Development of an indirect method for measuring porcine pancreatic lipase in human duodenal fluid.

Patients with exocrine pancreatic insufficiency are usually treated with porcine pancreatic enzymes but the bioavailability of these enzymes in the gut remains a matter of discussion. In order to determine the duodenal availability of porcine pancreatic lipase (PPL) present in pancreatic extracts (PE) taken orally, we developed a method for quantifying PPL in samples containing both PPL and human pancreatic lipase (HPL). Total pancreatic lipase activity measurements using the pH-stat technique and tributyrin as substrate were combined with an HPL-specific ELISA. Based on the known specific activity of the purified HPL, its activity was deduced from the ELISA measurements, and the PPL activity was obtained by subtracting the HPL activity from the total pancreatic lipase activity. This assay was established and validated using various samples containing pure PPL and recombinant HPL or PE, mixed or not with human duodenal juice. Samples collected in vivo from patients treated with PE were also tested. It was found that PPL did not affect the HPL ELISA, and the indirect PPL assay gave a measurement accuracy of 6.6% with the samples containing pure PPL and 10% with those containing PE. This assay was also used successfully to discriminate between PPL and the endogenous HPL present in the duodenal contents of patients with severe pancreatic insufficiency treated with PE. This method might provide a useful means of assessing the availability of PEs at their site of action, in the absence of a PPL-specific ELISA.

Inhibition of gastrointestinal lipolysis by Orlistat during digestion of test meals in healthy volunteers.

The inhibition of digestive lipases by the antiobesity drug Orlistat along with lipolysis levels and fecal fat excretion were measured in healthy humans. Orlistat was found to be a powerful gastric lipase inhibitor, achieving 46.6--91.4% enzyme inhibition and thus greatly reducing gastric lipolysis of solid and liquid meals (11--33% of respective controls). Gastric lipase inhibition by Orlistat was extremely fast (half-inhibition time < 1 min). Duodenal lipolysis was reduced significantly by Orlistat given with the solid meal (32.6--37.6% of controls) but was only slightly reduced by Orlistat given with the liquid meal (74.5--100% of controls). Human pancreatic lipase (HPL) inhibition was found to be high (51.2--82.6%), however, regardless of the meal. These paradoxical results were explained when in vitro lipolysis experiments were performed. The rates of HPL inhibition by Orlistat were found to be similar with both types of meals (half-inhibition time 5--6 min), but the preemulsified triglycerides of the liquid meal were rapidly hydrolyzed by HPL before the enzyme was significantly inhibited by Orlistat. With the solid meal, the rate of hydrolysis of the meal triglycerides by HPL was slower than the rate of HPL inhibition by Orlistat. As predicted from the previous results, the effects of Orlistat on fat excretion levels were found to be much greater with the solid (40.5--57.4% of ingested fat) than with the liquid (4.2--18.8%) test meal.

The specific activities of human digestive lipases measured from the in vivo and in vitro lipolysis of test meals.

BACKGROUND & AIMS: The lipolytic potential of digestive lipases in vivo has always been deduced so far from their in vitro activities under nonphysiologic conditions. In the present study, the specific activities of human gastric lipase (HGL) and pancreatic lipase (HPL) were measured on dietary triglycerides (TGs) during test meal lipolysis. METHODS: Healthy human volunteers ingested a liquid or solid meal. The specific activities of HGL and HPL were estimated from the lipase and free fatty acid (FFA) outputs at the postpyloric and duodenal levels, respectively. Based on the in vivo data, lipolysis was also performed in vitro by mixing the meal either with gastric juice and subsequently with pancreatic juice and bile or with purified HGL and HPL. FFAs were measured by thin-layer chromatography, and the specific activities of HGL and HPL were expressed as micromoles of FFA per minute per milligram of lipase. RESULTS: In vitro, the specific activities on the liquid meal TGs were 32 (gastric juice) and 34 (pure lipase) micromol x min(-1) x mg(-1) with HGL and 47 (pancreatic juice) and 43 (pure lipase) micromol x min(-1). mg(-1) with HPL. The specific activities on the solid meal TGs were 33 (gastric juice) and 32 (pure lipase) micromol x min(-1) x mg(-1) with HGL and 12 (pancreatic juice) and 15 (pure lipase) micromol x min(-1) x mg(-1) with HPL. The in vivo values obtained were in the same range. The secretory lipase outputs were 21.6+/-14.5 mg HGL and 253.5+/-95.5 mg HPL with the liquid test meal and 15.2+/-5.1 mg HGL and 202.9+/-96.1 mg HPL with the solid test meal. CONCLUSIONS: The specific activities of HGL and HPL on meal TGs were much lower than those measured in vitro under optimized assay conditions (1300-8000). However, these low specific activities are enough for the meal TGs to be completely lipolysed, given the amounts of HGL and HPL secreted during a meal.

A conformational transition between an open and closed form of human pancreatic lipase revealed by a monoclonal antibody.

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. Here we observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds. A single conformational mAb (248-31) fails to immunoprecipitate HPL in the presence of activator compounds and HPL covalently inhibited with diethyl p-nitrophenyl phosphate (DP.HPL). This loss of recognition of HPL by mAb 248-31 was probably due to the motion of the lid, since HPL(-lid) was always recognized in the presence or absence of activator compounds. Furthermore, two other mAbs (81-23 and 146-40) immunoprecipitated HPL similarly whether or not the activator compounds were present. MAb 248-31 therefore specifically recognizes HPL in the closed but not the open conformation.

Pancreatic lipase-related protein 1 (PLRP1) is present in the pancreatic juice of several species.

Pancreatic lipase-related protein 1 (PLRP1) was purified from human, canine, porcine and rat pancreatic juices. The four PLRP1s were identified using microsequencing methods after performing gel filtration on Ultrogel AcA-54 followed by chromatography on Heparin-Sepharose cation-exchanger. Polyclonal antibodies specific to human PLRP1 (HPLRP1) were raised in the rabbit using a synthetic decapeptide from HPLRP1. The results of Western blotting analysis showed that these antibodies recognized native HPLRP1 and recombinant HPLRP1 produced by insect cells, and cross-reacted only with rat PLRP1 (RPLRP1). No significant lipolytic activity was observed with native canine PLRP1 and recombinant HPLRP1 on various glycerides, phospholipid and vitamin esters, or on cholesterol esters. It was established for the first time that this protein is secreted in variable amounts by the adult exocrine pancreas of several species.

Reactivation of the totally inactive pancreatic lipase RP1 by structure-predicted point mutations.

Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 lipases.

Human pancreatic lipase: an exposed hydrophobic loop from the C-terminal domain may contribute to interfacial binding.

Epitope mapping was performed using four anti-HPL monoclonal antibodies (mAb's 81-23, 146-40, 315-25, and 320-24) directed against human pancreatic lipase (HPL). Three HPL mutants produced in insect cells were tested for this purpose: (i) N-HPL, which consists of only the N-terminal domain of HPL, (ii) HPL(-lid), in which a short loop consisting of 5 amino acid residues replaces the full-length 23-residue lid domain present in HPL, and (iii) N-GPLRP2/C-HPL chimera, a chimeric mutant consisting of the N-terminal domain of the guinea pig pancreatic lipase related protein 2 (GPLRP2) fused to the C-terminal domain of HPL. The C-terminal domain of HPL (C-HPL) was prepared in a pure form after performing chymotryptic digestion of HPL. The mAb 146-40 recognizes HPL, HPL(-lid), and N-HPL but not GPLRP2, N-GPLRP2/C-HPL chimera, or the C-HPL. The antibody mAb 146-40 therefore specifically recognizes the N-terminal domain of HPL, and the epitope recognized does not include the amphiphilic lid. On the other hand, mAb's 81-23, 315-25, and 320-24 react specifically to the C-terminal domain of HPL, since they recognize HPL, HPL(-lid), the N-GPLRP2/C-HPL chimera, and the C-HPL but not N-HPL or GPLRP2. It was further established that these three mAb's recognize the same conformational epitope, the structure of which is stabilized by the N-terminal domain in the presence of SDS at concentrations greater than its critical micellar concentration. This conformational epitope was found to be located in the vicinity of Met 397 and Arg 414. These two residues delineate a highly exposed peptide stretch extending from the HPL C-terminal domain, which includes a hydrophobic surface loop (beta5'). Kinetic studies on the HPL/mAb's complexes showed that the lipase activity was much lower in these complexes than in HPL. The results of the present study suggest for the first time that the beta5' loop from the C-terminal domain may be involved in the interaction of HPL with a lipid/water interface.

An inactive pancreatic lipase-related protein is activated into a triglyceride-lipase by mutagenesis based on the 3-D structure.

Both classical dog pancreatic lipase (DPL) and dog pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by the exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested: di- and tri-glycerides; phospholipids (PC); etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical pancreatic lipase (PL) structures determined in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 was suspected of being responsible for the absence of enzymatic activity by inducing a steric clash with one of the acyl chain observed in the structures of chiral C11 alkyl phosphonate inhibitors, bound to the classical PL. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of the three known pancreatic lipase-related protein 1 (PLRP1), whereas Ala and Pro residues are always present at the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic-related protein 1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on tributyrin (1800 U/mg) as well as trioctanoin (2250 U/mg) and its activity is low in the presence of taurodeoxycholate and stimulated in the presence of colipase. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the PLRP1 lipases.

An enzymatically active truncated form (-55 N-terminal residues) of rabbit gastric lipase. Correlation between the enzymatic activity and disulfide bond oxydo-reduction state.

Rabbit gastric lipase (RGL) was subjected to proteolysis with trypsin and led to cleavage occurring at three defined sites (Lys-4, Arg-55 and Arg-229). The tryptic hydrolysate contained four fragments: Gly-230-Lys-379 (T1), Gly-56-Arg-229 (T2), Ser-5-Arg-55 (T3), as well as a 45 kDa molecular form consisting of peptides T1 and T2 linked by a disulfide bridge. The tryptic hydrolysate of RGL as well as the 55 N-terminal amino acid deleted forms conserved 30% of the initial enzymatic activity in a tributyrin assay. Two out of the three cysteine residues which are present in all the known gastric lipases were found to be involved in a disulfide bridge. Unlike HGL, RGL appears to have a heterogenous pattern of cysteine residues. The 30% enzymatic activity of RGL persisting after trypsin treatment may be attributable to the 45 kDa molecular form (with the Cys-227-Cys-236 or Cys-227-Cys-244 disulfide bridge). Trypsin-treated HGL, which was completely inactivated, showed that a single location of the disulfide bridge existed between cysteine residues 236 and 244. It can be concluded that the existence of one disulfide bridge is necessary to maintain the lipase activity of the 45 kDa form of RGL.

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent alpha-hydroxyacid oxidizing enzyme family.

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzyme's active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent alpha-hydroxyacid oxidizing enzyme family.

Purification and molecular characterization of lamb pregastric lipase.

Lamb pregastric lipase (LPGL) was purified from pharyngeal tissues. The purification procedure was based on an aqueous extract containing 0.7% Tween 80 which was chromatographed on DEAE-cellulose anion-exchanger and adsorbed on HA-Ultrogel followed by gel filtration on Ultrogel AcA-54. The final enzymatic preparation, where the overall activity recovery was 3%, showed a single protein band on SDS-PAGE with a molecular mass of 50 kDa. LPGL is a glycoprotein containing approx. 14% (w/w) of carbohydrate. Extensive deglycosylation using peptide N-glycosidase F yielded a protein with an apparent molecular mass of 43 kDa. An uncontrolled proteolysis of LPGL during the purification lead to a 45 kDa form which was previously observed in human lysosomal acid lipase (HLAL) and rabbit gastric lipase (RGL). The labile bond X54-Leu55 was identified. Isoelectric focusing of LPGL reveals a major band corresponding to an isoelectric point of 4.8. The pure enzyme displayed specific activities of 950 U mg-1, 300 U mg-1 and 30 U mg-1 at pH 6.0, using tributyroylglycerol, trioctanoylglycerol and trioleoylglycerol as substrates, respectively. Using Western blot analysis, a cross-immunoreactivity of LPGL was observed with purified anti-human gastric lipase polyclonal antibodies. Determination of the amino-acid sequence of 62 residues revealed a high degree of homology with other known preduodenal lipases.

In vitro oxidative decarboxylation of L-2-hydroxy-4-methylthiobutanoic acid by L-2-hydroxy acid oxidase A from chicken liver.

The first step in the set of reactions responsible for the biological utilization of L-2-hydroxy-4-methylthiobutanoic acid, the methionine hydroxy analogue, in protein synthesis was investigated in vitro using pure L-2-hydroxy acid oxidase A from chicken liver. The reaction yielded no more than 20% of the corresponding alpha-keto acid, the well-known intermediate in methionine metabolism, and as much as 80% of the subsequent decarboxylation product, 3-methylthiopropionate, suggesting that L-2-hydroxy-4-methylthiobutanoic acid cannot be completely converted into methionine in vivo. It was therefore concluded that chicken liver L-2-hydroxy acid oxidase, a peroxisomal enzyme requiring flavin mononucleotide as a coenzyme, also has an oxidative decarboxylation activity in vitro, which was found to be NADH-dependent. The mechanism possibly underlying the successive conversion of the methionine hydroxy analogue into alpha-keto acid and 3-methylthiopropionate by this NADH:flavin oxidoreductase-decarboxylase activity is described.

Comparative oxidative rates of methionine hydroxy analogue in rat and chicken kidney.

1. L-2-HAOX A and L-2-HAOX B were purified from chicken and rat kidney with purification factors of 550 and 45, respectively. 2. It was established that both enzymes were tetrameric (M(r) = 169,000) and consisted of 40 kDa monomers. 3. While chicken kidney L-2-HAOX A is N-terminally blocked like spinach glycolate oxidase and chicken liver L-2-HAOX A, L-2-HOAX B begins with a Pro residue. 4. The kinetic parameters of L-HMB oxidation by L-2-HAOX A and B were determined. 5. The results are particularly interesting as regards L-HMB oxidation by L-2-HAOX B.

Purification and some characteristics of chicken liver L-2-hydroxyacid oxidase A.

The isozyme A of L-2-hydroxyacid oxidase is a peroxisomal flavoenzyme that catalyzes the oxidation of short-chain aliphatic L-2-hydroxyacids in many tissues of higher organisms. A new purification procedure allowed us to obtain a 1400-fold purified enzyme from chicken liver. The N-terminal amino acid of the polypeptide chain was found to be blocked as that of spinach glycolate oxidase, contrastingly with that of rat kidney isozyme B. Its amino acid composition was comparable to that of other known L-2-hydroxyacid oxidases. Despite different substrate specificity, some immunological identity was observed between chicken liver L-2-hydroxyacid isozyme A and rat kidney isozyme B.

The histidines reacting with ethoxyformic anhydride in porcine pancreatic lipase: their relationships with enzyme activity.

The activities of porcine pancreatic lipase (449 amino acid residues) toward two different substrates, p-nitrophenylacetate and tributyrylglycerol, and their dependence on histidine ethoxyformylation were studied. In parallel, the ethoxyformylation of the lipase fragment constituting the C-terminal sequence of lipase (residues 336 to 449) was also investigated. This fragment was found to have retained the ability of lipase to catalyse p-nitrophenylacetate hydrolysis. The first histidine to react either in lipase or in the lipase fragment was His-354. The activities of the two compounds toward p-nitrophenyl-acetate were lost but that of the enzyme toward tributyrylglycerol was almost entirely retained. When a larger excess of ethoxyformic anhydride was used for the lipase reaction, 2.8 histidine residues were ethoxyformylated and characterised as His-354, His-156 and His-75, which resulted in an 85% inhibition of the tributyrylglycerol hydrolysis by the enzyme. Hydroxylamine treatment reactivated most of the lipase and lipase fragment. This is the first demonstration that the two lipase activities are not associated with the same active site. The loss of activity toward triacylglycerol hydrolysis suggests that His-156 and/or His-75 belong(s) to the active site or that a conformational change resulting from the ethoxyformylation renders the lipase inactive.

N-terminal sequence extension in the glycosylated forms of human pancreatic stone protein. The 5-oxoproline N-terminal chain is O-glycosylated on the 5th amino acid residue.

The pancreatic stone protein isolated from human calculi (PSP) derives from the immunoreactive protein forms detected in human pancreatic juice (PSP S2-5) through the tryptic cleavage of the Arg-11-Ile-12 bond. Among the eleven amino acids of the PSP S2-5 N-terminal extension Z-E-A-Q-T-E-L-P-Q-A-R, the first residue is an oxoproline and the fifth, a threonine, bears the single carbohydrate chain of the protein molecules. Variations in the glycan chain composition account for the differences in the Mr of PSP S2-5. The PSP S2-5 forms are very soluble in aqueous solutions between the pH values 5.0-9.0, whereas the proteolysated form is scarcely soluble.

Acetylation of Lys-373 in porcine pancreatic lipase after reaction of the enzyme or its C-terminal fragment [corrected] with p-nitrophenyl acetate.

The reactions of lipase (449 amino acid residues) and lipase fragment (336-449) with p-nitrophenyl acetate have been studied from 2 different angles. In previous papers it has been shown that lipase and lipase fragment enzymatically hydrolyze p-nitrophenyl acetate. The amino acid residue of the catalytic site that is temporarily acetylated has not yet been characterized in lipase or lipase fragment. Besides this very fast enzymatic hydrolysis, acetylation reactions may take place on nucleophilic amino acid side-chain groups. In the present report, acetylated amino acid residues whose acetyl linkages were not cleaved after pH 7.5-8.5 incubations have been investigated. Several residues were acetylated in very low proportion, whereas lysine 373 was stoichiometrically acetylated in lipase and in lipase fragment. This specific acetylation may have been favored by the presence of a hydrophobic reversible binding site for p-nitrophenyl acetate near Lys-373. This acetylation did not greatly change the specific activity of lipase towards an emulsion of tributyrylglycerol in the presence of colipase, but under certain conditions it had an effect on the enzymatic hydrolysis of p-nitrophenyl acetate by the lipase fragment.

The disulfide bridges of the immunoreactive forms of human pancreatic stone protein isolated from pancreatic juice.

Following the complete sequence elucidation of human pancreatic stone protein (immunoreactive form PSP S1 isolated from pancreatic juice) [(1987) Eur. J. Biochem. 168, 201-207], the location of the three S-S bridges of the protein was investigated. The cystine-containing peptides, detected after the separation of the peptic or chymotryptic digests on SP-Sephadex or Sephadex G-50, were submitted to Edman degradation and/or to oxidation. The cysteic peptides after separation on SP-Sephadex or Sephadex G-50 were characterized by their amino acid compositions. The pairing of the half-cystines: Cys 3-Cys 14, Cys 31-Cys 129 and Cys 104-Cys 121 was determined. The same experiments carried out with PSP S2-5 (other immunoreactive forms) gave an identical characterization.

Complete amino acid sequence of an immunoreactive form of human pancreatic stone protein isolated from pancreatic juice.

The primary structure of a pancreatic stone protein form has been elucidated for the first time. The protein studied was the lowest-Mr form prepared from human pancreatic juice (PSP S1). The N-terminal sequence up to residue 65 had already been determined. The five peptides obtained after staphylococcal protease digestion of the carboxymethylated reduced and succinylated PSP S1 enabled the deduction of the entire sequence. The tryptic peptides arising from the digest of cyclohexanedione--treated PSP S1 and the amino acids released by carboxypeptidase P digestion of PSP S1 confirmed the data of the sequence. The peptides were purified by Sephadex filtration and, if required, by chromatography on DEAE-cellulose or thin-layer cellulose. The amino acid sequences of the peptides were determined with a sequencer. From the sequence data it was deduced that the PSP S1 polypeptide chain contains 133 amino acid residues and has a Mr of 15,000.