Lars Bläckberg

The complete digestion of human milk triacylglycerol in vitro requires gastric lipase, pancreatic colipase-dependent lipase, and bile salt-stimulated lipase.

Gastric lipase, pancreatic colipase-dependent lipase, and bile salt-stimulated lipase all have potential roles in digestion of human milk triacylglycerol. To reveal the function of each lipase, an in vitro study was carried out with purified lipases and cofactors, and with human milk as substrate. Conditions were chosen to resemble those of the physiologic environment in the gastrointestinal tract of breast-fed infants. Gastric lipase was unique in its ability to initiate hydrolysis of milk triacylglycerol. Activated bile salt-stimulated lipase could not on its own hydrolyze native milk fat globule triacylglycerol, whereas a limited hydrolysis by gastric lipase triggered hydrolysis by bile salt-stimulated lipase. Gastric lipase and colipase-dependent lipase, in combination, hydrolyzed about two thirds of total ester bonds, with monoacylglycerol and fatty acids being the end products. Addition of bile salt-stimulated lipase resulted in hydrolysis also of monoacylglycerol. When acting together with colipase-dependent lipase, bile salt-stimulated lipase contributed also to digestion of tri- and diacylglycerol. We conclude that digestion of human milk triacylglycerol depends on three lipases with unique, only partly overlapping, functions. Their concerted action results in complete digestion with free glycerol and fatty acids as final products.

Fatty acids generated by gastric lipase promote human milk triacylglycerol digestion by pancreatic colipase-dependent lipase

The concerted action of purified bovine gastric lipase and human pancreatic colipase-dependent lipase and colipase, or crude human pancreatic juice, in the digestion of human milk triacylglycerols was explored in vitro. Gastric lipase hydrolyzed milk triacylglycerol with an initially high rate but became severely inhibited already at low concentration of released fatty acid. In contrast, colipase-dependent lipase could not, by itself, hydrolyze milk triacylglycerol. However, a short preincubation of milk with gastric lipase, resulting in a limited lipolysis, made the milk fat triacylglycerol available for an immediate and rapid hydrolysis by pancreatic juice, and also for purified colipase-dependent lipase, provided colipase and bile salts were present. The same effect was obtained when incubation with gastric lipase was replaced by addition of long-chain fatty acid. Long-chain fatty acid increased the binding of colipase-dependent lipase to the milk fat globule. Binding was efficient only in the presence of both fatty acid and colipase. We conclude that a limited gastric lipolysis of human milk triacylglycerol, resulting in a release of a low concentration of long-chain fatty acids, is of major importance for the subsequent hydrolysis by colipase-dependent lipase in the duodenum.

Bile salt-stimulated lipase in human milk: evidence of activity in vivo and of a role in the digestion of milk retinol esters.

Summary: Human milk contains an enzyme which, in the presence of certain bile salts, has high lipase and esterase activity. These activities are lost on pasteurization of the milk. Bile salt-stimulated lipase was demonstrated in gastric contents during the first 2 hr after feeding fresh human milk to infants, indicating that it is stable in the stomach and passes with the milk into the duodenum where it is activated by bile salts.When infants were fed test meals of pasteurized human milk both esterase and lipase activities in their duodenal contents fell, presumably because the milk diluted their endogenous enzymes. In contrast, when fresh milk was fed, the esterase activity did not decrease. The difference in esterase activity 1 hr after feeding fresh and pasteurized milk corresponded to the activity in the milk.The bile salt-stimulated lipase of milk but not the pancreatic lipase is inhibited by eserine. After feeding fresh milk a fraction of the lipase activity of duodenal contents was inhibited by eserine; the amount so inhibited corresponded to the activity of the milk. Duodenal contents collected 1 hr after feeding fresh milk were found to hydrolyze retinol palmitate severalfold faster than duodenal contents obtained after feeding pasteurized milk; again the difference corresponded to the activity of the milk fed. It is concluded that the bile salt-stimulated milk lipase is active in the infants intestine and may contribute to the digestion of the milk lipids, particularly the retinol esters.Speculation: The bile salt-stimulated lipase has been found in milk only from primates; it is not present in lower animals. There are few examples of a new enzyme function which appears so late in evolution and an important physiologic role for this lipase is implicated. It may ensure an adequate lipolytic capacity in the intestine under conditions when the infants endogenous enzyme activities are critically low. Since the bile salt-stimulated lipase was found to enhance severalfold the rate at which retinol esters are hydrolyzed by intestinal contents of the newborn, it seems possible that it may considerably enhance the utilization of the retinol esters in milk. These are the main source of vitamin A for the newborn, and must be hydrolyzed before they are absorbed. This vitamin is necessary for normal growth and development but the amounts present in human milk are often barely sufficient to cover the needs of the newborn, particularly when the mother is malnourished. Vitamin A deficiency is common in many nonprivileged societies and a role for the bile salt-stimulated lipase in enhancing its absorption could be of great nutritional significance.

Bile salt-stimulated lipase in human milk and carboxyl ester hydrolase in pancreatic juice: Are they identical enzymes?

Human milk contains a lipase that readily hydrolyzes a variety of ester substrates [l-3]. A distinguishing feature of this lipase is that for activity against emulsified long-chain triacylglycerols it requires primary bile salts [ 1,2]. This so-called bile salt-stimulatedlipase (BSSL)is an evolutionary newcomer as a milk enzyme; so far it has been found only in milk from man and gorilla (review [4]). Recent data strongly suggest that it has important roles, both quantitative and qualitative, for the digestion of milk lipids in the intestine of the breast-fed infant ([5], review [6]). Human pancreatic juice contains an enzyme which has been designated carboxyl ester hydrolase (CEH) [7]. This enzyme has many properties in common with BSSL, e.g., stimulation by bile salts, ability to hydrolyze retinyl esters, inhibition by organophosphates (cf. [4,6] and [7,8]). It is the only enzyme in human pancreatic juice with activity against cholesteryl esters [8]. BSSL crossreacts immunochemically with a protein in human pancreatic juice [9]. We have now compared immunochemical, molecular and kinetic properties of bile salt-stimulated lipase and carboxyl ester hydrolase. We conclude that the 2 enzymes are very similar but not identical.

Isolation of lactoferrin from human whey by a single chromatographic step

Human milk, in csntrast PO bovine milk, is rich in the iron-binding protein lactoferrin. Lactoferrin is a single-chain glycoprotein with est. mol. wt 75 000-85 000. It has two irou”bind~~ sites, each birding one atom of ferric iron [I-4], In milk it is normal& only partialIy saturated with iron f5]. Experiments in vitro indicate that lactoferrin is an important c~mpanent of the bacteriostatic system present in human milk. Lactoferrin, by binding iron, prevents growth of iron-re~ui~~g bacteria in the m.i& The bacteriostatic effect is au~ented by specific antibacterial antibodies in the milk while it is abolished by denaturation of the protein or saturating it with iron [S,C;]. To what extent this bacteriostatic effect is really operating in viva, e.g., in the gastrointestinal tract of the breast fed infant, remains to be shown, although, this hypothesis is supported by data from in viva experiments in guinea pigs [S]. Whether or not the high content of lact~ferrin is part of an explanation why newborns absorb iron more efficiently from human milk than from bovine milk or formulas f7] is presently not known. Further studies of the physiological role of Iactoferrin wil1 require large amounts of the isolated pro+ tein. Several methods for its isolation have been described but most of them are rather laborious [2,3,8,9]. This paper ~monstrates that lactoferrin can be isolated from human whey in pure form in a high yield by a one stew-chromatography on hepar&Sepharose. 2. I. Preparation of whey proteins Mature human milk, collected by a brea&pump from one mother over 24 h, was stored at 4°C until the end of the period, then pooled and frozen at -20°C until used. The miIk was thawed at 4”C, centrifuged at IO 000 X g for 40 min and, after removal of the cream, the skim milk was adjusted to pH 4.7 with MC1 and the caseins precipitated by heating the milk to 40°C for 30 min. After another cent~fngatio~ at IO 000 X g for 40 ruin the whey was decanted.

Pasteurization of mother's own milk reduces fat absorption and growth in preterm infants

Aim: A randomized study was conducted to evaluate whether pasteurized milk (Holder pasteurization 62.5°C, 30 min) reduces fat absorption and growth in preterm infants.

Digestion of Human Milk Lipids: Physiologic Significance of sn-2 Monoacylglycerol Hydrolysis by Bile Salt-Stimulated Lipase

Summary: The bile salt-stimulated lipase secreted with human milk was found to be devoid of positional specificity, i.e., it hydrolzed emulsified triacylglycerols to glycerol and fatty acids. It also hydrolyzed micellar sn-2 monoacylglycerols. This is in contrast to pancreatic lipase which has a pronounced preferrance for hydrolysis of sn-1 and sn-3 ester bonds. When the two enzymes were operating together, as in the intestine of the infant fed raw human milk, the sn-2 monoacylglycerols formed by pancreatic lipase served as an excellent substrate for bile salt-stimulated lipase. Thus, the end products of triacylglycerol hydrolysis became glycerol and fatty acids and not sn-2 monoacylglycerol and fatty acids. The bile salt-stimulated lipase also catalyzed incorporation of fatty acids into acylglycerols to a much lesser extent than did pancreatic lipase. Together these two effects of bile salt-stimulated lipase have a promoting effect on the overall process of intraluminal lipolysis.In newborn infants, with low intraduodenal bile salt concentrations, glycerol and fatty acids also should be more readily aborbed than monoacylglycerol and fatty acids. Thus, by serving as a complement to pancreatic lipase, bile salt-stimulated lipase can ensure efficient utilization of milk lipids also in infants with immature endogenous mechanisms for fat digestion and absorption.

Human milk bile salt-stimulated lipase: Functional and molecular aspects

In breast-fed infants, digestion of milk triglycerides, the major source of energy and long-chain polyunsaturated fatty acids, is catalyzed by a concerted action of gastric lipase, colipase-dependent pancreatic lipase, and bile salt-stimulated lipase (BSSL). The major part of BSSL is present in the milk and the lesser part originates in the infants exocrine pancreas. Gastric lipase is important in initiating digestion of milk fat globule triglycerides in the stomach. BSSL shifts the final products of triglyceride digestion from monoglyceride and free fatty acid (the products of colipase-dependent pancreatic lipase) to glycerol and free fatty acid, which may promote efficient absorption. Moreover, BSSL is likely to promote efficient use of milk cholesteryl- and fat-soluble vitaminesters and long-chain polyunsaturated fatty acids (> C18). The cDNA sequence has shown that BSSL has a unique primary structure. The N-terminal half is highly conserved between species and shows striking homology to typical esterases, for example, acetylcholine esterase. In contrast, the C-terminal half, containing 16 proline-rich repeats of 11 amino acid residues, is unique to BSSL. Using several recombinant variants of BSSL, we have found that these unique repeats and the glycosylation are completely dispensable for activity. Thus all typical properties of BSSL reside in the N-terminal half of the molecule.

Digestion of ceramide by Human milk bile salt-stimulated lipase

BACKGROUND There is a renewed interest in metabolism of sphingolipids because of their role in signal transduction. Sphingomyelin is the dominating phospholipid in human milk but its metabolism and possible function in the gastrointestinal tract of breast fed infants is unknown. We explored whether bile salt-stimulated milk lipase has a role in sphingolipid metabolism. METHODS In vitro assays of sphingomyelinase and ceramidase activities, using radiolabeled substrates, human milk samples and purified native and recombinant variants of bile salt-stimulated milk lipase with or without known activators or inhibitors. RESULTS Human whey and purified lipase catalysed hydrolysis of palmitoyl-labeled ceramide with the highest rate around pH 8.5-9.0. 1 mg of lipase hydrolysed 0.7 micromol ceramide in one hour at pH 8.5 in presence of 4 mM bile salt. The activity of whey was inhibited by antibodies towards human bile salt-stimulated milk lipase, indicating that this lipase accounted for virtually all ceramidase activity in the milk. In contrast, bile salt-stimulated milk lipase showed no activity against sphingomyelin. However we give evidence of a separate, hitherto unknown, acid sphingomyelinase in human milk. Under the used in vitro conditions this sphingomyelinase could account for hydrolysis of half of milk sphingomyelin in one hour. CONCLUSIONS Human milk bile salt-stimulated milk lipase hydrolyses ceramide and may thus have a role in sphingomyelin digestion, but only after initial hydrolysis to ceramide and phosphorylcholine. Part of the latter could be carried out in the stomach by the acid milk sphingomyelinase now described. We speculate that these two milk enzymes may be of importance for optimal use of human milk sphingolipids.

Hydrolysis of human milk fat globules by pancreatic lipase: role of colipase, phospholipase A2, and bile salts.

Human milk fat globules were used to explore how dietary triglycerides are hydrolyzed by pancreatic lipase. These triglycerides were hydrolyzed very slowly by lipase alone as if the surface layer of proteins and phospholipids impeded the action of the enzyme. The inhibition of lipase activity could be overcome by addition either of colipase or of pancreatic phospholipase A2. Colipase enhanced triglyceride hydrolysis in a dose-dependent manner whether bile salts were present or not. Bile salts had no effect on the activity of pancreatic lipase alone but further enhanced the activity at all concentrations of colipase tested. Bile salts were a prerequisite to relieve inhibition of lipase activity by phospholipase A2. Human milk fat globules exposed to phospholipase A2 should be representative of a physiological substrate for pancreatic lipase. A major new observation was that bile salts, even at high concentrations, stimulated triglyceride hydrolysis of such phospholipase-treated globules by pancreatic lipase also in the absence of colipase.