP), an irreversible inhibitor of carnitine palmitoyltransferase I, was given orally to male rats 2, 4, and 6 h prior to an intravenous infusion of the saturated fatty acid [U-14C]palmitic acid (PA) or the polyunsaturated fatty acid [1-14C]arachidonate (AA). With [U-14C]PA, MEP (10-25 mg/kg) increased brain organic radioactivity 2-fold and decreased brain aqueous radioactivity 3- to 5-fold relative to control values at all pretreatment times. The effect was due to prolongation of the plasma integral of [U-14C]PA due to peripheral inhibition of beta-oxidation, and to direct inhibition of beta-oxidation of the tracer within the brain. MEP had no effect on brain organic radioactivity after infusion of [1-14C]AA. Increasing the interval between MEP administration and [U-14C]PA infusion from 2 to 6 h resulted in a dramatic redistribution of [U-14C]PA within brain lipids. The percentage of radioactivity in phospholipids decreased from 65 to 33%, whereas that in the free fatty acid fraction increased from 10 to 47% and that in triglycerides was elevated 2-3 fold over control levels. These results indicate that MEP may facilitate the use of radiolabeled PA as an in vivo probe of brain lipid metabolism using quantitative autoradiography or positron emission tomography.
Asunto(s)
Ácido Araquidónico/metabolismo , Encéfalo/metabolismo , Metabolismo de los Lípidos , Ácidos Palmíticos/metabolismo , Animales , Radioisótopos de Carbono , Relación Dosis-Respuesta a Droga , Compuestos Epoxi/farmacología , Ayuno , Lípidos/sangre , Masculino , Oxidación-Reducción/efectos de los fármacos , Ácido Palmítico , Propionatos/farmacología , Ratas , Ratas Endogámicas F344RESUMEN
Regional cerebral 'incorporation coefficients' k* of each of 3 labeled long-chain fatty acids -[9,10-3H]palmitate ([3H]PA), [1-14C]arachidonate ([14C]AA) and [1-14C]docosahexaenoate ([14C]DHA)-were measured using quantitative autoradiography in 11 bilateral brain visual areas of 3.5-month-old awake, hooded, Long-Evans rats, and were compared with regional cerebral metabolic rates for glucose (rCMRglc). The rats, which had undergone unilateral orbital enucleation at 15 days of age, were studied either in the dark with eyelids of the intact eye sutured, or when stimulated in a light box with the intact eye open. rCMRglc did not differ between homologous contralateral and ipsilateral visual areas in the dark or during stimulation, but was elevated bilaterally by 25% or more in many visual areas during stimulation compared with dark. Contralateral compared with ipsilateral k* was lower for each fatty acid tracer in superficial gray of the superior colliculus (in dark and during stimulation) and dorsal nucleus of lateral geniculate body (during stimulation). In the dark, k* for [3H]PA was correlated significantly with rCMRglc for the 22 visual areas studied, whereas during stimulation k* for [14C]AA was correlated with rCMRglc. These results suggest that central neuroplastic changes following chronic unilateral enucleation are accompanied by reduced incorporation of [3H]PA, [14C]AA and [14C]DHA into contralateral brain ares that normally receive crossed retinofugal fibers, and by symmetry of rCMRglc in the dark but increased bilateral symmetrical responsiveness of rCMRglc to visual stimulation of the intact eye.
Asunto(s)
Encéfalo/metabolismo , Desoxiglucosa/metabolismo , Ácidos Grasos/metabolismo , Lateralidad Funcional/fisiología , Privación Sensorial , Vías Visuales/fisiología , Animales , Ácido Araquidónico/metabolismo , Autorradiografía , Ácidos Docosahexaenoicos/metabolismo , Glucosa/metabolismo , Masculino , Plasticidad Neuronal/fisiología , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Estimulación Luminosa , Ratas , Factores de TiempoRESUMEN
Parenterally administered domoic acid, a structural analog of the excitatory amino acids glutamic acid and kainic acid, has specific effects on brain histology in rats, as measured using different anatomic markers. Domoic acid-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex, and different anatomic markers can detect these neurotoxic effects to varying degrees. Here we report effects of domoic acid administration on quantitative indicators of brain metabolism and gliosis. Domoic acid, 2.25 mg/kg i.p., caused stereotyped behavior and convulsions in approximately 60% of rats which received it. Six to eight days after domoic acid or vehicle administration, the animals were processed to measure regional brain incorporation of the long-chain fatty acids [1-(14)C]arachidonic acid ([14C]AA) and [9,10-(3)H]palmitic acid ([3H]PA), or regional cerebral glucose utilization (rCMRglc) using 2-[1-(14)C]deoxy-D-glucose, by quantitative autoradiography. Others rats were processed to measure brain glial fibrillary acidic protein (GFAP) by enzyme-linked immunosorbent assay. Domoic acid increased GFAP in the anterior portion of cerebral cortex, the caudate putamen and thalamus compared with vehicle. However, in rats that convulsed after domoic acid GFAP was significantly increased throughout the cerebral cortex, as well as in the hippocampus, septum, caudate putamen, and thalamus. Domoic acid, in the absence of convulsions, decreased relative [14C]AA incorporation in the claustrum and pyramidal cell layer of the hippocampus compared with vehicle-injected controls. In the presence of convulsions, relative [14C]AA incorporation was decreased in hippocampus regions CA1 and CA2. Uptake of [3H]PA into brain was unaffected. Relative rCMRglc decreased in entorhinal cortex following domoic acid administration with or without convulsions. These results suggest that acute domoic acid exposure affects discrete brain circuits by inducing convulsions, and that domoic acid-induced convulsions cause chronic effects on brain function that are reflected in altered fatty acid metabolism and gliosis.
Asunto(s)
Encéfalo/efectos de los fármacos , Ácido Kaínico/análogos & derivados , Neurotoxinas/toxicidad , Animales , Ácido Araquidónico/metabolismo , Autorradiografía , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patología , Radioisótopos de Carbono , Desoxiglucosa/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glucosa/metabolismo , Infusiones Parenterales , Ácido Kaínico/administración & dosificación , Ácido Kaínico/toxicidad , Masculino , Neurotoxinas/administración & dosificación , Especificidad de Órganos , Ácido Palmítico/metabolismo , Ratas , Ratas Endogámicas F344 , Convulsiones/inducido químicamente , Convulsiones/patología , TritioRESUMEN
Positron emission tomography (PET) was used to determine brain incorporation coefficients k* of [1-11C]arachidonate in isoflurane-anesthetized rhesus monkeys, as well as cerebral blood flow (CBF) using [15O]water. Intravenously injected [1-11C]arachidonate disappeared from plasma with a half-life of 1.1 min, whereas brain radioactivity reached a steady-state by 10 min. Mean values of k* were the same whether calculated by a single-time point method at 20 min after injection began, or by least-squares fitting of an equation for total brain radioactivity to data at all time points. k* equalled 1.1-1.2 x 10(-4) ml x s(-1) x g(-1) in gray matter and was unaffected by a 2.6-fold increase in CBF caused by hypercapnia. These results indicate that brain incorporation of [1-11C]arachidonate can be quantified in the primate using PET, and that incorporation is flow-independent.
Asunto(s)
Ácido Araquidónico/metabolismo , Encéfalo/metabolismo , Circulación Cerebrovascular/fisiología , Hipercapnia/metabolismo , Fosfolípidos/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Radioisótopos de Carbono , Femenino , Análisis de los Mínimos Cuadrados , Macaca mulatta , Masculino , Ensayo de Unión Radioligante , Valores de Referencia , Tomografía Computarizada de EmisiónRESUMEN
Human milk contains a lipase (bile salt-stimulated lipase) that is considered to have an important role in infant fat digestion. In this study we compared the characteristics of bile salt-stimulated lipase activity in milk samples from mothers delivering prematurely (26-30 and 31-37 weeks of gestation) and in milk from mothers delivering at term (38-42 weeks of gestation). Preterm milks were collected at day 1-5 and during week 6 of lactation. Term milks were collected during week 6 of lactation. The characteristics of the enzyme (kinetics, enzyme concentration, pH optimum, and pH stability, effects of bile salt structure and concentration, eserine inhibition) were identical regardless of length of pregnancy or duration of lactation. Bile salt-stimulated lipase had a neutral to alkaline pH optimum (pH 7.3-8.6), was stable for 1 h at a wide pH range (pH 3.1-8.6), was active only in the presence of primary bile salts, and was inhibited by eserine. The data indicate that, following parturition at as early as 26 weeks of gestation, the mammary glands synthesizes bile salt-stimulated lipase with identical characteristics as does the mammary gland after a full-term pregnancy.
Asunto(s)
Recien Nacido Prematuro , Lipasa/análisis , Leche Humana/enzimología , Esterol Esterasa , Ácidos y Sales Biliares/fisiología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Recién Nacido , Cinética , Fisostigmina/farmacologíaRESUMEN
This study examined the diurnal and within-feed variations in the two lipases of human milk, bile-salt-stimulated lipase (BSSL) and lipoprotein lipase (LPL). In addition, all milks were also analyzed for triglyceride content. As compared to BSSL, LPL activity was more variable both among mothers and among different samples from the same mother (largest variation, 0-2.8 U/ml milk). The only significant variation in lipase activity was in LPL activity during a 24-h period (0.96 +/- 0.113, 0.52 +/- 0.21, and 0.57 +/- 0.092 U/ml milk at 0801-1600, 0001-0800, and 1601-2400 h, respectively) (p less than 0.05). Triglyceride content showed no significant diurnal variation but did increase significantly during a single feeding (31 +/- 9.4, 52 +/- 11.7, and 72 +/- 14.8, mumoles triglyceride/ml milk for fore-, mid-, and hind-milks, respectively) (p less than 0.05). There was no correlation between either LPL or BSSL activity and triglyceride content of the milks. Our data indicate that milk obtained for banking or research purposes will contain similar levels of BSSL activity irrespective of diurnal or within-feed timing of milk collections.
Asunto(s)
Ritmo Circadiano , Lipoproteína Lipasa/metabolismo , Leche Humana/análisis , Triglicéridos/análisis , Humanos , Leche Humana/enzimologíaRESUMEN
Human milk contains two lipases, bile salt-stimulated lipase (BSSL) and lipoprotein lipase (LPL). In the mammary gland, LPL provides long-chain fatty acid for milk fat synthesis. LPL has no known function in milk, but has been implicated in milk fat hydrolysis during cold storage. BSSL may have an important role in infant fat digestion. The aims of the present studies were to assess (1) the methodological validity of using whole milk to analyze BSSL activity, (2) the longitudinal variation of BSSL and LPL activity in the milk of mothers delivering premature and full-term infants, and (3) the stability of BSSL and LPL activity during cold storage. Diluted whole milk and purified BSSL were shown to have similar characteristics. LPL activity was equally stable at -20 and -70 degrees C, whereas BSSL activity was higher in milks stored at -70 than at -20 degrees C (38.8 +/- 0.88 vs 33.3 +/- 0.87 U/ml milk, respectively; 1U = 1 mumol free fatty acid release/min). Levels of BSSL activity in preterm and term milk were similar. LPL activity tended to be higher in term milk. Overall, BSSL activity showed significant longitudinal variation, being highest at 1 and 3 weeks of lactation (43.2 +/- 0.04 and 42.6 +/- 1.03 U/ml milk, respectively). For LPL, the longitudinal pattern of activity depended upon the length of pregnancy. Implications for infant nutrition and mammary gland biology are discussed.
Asunto(s)
Edad Gestacional , Lactancia/fisiología , Lipasa/análisis , Leche Humana/enzimología , Esterol Esterasa , Femenino , Humanos , Lipoproteína Lipasa/análisis , Embarazo , Factores de TiempoRESUMEN
Changes in lipid composition of mammary secretions of five women were studied at two prepartum periods and compared with composition of colostrum, transitional, and mature human milk. Fat content was approximately 1 g/dl during early (-42.0 days before parturition) and late (-9.5 days) prepartum periods and increased to 3-4 g/dl in colostrum (3.0 days post partum), transitional (7.2 days), and mature milk (56.2 days). Most of the lipid present was triglyceride either pre- (93%) or post- (97%) partum. All fat globule core lipids, with the exception of 1,3-diglycerides, increased from prepartum concentrations to levels usually found in milk. Prepartum secretions contained higher amounts of the membrane components, phospholipid (3.2 g/dl), cholesterol (2.3 g/dl), and cholesteryl ester (1.1 g/dl), which declined postpartum to 0.65, 0.37, and 0.09 g/dl, respectively. Thus, the content of core lipids exhibited an opposite pattern to the content of membrane lipids pre- and postpartum. With regard to synthesis of fatty acids, prepartum secretory mechanisms appeared to be very similar to those occurring postpartum since fatty acid composition of prepartum secretions closely resembled that of postpartum milk.
Asunto(s)
Mama/metabolismo , Exudados y Transudados/análisis , Lípidos/análisis , Leche Humana/análisis , Colesterol/análisis , Calostro/análisis , Ésteres/análisis , Ácidos Grasos/análisis , Femenino , Humanos , Recién Nacido , Lípidos de la Membrana/análisis , Fosfolípidos/análisis , Periodo Posparto , EmbarazoRESUMEN
An experimental method and its associated mathematical model are described to quantitate in vivo incorporation rates into and turnovers of fatty acids (FAs) within stable brain metabolic compartments, particularly phospholipids. A radiolabeled FA is injected i.v. in a rat, and arterial plasma unacylated FA radioactivities and unlabeled concentrations are sampled until the animal is killed after 15 min, when the brain is analyzed biochemically or with quantitative autoradiography. Unbound unacylated label in blood easily crosses the blood-brain barrier; rapidly equilibrates in the unacylated FA, acyl-CoA and phosphatidate-diacylglycerol brain pools; then is incorporated into phospholipids and other stable metabolic compartments. Uptake and incorporation of labeled FAs are independent of cerebral blood flow at constant brain blood volume. Different labeled FAs enter specific sn positions of different brain phospholipids, suggesting that a combination of probes can be used to investigate metabolism of these phospholipids. Thus, [9,10-3-H]palmitate preferentially labels the sn1 position of phosphatidylcholine; [1-14C]arachidonate the sn2 positions of phosphatidylinositol and phosphatidylcholine; and [1-14C]docosahexaenoate the sn2 positions of phosphatidylethanolamine and phosphatidylcholine. The FA model provides an operational equation for rates of incorporation of FAs into brain phospholipids, taking into account intracerebral recycling and de novo synthesis of the FA, as well as entry into brain of FA from acylated blood sources. The equation is essentially independent of specific details of the proposed model, and can be used to calculate turnovers and half-lives of FAs within different phospholipid classes. For the model to be most applicable, experiments should satisfy conditions for pulse-labeling of the phospholipids, with brain sampling times short enough to minimize exchange of label between stable metabolic compartments. A 15-20 min sampling time satisfies these criteria. The FA method has been used to elucidate the dynamics of brain phospholipids metabolism in relation to brain development, brain tumor, chronically reduced auditory input, transient ischemic insult, axotomy with and without nerve regeneration, and cholinergic stimulation in animals with or without a chronic unilateral lesion of the nucleus basalis magnocellularis.
Asunto(s)
Encéfalo/metabolismo , Circulación Cerebrovascular/fisiología , Ácidos Grasos/metabolismo , Fosfolípidos/metabolismo , Animales , Autorradiografía , Ácidos Grasos/sangre , Semivida , Humanos , Modelos NeurológicosRESUMEN
Frozen storage is often used by milk banks to preserve expressed human milk for later use. Optimal storage and handling conditions which ensure minimum alteration of lipid composition have not been well defined. Therefore we investigated the effect of rapid freeze-thawing and storage conditions (-20 and -70 degrees C) on the free fatty acid (FFA) levels and on the activities of lipoprotein lipase (LPL) and bile salt-stimulated lipase (BSSL) in human milk. Since during mechanical expression leakage of serum components into milk may occur, we also investigated the effect of the presence of serum on human milk LPL during storage. Lipase activity levels were unaffected by rapid freeze-thawing (x3) followed by storage for 1 month at -20 or -70 degrees C. LPL activity (nmol FFA released/ml milk/min) was 414 +/- 128, 451 +/- 37, and 351 +/- 20 and BSSL activity (mumol FFA/ml milk/min) was 5.7 +/- 0.7, 5.5 +/- 0.8, and 5.7 +/- 0.2 in fresh, freeze-thawed, and stored milk, respectively. FFA levels (% of total lipid) were 3.01 +/- 1.05 and 10.3 +/- 1.6 in fresh-frozen milk stored at -70 and -20 degrees C for 5 months, and 3.78 +/- 1.08 and 13.60 +/- 1.25 in specimens of freeze-thawed (x3) before storage at -70 or -20 degrees C. Addition of serum had no effect on milk LPL at either temperature. We conclude that LPL and BSSL remain fully active during frozen storage of human milk and that milk fat is hydrolyzed at -20 degrees C but not at -70 degrees C. We suggest that banked human milk be stored routinely at -70 degrees C.