Journal of the Pancreas Open Access

Keywords

Lymph; Pancreatitis, Acute Necrotizing; Proteomics; Rats

Abbreviations

IPI: International Protein Index; LC-MS/MS: liquid chromatography-tandem mass spectrometry; MODS: multiple organ dysfunction syndrome; MS: mass spectrometry; NBF: neutral buffered formalin; XO: xanthine oxidase

INTRODUCTION

Acute pancreatitis is a common inflammatory disease that remains a significant clinical challenge. For the third of patients who develop severe acute pancreatitis, the risk of mortality remains high at 20-30% [1, 2] despite improvements in resuscitation and intensive care support [3, 4]. The mortality is due to multiple organ failure, and this has a bimodal time course distribution. Early deaths, during the first week, are due to a fulminant cytokine mediated systemic inflammatory response syndrome and multiple organ dysfunction (MODS), without an overt septic focus [5]. Later deaths, after 2 or more weeks, are due to MODS associated with infection of necrotic pancreas [6]. Many pathophysiological processes in acute pancreatitis have been described, but the critical factors that drive the MODS have yet to be fully elucidated [1].

There is a body of experimental work, largely derived from rodent studies, suggesting that mesenteric lymph, collected during critical illness, contains toxic factors [7, 8, 9, 10, 11] that contribute to the development of MODS and might be more important than translocated bacteria [12, 13]. Disease conditioned mesenteric lymph is reported to be toxic and associated with neutrophil dysfunction [14, 15], bone marrow suppression [16], and damage to pulmonary epithelial and endothelial cells [17, 18]. Indeed, Magnotti et al. reported that it was disease conditioned mesenteric lymph and not portal venous blood that caused increased endothelial cell permeability and lung injury [19]. This should not be a surprise because the anatomical route of the mesenteric lymph to the subclavian vein via the thoracic duct bypasses the liver. Therefore, unlike portal blood, mesenteric lymph is able to avoid any hepatic first-pass modification or detoxification, and potentially ‘toxic’ factors are instead delivered directly to distant organs (especially the heart and lungs). We have recently reported an increase in the histological severity of experimental acute pancreatitis with the peripheral administration of mesenteric lymph conditioned by mild intestinal ischaemia and reperfusion [1]. Other studies have demonstrated a protection against MODS in a model of hypovolaemic shock when mesenteric lymph is excluded by division [20] or ligation [21] of the main rodent mesenteric lymph duct or by diversion of the thoracic duct [16]. The toxic factors in mesenteric lymph that are responsible for these effects have yet to be identified, although some recent work has suggested the toxic factors are largely carried in the aqueous or protein fraction of mesenteric lymph [8] and that pancreatic enzymes may contribute to the toxicity of disease conditioned mesenteric lymph [10, 22, 23, 24, 25, 26].

We recently published the first comprehensive description of normal rodent mesenteric lymph in the fasted and fed states using the advanced proteomic techniques of isobaric tags (iTRAQTM Reagent Multi- Plex Kit, Applied Biosystems, Foster City, CA, USA) for relative protein quantitation together with LCMS/ MS (liquid chromatography-tandem mass spectrometry) for the identification of the component proteins [27]. The aim of this current study was to use these state-of-the-art proteomic techniques to provide the first comprehensive description of the mesenteric lymph rodent proteome associated with acute pancreatitis, and to determine whether there were any significant increases in the relative abundance of detected proteins compared with sham control mesenteric lymph.

METHODS

Animals

Sixteen inbred male Wistar rats (466±2.9 g; mean±SEM) fed a standard 18% plant protein derived rodent diet (Harlan Teklad 2018, Madison, WI, USA), were randomised to two groups. Group 1 (acute pancreatitis, n=8) had 90 minutes of acute pancreatitis followed by collection of mesenteric lymph for a further 60 minutes. Group 2 (sham control, n=8) had matched interventions and lymph collection to the pancreatitis group but without the induction of acute pancreatitis. In each case the surgery commenced at the same time each day (09:00) and animals were fed ad libitum.

Acute Pancreatitis Model

We used an established model of acute pancreatitis [28, 29, 30, 31]. General anaesthesia was induced by isoflurane (2-5%; 2 L/min O2 via nasal cone). A tracheostomy was inserted (modified 14g angiocath) and connected to a small animal ventilator (Kent Scientific Corporation, Torrington, CT, USA). Balanced general anaesthesia was maintained with isoflurane (2-3.5%) and buprenorphine (0.05 μg/kg, s.c., Temgesic®, Reckitt and Coleman, Hull, England). The fraction of inspired oxygen/air was 40%; the respiratory rate was 50-80 breaths per minute; and the peak inspiratory pressures 11-15 cmH2O kept the expired CO2 at 35-45 mL/L as measured by a capnograph (Pryon Corporation, Menomonee Falls, WI, USA). Body temperature was maintained between 36-38°C by use of a warming plate. Maintenance fluid (0.9% sodium chloride, NaCl) was infused at 1-2 mL/h for the duration of the experiment via a femoral intravenous line. Mean arterial pressure was maintained between 80 and 100 mmHg with the use of intravenous NaCl and monitored using a solid-state 2F pressure transducer (Millar Instruments Inc., Houston, TX, USA) placed in the right femoral artery.

The common pancreatic duct was cannulated with a 24g angiocath passed transduodenally into the pancreato-biliary duct through a 1.5 cm abdominal midline incision. The rostral part of the animal was raised 60° to the horizontal for 5 min to allow the biliary tree to drain (about 0.1 mL). During the last 2 min of this procedure, the common hepatic bile duct was occluded at the hilum of the liver (Biemer atraumatic vascular clip, AESCULAP, Center Valley, PA, USA).

Sodium taurocholate (4% w/v in 0.9% NaCl; 0.1 mL/100 g BW; Sigma Aldrich Pty Ltd., Castle Hill, New South Wales, Australia) was infused at 0.1 mL/min by a controlled infusion pump (Genie Precision Pump, Kent Scientific, Torrington, CT, USA). The Biemer clip and angiocath were removed upon completion of the infusion, and the common pancreatic duct was ligated to prevent reflux of taurocholate into the duodenum.

Severe acute pancreatitis was allowed to develop over a 90-minute period. We chose this relatively early time point and careful control of the blood pressure because we wanted to minimise the risk of hypotension and reflex splanchnic vasoconstriction resulting in an intestinal ischaemia-reperfusion injury that is known to occur during the course of severe acute pancreatitis [32, 33]. An intestinal ischaemia-reperfusion injury could potentially have altered the mesenteric lymph composition and confounded our results.

Collection of Mesenteric Lymph

After 90 minutes elapsed from the induction of pancreatitis, the duodenum and intestines were reflected to the left thus exposing the base of the mesentery. The mesenteric lymph duct was then cleared of surface peritoneum and fat. Silastic tubing (0.96 mm internal diameter, pre-soaked in 70% (v/v) ethanol, rinsed Milli-QTM (Millipore, Billerica, MA, USA) water, 18 MΩ) was drawn through the right posterolateral abdominal wall using a 14g angiocatheter. The mesenteric lymph duct was cannulated with the silastic tube and secured in place with a drop of cyanoacrylate tissue glue (Aesculap Inc., Center Valley, PA, USA). The intestines were then returned to their original position and the abdomen closed. Mesenteric lymph was collected for the following 60 minutes. Collection was performed directly into sterile ice-cold siliconised Eppendorff tubes pre-loaded with protease inhibitors (final: 16.7 μM bestatin, 8.3 μM pepstatin and 5 mM EGTA; Sigma Aldrich Pty Ltd, Castle Hill, New South Wales, Australia). We chose to use Eppendorf tubes preloaded with protease inhibitors to prevent any ex-vivo protein modification of the mesenteric lymph samples. At the end of the experiment the mesenteric lymph was centrifuged (1,700 g, 4°C, 10 min) to remove any cellular material then immediately stored at -80ºC until analysis.

Histology and Assays

At the end of the mesenteric lymph collection (150 minutes from the start of the experimental protocol), animals were euthanised for collection of organs and blood. A 1 cm3 piece of the pancreatic tail was fixed (10% neutral buffered formalin, (NBF)), and histological severity scoring was performed by a blinded consultant histopathologist on 5 μm thick longitudinal paraffin sections using haematoxylin and eosin stain. Pancreatic histology was assessed using a published 5 point scale (from 0=normal to 4=severe) for each of the following criteria: leukocyte infiltration, pancreatic oedema, haemorrhage, fat necrosis, and acinar necrosis for a total score out of 20 [34].

A 5 cm length of small intestine, 20 cm from the caecum, was fixed (10% NBF) and histological severity scoring was performed by a blinded consultant histopathologist on 5 μm thick longitudinal paraffin sections using haematoxylin and eosin stain. The small intestine histology was assessed on a published 6 point scale (from 0, normal to 5, severe) for mucosal injury, inflammation and haemorrhage respectively for a total score out of 15 [35].

Biochemical assays were performed on rodent serum using a Roche/Hitachi MODULAR® analytical system (Roche Diagnostics GmbH, Mannheim, Germany) in accordance with the manufacture’s methods.

Depletion of the Major Proteins in Mesenteric Lymph

IgY immunoaffinity columns were used to deplete the most abundant proteins and enhance the detection of lower abundance proteins [36]. In this study, the expected major abundant proteins of mesenteric lymph (albumin, IgG, fibrinogen, transferrin, alpha1- antitrypsin, and haptoglobin) were depleted using ProteomeLab IgY-R7 affinity spin columns (Beckman Coulter, Fullerton, CA, USA). Each of the samples from the 16 rats was individually depleted. The protein concentration of the mesenteric lymph samples was determined using the EZQ® protein assay (Molecular Probes, Eugene, OR, USA). The depleted samples were concentrated by ultrafiltration using Vivaspin 4 concentrators with a 5 kDa polyethersulfone filter (Sartorius AG, Goettingen, Germany).

LC-MS/MS Based Proteomics

The mesenteric lymph samples underwent LC-MS/MS based proteomics both with and without immunodepletion of the top 6 most abundant proteins. Each sample underwent reduction (incubation of 100 μg protein with 10 mM DTT at 56°C for one hour) and alkylation (incubation with 20 mM iodoacetamide at pH 8.0 in the dark for one hour). Protein was then digested by incubation with 1 μL trypsin (Promega, Madison, WI, USA) at 1 mg/mL and incubated at 37°C overnight. The peptides were then desalted on 10 mg Oasis SPE cartridges (Waters Corporation, Taunton, MA, USA), eluted with 70% acetonitrile and completely dried using a speed vacuum concentrator (Thermo Savant, Holbrook, NY, USA).

iTRAQTM has previously been evaluated and validated against SDS-PAGE and western blotting as a method of tracking relative concentrations of proteins in four different samples [37, 38]. The dried protein digests were reconstituted with 30 μL of dissolution buffer from the iTRAQTM and labelled with iTRAQTM reagents according to the manufacturer’s instructions. Labelled material was then combined, acidified by addition of 10% (v/v) formic acid, concentrated to approximately 200 μL, and then diluted to 2 mL with 0.1% formic acid. This sample was desalted as above, the eluate then concentrated to 100 μL, and finally diluted to 270 μL with 0.1% (v/v) formic acid.

Samples were then fractionated on-line on a BioSCX II 0.3x35 mm column (Agilent Technologies, Santa Clara, CA, USA). A 20 salt-step protocol was performed using 10 μL injections of 10, 20, 40, 60, 70, 80, 90, 100, 110, 120, 130, 140, 160, 180, 200, 220, 240, 260, 400 and 500 mM KCl. Peptides were captured on a 0.3x5 mm PepMap cartridge (LC Packings, Dionex Corporation, Sunnyvale, CA, USA) before being separated on a C18 300SB 0.3x100 mm Zorbax column (Agilent Technologies, Santa Clara, CA, USA). The HPLC gradient between Buffer A (0.1% formic acid in water) and Buffer B (0.1% formic acid in acetonitrile) was formed at 6 μL/min as follows: 10% B for the first 3 min, increasing to 35% B by 80 min, increasing to 95% B by 83 min, held at 95% until 91 min, back to 10% B at 91.5 min and held there until 100 min. The liquid chromatography effluent was directed into the ion spray source of a QSTAR XL hybrid mass spectrometer (Applied Biosystems, Foster City, CA, USA) scanning from 300-1,600 m/z. The three most abundant, multiply-charged peptides were selected for MS/MS analysis (80-1,600 m/z). The mass spectrometer and HPLC system were under the control of the Analyst QS software package (Applied Biosystems, Foster City, CA, USA).

Sequence Database Searches

ProteinPilot (version 1.0, Applied Biosystems, Foster City, CA, USA) [39] was used to search the MS/MS data against the International Protein Index (IPI) Rat database v3.27 (https://www.ebi.ac.uk/IPI/IPIhelp.html) with the following search parameters: Cys alkylation - Iodoacetamide; Digestion - Trypsin; Instrument - QSTAR ESI; Search Effort - Rapid. The data were also searched against the above database using Mascot 2.0.5 software (Matrix Science, London, UK), and a similar set of protein hits obtained (data not shown). Proteins that were identified as potentially hypothetical by the ProteinPilot IPI Rat database v3.27 search were then subjected to a NCBI Basic Local Alignment Search Tool search against the ‘UniProt Clusters 100%’ database (BLAST; https://www.ncbi.nlm.nih.gov/blast/).

Validation of Protein Identifications

A search of the IPI Rat database v3.27 with the reversed amino acid sequence of each entry was carried out to determine the minimum required ProteinPilot score for the proteins that would yield an overall confidence greater than 97%. Protein matches were considered valid if their ProteinPilot scores were equal to or above the minimum required score for each run.

Validation of Protein Changes

After immunodepletion and LC-MS/MS, only small volumes (10-20 μL per animal) of the original mesenteric lymph samples were available for crossvalidation of protein changes reported by iTRAQTM LC-MS/MS. Despite the small sample volumes, we were able to measure albumin, pancreatic amylase, and lipase in mesenteric lymph using commercial reagents (Pointe Scientific, Canton, MI, USA) on a COBAS MIRA analyser (Roche, Basel, Switzerland) in accordance with the manufacturer’s instructions.

STATISTICS

Protein abundance was calculated from the peptide summary data generated by ProteinPilot. Strict criteria were applied when calculating the protein abundance from peptide data - peptides identified as belonging to more than one protein were eliminated, and any spectra below the confidence threshold set by ProteinPilot were also eliminated. The remaining peak areas were log-transformed and, for each sample, average log peak areas were calculated from the spectra within each reporter region for every identified protein. Differences in relative abundance were calculated as differences in log peak areas (acute pancreatitis - sham) and reported as fold differences between the two.

The statistical analysis was carried out using the LIMMA package v2.9.17 [40] in the R software v2.6.1 (R Development Core Team, 2007) [41]. The analysis for differential expression was performed on a proteinby- protein basis using a linear model that included run, label and treatment effects. A moderated t-statistic, in which the standard errors were moderated across proteins using a Bayesian model, was used for the significance analysis [42]. P values were adjusted for multiple testing using Benjamini and Hochberg’s false discovery rate setting the expected proportion of false discoveries to 5% [43]. Changes in protein abundance with an adjusted P value less than 0.05 were considered significant.

For non-proteomic data, such as histology scores and biochemical parameters, the non-parametric Mann- Whitney U test was used to derive statistical significance and two-tailed P value less than 0.05 was considered significant.

Bioinformatics

Proteins that were found to have a statistically significant difference in abundance between sham and acute pancreatitis conditioned mesenteric lymph were then further analysed for functional and biological relevance. With the help of Gaggle [44], an opensource Java software environment, and Gene Ontology (GO) provided free by the Gene Ontology Consortium (https://www.geneontology.org/index.shtml) [45], these proteins were classified by their molecular function and cellular location.

ETHICS

This study was approved by the University of Auckland Animal Ethics Committee. All animals received humane care in keeping with the “Guide for Care and Use of Laboratory Animals (1996)” prepared by the National Academy of Sciences.

RESULTS

Acute Pancreatitis Model

The taurocholate model produced acute pancreatitis with the expected elevation in serum amylase (3,829±302 U/L vs. 2,053±81 U/L; sham vs. acute pancreatitis, respectively; P<0.001) and serum lipase (4,377±1,088 U/L vs. 310±61 U/L; sham vs. acute pancreatitis, respectively; P<0.001) (Table 1). The histology of the pancreas confirmed severe acute pancreatitis (Table 1). There was no significant difference between the two groups for histology of the intestine.

Mesenteric Lymph Proteomics

There were 245 proteins identified in mesenteric lymph from all the mass spectrometry runs (with and without depletion) that met the validity criteria. All of the proteins were identified in both experimental groups, and there were no proteins unique to either experimental group. A non-redundant list of these proteins is provided (Supplementary Table 1).

Forty-seven of the 245 identified proteins (19.2%) were listed as potentially hypothetical proteins according to the International Protein Index (Rat database v3.27). These proteins were then subjected to a NCBI BLASTP analysis. Thirty-five proteins (14.3%) were confirmed as hypothetical but 12 proteins were not, being identical to other rat proteins (Supplementary Table 2). Nine of these 35 proteins were previously identified in a recent proteomic study of normal mesenteric lymph [27] but continue to be listed as hypothetical according to the International Protein Index (Rat database v3.27) (Supplementary Table 2).

Prior to the immunoaffinity depletion, the 6 proteins removed by this process (albumin, fibrinogen, transferrin, alpha1-antitrypsin, IgG and haptoglobin) were investigated using mass spectrometry data and there were no statistically significant differences between the two experimental groups (Supplementary Table 1).

There was a statistically significant increase in the relative abundance of 8 proteins in the mesenteric lymph of the acute pancreatitis experimental group after immunoaffinity depletion (Table 2). An additional 2 proteins (hemoglobin beta chain complex and phosphoglycerate mutase 1) had changes in their relative abundance that approached significance (P<0.05 and an adjusted P<0.10).

The 8 proteins that were significantly increased in acute pancreatitis conditioned mesenteric lymph were then classified by their cellular location and molecular function using the Gene Ontology classification system. Seven of the proteins were extracellular pancreatic enzymes and one was cytosolic (Table 2). In regards to their molecular function, all 8 were catabolic enzymes and 4 were also ion binding (Table 2). Of the 7 extracellular pancreatic catabolic enzymes, four had peptidase activity (carboxypeptidase B1, chymotrypsinogen B, carboxypeptidase A2 and cationic trypsinogen), one had ester hydrolase activity (pancreatic lipase), one had endoribonuclease activity (ribonuclease), and one acted on glycosyl bonds (pancreatic amylase 2).

The results of the specific biochemical assays performed for albumin, pancreatic amylase and lipase in mesenteric lymph are consistent with the LCMS/ MS findings. Albumin was not different between the two groups (mean±SEM; 14.7±2.8 g/L vs. 14.0±1.8 g/L; acute pancreatitis vs. sham, respectively; P=0.867) while both pancreatic amylase (7,024±2,079 U/L vs. 901±196 U/L; acute pancreatitis vs. sham, respectively; P<0.001) and lipase (1,424±367 U/L vs. 272±132 U/L acute pancreatitis vs. sham, respectively; P=0.036) were significantly increased

DISCUSSION

This study provides the first comprehensive description of the changes that occur in the proteome of mesenteric lymph during acute pancreatitis. A total of 245 proteins were identified in mesenteric lymph using strict acceptance criteria and with greater than 97% confidence. All identified proteins were present in both the acute pancreatitis and sham control groups. There were 8 proteins that were significantly more abundant in acute pancreatitis conditioned mesenteric lymph. All 8 of these proteins were catabolic enzymes with 7 being secreted pancreatic catabolic enzymes. Also identified in the mesenteric lymph of both groups were 35 hypothetical proteins.

Severe acute pancreatitis is associated with hypotension and reflex splanchnic vasoconstriction resulting an intestinal ischaemia-reperfusion injury [32, 33]. It has previously been hypothesized that pancreatic enzymes which are normally present in the intestinal lumen in high concentration may be able to pass through a compromised intestinal barrier and cause remote organ injury [22, 46]. In the current study, we controlled the mean arterial pressure to help prevent ischaemic injury of the intestine, and this was confirmed by the normal intestinal histology scores in the acute pancreatitis group. Thus, we show for the first time that high levels of several pancreatic catabolic enzymes are present in acute pancreatitis conditioned mesenteric lymph early in disease process in the presence of normal intestinal histology.

A strength of this study is the use of state-of-the-art iTRAQTM LC-MS/MS techniques used to define the proteome of acute pancreatitis conditioned mesenteric lymph. Previous studies have used enzyme specific biochemical methods to identify amylase, lipase and trypsin in the thoracic duct lymph of animals [47] and humans [7, 26] with acute pancreatitis. In addition to confirming the presence of these three catabolic enzymes, we report for the first time the presence of ribonuclease 1, carboxypeptidase B1, chymotrypsinogen B and carboxypeptidase A2 in acute pancreatitis conditioned mesenteric lymph. The most abundant protein class identified in normal mesenteric lymph was previously reported to be protease inhibitors [27]. It is striking that despite a substantial increase in the relative abundance of proteases in acute pancreatitis conditioned mesenteric lymph identified here, there was no concomitant rise in relative abundance of protease inhibitors.

A study published in 2008 by Mole et al. used two different proteomic techniques to investigate acute pancreatitis conditioned mesenteric lymph [9]. The SELDI-TOF (surface-enhanced laser desorption - ionization time-of-flight mass spectrometry) technique generated spectra that differentiated acute pancreatitis conditioned mesenteric lymph from sham mesenteric lymph, but this could not perform identification of individual proteins [48]. In a separate experiment they used 2D-PAGE (two-dimensional gel electrophoresis) to identify just 4 proteins (transferrin, haptoglobin, alpha1-protease inhibitor and apolipoprotein A1) that showed a relative increase or decrease in acute pancreatitis conditioned mesenteric lymph. Using these techniques it was not possible for Mole et al. to demonstrate any numerical fold change data or statistical measures of significance. If immunodepletion had been used to deplete the major abundant proteins prior to 2D-PAGE it might have been possible to achieve a higher level of resolution and identification of additional protein changes. Another limitation of the study by Mole et al. [9] is that mean arterial pressure was not controlled. This raises the possibility that the reported proteomic changes in acute pancreatitis conditioned mesenteric lymph might have been due, at least in part, to concomitant hypotension and intestinal ischaemia.

Pancreatic enzymes contribute to the development of distant organ injury and MODS by the proteolytic cleavage of cellular membranes and extracellular proteins, and by activating leucocytes to generate reactive oxygen species (ROS) [49, 50, 51, 52, 53]. Pancreatic proteases are also thought to contribute to the generation of ROS by the limited proteolytic conversion of the enzyme xanthine dehydrogenase to xanthine oxidase (XO) [54]. In the oxidase form, this enzyme produces the superoxide anion and thus generates ROS [55]. It is recognized that pancreatic proteases are not the only factor responsible for the development of MODS in acute pancreatitis. Pancreatic amylase has also been implicated as a potentially toxic factor. It is now thought that high levels of pancreatic amylase disrupt the binding of tissue XO by hydrolyzing the internal alpha1-4 linkages of some of the glycoproteins present in the extracellular space. Once mobilized, XO is able to concentrate in distant organs with low intrinsic XO activity and produce ROS contributing to organ dysfunction [56].

There is evidence that pancreatic enzymes contribute to the toxicity of mesenteric lymph in acute pancreatitis and other critical illnesses. In a human study of severe acute pancreatitis, diversion of trypsin rich thoracic duct lymph reduced lung injury [26]. In the setting of haemorrhage it was found in animal models that shock conditioned mesenteric lymph caused neutrophil dysfunction [14, 15], bone marrow suppression [16], and damage to endothelial cells of the pulmonary microvasculature [17, 18]. These effects were prevented by either ligation of the pancreatic duct prior to the induction of shock [10, 22] or by the intraintestinal inhibition of pancreatic serine proteases [23, 24, 25, 50] thereby implicating pancreatic proteases as toxic factors in mesenteric lymph.

The findings of this study support the proposal that pancreatic catabolic enzymes in mesenteric lymph during acute pancreatitis could be therapeutic targets. The history of intravenous anti-protease treatment in acute pancreatitis, using gabexate and nafamostat, is disappointing [57]. After more than 70 clinical trials and several meta-analyses, there is no convincing evidence to recommend the use of intravenous protease inhibition in acute pancreatitis [58]. Of the 16 recently published clinical guidelines there are only two, from Japan and China, that recommend the use of intravenous protease inhibitors [58, 59], and not on the basis of high level evidence. The findings of the present study would suggest that protease inhibition might be more effective if given by the enteral rather than the intravenous route, especially if it were lymphotropic and concentrated in mesenteric lymph. To our knowledge, there is only one clinical trial that investigated the use of oral protease inhibition (FOY 305) in acute pancreatitis [60] and showed significant improvement in abdominal pain scores and urinary amylase in the treatment arm. Further studies to evaluate the efficacy of protease inhibition delivered by the enteral route appear to be justified in this context. There have been previous reports of enteral protease inhibitor treatment improving haemodynamic parameters, reducing intestinal injury and leukocyte activation in models of intestinal ischaemia-reperfusion injury [24, 25] and septic shock [61].

One of the challenges of proteomics is that high abundance proteins mask low abundance proteins when compositional analysis is attempted. This problem was addressed in the present study by the immunodepletion of the highly abundant proteins using a validated method [36, 62, 63, 64, 65]. Unfortunately, unintentional protein loss inevitably occurs during immunodepletion because of non-specific binding to the column, specific binding to immunoglobulin with structural homology to the proteins being depleted, and/or binding to the proteins that are being depleted [36, 62]. Given the modest amount of lymph that can be collected from a rat during the experimental protocol (750-1,000 μL), only 10-20 μL of the original sample is left after immunodepletion and LC-MS/MS for further evaluation thus prohibiting further analyses by complementary gel-based proteomic methods. The protein fraction of mesenteric lymph is unlikely to contain all of the factors responsible for the toxicity found in acute pancreatitis and other critical illnesses, although it has been found to be more toxic than the lipid fraction [8]. Delineating the composition of the lipid fraction of acute pancreatitis conditioned mesenteric lymph is the focus of further studies.

CONCLUSION

This is the first comprehensive description of the proteome of mesenteric lymph conditioned by acute pancreatitis. It has demonstrated a significant increase in the relative abundance of 8 proteins amongst the 245 proteins identified using state-of-the-art iTRAQTM based LC-MS/MS techniques, 7 of which are secreted pancreatic catabolic enzymes. This study provides a clear rationale for further research to investigate the efficacy of enteral protease inhibitors in the treatment of acute pancreatitis.

Grant support

Royal Australasian College of Surgeons, University of Auckland Research Council, Health Research Council of New Zealand

Financial disclosures

None

References

1. Flint RS, Phillips AR, Power SE, Dunbar PR, Brown C, Delahunt B, et al. Acute pancreatitis severity is exacerbated by intestinal ischemia-reperfusion conditioned mesenteric lymph. Surgery 2008; 143:404-13. [PMID 18291262]
2. McKay CJ, Imrie CW. The continuing challenge of early mortality in acute pancreatitis. Br J Surg 2004; 91:1243-4. [PMID 15382103]
3. Neoptolemos JP, Raraty M, Finch M, Sutton R. Acute pancreatitis: the substantial human and financial costs. Gut 1998; 42:886-91. [PMID 9691932]
4. Flint R, Windsor J, Bonham M. Trends in the management of severe acute pancreatitis: interventions and outcome. ANZ J Surg 2004; 74:335-42. [PMID 15144253]
5. Davies MG, Hagen PO. Systemic inflammatory response syndrome. Br J Surg 1997; 84:920-35. [PMID 9240130]
6. Vege SS, Chari ST. Organ failure as an indicator of severity of acute pancreatitis: time to revisit the Atlanta classification. Gastroenterology 2005; 128:1133-5. [PMID 15825098]
7. Fanous MY, Phillips AJ, Windsor JA. Mesenteric lymph: the bridge to future management of critical illness. JOP. J Pancreas (Online) 2007; 8:374-99. [PMID 17625290]
8. Dayal SD, Hauser CJ, Feketeova E, Fekete Z, Adams JM, Lu Q, et al. Shock mesenteric lymph-induced rat polymorphonuclear neutrophil activation and endothelial cell injury is mediated by aqueous factors. J Trauma 2002; 52:1048-55. [PMID 12045629]
9. Mole DJ, McFerran NV, Collett G, O'Neill C, Diamond T, Garden OJ, et al. Tryptophan catabolites in mesenteric lymph may contribute to pancreatitis-associated organ failure. Br J Surg 2008; 95:855-67. [PMID 18473343]
10. Caputo FJ, Rupani B, Watkins AC, Barlos D, Vega D, Senthil M, Deitch EA. Pancreatic duct ligation abrogates the trauma hemorrhage-induced gut barrier failure and the subsequent production of biologically active intestinal lymph. Shock 2007; 28:441-6. [PMID 17558354]
11. Berezina TL, Zaets SB, Mole DJ, Spolarics Z, Deitch EA, Machiedo GW. Mesenteric lymph duct ligation decreases red blood cell alterations caused by acute pancreatitis. Am J Surg 2005; 190:800-4. [PMID 16226961]
12. Adams CA Jr, Xu DZ, Lu Q, Deitch EA. Factors larger than 100 kd in post-hemorrhagic shock mesenteric lymph are toxic for endothelial cells. Surgery 2001; 129:351-63. [PMID 11231464]
13. Deitch EA. Bacterial translocation or lymphatic drainage of toxic products from the gut: what is important in human beings? Surgery 2002; 131:241-4. [PMID 11894026]
14. Caruso JM, Feketeova E, Dayal SD, Hauser CJ, Deitch EA. Factors in intestinal lymph after shock increase neutrophil adhesion molecule expression and pulmonary leukosequestration. J Trauma 2003; 55:727-33. [PMID 14566130]
15. Adams JM, Hauser CJ, Adams CA Jr, Xu DZ, Livingston DH, Deitch EA. Entry of gut lymph into the circulation primes rat neutrophil respiratory burst in hemorrhagic shock. Crit Care Med 2001; 29:2194-8. [PMID 11700422]
16. Deitch EA, Forsythe R, Anjaria D, Livingston DH, Lu Q, Xu DZ, Redl H. The role of lymph factors in lung injury, bone marrow suppression, and endothelial cell dysfunction in a primate model of trauma-hemorrhagic shock. Shock 2004; 22:221-8. [PMID 15316391]
17. Deitch EA, Adams CA, Lu Q, Xu DZ. Mesenteric lymph from rats subjected to trauma-hemorrhagic shock are injurious to rat pulmonary microvascular endothelial cells as well as human umbilical vein endothelial cells. Shock 2001; 16:290-3. [PMID 11580112]
18. Lu Q, Xu DZ, Davidson MT, Haskó G, Deitch EA. Hemorrhagic shock induces endothelial cell apoptosis, which is mediated by factors contained in mesenteric lymph. Crit Care Med 2004; 32:2464-70. [PMID 15599152]
19. Magnotti LJ, Upperman JS, Xu DZ, Lu Q, Deitch EA. Gutderived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg 1998; 228:518-27. [PMID 9790341]
20. Adams CA Jr, Hauser CJ, Adams JM, Fekete Z, Xu DZ, Sambol JT, Deitch EA. Trauma-hemorrhage-induced neutrophil priming is prevented by mesenteric lymph duct ligation. Shock 2002; 18:513-7. [PMID 12462558]
21. Sambol JT, Xu DZ, Adams CA, Magnotti LJ, Deitch EA. Mesenteric lymph duct ligation provides long term protection against hemorrhagic shock-induced lung injury. Shock 2000; 14:416-9. [PMID 11028566]
22. Cohen DB, Magnotti LJ, Lu Q, Xu DZ, Berezina TL, Zaets SB, et al. Pancreatic duct ligation reduces lung injury following trauma and hemorrhagic shock. Ann Surg 2004; 240:885-91. [PMID 15492572]
23. Deitch EA, Shi HP, Lu Q, Feketeova E, Xu DZ. Serine proteases are involved in the pathogenesis of trauma-hemorrhagic shockinduced gut and lung injury. Shock 2003; 19:452-6. [PMID 12744489]
24. Fitzal F, DeLano FA, Young C, Rosario HS, Schmid-Schönbein GW. Pancreatic protease inhibition during shock attenuates cell activation and peripheral inflammation. J Vasc Res 2002; 39:320-9. [PMID 12187122]
25. Fitzal F, DeLano FA, Young C, Schmid-Schönbein GW. Improvement in early symptoms of shock by delayed intestinal protease inhibition. Arch Surg 2004; 139:1008-16. [PMID 15381622]
26. Dugernier T, Reynaert MS, Deby-Dupont G, Roeseler JJ, Carlier M, Squifflet JP, et al. Prospective evaluation of thoracic-duct drainage in the treatment of respiratory failure complicating severe acute pancreatitis. Intensive Care Med 1989; 15:372-8. [PMID 2553789]
27. Mittal A, Middleditch M, Ruggiero K, Buchanan CM, Jullig M, Loveday B, et al. The proteome of rodent mesenteric lymph. Am J Physiol Gastrointest Liver Physiol 2008; 295:G895-903. [PMID 18772360]
28. Zhang XP, Ye Q, Jiang XG, Ma ML, Zhu FB, Zhang RP, Cheng QH. Preparation method of an ideal model of multiple organ injury of rat with severe acute pancreatitis. World J Gastroenterol 2007; 13:4566-73. [PMID 17729407]
29. Aho HJ, Nevalainen TJ, Aho AJ. Experimental pancreatitis in the rat. Development of pancreatic necrosis, ischemia and edema after intraductal sodium taurocholate injection. Eur Surg Res 1983; 15:28-36. [PMID 6840152]
30. Aho HJ, Koskensalo SM, Nevalainen TJ. Experimental pancreatitis in the rat. Sodium taurocholate-induced acute haemorrhagic pancreatitis. Scand J Gastroenterol 1980; 15:411-6. [PMID 7433903]
31. Mittal A, Flint RJ, Fanous M, Delahunt B, Kilmartin PA, Cooper GJ, et al. Redox status of acute pancreatitis as measured by cyclic voltammetry: initial rodent studies to assess disease severity. Crit Care Med 2008; 36:866-72. [PMID 18431274]
32. Flint R, Windsor J. The role of the intestine in the pathophysiology and management of severe acute pancreatitis. HPB (Oxford) 2003; 5:69-85. [PMID 18332961]
33. Sakagami J, Kataoka K, Sogame Y, Usui N, Mitsuyoshi M. Ultrasonographic splanchnic arterial flow measurement in severe acute pancreatitis. Pancreas 2002; 24:357-64. [PMID 11961488]
34. Schmidt J, Lewandrowski K, Fernandez-del Castillo C, Mandavilli U, Compton CC, Warshaw AL, Rattner DW. Histopathologic correlates of serum amylase activity in acute experimental pancreatitis. Dig Dis Sci 1992; 37:1426-33. [PMID 1380425]
35. Lane JS, Todd KE, Lewis MP, Gloor B, Ashley SW, Reber HA, et al. Interleukin-10 reduces the systemic inflammatory response in a murine model of intestinal ischemia/reperfusion. Surgery 1997; 122:288-94. [PMID 9288134]
36. Huang L, Harvie G, Feitelson JS, Gramatikoff K, Herold DA, Allen DL, et al. Immunoaffinity separation of plasma proteins by IgY microbeads: meeting the needs of proteomic sample preparation and analysis. Proteomics 2005; 5:3314-28. [PMID 16041669]
37. Wiese S, Reidegeld KA, Meyer HE, Warscheid B. Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research. Proteomics 2007; 7:340-50. [PMID 17177251]
38. Aggarwal K, Choe LH, Lee KH. Shotgun proteomics using the iTRAQ isobaric tags. Brief Funct Genomic Proteomic 2006; 5:112- 20. [PMID 16772272]
39. Kersey PJ, Duarte J, Williams A, Karavidopoulou Y, Birney E, Apweiler R. The International Protein Index: an integrated database for proteomics experiments. Proteomics 2004; 4:1985-8. [PMID 15221759]
40. Smyth GK. Limma: linear models for microarray data. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor:397-420. Gentleman R, Carey V, Dudoit S, Irizarry R, Huber W (eds), Springer, New York.
41. Ihaka R, Gentleman R. R: A language for data analysis and graphics. J Comput Graph Stat 1996; 5:299-314.
42. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 2004; 3:Article3. [PMID 16646809]
43. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Statist Soc Ser B (Methodological) 1995; 57:289-300.
44. Shannon PT, Reiss DJ, Bonneau R, Baliga NS. The Gaggle: an open-source software system for integrating bioinformatics software and data sources. BMC Bioinformatics 2006; 7:176. [PMID 16569235]
45. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25-9. [PMID 10802651]
46. Schmid-Schönbein GW, Hugli TE. A new hypothesis for microvascular inflammation in shock and multiorgan failure: selfdigestion by pancreatic enzymes. Microcirculation 2005; 12:71-82. [PMID 15804975]
47. Sim DN, Duprez A, Anderson MC. Alterations of the lymphatic circulation during acute experimental pancreatitis. Surgery 1966; 60:1175-82. [PMID 5926539]
48. Cho WC. Research progress in SELDI-TOF MS and its clinical applications. Sheng Wu Gong Cheng Xue Bao (Chinese Journal of Biotechnology) 2006; 22:871-6. [PMID 17168305]
49. Kistler EB, Hugli TE, Schmid-Schönbein GW. The pancreas as a source of cardiovascular cell activating factors. Microcirculation 2000; 7:183-92. [PMID 10901497]
50. Mitsuoka H, Kistler EB, Schmid-Schönbein GW. Protease inhibition in the intestinal lumen: attenuation of systemic inflammation and early indicators of multiple organ failure in shock. Shock 2002; 17:205-9. [PMID 11900339]
51. Schmid-Schönbein GW, Hugli TE, Kistler EB, Sofianos A, Mitsuoka H. Pancreatic enzymes and microvascular cell activation in multiorgan failure. Microcirculation 2001; 8:5-14. [PMID 11296853]
52. Mitsuoka H, Schmid-Schönbein GW. Mechanisms for blockade of in vivo activator production in the ischemic intestine and multiorgan failure. Shock 2000; 14:522-7. [PMID 11092684]
53. Mitsuoka H, Kistler EB, Schmid-Schonbein GW. Generation of in vivo activating factors in the ischemic intestine by pancreatic enzymes. Proc Natl Acad Sci U S A 2000; 97:1772-7. [PMID 10677533]
54. Closa D, Bulbena O, Hotter G, Roselló-Catafau J, Fernández- Cruz L, Gelpí E. Xanthine oxidase activation in cerulein- and taurocholate-induced acute pancreatitis in rats. Arch Int Physiol Biochim Biophys 1994; 102:167-70. [PMID 7528065]
55. Mittal A, Phillips AR, Loveday B, Windsor JA. The potential role for xanthine oxidase inhibition in major intra-abdominal surgery. World J Surg 2008; 32:288-95. [PMID 18074171]
56. Granell S, Bulbena O, Genesca M, Sabater L, Sastre J, Gelpi E, Closa D. Mobilization of xanthine oxidase from the gastrointestinal tract in acute pancreatitis. BMC Gastroenterol 2004; 4:1. [PMID 14728722]
57. Singh VP, Chari ST. Protease inhibitors in acute pancreatitis: lessons from the bench and failed clinical trials. Gastroenterology 2005; 128:2172-4. [PMID 15940654]
58. Kitagawa M, Hayakawa T. Antiproteases in the treatment of acute pancreatitis. JOP. J Pancreas (Online) 2007; 8(4 Suppl):518-25. [PMID 17625309]
59. Loveday BP, Mittal A, Phillips A, Windsor JA. Minimally invasive management of pancreatic abscess, pseudocyst, and necrosis: a systematic review of current guidelines. World J Surg 2008; 32:2383-94. [PMID 18670801]
60. Tanaka N, Tsuchiya R, Ishii K. Comparative clinical study of FOY and Trasylol in acute pancreatitis. Adv Exp Med Biol 1979; 120B:367-78. [PMID 229707]
61. Fitzal F, Delano FA, Young C, Rosario HS, Junger WG, Schmid-Schönbein GW. Pancreatic enzymes sustain systemic inflammation after an initial endotoxin challenge. Surgery 2003; 134:446-56. [PMID 14555932]
62. Brand J, Haslberger T, Zolg W, Pestlin G, Palme S. Depletion efficiency and recovery of trace markers from a multiparameter immunodepletion column. Proteomics 2006; 6:3236-42. [PMID 16645986]
63. Ogata Y, Charlesworth MC, Higgins L, Keegan BM, Vernino S, Muddiman DC. Differential protein expression in male and female human lumbar cerebrospinal fluid using iTRAQ reagents after abundant protein depletion. Proteomics 2007; 7:3726-34. [PMID 17853512]
64. Liu X, Valentine SJ, Plasencia MD, Trimpin S, Naylor S, Clemmer DE. Mapping the human plasma proteome by SCX-LCIMS- MS. J Am Soc Mass Spectrom 2007; 18:1249-64. [PMID 17553692]
65. Hu S, Loo JA, Wong DT. Human body fluid proteome analysis. Proteomics 2006; 6:6326-53. [PMID 17083142]