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Variable number of tandem repeat (VNTR) sequences in the genome can have functional consequences that contribute to human disease. This is the case for the CEL gene, which is specifically expressed in pancreatic acinar cells and encodes the digestive enzyme carboxyl ester lipase. Rare single-base deletions (DELs) within the first (DEL1) or fourth (DEL4) VNTR segment of CEL cause maturity-onset diabetes of the young, type 8 (MODY8), an inherited disorder characterized by exocrine pancreatic dysfunction and diabetes. Studies on the DEL1 variant have suggested that MODY8 is initiated by CEL protein misfolding and aggregation. However, it is unclear how the position of single-base deletions within the CEL VNTR affects pathogenic properties of the protein. Here, we investigated four naturally occurring CEL variants, arising from single-base deletions in different VNTR segments (DEL1, DEL4, DEL9, and DEL13). When the four variants were expressed in human embryonic kidney 293 cells, only DEL1 and DEL4 led to significantly reduced secretion, increased intracellular aggregation, and increased endoplasmic reticulum stress compared with normal CEL protein. The level of O-glycosylation was affected in all DEL variants. Moreover, all variants had enzymatic activity comparable with that of normal CEL. We conclude that the longest aberrant protein tails, resulting from single-base deletions in the proximal VNTR segments, have highest pathogenic potential, explaining why DEL1 and DEL4 but not DEL9 and DEL13 have been observed in patients with MODY8. These findings further support the view that CEL mutations cause pancreatic disease through protein misfolding and proteotoxicity, leading to endoplasmic reticulum stress and activation of the unfolded protein response., SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction Abdominal pain is common and is associated with high disease burden and health care costs in pediatric acute recurrent and chronic pancreatitis (ARP/CP). Despite the strong central component of pain in ARP/CP and the efficacy of psychological therapies for other centralized pain syndromes, no studies have evaluated psychological pain interventions in children with ARP/CP. The current trial seeks to 1) evaluate the efficacy of a psychological pain intervention for pediatric ARP/CP, and 2) examine baseline patient-specific genetic, clinical, and psychosocial characteristics that may predict or moderate treatment response. Methods This single-blinded randomized placebo-controlled multicenter trial aims to enroll 260 youth (ages 10–18) with ARP/CP and their parents from twenty-one INSPPIRE ( IN ternational S tudy Group of P ediatric P ancreatitis: I n search for a cu RE) centers. Participants will be randomly assigned to either a web-based cognitive behavioral pain management intervention (Web-based Management of Adolescent Pain Chronic Pancreatitis; WebMAP; N = 130) or to a web-based pain education program (WebED; N = 130). Assessments will be completed at baseline (T1), immediately after completion of the intervention (T2) and at 6 months post-intervention (T3). The primary study outcome is abdominal pain severity. Secondary outcomes include pain-related disability, pain interference, health-related quality of life, emotional distress, impact of pain, opioid use, and healthcare utilization. Conclusions This is the first clinical trial to evaluate the efficacy of a psychological pain intervention for children with CP for reduction of abdominal pain and improvement of health-related quality of life. Findings will inform delivery of web-based pain management and potentially identify patient-specific biological and psychosocial factors associated with favorable response to therapy. Clinical Trial Registration #: NCT03707431.

Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults, whereas a homolog, pancreatic lipase-related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar, yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIPRP2 cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified, and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the surface loops tested, the lid domain and the β5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid domain or β5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP β5-loop and decreased by the PNLIP lid domain. The source of the β9-loop had minimal effect on activity. We conclude that the lid domain and β5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2.

Congenital pancreatic triglyceride lipase (PNLIP) deficiency is a rare disorder with uncertain genetic background as most cases were described before gene sequencing was readily available. Recently, two brothers with PNLIP deficiency were found to carry a homozygous missense mutation, c.662C>T (p.T221M) in the PNLIP gene (J. Lipid Res. 2014. 55:307–312). Molecular modeling suggested the substitution would change the orientation of residues in the catalytic site and disrupt the function of p.T221M PNLIP. To test the effect of the p.T221M mutation on PNLIP function, we expressed wild-type and p.T221M PNLIP in human embryonic kidney (HEK) 293A cells and dexamethasone-differentiated AR42J rat acinar cells. In both cellular models, wild-type PNLIP was secreted into the conditioned medium where it was readily detectable by protein staining, immunoblot or lipase activity assays. In contrast, mutant p.T221M was not secreted into the medium, but it was present in cell lysates where it accumulated in the insoluble fraction. Intracellular retention of mutant p.T221M resulted in endoplasmic reticulum (ER) stress as measured by elevated XBP1 splicing and increased levels of ER chaperones. Our results demonstrate that the presence of methionine at position 221 in the PNLIP protein sequence causes misfolding and aggregation of the p.T221M mutant inside the cell. The consequent loss of enzyme secretion adequately explains the clinical phenotype of PNLIP deficiency reported for homozygous carriers of p.T221M. Furthermore, the ability of mutant p.T221M to induce ER stress suggests that this form of PNLIP deficiency might cause acinar cell damage as well.

Pancreatitis-Panniculitis-Polyarthritis (PPP) syndrome is rare and its physiopathology unclear. A 6-year old boy suffered of traumatic pancreatitis complicated by PPP syndrome. Extensive investigations demonstrated high levels of pancreatic lipase and fatty acids in the affected peripheral tissues. These findings support the sequence of peripheral lipolysis and fatty acid accumulation inducing tissue inflammation.

Description Total pancreatectomy with islet autotransplantation (TPIAT) is a surgical procedure used to treat severe complications of chronic pancreatitis or very high risk of pancreatic cancer while reducing the risk of severe diabetes mellitus. However, clear guidance on indications, contraindications, evaluation, timing, and follow-up are lacking. Methods A working group reviewed the medical, psychological, and surgical options and supporting literature related to TPIAT for a consensus meeting during PancreasFest . Results Five major areas requiring clinical evaluation and management were addressed: These included: 1) indications for TPIAT; 2) contraindications for TPIAT; 3) optimal timing of the procedure; 4) need for a multi-disciplinary team and the roles of the members; 5) life-long management issues following TPIAP including diabetes monitoring and nutrition evaluation. Conclusions TPIAT is an effective method of managing the disabling complications of chronic pancreatitis and risk of pancreatic cancer in very high risk patients. Careful evaluation and long-term management of candidate patients by qualified multidisciplinary teams is required. Multiple recommendations for further research were also identified.

Genetically engineered mice have been employed to understand the role of lipases in dietary fat digestion with the expectation that the results can be extrapolated to humans. However, little is known about the properties of mouse pancreatic triglyceride lipase (mPTL) and pancreatic lipase-related protein-2 (mPLRP2). In this study, both lipases were expressed in Pichia Pastoris GS115, purified to near homogeneity, and their properties were characterized. Mouse PTL displayed the kinetics typical of PTL from other species. Like mPTL, mPLRP2 exhibited strong activity against various triglycerides. In contrast to mPTL, mPLRP2 was not inhibited by increasing bile salt concentration. Colipase stimulated mPLRP2 activity 2- to 4-fold. Additionally, mPTL absolutely required colipase for absorption to a lipid interface, whereas mPLRP2 absorbed fully without colipase. mPLRP2 had full activity in the presence of BSA, whereas BSA completely inhibited mPTL unless colipase was present. All of these properties of mPLRP2 differ from the properties of human PLRP2 (hPLRP2). Furthermore, mPLRP2 appears capable of compensating for mPTL deficiency. These findings suggest that the molecular mechanisms of dietary fat digestion may be different in humans and mice. Thus, extrapolation of dietary fat digestion in mice to humans should be done with care.

Because dietary fats provide an important source of energy in the newborn, the efficient digestion of dietary fats is critical to their well-being. Despite the importance of dietary fat digestion, newborns have a deficiency of pancreatic triglyceride lipase, the predominant digestive lipase in adults. The efficient dietary fat digestion in newborns suggests that other lipases must compensate for the lack of pancreatic triglyceride lipase. In this study, we test the hypothesis that breast milk, pancreatic carboxyl ester lipase (CEL), or both contribute to dietary fat digestion in the newborn. To test this hypothesis, we determined the amount and composition of fecal fat in wild-type and CEL-deficient newborns nursed by either wild-type or CEL-deficient dams. We tested all genetic permutations of the nursing pairs. An interaction between the genotype of the dam and of the pup determined the amount of fecal fat (P < 0.001). Fecal fat was highest in CEL-deficient pups nursed by CEL-deficient dams. Furthermore, only the feces from the CEL-deficient pups nursed by CEL-deficient dams contained undigested lipids. Even with increased fecal fats, the CEL-deficient pups had normal weight gain. Our results demonstrate that CEL contributes significantly to dietary triglyceride digestion whether it originates from mother's milk or pancreatic secretions. However, only the absence of both mother's milk and pancreatic CEL produces fat maldigestion. The absence of a single CEL source makes no difference in the efficiency of dietary fat absorption.

Type 2 diabetes mellitus is a multifactorial and polygenic disorder with increasing prevalence. Recently, a polymorphism in the gene encoding procolipase, a cysteine for arginine substitution at position 92, was associated with type 2 diabetes in two human populations. Because procolipase plays a critical role in dietary fat metabolism, polymorphisms that affect the function of procolipase could influence the development of type 2 diabetes. We hypothesized that the Arg92Cys polymorphism has functional consequences. To test our hypothesis, we expressed recombinant cysteine 92 (Cys92) procolipase in a yeast expression system and compared the function and stability of purified Cys92 with that of the more common arginine 92 (Arg92) procolipase. Cys92 fully restored the activity of bile-salt inhibited lipase with short- and medium-chain triglycerides but only had 50% of Arg92 function with long-chain triglycerides. After storage at 4 degrees C, Cys92 lost the ability to restore pancreatic triglyceride lipase activity with medium- and long-chain triglycerides. The loss of function correlated with the inability of Cys92 to anchor lipase on an emulsion surface and oxidation of the cysteine. No detectable degradation or intramolecular disulfide formation occurred in Cys92 after storage. Our findings demonstrate that the Arg92Cys polymorphism decreases the function of Cys92 colipase. This change may contribute to the development of type 2 diabetes.

Pancreatic triglyceride lipase (PTL) and its protein cofactor, colipase, are required for efficient dietary triglyceride digestion. In addition to PTL, pancreatic acinar cells synthesize two pancreatic lipase related proteins (PLRP1 and PLRP2), which have a high degree of sequence and structural homology with PTL. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, behavior in bile salts and dependence on colipase. Each protein has a globular amino-terminal (N-terminal) domain, which contains the catalytic site for PTL and PLRP2, and a beta-sandwich carboxyl-terminal (C-terminal) domain, which includes the predominant colipase-binding site for PTL. Inactive and active conformations of PTL have been described. They differ in the position of a surface loop, the lid domain, and of the beta5-loop. In the inactive conformation, the lid covers the active site and, upon activation by bile salt micelles and colipase or by lipid-water interfaces, the lid moves dramatically to open and configure the active site. After the lid movement, PTL and colipase create a large hydrophobic plateau that can interact with the lipid-water interface. A hydrophobic surface loop in the C-terminal domain, the beta5' loop, may also contribute to the interfacial-binding domain of the PTL-colipase complex.

In vitro, pancreatic triglyceride lipase requires colipase to restore activity in the presence of inhibitors, like bile acids. Presumably, colipase performs the same function in vivo, but little data supports that notion. Other studies suggest that colipase or its proform, procolipase, may have additional functions in appetite regulation or in fat digestion during the newborn period when pancreatic triglyceride lipase is not expressed. To identify the physiological role of procolipase, we created a mouse model of procolipase deficiency. TheClps −/− mice appeared normal at birth, but unexpectedly 60% died within the first 2 weeks of life. The survivors had fat malabsorption as newborns and as adults, but only when fed a high fat diet. On a low fat diet, the Clps −/−mice did not have steatorrhea. The Clps −/−pups had impaired weight gain and weighed 30% less thanClps+/+ or Clps +/−littermates. After weaning, the Clps −/− mice had normal rate of weight gain, but they maintained a reduced body weight compared with normal littermates even on a low fat diet. Despite the reduced body weight, the Clps −/− mice had a normal body temperature. To maintain their weight gain in the presence of steatorrhea, the Clps −/− mice had hyperphagia on a high fat diet. Clps −/− mice had normal intake on a low fat diet. We conclude that, in addition to its critical role in fat digestion, procolipase has essential functions in postnatal development and in regulating body weight set point.

Pancreatic triglyceride lipase (PTL) requires colipase for activity. Various constituents in meals and in bile, particularly bile acids, inhibit PTL. Colipase restores activity to lipase in the presence of inhibitory substances like bile acids. Presumably, colipase functions by anchoring and orienting PTL at the oil-water interface. The x-ray structure of the colipase.PTL complex supports this model. In the x-ray structure, colipase has a hydrophobic surface positioned to bind substrate and a hydrophilic surface, lying opposite the hydrophobic surface, with two putative lipase-binding domains, Glu(45)/Asp(89) and Glu(64)/Arg(65). To determine whether the hydrophilic surface interacts with PTL in solution, we introduced mutations into the putative PTL binding domains of human colipase. Each mutant was expressed, purified, and assessed for activity against various substrates. Most of the mutants showed impaired ability to reactivate PTL, with mutations in the Glu(64)/Arg(65) binding site causing the greatest effect. Analysis indicated that the mutations decreased the affinity of the colipase mutants for PTL and prevented the formation of PTL.colipase complexes. The impaired function of the mutants was most apparent when assayed in micellar bile salt solutions. Most mutants stimulated PTL activity normally in monomeric bile salt solutions. We also tested the mutants for their ability to bind substrate and anchor lipase to tributyrin. Even though the ability of the mutants to anchor PTL to an interface decreased in proportion to their activity, each mutant colipase bound to tributyrin to the same extent as wild type colipase. These results demonstrate that the hydrophilic surface of colipase interacts with PTL in solution to form active colipase.PTL complexes, that bile salt micelles influence that binding, and that the proper interaction of colipase with PTL requires the Glu(64)/Arg(65) binding site.

Pancreatic triglyceride lipase (PTL) and the homologous pancreatic lipase related protein 2 (PLRP2) provide a unique opportunity to understand the molecular mechanism of lipolysis. They differ in substrate specificity, sensitivity to bile salts, and colipase dependence despite their close amino acid and tertiary structure identity. One important structure, present in both lipases, is the lid which occupies different positions in the inactive and active forms of PTL. We investigated the role of the lid in lipase function by site-specific mutagenesis. By exchanging the lids between PTL and PLRP2, we created two chimeric lipases. Additionally, we made multiple substitution mutations in the PTL lid. PLRP2 with the PTL lid had kinetic properties similar to PLRP2. PTL with the PLRP2 lid was greatly impaired and had no activity at micellar bile salt concentrations even in the presence of colipase. Both chimeras showed interfacial activation suggesting that the closed lid position was maintained. A series of substitution mutations were made in positions Arg257 and Asp258. These mutations demonstrated the importance of these two residues to maintaining the normal activity, triglyceride acyl chain specificity, and colipase interaction of PTL. The preserved interfacial activation in the chimeras, the similar crystal structure of the two lids in the closed position, and the importance of Arg257 and Asp258 in mediating the open conformation of the lid argue that the position of the open lid influences the differences in activity against triglycerides, in sensitivity to bile salts, and in colipase dependence between PTL and PLRP2. —Yang, Y., and M. E. Lowe. The open lid mediates pancreatic lipase function.

The pancreas secretes several different lipases. The most abundant is pancreatic triglyceride lipase (PTL). The pancreas also synthesizes two homologues of PTL, the pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Cytotoxic T-lymphocytes also express PLRP2 under certain conditions. We sought to determine the role of PLRP2 in fat absorption and in T-cell cytotoxicity by creating a PLRP2-deficient mouse. Adult PLRP2-deficient mice had normal fat absorption. In contrast, suckling PLRP2-deficient mice had fat malabsorption evidenced by increased fecal weight, increased fecal fats, and the presence of undigested and partially digested dietary triglycerides in the feces. As a result, the PLRP2-deficient pups had a decreased rate of weight gain. To assess T cell cytotoxicity, we immunized PLRP2-deficient mice with a mastocytoma cell line, P815, and determined the ability of splenocytes from the immunized mice to kill P815 cells in a 51Cr release assay. PLRP2-deficient cells had deficient killing activity in this assay, and PLRP2-deficient splenocytes released fewer fatty acid from the target cells than did control cells. Our results provide the first evidence of a physiological function for PLRP2. PLRP2 participates in T cell cytotoxicity, and PLRP2 performs a crucial role in the digestion of dietary fats in suckling animals.

Background & Aims: Procolipase, the cofactor for pancreatic lipase, was recently found in the rat stomach using immunohistochemistry. The aim of this study was to determine the sequence of rat gastric procolipase, to evaluate the expression and secretion during high-fat feeding, and to find out the conditions for activation of gastric procolipase to form colipase and enterostatin. Methods: Gastric procolipase was cloned from a rat complimentary DNA (cDNA) library using a 32 Plabeled pancreatic procolipase probe for screening. For the expression of gastric procolipase, rats were fed a high-fat diet for 0, 1, 2, 5, and 14 days. Gastric mucosa was collected for isolation of RNA and gastric juice for measurement of procolipase. After treatment with pepsin, HCl, and trypsin, gastric juice was analyzed on high-performance liquid chromatography for identification of enterostatin. Results: The cDNA sequence for gastric procolipase was identical to that of pancreatic procolipase. High-fat diet decreased the expression of gastric procolipase. Enterostatin was present in the gastric juice, with pepsin and acid involved in the cleavage of gastric procolipase. Conclusions: Gastric procolipase is activated to re-lease colipase and enterostatin. The role of gastric colipase may be to prepare lipase-catalyzed fat digestion already in the stomach. Gastric enterostatin may be involved in the onset of early satiety. GASTROENTEROLOGY 1998;115:1179-1185

Pancreatic lipase is characterized by increased activity against water-insoluble substrates and by dependence on another protein, colipase, for binding to the substrate interface. In most models of pancreatic lipase activity, colipase functions to anchor lipase on the substrate interface. Recent studies of the x-ray crystal structure of the complex between colipase and lipase suggest another function for colipase in maintaining the active conformation of lipase. We tested this hypothesis by introducing mutations into colipase at position 15, a residue that contacts the lid domain lipase in the open conformation. Multiple mutant colipases were expressed and shown to have decreased activity. To further investigate the function of the interaction between Glu15 of colipase and lipase, we examined one mutant, E15R, in detail. This mutant had 175-fold less activity compared with wild-type colipase. Although E15R had decreased activity, it was as effective as wild-type lipase in anchoring lipase to mixed emulsions of bile salt and tributyrin. The importance of the interaction with the lid domain was tested by determining the activity of E15R with lid deletion mutants of lipase. E15R was as active as wild-type colipase with these mutant lipases. These results indicate that Glu15 is critical for activity of the colipase-lipase complex at an interface and that colipase has a function in lipolysis in addition to anchoring lipase to an interface. We propose that this function is to stabilize the lid domain of lipase in the open conformation, thereby facilitating lipolysis.

The pancreatic lipase family contains three subfamilies, the ‘classical’ lipases and the pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2). Galactolipids are present in membranes of leaves and vegetables and consist of digalactosyldiacylglycerol (DGalDG) monogalactosyldiacylglycerol (MGalDG) and sulfoquinovosyldiacylglycerol (SQDG). These lipids were incubated with PLRP2 from guinea-pig (GPLRP2) and rat (RPLRP2). In the presence of bile salts (DGalDG) was efficiently hydrolyzed by GPLRP2 and, although less efficiently, by RPLRP2 to digalactosylmonoacylglycerol (DGalMG), free fatty acids and water-soluble galactose-containing compounds. Also, MGalDG and SQDG were hydrolyzed by GPLRP2 and RPLRP2. These data suggest a possible role of PLRP2 in the digestion of dietary galactolipids.

The catalytic mechanism for members of the lipase gene family incorporates a serine-histidine-acidic group triad. In general, the acidic group is an aspartate, Asp177 in human pancreatic lipase, but glutamate is found in some lipases. Previously, we demonstrated that site-specific mutagenesis of Asp177 to Glu177 produced a mutant human pancreatic lipase with near normal activity against triolein, thereby, raising questions about the role of Asp177 in the catalytic triad and about the evolutionary pressure which selected Asp over Glu in the catalytic mechanism. To address these questions, we constructed and expressed mutants of Asp177 and Asp206, another acidic residue that could participate in the catalytic triad. The Glu177 mutant had a substrate specificity, specific activity, pH profile, colipase dependance, and interfacial activation comparable to the native lipase, Asp177. Several mutants of Asp206 were normally active, thus, confirming the important role of Asp177 in pancreatic lipase function. Additionally, we found that the Glu177 mutant had increased susceptibility to proteases and to urea denaturation. These findings demonstrated decreased conformational stability of the mutant lipase and provided an explanation for the preference of aspartate in the catalytic triad of human pancreatic lipase.

Suckling mice express colipase before the expression of pancreatic triglyceride lipase. Yet, efficient fat digestion in newborns requires colipase, suggesting that colipase may act as a cofactor for another lipase such as pancreatic lipase-related protein 2 (PLRP2). We determined whether PLRP2 or another lipase depends on colipase for maximal activity in newborn mice by analyzing extracts from the pancreas of 4-d-old colipase-deficient and PLRP2-deficient mice. Pancreatic extracts from colipase-deficient pups had lipase activity that was stimulated onefold by the addition of exogenous colipase (P < 0.001). The activity was completely inhibited by an antibody against pancreatic triglyceride lipase that also recognizes PLRP2. In contrast, pancreatic extracts from PLRP2-deficient pups had significantly lower baseline activity and no colipase-dependent activity. The baseline activity was not inhibited by the anti-pancreatic triglyceride lipase antibody or an antibody against carboxyl ester lipase. We next separated the extracts into two fractions, one containing PLRP2 and the other devoid of PLRP2. All of the colipase-dependent activity segregated with the PLRP2-containing fraction, consistent with the conclusion that PLRP2 is the major colipase-dependent lipase in the pancreas of newborns.

The pancreas contains three homologous proteins, colipase-dependent pancreatic lipase (PL) and two recently described pancreatic lipase-related proteins, PLRP1 and PLRP2. Rat (r) PLRP2 was first identified as a zymogen granule membrane protein, GP-3. Subsequently, we showed that rPLRP2 could cleave fatty acids from triglycerides, but the kinetic properties of rPLRP2 have not been further investigated. To further characterize rPLRP2, we expressed the recombinant enzyme in a baculovirus system, purified the secreted protein, and measured its kinetic properties. rPLRP2 had a broad pH optimum and the curve was similar to that of rPL. At pH 7.5, rPLRP2 cleaved short, medium, and long chain triglycerides by a kinetic mechanism that did not include interfacial activation. The activity against these substrates was not affected by bile salts. In particular, rPLRP2 did not show the bile salt inhibition typical of PL. Although colipase increased rPLRP2 activity in the presence of bile salts, the increase was only 2- to 5-fold compared to the absolute requirement for colipase that rPL had under these conditions. Finally, rPLRP2 could hydrolyze phospholipids, a substrate poorly hydrolyzed by PL. Our characterization of rPLRP2 demonstrates clear differences among the kinetic properties of rPLRP2 and rPL, rPLRP2, and PLRP2 homologues isolated from guinea pig and coypu pancreas. These findings have important implications for the physiological function of rPLRP2.

Serum amylase and lipase frequently rise during bouts of acute pancreatitis, and measurement of these enzymes provides important diagnostic information. We report a pediatric patient with persistent elevations of serum lipase resulting from macrolipasemia, a complex of lipase with IgG, rather than pancreatitis.

In this study, the essential serine residue and 2 other amino acids in human pancreatic triglyceride lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) were tested for their contribution to the enzyme's catalytic site or interfacial binding site. By site-specific mutagenesis of the cDNA for human pancreatic lipase, amino acid substitutions were made at Ser153, His264, and Asp177. The mutant cDNAs were expressed in transfected COS-1 cells. Both the medium and the cells were examined for the presence of pancreatic lipase by Western blot analysis. The activity of the expressed proteins against triolein and the interfacial binding was measured. Proteins with mutations in Ser153 were secreted by the cells and bound to interfaces but had no detectable activity. Changing His264 to a leucine or Asp177 to an asparagine also produced inactive lipase. Substituting glutamic acid for Asp177 produced an active protein. These results demonstrate that Ser153 is involved in the catalytic site of pancreatic lipase and is not crucial for interfacial binding. Moreover, the essential roles of His264 and Asp177 in catalysis were demonstrated. A Ser-His-Asp catalytic triad similar to that present in serine proteases is present in human pancreatic lipase.

Rat intestinal alkaline phosphatase (IAP) has been purified and proteolytic fragments sequenced. A cDNA library was constructed from duodenal poly(A)+ RNA and screened for IAP positive clones by a full-length cDNA clone-encoding human IAP. A full length rat IAP clone (2237 bp) was isolated and sequenced, revealing a predicted primary sequence of 519 amino acids (61.974 kDa) with an additional signal peptide of 20 amino acids. 80% of amino acids from residues 1–474 were identical when compared with the human IAP, but there was only 31% identity in the COOH-terminal 45 amino acids. The homology diverges just before the putative binding site for the phosphatidylinositol-glycan (PI-glycan) anchor. The resulting peptide in rat AP contains five hydrophilic amino acids not present in the primary structure of human IAP. Binding of a synthetic 48-mer encoding a portion of this unique and divergent region (residues 476–491) was compared with that of the full-length clone on Northern blots of rat intestinal RNA. Two mRNAs, 3.0 and 2.7 kb, were detected by both probes, confirming earlier results, but the 48-mer bound preferentially to the 3.0 kb mRNA. The protein product of the full-length cDNA in a cell-free system was 62 kDa, corresponding with the smaller of the two IAP proteins produced by rat duodenal RNA. The cDNA transfected into COS-1 cells produced a membrane-bound IAP that was released by phosphatidylinositol-specific phospholipase (PI-PLC). These data provide definitive evidence that IAP is anchored by PI-glycan and conclusively demonstrate that the unique COOH-terminal structure encoded by this rat mRNA supports the addition of a PI-glycan anchor.

Pediatric Acute Recurrent and Chronic Pancreatitis: Report From INSPPIRE Consortium Aliye Uc, Elizabeth H. Yen, Michael Wilschanski, Steven Werlin, David Troendle, Sarah J. Schwarzenberg, John Pohl, Chee Y. Ooi, Veronique D. Morinville, Soma Kumar, Sohail Z. Husain, Ryan Himes, Melvin B. Heyman, Tanja Gonska, Matthew J. Giefer, Cheryl E. Gariepy, Steven D. Freedman, Douglas S. Fishman, Peter R. Durie, Heather Davis, Melena Bellin, Bradley A. Barth, Monika Ahuja, Mark Lowe

Objective To determine the etiologic factors and outcome of acute pancreatitis in children under age 3 years. Study design This was a retrospective study of children under age 3 years with acute pancreatitis between January 1995 and December 2004. Stringent diagnostic criteria were used. Demographic and clinical data were collected, and etiology and outcome were recorded. The study was approved by the University of Pittsburgh's Institutional Review Board. Results Of 109 cases, 87 met the diagnostic criteria. Median age was 20 months (range, 1 week to 35 months). AP was associated with multisystem disease in 29 cases (34%), with hemolytic uremic syndrome (HUS) being common. Pancreatitis was associated with systemic infections in 16 cases (18%) and was idiopathic in 15 cases (17%). Biliary disease played an important etiologic role (9%), as did trauma (8%). Pancreatitis was mild in 76 cases (87.3%) and severe in 3 cases (3.4%). Conclusions AP is commonly associated with multisystem disease, particularly with HUS. Idiopathic pancreatitis and pancreatitis associated with biliary disease are seen in children under age 3 years. Trauma is a less frequent cause of pancreatitis, and severe pancreatitis is rare in this age group.

The major polypeptide (43,000 daltons) of the rat liver receptor for asialoglycoproteins was isolated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Antibodies raised in a goat against this SDS-treated polypeptide exhibited marked cross-reactivity toward the SDS-denatured forms of the two other prominent polypeptides (54,000 and 64,000 daltons) of the receptor preparation. Monoclonal antibodies directed against the receptor were prepared using the spleen cells of mice immunized with the soluble, active receptor purified by affinity chromatography. The most extensively characterized of the monoclonal antibodies, designated D3-5D3, recognized the solubilized receptor and bound to the exterior surface of isolated rat hepatocytes. The binding of D3-5D3 to hepatocytes prevented subsequent binding of the ligand, 125I-asialo-orosomucoid. Conversely, occupation of the receptor with ligand inhibited binding of 125I-IgG prepared from D3-5D3 ascites fluid. The secondary structure of the receptor appears to be critical for recognition by D3-5D3, since denaturation of the receptor with 1% SDS, 5% beta-mercaptoethanol at 100 degrees C abolished antibody binding. Under less denaturing conditions (0.1% SDS, 25 degrees C), antigenic reactivity was retained by the receptor. Preparative electrophoresis using the latter conditions permitted the demonstration that D3-5D3 recognized a unique determinant that is present in each of the three polypeptides.