Differences in the renal handling of pancreatic and salivary amylase in the rat.

Plasma activity and excretion of pancreatic (P) amylase in the rat was found to be negligible. In contrast, the excretion rate of salivary (S) amylase was substantial and variable, depending on diuresis. P-amylase had a higher isoelectric point, a greater sieving coefficient, and a shorter half-life than S-amylase. A bolus injection of 125I-labelled enzymes was followed by the appearance of 125I-labelled enzyme- as well as protein-free 125I activity in the urine. The enzyme loss was smaller and the fraction of protein-free 125I activity higher following injection of P-amylase. The affinity of P-amylase to paraffin oil exceeded that of S-amylase in partition experiments with water and paraffin oil in vitro. It is concluded that both renal filtration and reabsorption of P-amylase exceed those of S-amylase. This might be due to the higher lipophility of P-amylase in comparison to the salivary type.     

Clinical value of serum immunoreactive trypsin concentration.

The clinical value of estimation of serum concentrations of immunoreactive trypsin was evaluated by studying 46 healthy controls, 23 controls in hospital, 44 patients with chronic pancreatic disease, and 184 patients with non-pancreatic conditions in which pancreatic disease commonly enters into the differential diagnosis. Serum trypsin concentration had a log normal distribution in the controls, and the calculated normal range was considerably wider than that previously reported. The concentration was abnormal in only 13 out of 27 patients with chronic pancreatitis and was extremely variable in patients with pancreatic cancer. Abnormal results occurred in 11% of the patients with non-pancreatic disease. Eighteen patients had a subnormal trypsin concentration, of whom six did not have pancreatic disease and 12 had either chronic pancreatitis or pancreatic cancer. There was no correlation between serum trypsin concentration and mean tryptic activity as measured by the Lundh test. Of 11 patients with pancreatic steatorrhoea, only seven had subnormal trypsin concentrations. There results suggest that the serum concentrations of immunoreactive trypsin has a low specificity and sensitivity for pancreatic disease and does no reflect the degree exocrine insufficiency in patients with proved chronic pancreatitis.     

Circulatory turnover of pancreatic amylase.

Radiolabeled amylase and unlabeled pancreatic amylase were infused into pigs in order to determine the plasma half-life of the enzyme. Regardless of the parameter measured (radioactivity, enzyme activity or concentration), the plasma removal curves could be resolved into three components when subjected to tracer analysis. The plasma half-life was estimated to be approximately 3 hr. Through the use of a recently developed radioimmunoassay specific for porcine pancreatic amylase, the plasma concentration of amylase was calculated at 2.4 mug/ml. Knowing the plasma concentration and half-life of amylase we determined the circulatory turnover of the enzyme. Over a 2.4-hr period, 9.6 mug of pancreatic amylase/ml of plasma must re-enter the circulation to maintain the enzyme at constant levels.     

Inflammation and Cancer V. Chronic pancreatitis and pancreatic cancer

Pancreatic inflammation appears to increase the risk of pancreatic cancer. This observation is striking in the hereditary pancreatitis kindreds but also occurs in alcoholic, idiopathic, and tropical chronic pancreatitis and cystic fibrosis. However, the mutations associated with hereditary pancreatitis or cystic fibrosis are not found in sporadic pancreatic adenocarcinomas, suggesting that the effects are indirect by causing recurrent pancreatitis and chronic inflammation. The process of mutation accumulation and clonal expansion that is required for development of invasive pancreatic adenocarcinoma must therefore be accelerated in chronic pancreatitis to account for the high incidence of pancreatic cancer in these patients.     

Microvascular density in chronic pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic adenocarcinoma represent two pathologic phenomena with marked production of connective tissue stroma containing numerous small blood vessels. The aim of this study was to characterise quantitatively the vascular supply of pancreatic adenocarcinoma and fragments of the periductal tissue collected from patients with chronic pancreatitis. The study material included 18 cases of pancreatitis and 22 cases of pancreatic ductal adenocarcinoma. Microvessels were marked using monoclonal anti-CD34 antibodies. The number of blood vessels in the fibrous stroma was significantly higher in the chronic pancreatitis samples compared to the pancreatic carcinoma group (mean vessel count 298 and 194 vessel/mm2; median 251 and 187 vessel/mm2 respectively; p<0.01). Distributions of the vascular diameter in both studied groups were very similar. The obtained results suggest that the development of vascular network accompanying chronic pancreatitis is more effective in some parts of pancreas compared to angiogenic intensity in pancreatic adenocarcinoma.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

Renal handling of calcium in hypoparathyroidism.

Treatment of hypoparathyroidism usually requires the use of pharmacological doses of parent vitamin D or near physiological amounts of the hydroxylated metabolites, calcitriol or alphacalcidol. Vitamin D intoxication and hypercalcaemia may be a problem but can be minimised by the use of small doses of vitamin D or its metabolites combined with large amounts of oral calcium. The response to treatment can be easily monitored by measuring serum and urinary calcium and creatinine concentrations. This allows the derivation of two simple indices reflecting calcium load presented to the kidney (calcium excretion in mmol/l glomerular filtrate) and renal tubular calcium reabsorption (TmCa/GFR). These can be used to predict the requirement of calcium supplements and also identify those patients at particular risk of hypercalcaemia.     

Rational design and molecular engineering of peptide aptamers to target human pancreatic trypsin in acute pancreatitis

Human pancreatic trypsin (hPT) is an established target for acute pancreatitis (AP) therapeutics. Here, a bioinformatics protocol of protein docking, peptide refinement, dynamics simulation and affinity analysis was described to perform rational design and molecular engineering of hPT peptide aptamers. Protein docking was employed to model the intermolecular interactions between hPT and its cognate inhibitory protein, the human pancreatic trypsin inhibitor (hTI). A number of peptide fragments were cut out from the interaction sites of docked hPT–hTI complexes, from which a decapeptide fragment ¹³LNGCTLEYRP²² was found to exhibit potent inhibition against hPT (K _i = 5.3 ± 0.8 μM). We also carried out alanine scanning and virtual mutagenesis to systematically examine the independent contribution of peptide residues to binding affinity, and the harvested knowledge were then used to guide modification and optimization of the decapeptide fragment. Subsequently, inhibition studies of nine promising candidates against recombinant hPT were conducted, from which four samples were successfully identified to have high or moderate potency (K _i < 10 μM). In particular, the peptides LQVCTLEYCN and LQICTLEYCT were found to inhibit hPT activity significantly (K _i = 0.23 ± 0.04 and 0.85 ± 0.18 μM, respectively). Structural analysis of hPT–peptide complex systems unraveled diverse chemical interactions such as hydrogen bonds, salt bridges and hydrophobic forces across the complex interfaces.     

Possible mechanisms of normal amylase activity in hyperlipemic pancreatitis.

Lipemic serum from three patients with acute pancreatitis and type IV hyperlipemia was fractionated into very-low-density lipoproteins and clear serum. Amylase activity (determined by the Phadebas method) in the component fractions did not exceed that in the original lipemic serum. Addition of these fractions or VLDL and chylomicrons from asymptomatic patients with hyperlipemia to nonlipemic serum from patients with "routine acute pancreatitis" did not inhibit amylase activity or alter the electrophoretic mobility of amylase isoenzymes. Therefore the normal amylase activity often observed in hyperlipemic pancreatitis does not result from an inhibition of amylase activity by serum lipoproteins.     

The role of interleukin-18 in pancreatitis and pancreatic cancer

Originally described as an interferon (IFN)-γ-inducing factor, interleukin (IL)-18 has been reported to be involved in Th1 and Th2 immune responses, as well as in activation of NK cells and macrophages. There is convincing evidence that IL-18 plays an important role in various pathologies (i.e. inflammatory diseases, cancer, chronic obstructive pulmonary disease, Crohn's disease and others). Recently, IL-18 has also been shown to execute specific effects in pancreatic diseases, including acute and chronic pancreatitis, as well as pancreatic cancer. The aim of this study was to give a profound review of recent data on the role of IL-18 and its potential as a therapeutic target in pancreatic diseases. The existing data on this topic are in part controversial and will be discussed in detail. Future studies should aim to confirm and clarify the role of IL-18 in pancreatic diseases and unravel their molecular mechanisms.     

TPS and CA 19-9 measurements in the follow-up of patients with pancreatic cancer and chronic pancreatitis

The aim of this study was to assess the value of TPS and CA 19-9 in a long-term follow-up analysis of 11 patients with chronic pancreatitis (CP) and 15 patients with pancreatic cancer (PC). In all monitored patients with chronic pancreatitis the initial TPS level was below 200 U/L, whereas CA 19-9 was elevated in two of them. In one patient a dramatic increase in the TPS concentration (820 U/L) was measured at the last follow-up visit (after 8.6 months), which led to the detection of PC. In all patients with PC the preoperative TPS level exceeded 200 U/L, whereas CA 19-9 was elevated in only nine patients. After the Kausch-Whipple operation 11 patients showed no evidence of disease and in eight of these patients both TPS and CA 19-9 were within the reference range; however, in three patients liver metastases were detected after 8-24 months from the last tumor marker measurement. In four of the 15 patients both markers were elevated at the end of the follow-up period and distant metastases were clinically confirmed. Our results indicate that in patients with CP and PC undergoing long-term follow-up, TPS reflects the clinical status of patients more accurately than CA 19-9.     

Discrimination of pancreatic cancer and pancreatitis by LC-MS metabolomics

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the fifth most common cause of cancer-related death in Europe with a 5-year survival rate of <5%. Chronic pancreatitis (CP) is a risk factor for PDAC development, but in the majority of cases malignancy is discovered too late for curative treatment. There is at present no reliable diagnostic marker for PDAC available.

Objectives

The aim of the study was to identify single blood-based metabolites or a panel of metabolites discriminating PDAC and CP using liquid chromatography-mass spectrometry (LC-MS).

Methods

A discovery cohort comprising PDAC (n?=?44) and CP (n?=?23) samples was analyzed by LC-MS followed by univariate (Student’s t test) and multivariate (orthogonal partial least squares-discriminant analysis (OPLS-DA)) statistics. Discriminative metabolite features were subject to raw data examination and identification to ensure high feature quality. Their discriminatory power was then confirmed in an independent validation cohort including PDAC (n?=?20) and CP (n?=?31) samples.

Results

Glycocholic acid, N-palmitoyl glutamic acid and hexanoylcarnitine were identified as single markers discriminating PDAC and CP by univariate analysis. OPLS-DA resulted in a panel of five metabolites including the aforementioned three metabolites as well as phenylacetylglutamine (PAGN) and chenodeoxyglycocholate.

Conclusion

Using LC-MS-based metabolomics we identified three single metabolites and a five-metabolite panel discriminating PDAC and CP in two independent cohorts. Although further study is needed in larger cohorts, the metabolites identified are potentially of use in PDAC diagnostics.

     

MTHFR C677T polymorphism in chronic pancreatitis and pancreatic adenocarcinoma

Chronic pancreatitis and pancreatic adenocarcinoma are extensively studied as common and potentially lethal disorders. However, their causes and genetic background in most cases remain unclear. The C677T polymorphism in 5',10'-methylenetetrahydrofolate reductase (MTHFR) gene may modulate the risk of pancreatic disorders. In this study, we tested whether MTHFR C677T polymorphism is associated with chronic pancreatitis and pancreatic adenocarcinoma in the Serbian population. DNA was extracted from blood samples of 51 chronic pancreatitis patients, 21 pancreatic adenocarcinoma patients, and a control group consisting of 50 healthy smokers. The MTHFR C677T polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Although, no statistically significant differences were observed in the distribution of MTHFR genotype or allele frequencies between patients and control groups, the results showed an increased frequency of homozygotes for MTHFR C677T polymorphism in chronic pancreatitis patients (14%) and a decreased frequency in pancreatic adenocarcinoma patients (5%) in comparison to the control group (8%). We speculate that the MTHFR C677T polymorphism could act as a possible risk factor for chronic pancreatitis and a possible protective factor in pancreatic adenocarcinoma. This observation needs further investigation in prospective studies on a larger number of patients, in which the effect of other genetic and environmental factors should also be taken into consideration.     

Purification and characterization of ovine pancreatic alpha - amylase.

Ovine pancreatic amylase has been purified from pancreas homogenate by ammonium sulfate, acetone precipitation, DEAE-cellulose chromatography and finally by specific adsorption on polydextran gel. The enzyme is homogeneous and found as a single form as shown by disc electrophoresis, SDS gel electrophoresis, electrofocusing and ultracentrifugation. Its specific activity is similar to that of porcine amylase. The amino acid composition indicates a high content in aromatic and acidic amino acids as for the porcine enzyme; however the methionine and half cystine content differ widely. The N-terminal end is blocked. Also ovine amylase is glycosylated. The molecular weight (56,000-58,000) is slightly higher than for the porcine enzyme. The isoelectric point is acidic (pI = 3.2).     

Molecular size distribution of immunoreactive trypsin and renal tubular dysfunction: role in trypsin plasma-urine transfer

In order to investigate the role of circulating free trypsinogen and renal tubular dysfunction in affecting trypsin plasma-urine transfer, serum immunoreactive trypsin (IRT), its urinary output, IRT molecular size distribution, filtrable immunoreactive trypsin, gamma-glutamyltransferase and alpha-glucosidase outputs were studied in 6 control subjects, 9 patients with pancreatic cancer and 15 with chronic pancreatitis. The majority of immunoreactivity was always eluted at a molecular weight of about 24,000 and might therefore be considered as free trypsinogen. Variable amounts of IRT at higher molecular weights, possibly represented by trypsin-inhibitor complexes, were also detected. Increasing IRT levels were generally accounted for by free trypsinogen, regardless of the nature of the disease. Unlike serum free trypsinogen levels, renal tubular damage, evaluated by means of the excretion of two high-molecular weight urinary enzymes, seems to play a prominent role in explaining trypsin plasma-urine transfer.