Mark E. Lowe

Human Pancreatic Digestive Enzymes

A primary function of the pancreas is to produce digestive enzymes that are delivered to the small intestine for the hydrolysis of complex nutrients. Much of our understanding of digestive enzymes comes from studies in animals. New technologies and the availability of the sequence of the human genome allow for a critical review of older reports and assumptions based on animal studies. This report updates our understanding of human pancreatic digestive enzymes with a focus on new insights into the biology of human proteases, lipases and amylases.

Fibronectin alters the phenotypic properties of cultured chick embryo chondroblasts.

The state of chick embryo chondroblasts in culture was found to be sensitive to both fibronectin and another substance(s) (activity A) which could be extracted from chick embryo fibroblasts with 1 M urea or from conditioned medium. In the presence of either of these activities at concentrations of 25-150 micrograms/ml, chondroblasts, which normally grow as mixed cultures of floating and adherent cells, all immediately became attached to the tissue culture dish and spread. After several days, the morphology of these typically epithelioid cells became fibroblastic. This did not involve a selection process, since the effect was reversible. The synthetic program of these cells was also dramatically modified: the cultures no longer synthesized the chondroblast-unique type IV sulfated proteoglycan and began synthesizing alpha 2 collagen chains typical of fibroblastic or early limb bud cells. Fibronectin was resolved from activity A by gelatin affinity chromatography or gel filtration. Both activities were trypsin-sensitive. The two activities differed, however, on the basis of how the protein fractions in which they were found migrated in SDS-polyacrylamide gels, their specific activities and their effects on cell morphology and cell growth.

Definitions of pediatric pancreatitis and survey of present clinical practices.

Objectives: There is limited literature on acute pancreatitis (AP), acute recurrent pancreatitis (ARP), and chronic pancreatitis (CP) in children. The International Study Group of Pediatric Pancreatitis: In Search for a Cure (INSPPIRE) consortium was formed to standardize definitions, develop diagnostic algorithms, investigate disease pathophysiology, and design prospective multicenter studies in pediatric pancreatitis. Methods: Subcommittees were formed to delineate definitions of pancreatitis, and a survey was conducted to analyze present practice. Results: AP was defined as requiring 2 of the following: abdominal pain compatible with AP, serum amylase and/or lipase values ≥3 times upper limits of normal, and imaging findings of AP. ARP was defined as ≥2 distinct episodes of AP with intervening return to baseline. CP was diagnosed in the presence of typical abdominal pain plus characteristic imaging findings, or exocrine insufficiency plus imaging findings, or endocrine insufficiency plus imaging findings. We found that children with pancreatitis were primarily managed by pediatric gastroenterologists. Unless the etiology was known, initial investigations included serum liver enzymes, triglycerides, calcium, and abdominal ultrasound. Further investigations (usually for ARP and CP) included magnetic resonance or other imaging, sweat chloride, and genetic testing. Respondents’ future goals for INSPPIRE included determining natural history of pancreatitis, developing algorithms to evaluate and manage pancreatitis, and validating diagnostic criteria. Conclusions: INSPPIRE represents the first initiative to create a multicenter approach to systematically characterize pancreatitis in children. Future aims include creation of patient database and biologic sample repository.

Increasing Incidence of Acute Pancreatitis at an American Pediatric Tertiary Care Center: Is Greater Awareness Among Physicians Responsible?

Objectives: Studies show an increased incidence of adult acute pancreatitis (AP) in recent decades. The aim was to review pediatric AP incidence. Methods: Retrospective review of computerized databases at the Childrens Hospital of Pittsburgh from 1993 to 2004. The International Classification of Diseases, Ninth Revision, code 5770 Acute Pancreatitis was used; results were tabulated by discharge year and month. The incidence of AP was compared with orders for amylase and lipase testings and with the catchment population. Results: Over the study period, there were a total of 1021 discharge diagnoses of AP (731 first diagnoses). The diagnosis of AP increased from a low of 28 total cases (21 first diagnoses) in 1993 to a high of 141 total cases (109 first diagnoses) in 2004. The catchment population decreased from 882,000 to 826,500. The estimated incidences of first AP admission were 2.4 to 13.2 per 100,000 children (years 1993-2004; r2 = 0.8339). Linear regression analysis suggests that increased testing for amylase and lipase could account for 94% of the change in all AP admissions (P = 5.1 × 10−7). Conclusions: The increased incidence of AP at the Childrens Hospital of Pittsburgh from 1993 to 2004 may have been primarily driven by increased testing for the disease.

Pancreatic triglyceride lipase and colipase: Insights into dietary fat digestion

Dietary fats have an impact on health and disease. A pancreatic exocrine protein, pancreatic triglyceride lipase, is essential for the efficient digestion of dietary fats. This enzyme requires another pancreatic exocrine protein, colipase, for full activity in the gut lumen. In addition to its importance in fat digestion, pancreatic triglyceride lipase has potential applications in medical therapy, medical diagnostics, and industry. This potential stimulated interest in lipases; radiograph during the last few years, studies applying the technologies of molecular biology and radiograph crystallography greatly increased our knowledge about pancreatic triglyceride lipase and colipase protein structure, enzyme mechanism, and gene structure. This review focuses on these recent advances and discusses models for the kinetic properties of pancreatic triglyceride lipase and for the interaction of pancreatic triglyceride lipase with colipase.

What Have We Learned About Acute Pancreatitis in Children

Pediatric pancreatitis has received much attention during the past few years. Numerous reports have identified an increasing trend in the diagnosis of acute pancreatitis in children and key differences in disease presentation and management between infants and older children. The present review provides a brief, evidence-based focus on the latest progress in the clinical field. It also poses important questions for emerging multicenter registries to answer about the natural history and management of affected children with pancreatitis.

Decreased Neonatal Dietary Fat Absorption and T Cell Cytotoxicity in Pancreatic Lipase-related Protein 2-Deficient Mice

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.

Structure and activity of rat pancreatic lipase-related protein 2.

The pancreas expresses several members of the lipase gene family including pancreatic triglyceride lipase (PTL) and two homologous proteins, pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Despite their similar amino acid sequences, PTL, PLRP1, and PLRP2 differ in important kinetic properties. PLRP1 has no known activity. PTL and PLRP2 differ in substrate specificity, bile acid inhibition, colipase requirement, and interfacial activation. To begin understanding the structural explanations for these functional differences, we solved the crystal structure of rat (r)PLRP2 and further characterized its kinetic properties. The 1.8 Å structure of rPLRP2, like the tertiary structure of human PTL, has a globular N-terminal domain and a β-sandwich C-terminal domain. The lid domain occupied the closed position, suggesting that rPLRP2 should show interfacial activation. When we reexamined this issue with tripropionin as substrate, rPLRP2 exhibited interfacial activation. Because the active site topology of rPLRP2 resembled that of human PTL, we predicted and demonstrated that the lipase inhibitors E600 and tetrahydrolipstatin inhibit rPLRP2. Although PTL and rPLRP2 have similar active sites, rPLRP2 has a broader substrate specificity that we confirmed using a monolayer technique. With this assay, we showed for the first time that rPLRP2 prefers phosphatidylglycerol and ethanolamine over phosphatidylcholine. In summary, we confirmed and extended the observation that PLRP2 lipases have a broader substrate specificity than PTL, we demonstrated that PLRP2 lipases show interfacial activation, and we solved the first crystal structure of a PLRP2 lipase that contains a lid domain.

Total pancreatectomy and islet autotransplantation in chronic pancreatitis: recommendations from PancreasFest.

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.

Pharmacology and Safety of Glycerol Phenylbutyrate in Healthy Adults and Adults with Cirrhosis

Phenylbutyric acid (PBA), which is approved for treatment of urea cycle disorders (UCDs) as sodium phenylbutyrate (NaPBA), mediates waste nitrogen excretion via combination of PBA‐derived phenylacetic acid with glutamine to form phenylactylglutamine (PAGN) that is excreted in urine. Glycerol phenylbutyrate (GPB), a liquid triglyceride pro‐drug of PBA, containing no sodium and having favorable palatability, is being studied for treatment of hepatic encephalopathy (HE). In vitro and clinical studies have been performed to assess GPB digestion, safety, and pharmacology in healthy adults and individuals with cirrhosis. GPB hydrolysis was measured in vitro by way of pH titration. Twenty‐four healthy adults underwent single‐dose administration of GPB and NaPBA and eight healthy adults and 24 cirrhotic subjects underwent single‐day and multiple‐day dosing of GPB, with metabolites measured in blood and urine. Simulations were performed to assess GPB dosing at higher levels. GPB was hydrolyzed by human pancreatic triglyceride lipase, pancreatic lipase‐related protein 2, and carboxyl‐ester lipase. Clinical safety was satisfactory. Compared with NaPBA, peak metabolite blood levels with GPB occurred later and were lower; urinary PAGN excretion was similar but took longer. Steady state was achieved within 4 days for both NaPBA and GPB; intact GPB was not detected in blood or urine. Cirrhotic subjects converted GPB to PAGN similarly to healthy adults. Simulations suggest that GPB can be administered safely to cirrhotic subjects at levels equivalent to the highest approved NaPBA dose for UCDs. Conclusion: GPB exhibits delayed release characteristics, presumably reflecting gradual PBA release by pancreatic lipases, and is well tolerated in adults with cirrhosis, suggesting that further clinical testing for HE is warranted. (HEPATOLOGY 2010;)