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O Hernell

Publications and source records attributed to O Hernell.

At least 91 records · Page 5Linked to original sources
PubMed1990

cDNA cloning of human-milk bile-salt-stimulated lipase and evidence for its identity to pancreatic carboxylic ester hydrolase.

We have isolated and sequenced cDNA clones covering the entire coding sequence of human-milk bile-salt-stimulated lipase, as well as 996 nucleotides of the 3' end of the pancreatic enzyme carboxylic ester hydrolase. The deduced amino acid sequence of the lipase starts with a 23-residue leader peptide. The open reading frame continues with 722 amino acid residues. The sequence contains in the C-terminal part a proline-rich repeat, 16 repeats of 11 amino acid residues each. The mRNA was estimated to be approximately 2500 nucleotides from Northern blot and of similar size in mammary and pancreatic tissues. Data obtained indicate that the lipase and the carboxylesterase are identical and coded for by the same gene. The cDNA is 2428 bases long, which indicates that a near full-length copy of the transcript has been isolated. Comparisons with other enzymes show that the lipase is a new member of the supergene family of serine hydrolases. It is not only closely related (and in its N-terminal half virtually identical) to lysophospholipase from rat pancreas and cholesterol esterase from bovine pancreas, but also shows a high degree of similarity to several esterases, e.g. acetylcholine esterase. In contrast, no such similarity could be found to typical lipases.

Adipose Tissue↗
PubMed1990

Cloning and sequencing of a cDNA encoding human milk beta-casein.

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.

Amino Acid Sequence↗
PubMed1990

Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 1. Phase behavior and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings.

We developed equilibrium phase diagrams corresponding to aqueous lipid compositions of upper small intestinal contents during lipid digestion and absorption in adult human beings. Ternary lipid systems were composed of a physiological mixture of bile salts (BS), mixed intestinal lipids (MIL), principally partially ionized fatty (oleic) acid (FA) plus racemic monooleylglycerol (MG), and cholesterol (Ch), all at fixed aqueous-electrolyte concentrations, pH, temperature, and pressure. The condensed phase diagram for typical physiological conditions (1 g/dL total lipids, FA:MG molar ratio of 5:1, pH 6.5, 0.15 M Na+ at 37 degrees C) was similar to that of a dilute model bile [BS/lecithin (PL)/Ch] system [Carey, M. C., & Small, D. M. (1978) J. Clin. Invest. 61, 998-1026]. We identified two one-phase zones composed of mixed micelles and lamellar liquid crystals, respectively, and two two-phase zones, one composed of Ch monohydrate crystals and Ch-saturated micelles and the other of physiologic relevance composed of Ch- and MIL-saturated mixed micelles and unilamellar vesicles. A single large three-phase zone in the system was composed of Ch-saturated micelles, Ch monohydrate crystals, and liquid crystals. Micellar phase boundaries for otherwise typical physiological conditions were expanded by increases in total lipid concentration (0.25-5 g/dL), pH (5.5-7.5), and FA:MG molar ratio (5-20:1), resulting in a reduction of the size of the physiological two-phase zone. Mean particle hydrodynamic radii (Rh), measured by quasielastic light scattering (QLS), demonstrated an abrupt increase from micellar (less than 40 A) to micelle plus vesicle sizes (400-700 A) as this two-phase zone was entered. With relative lipid compositions within this zone, unilamellar vesicles formed spontaneously following coprecipitation, and their sizes changed markedly as functions of time, reaching equilibrium values only after 4 days. Further, vesicle Rh values were influenced appreciably by MIL:mixed bile salt (MBS) ratio, pH, total lipid concentration, and FA:MG ratio, but not by Ch content. In comparison, micellar systems equilibrated rapidly, and their Rh values only slightly influenced by physical-chemical variables of physiological importance. In contrast to the BS-PL-Ch system [Mazer, N. A., & Carey, M. C. (1983) Biochemistry 22, 426-442], no divergence in micellar sizes occurred as the micellar phase boundary was approached. The ionization state of FA at simulated "intestinal" pH values (5.5-7.5) in the micellar and physiologic two-phase zones was principally that of 1:1 sodium hydrogen dioleate, an insoluble swelling "acid soap" compound. By phase separation and analysis, tie-lines for the constituent phase in the two-phase zone demonstrated that the mixed micelles were saturated with MIL and Ch and the coexisting vesicles were saturated with MBS, but not with Ch.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1990

Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 2. Phase analysis and aggregation states of luminal lipids during duodenal fat digestion in healthy adult human beings.

Following the feeding of a triacylglycerol-rich meal to healthy adult human beings, duodenal contents were aspirated for ex vivo chemical and physical-chemical analyses. The aspirates were collected during established lipid digestion and absorption into a "cocktail" of chemical inhibitors that rapidly inhibited ex vivo lipolysis. Following ultracentrifugation, the lipids separated into a floating oil layer, several interfacial layers, a "clear" or turbid "subphase", and a precipitated "pellet". By chemical and phase analyses, the floating layer was composed of oil-in-water emulsion particles with cores of triacylglycerol (TG), diacylglycerols (DG), and cholesteryl esters (CE) emulsified with a surface coat of partially ionized fatty acids (FA), monoacylglycerols (MG), diacylphosphatidylcholine (PL), and bile salts (BS). The interfacial layers contained similar emulsion particles dispersed among excess emulsifier which adopted a lamellar liquid-crystalline structure. Precipitated pellets were composed principally of emulsifying lipids, with smaller amounts of crystalline calcium soaps and BS. Relative lipid compositions of all but three subphases fell within a two-phase region of the condensed ternary phase diagram (Staggers et al., 1990, companion paper) where saturated mixed micelles composed of BS, FA "acid-soaps", MG, PL, cholesterol (Ch), and traces of DG (and TG) coexisted with unilamellar liquid-crystalline vesicles composed of the same lipids. Attempts to achieve clean separation of vesicles from micelles by repeat ultracentrifugation failed. Compared with the structure and sizes of lipid particles in equilibrated model systems (Staggers et al., 1990), quasielastic light scattering (QLS) analysis revealed that ex vivo micellar sizes (mean hydrodynamic radii, Rh) were similar (less than or equal to 40 A), whereas unilamellar vesicle sizes (Rh = 200-600 A) were appreciably smaller. Two-component QLS analysis of the subphases showed that much larger proportions of lipids were solubilized by micelles than were dispersed as unilamellar vesicles. When followed as functions of time, vesicles frequently dissolved spontaneously into mixed micelles, indicating that, in the nonequilibrium in vivo conditions, the constituent micellar phase was often unsaturated with lipids. These results are consistent with the hypothesis that, during hydrolysis of emulsified DG and TG by luminal lipases, unilamellar vesicles originate in lamellar liquid crystals that form at emulsion-water interfaces in the upper small intestine. In a BS-replete environment, unilamellar vesicles probably represent the primary dispersed product phase of human fat digestion and facilitate the dissolution of lipolytic products into unsaturated mixed micelles.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1990

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.

Bile Acids and Salts↗
PubMed1989

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 Acids and Salts↗
PubMed1987

Bovine pregastric lipase: a model for the human enzyme with respect to properties relevant to its site of action.

Preduodenal lipolysis is considered to promote efficient lipid digestion in the neonatal period. The lipase(s) responsible may be of pregastric or gastric origin depending upon the species. We have previously reported on purification and molecular characterization of a pregastric lipase from calf. Antibodies to this bovine enzyme crossreact with a protein of similar size in human gastric contents and also inhibit its lipolytic activity. Since the bovine and human enzymes also have similar kinetic properties, the view is favoured that the bovine enzyme can be used as a model for physiological studies relevant to human neonates. In contrast to the lipases operating in the small intestine pregastric lipase has the unique property of initiating the hydrolysis of human milk fat globule triacylglycerol. In order to do this no cofactor is required. Pregastric lipase was stable at low pH and had an acid-pH optimum. Furthermore, it was extremely resistant to pepsin. In contrast, pancreatic proteinases, i.e. trypsin and chymotrypsin, inactivated the enzyme. The rate of inactivation was increased in the presence of bile salts which by themselves could inhibit enzyme activity. Thus, pregastric lipase is ideally suited for activity in the stomach but will not, under healthy conditions, contribute to lipid digestion in the duodenum.

Animals↗
PubMed1987

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 micrograms/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.

Bile Acids and Salts↗
PubMed1987

Carboxylic ester hydrolase: a serum marker of acute pancreatitis.

By use of an enzyme-linked immunosorbent assay we established serum reference values of carboxylic ester hydrolase, a pancreatic secretory lipolytic enzyme, and explored to see if a raised serum level is indicative of acute pancreatitis. Postoperative elevation of carboxylic ester hydrolase was observed in seven out of ten patients who underwent pancreatic surgery. Serum levels of carboxylic ester hydrolase and amylase were determined in 129 patients admitted due to abdominal emergency conditions. Amylase was elevated in 27 patients, and in 20 of these raised carboxylic ester hydrolase levels affirmed the diagnosis acute pancreatitis. In five out of the seven patients with elevated amylase alone no etiologic factor of acute pancreatitis was found. Another 11 patients had raised carboxylic ester hydrolase levels without concomitant elevation of amylase. In all these patients, a likely cause of pancreatic inflammation was identifiable. Hence, a raised carboxylic ester hydrolase level, even in presence of normal amylase, could be indicative of acute pancreatic inflammation.

Abdomen↗
PubMed1986

Killing of Giardia lamblia by human milk lipases: an effect mediated by lipolysis of milk lipids.

Killing of Giardia lamblia by fresh human milk requires the presence of bile salt, a known activator of bile salt-stimulated lipase, the major lipase in human milk. Purified enzyme did not kill the parasite even in the presence of activator unless milk lipids were also present in the reaction mixture. Free fatty acids had a marked giardiacidal effect, a phenomenon supporting the view that fatty acids, released during hydrolysis of milk triglycerides, are responsible for the killing of G. lamblia by human milk. Bile salt-independent lipolysis took place in milk during storage at 4 C. This lipolysis correlated strongly with activity of lipoprotein lipase, also present in human milk. During such storage, raw human as well as bovine milk developed giardiacidal activity that could be prevented by inactivation or inhibition of the milk lipases by pasteurization or addition of eserine to the milk, respectively, before storage.

Bile Acids and Salts↗
PubMed1985

Purification and molecular characterization of bovine pregastric lipase.

A pregastric lipase was purified from calf pharyngeal tissues. The purification procedure was based on chromatographies on octyl-Sepharose and lentil-lectin-Sepharose followed by gel filtration. The final preparation, with an overall recovery of 26% of activity, gave a single protein band on dodecyl sulfate/polyacrylamide gel electrophoresis with a Mr of 55000. The Mr on gel filtration was 44-48000. The discrepancy may be due to the fact that pregastric lipase is a glycoprotein containing approximately 10% (w/w) of carbohydrate. The pI was around 7.0 and the enzyme protein is characterized by a high content of branched, aliphatic amino acid residues. The NH2-terminal amino acid sequence is: H2N-Phe-Leu/(Ile)-Gly-. Rabbit antibodies to the purified preparation detected only one component in the crude starting material in immuno-blotting experiments. Preincubation with antiserum resulted in loss of enzyme activity, showing that the antibodies were directed against the lipase.

Amino Acids↗
PubMed1985

On the source of bile salt-stimulated lipase in human milk: a study based on serum concentrations as determined by sandwich enzyme-linked immunosorbent assay technique.

Human milk contains a bile salt-stimulated lipase (BSSL) which is considered to contribute significantly to lipid digestion in newborns. Human pancreatic juice contains an enzyme, carboxyl ester hydrolase (CEH), that is immunochemically and functionally identical to the milk enzyme. It has not been clear, however, whether the milk enzyme is synthesized within the mammary gland or whether the pancreatic enzyme is transported to the mammary gland for secretion. In this study we describe an enzyme-linked immunosorbent assay (ELISA) for determinations of BSSL and CEH in serum. Serum samples from healthy adults of both sexes contain significant amounts of enzyme (3.2 +/- 1.7 micrograms/L). Lactating women did not have higher serum levels, which would be expected if the milk enzyme were a product of the pancreas, and it was calculated that the concentrations found could not sufficiently account for the amounts secreted in the milk. We therefore conclude that BSSL is synthesized within the mammary gland. This conclusion is further strengthened by our findings that serum of breast-fed newborns contains very little if any enzyme. Since macromolecular absorption is high early in life, and witch's milk contains high enzyme levels, again one would expect high serum levels if BSSL were a pancreatic product.

Adult↗
PubMed1985

Gliadin-specific serum immunoglobulins A, E, G, and M in childhood: relation to small intestine mucosal morphology.

An enzyme-linked immunosorbent assay technique was developed to determine serum antigliadin antibodies of the IgA, IgE, IgG, and IgM classes. The antibody level of each serum specimen was expressed as an index value, i.e., optical density of test serum/optical density of cutoff, where cutoff was calculated for each immunoglobulin class as the mean + 3 SD for six healthy controls. Indices for each immunoglobulin class were determined in 69 children who were admitted for their first small intestinal mucosal biopsy due to either symptoms of malabsorption compatible with celiac disease, or short stature without other symptoms. Especially raised levels of antigliadin IgA antibodies in serum correlated strongly with villous atrophy and in infants less than or equal to 3 years of age were invariably elevated above controls, provided they were on a gluten-containing diet. Raised levels of IgG and IgE antibodies to gluten were often seen in children with normal mucosal morphology, i.e., when symptoms were due to other gastrointestinal disorders than celiac disease. It is concluded that determination of antigliadin IgA antibodies in children less than or equal to 3 years is a useful screening test before small intestinal biopsy, especially in children where the indication for biopsy is not otherwise obvious. The method can also be used to assess the results of therapy and, conceivably, compliance.

Atrophy↗