Bile Salt-stimulated Lipase Research Articles

Lipid-Lipid Interactions as Regulators of Carboxylester Lipase Activity

The hydrolysis of 1,3-dioleoylglycerol and related substrates by mammalian pancreatic carboxylester lipases was studied. Mixed lipid films of substrates with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at the argon-buffer interface were exposed to relatively high levels of monomeric porcine pancreatic carboxylester lipase for a brief period. With either 1,3-dioleoylglycerol, 1,2-dioleoylglycerol, trioleoylglycerol, or oleoylmethanol as a substrate, the percentage of substrate hydrolysis increased abruptly from near zero to near 100% with increasing proportion of substrate in the film. The phospholipid was not hydrolyzed. Using 1,3-dioleoylglycerol as the substrate with either the dimeric, porcine pancreatic carboxylester lipase, human pancreatic carboxylester lipase, or human milk bile salt-stimulated lipase gave results identical to those obtained with the porcine monomer. Hydrolysis of 1,3-dioleoylglycerol by porcine monomeric carboxylester lipase was independent of the initial surface pressure of the film. However, a strong correlation was observed between hydrolysis and interfacial lipid composition at all surface pressures, even if bulk 1,3-dioleoylglycerol was also present. The ultrasensitive dependence of hydrolysis on interfacial lipid composition, i.e. lipid-lipid interactions, suggests that such "switching" may contribute to the regulation of diacylglycerol levels in cells where they function in signal transduction.

Lipases in human milk: effect of gestational age and length of lactation on enzyme activity.

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.

Anti-coccidial activity of human milk

Sporozoites of Eimeria tenella, an important pathogen of poultry, were killed in vitro in a time- and concentration-dependent manner by exposure to diluted concentrations of normal human milk. Sodium cholate (bile salt stimulator) potentiated the anti-coccidial activity. The anti-coccidial activity was not found in the milk of lower mammals (cow, sheep, goat, dog). The component in human milk showing the activity was initially hypothesized to be a bile salt-stimulated lipase. However, testing of purified lipase (with or without sodium cholate) indicated no anticoccidial activity. Consequently, we theorize that the active component may be a free fatty acid.

Separation of Proteins in Human Milk

Abstract A separation of proteins in human milk whey using high performance ion exchange chromatography is reported. Three chromatographic peaks were identified on the basis of retention times as bile salt-stimulated lipase (BSSL), lactoferrin, and α-lactalbumin. Implications for the purification of BSSL are discussed and suggestions for future purification procedures are made.

22 PHYSIOLOGICAL ROLE OF PREGASTRIC LIPASE

Three lipases are involved in the digestion of milk triglycerides in breast-fed infants. Neither colipase-dependent lipase secreted from the pancreas, nor bile salt-stimulated lipase provided by the milk could by themselves, even in the presence of their respective cofactors, initiate triglyceride digestion. This was a unique functional property of pregastric (lingual) lipase, the lipase operating in the stomach. A major quantitative function of this lipase is, however, unlikely since it was inhibited by a fatty acid concentration corresponding to hydrolysis of only a few percent of the triglycerides. Interestingly, this low degree of hydrolysis had dramatic effects. The remaining globule triglycerides were easily hydrolyzed by the two other lipases. This qualitative function of pregastric lipase was mediated by the fatty acids released; addition of free fatty acid to a corresponding concentration was as effective. Marked differencies between various fatty acids were observed regarding both inhibition of pancreatic lipase and initiation of hydrolysis by pancreatic lipase. Interestingly, in both respects long-chain unsaturated fatty acids were most effective. The reason why intact milk fat globule triglycerides were resistant to hydrolysis by pancreatic lipase was an inability of the lipase, even in the presence of its cofactor colipase, to bind to the globule surface. The effects of fatty acids was to mediate this binding. We conclude that generation of a low amount of fatty acid already in the stomach is the physiological role of pregastric lipase.

Heat Stability of the Bile Salt-Stimulated Lipase in Human Milk Fortified with Sodium Taurocholate

Time-temperature relationships for heat-inactivation of the bile salt-stimulated lipase activity were compared in whole human milk and in the same product fortified to 9 mM/ml with sodium taurocholate. Heat treatments were varied from 45 to 70°C for times ranging from 15s to 40 min. Enzyme activity was more heat stable in human milk fortified with taurocholate than in control samples. The temperature required for the onset of heat inactivation at 30-min holding time was increased from 45°C for control samples to 60°C following addition of taurocholate. A temperature differential of approximately 12°C was required in the fortified milks to produce inactivation equivalent to that observed in the control milks over the heating range studied.

Bile Salt-Stimulated Lipase of Human Milk

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.

Bile salt-stimulated lipase in human milk: Evidence for its synthesis in the lactating mammary gland

Human milk contains many enzymes and other biologically active proteins. One of the enzymes, the bile salt-stimulated lipase, constitutes as much as 1% of the milk proteins. Its importance for efficient utilization of milk lipids by the breast-fed infant is now well established. However, whether the lipase protein is a product of protein synthesis within the mammary gland has up till now been an unanswered question. Using biopsy material from lactating human mammary gland we have now demonstrated that the enzyme is synthesized within the gland. This was done by immunoprecipitation of [ 35S]methionine-labelled protein from tissue pieces. By activity determination we could also determine the amount of enzyme stored in the gland. It was concluded that bile salt-stimulated lipase accounted for 1.3 μg/mg tissue protein. Finally, from this figure it could be calculated that about 10–15% of the total protein present in the tissue was milk protein.

BREAST MILK JAUNDICE REVISITED: NO ROLE FOR B-GLOCORONIDASE OR “UNSTIMULATED LIPASE”

To date, the milk factor(s) repsonsible for breast milk jaundice (BMJ) has not been found. The lipases of human milk, lipoprotein lipase (LPL) and bile salt-stimulated lipase (BSSL), require specific activators, apoprotein CII for LPL and primary bile salts for BSSL. It has been suggested that, in BMJ milks, one or both lipases are active in the absence of activator [unstimulated lipase activity (USL) (Pediatr Res 14:1328, 1980)]. More recently, It has been reported that milk β-glucuronldase (β-glu) may be the causative agent (Lancet 1:644, 1986). We have reexamined these questions by analyzing β-glu activity and lipase activity [using highly sensitive techniques (Biochim Biophys Acta 878:209, 1986) for quantitation of stimulated and unstimulated LPL and BSSL activity] in milk samples from 13 mothers of infants with BMJ and 4 mothers of healthy infants. The following results (means and ranges, expressed as umol free fatty acids/min/ml milk for lipases and modified Sigma units/ml for β-glu) were obtained: Within-feed variation in milk collection does not affect the level of β-glu, the activity being 100 (48-189), 115 (48-233), and 132 (54-230) units in fore, mid and hind milk, respectively. These data show that the levels of β-glu, BSSL, LPL and USL activity are not higher in BMJ than in normal milks; therefore, a role for these enzymes in the etiology of BMJ is unlikely. (Supported by NIH grant HO 20833.)

Breast milk composition and bile salt-stimulated lipase in well-nourished and under-nourished Nigerian mothers.

Breast milk was analysed in 9 under-nourished Nigerian mothers and 23 well-nourished mothers who served as controls. Milk from the under-nourished mothers contained adequate amounts of lactose and total triglycerides, but had significantly lower bile salt-stimulated lipase activity (BSSL); their mean BSSL activity was only about 50% of the activity in milk from the control group. Total milk protein was also significantly lower than for the controls (1.45 vs. 1.09 g/dl, respectively; P less than 0.01). Our findings may have nutritional implications for breast-fed infants of under-nourished nursing mothers.

Bile salt-stimulated lipase activity in human colostrum from mothers of infants of different gestational age and birthweight.

The bile salt-stimulated lipase (BSSL) activity was measured in milk collected at 3-4 days postpartum (colostrum) from 36 mothers divided into three groups according to gestational age and birthweight of their infants. BSSL activity changed with the length of gestation. Preterm colostrum presented a mean activity significantly higher than the term groups (small-for-gestational age and appropriate-for-gestational age) which had similar values. The ratio of BSSL activity to the estimated fat content was 6.33 in colostrum of mothers who delivered preterm and 4.20 in colostrum of both groups of term mothers. These data suggest that preterm colostrum has a higher fat digesting potential than term colostrum and that it is the gestational age rather than the adequacy or non-adequacy of birthweight to gestational age that may influence the BSSL activity.

Giardia lamblia: The role of conjugated and unconjugated bile salts in killing by human milk

Killing of Giardia lamblia trophozoites by nonimmune human milk in vitro is dependent upon the presence of cholate which activates the milk bile salt-stimulated lipase to cleave fatty acids from milk triglycerides. In the present studies, conjugated bile salts, which predominate in vivo, displayed striking differences from unconjugated bile salts in ability to support killing by milk. Human milk killed >99% of the parasites in the presence of cholate, but not glycocholate or taurocholate. In contrast, after brief sonication which disrupts milk fat globules, milk killed G. lamblia after addition of either conjugated or unconjugated bile salts. Whereas cholate stimulated milk lipase to cleave triglycerides of either unsonicated or sonicated human milk, glycocholate or taurocholate stimulated lipolysis only in sonicated milk. Since the concentration of bile salts in the small intestine fluctuates, the effect of this variable on killing was examined. Each bile salt at and above its critical micellar concentration increased Giardia survival of human milk probably because it sequestered released fatty acids in micelles. This partial protection could be overcome by increasing the milk concentration. Human hepatic and gall bladder bile and artificial bile also activated human milk to kill at low concentrations but partly protected the parasite at higher concentrations. These studies show that conjugated bile salts can activate the bile salt-stimulated lipase of sonicated human milk to release fatty acids; and kill G. lamblia. Conversely, bile salts in concentrations above their critical micellar concentration sequester fatty acids and interfere with killing. Thus, nonimmune host secretions such as milk and bile may affect the course of infection by G. lamblia.

Intestinal mucus protects Giardia lamblia from killing by human milk.

We have previously shown that nonimmune human milk kills Giardia lamblia trophozoites in vitro. Killing requires a bile salt and the activity of the milk bile salt-stimulated lipase. We now show that human small-intestinal mucus protects trophozoites from killing by milk. Parasite survival increased with mucus concentration, but protection was overcome during longer incubation times or with greater milk concentrations. Trophozoites preincubated with mucus and then washed were not protected. Protective activity was associated with non-mucin CsCl density gradient fractions. Moreover, it was heat-stable, non-dialyzable, and non-lipid. Whereas whole mucus inhibited milk lipolytic activity, protective mucus fractions did not inhibit the enzyme. Furthermore, mucus partially protected G. lamblia trophozoites against the toxicity of oleic acid, a fatty acid which is released from milk triglycerides by lipase. These studies show that mucus protects G. lamblia both by inhibiting lipase activity and by decreasing the toxicity of products of lipolysis. The ability of mucus to protect G. lamblia from toxic lipolytic products may help to promote intestinal colonization by this parasite.

Lipid metabolism in premature infants.

Fats provide 40-50% of the total calories in human milk or infant formula. The milk secreted by mothers of preterm infants differs in fat composition from that of mothers of full-term infants in having higher levels of medium chain fatty acids (C12, C14) throughout the first 3 months of lactation, and higher levels of long chain polyenoic fatty acids during the first 3 months of lactation. These differences in composition benefit the preterm infant by providing higher levels of rapidly absorbed medium chain fatty acids and long chain polyenoic fatty acids needed for brain development. Fat digestion: The low levels of pancreatic lipase and bile salts in the preterm infant are compensated for by lipolysis in the stomach by lingual and gastric lipase and by the intestinal hydrolysis of fat through the action of human milk bile salt-stimulated lipase. Fat digestion is efficient in the preterm infant who absorbs about 80-90% of ingested fat. Lipid clearing from the circulation depends upon the activity of the enzymes lipoprotein lipase, hepatic lipase, and lecithin-cholesterol acyltransferase. The activity of these enzymes is lower or equal to that of term infants, depending upon the degree of prematurity and the nutritional regimen, especially in parenterally fed infants.

Diurnal and within-feed variations in lipase activity and triglyceride content of human milk.

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.

Human milk kills Giardia lamblia by generating toxic lipolytic products.

This study supports our previous hypotheses that normal human milk kills Giardia lamblia trophozoites in vitro and that this killing is due to the release of free fatty acids (FFAs) from milk triglycerides by action of the bile salt-stimulated lipase (BSL) of human milk. Heat-stable killing of G. lamblia was generated when normal human milk was preincubated with sodium cholate, which activates BSL. Moreover, both the skim-milk (containing BSL) and cream (containing mainly triglycerides) fractions were required to kill G. lamblia. We measured the toxicity of FFA and other products of lipolysis to G. lamblia. cis-Unsaturated FFAs (LD50 less than 12 microM), three of four monoglycerides, and four of five lysophosphatidylcholines were toxic to the parasites (LD50 less than 100 microM). In contrast, the parasites were not harmed by the corresponding diglycerides, phosphatidylcholines, triolein, or glycerol. Thus, products of lipid hydrolysis in the normal digestive tract are toxic to G. lamblia. We also demonstrated that albumin and conjugated bile salts, which bind FFA, partially protected trophozoites from killing by oleic acid.

Diurnal and Within‐Feed Variations in Lipase Activity and Triglyceride Content of Human Milk

SummaryThis study examined the diurnal and withinfeed 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 < 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, pimoles triglyceride/ml milk for fore‐, mid‐, and hind‐milks, respectively) (p < 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.

Bile salt-stimulated lipase in non-primate milk: longitudinal variation and lipase characteristics in cat and dog milk

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 μ jmol [ 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.

Studies in bile salt solutions: XVII. The effect of proteins on bile-salt-stimulated human milk lipase activity against 4-nitrophenylacetate

Bovine serum albumin, porcine pancreatic lipase, and horse heart myoglobin, but not any of bee venom melittin, human milk lysozyme, human milk lactotransferrin, human milk α-lactalbumin, human colostrum immunoglobulin A (IgA), or chicken egg white lysozyme, were found to catalyze the decomposition of 4-nitrophenylacetate (PNPA) in 0.1 M Tris, pH 7.5, 298 K, in the presence (2 m M) and absence of sodium taurocholate. The effect of each of these proteins (except bovine serum albumin) on the activity of bile salt-stimulated human milk lipase against PNPA was tested under the same conditions. In the absence of bile salt, human milk lysozyme had no effect, horse heart myoglobin was catalytic, and the other proteins were inhibitory. In the presence of bile salt, porcine pancreatic lipase and human milk lactotransferrin were slightly inhibitory, chicken egg white lysozyme was slightly catalytic, and the other proteins had no effect. A mechanism is proposed in which the bile salt competes with the enzyme for the protein interface. It is postulated that the binary bile salt-enzyme complex is active, whereas the binary protein-enzyme complex is not.

Within-Day Variation of Lipolytic Activity in Human Milk

Milk was collected from six mothers during their 8th wk of lactation to determine the diurnal variation of bile salt-stimulated lipase and serum-stimulated lipase. On the day of collection, one breast was completely emptied at 0600, 1000, 1400, 1800, and 2000h. Activities of bile-stimulated lipase and serum-stimulated lipase in the milk were determined. Activity of bile-stimulated lipase was constant throughout the day, whereas serum-stimulated lipase activity varied significantly with time. Both activities differed significantly in milk from different women. Time of collection must be considered when measuring activity of serum-stimulated lipase but is not a major variable when measuring activity of bile-stimulated lipase in human milk. These results may be helpful when designing future studies involving these enzymes.