ToxSci Advance Access originally published online on December 14, 2006
Toxicological Sciences 2007 96(1):184-193; doi:10.1093/toxsci/kfl190
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Evaluation of Two Novel Peptide Safety Markers for Exocrine Pancreatic Toxicity
Pfizer Global Research and Development, Drug Safety Research and Development, Chesterfield, Missouri 63017
1 To whom correspondence should be addressed at Pfizer Global Research and Development, Drug Safety Research and Development, 700 Chesterfield Parkway W, Mail Zone T1A, Chesterfield, Missouri 63017. Fax: (314) 274-4426. E-mail: jennie.walgren{at}pfizer.com.
Received August 29, 2006; accepted November 29, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Current markers of exocrine pancreatic toxicity have historically been poor indicators for both early diagnosis of disease and prediction of disease severity. Recently we identified two peptide markers (RA1609 and RT2864) of pancreatic toxicity that are target organ specific. In order to evaluate sensitivity of these markers versus current standard tests for pancreatic damage (i.e., lipase), we measured amylase and lipase, as well as RA1609 and RT2864 marker levels, in serum from rats treated with four doses (50–200 mg/kg) of the model pancreatic toxicant cyanohydroxybutene (CHB). In addition, to determine whether these peptide markers could detect pancreatic injury induced by different toxicants and in different species, we measured RA1609 and RT2864 marker levels in rats treated with the pancreatic toxicant caerulein, and in mice treated with CHB. RA1609 and RT2864 peptide markers proved to be more sensitive than amylase or lipase in detecting pancreatic damage, especially at an early time point (8 h) following CHB administration. The peptide markers also accurately predicted pancreatic injury induced by caerulein in rats. These markers were sensitive in detecting very mild pancreatic damage following CHB administration in mice, which are less susceptible to CHB-induced pancreatic toxicity. In addition, a species comparison of the RA1609 albumin fragment sequence indicated that cleavage of albumin from pancreatic proteases produces a similar fragment marker in several species, including humans. To determine whether the comparable human albumin fragment could be detected in sera from pancreatitis patients, we analyzed sera from normal individuals and from patients with diabetes, vasculitis, pancreatic cancer, and pancreatitis. It was found that markers corresponding to the fragments found in rat serum (RA1609 and RT2864) were present in human serum, and changes in these were indicative of and specific to pancreatitis. In conclusion, the RA1609 and RT2864 peptides are sensitive indicators of exocrine pancreatic damage that may be useful as safety markers for general pancreatic toxicity in multiple species.
Key Words: Pancreatitis; human; CHB; caerulein; SELDI; safety biomarkers.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exocrine pancreatic injury induces zymogen depletion from pancreatic acinar cells and can result in inflammation and autodigestion of the pancreas and other surrounding organs. Patients with pancreatic disease such as acute pancreatitis often experience severe abdominal pain resulting from the local and/or systemic inflammatory processes. The most common causes of acute pancreatitis in humans are alcoholism and gallstones (Vlodov and Tenner, 2001
). There are, however, several drugs or drug classes that have been implicated in the development of exocrine pancreatic injury as well. These include azathioprine, estrogens, furosemide, methyldopa, pentamidine, procainamide, sulfonamides, and thiazide diuretics (Scarpelli, 1989; Vlodov and Tenner, 2001). The ability to monitor for pancreatic injury both in drug development and in the clinic has often been a difficult issue due to the poor predictive ability of markers currently available for acute pancreatitis. The markers most commonly used to diagnose pancreatitis include serum amylase and serum lipase levels; a level two to three times the upper limit of normal indicates a pancreatic attack. Unfortunately, amylase is not specific to pancreatic injury, neither marker is always reliable at early stages of the disease, and their magnitude is not indicative of disease severity.
The earliest events in the development of acute pancreatitis are premature activation of proteases and the development of localized edema (Lerch and Gorelick, 2000; Vlodov and Tenner, 2001). It has been demonstrated in both in vitro and in vivo studies that trypsinogen activation precedes acinar cell injury (Saluja et al., 1999) and that protease inhibitors such as trypsin (Saluja et al., 1999) or elastase (Yamano et al., 1998) inhibitors can protect against cell injury (Saluja et al., 1999) and edema and hemorrhage in pancreatitis (Yamano et al., 1998). Clinical studies as well as studies in animal models have shown that markers tied to trypsin activation, such as urinary trypsin activation peptide, have very good diagnostic capability in the first 48 h following acute pancreatitis (Frossard, 2001; Wang et al., 2001). In addition, related markers such as serum trypsinogen-2 and urinary pancreatic secretory trypsin inhibitor have shown some promise in prognosis of disease severity (Lempinen et al., 2002). Finding serum markers which are tied to these initiating events may be helpful in identifying early markers of pancreatic injury that coincide with acinar cell damage. Such markers would be beneficial clinically in predicting endoscopic retrograde cholangiopancreatography–induced pancreatitis and also could serve as safety markers both in preclinical and clinical studies.
In previous studies we identified two peptide biomarkers (RA1609 and RT2864) in sera from rats treated with the pancreatic toxicant cyanohydroxybutene (CHB) that result from the cleavage of serum albumin and trypsin during trypsin digest incubations of sera from animals with pancreatic injury (Walgren, Mitchell, Whiteley, and Thompson manuscript in review). These peptides therefore appear to be pancreas specific and tied to enzyme release during acinar cell degranulation, and should be applicable to general exocrine pancreatic damage across species. The following studies further characterize the potential usefulness of these two biomarkers in detecting pancreatic damage from caerulein-induced pancreatitis in rats. Finally, the ability of these two biomarkers to detect pancreatic damage in multiple species, including mouse and human, was assessed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials
Male Sprague-Dawley IGS rats and male Swiss Webster mice were purchased from Charles River Laboratories (Wilmington, MA). CHB was synthesized by Gateway Chemical Technologies (St Louis, MO). Collagenase Type 4 was purchased from Worthington Biochemical (Lakewood, NJ). Ion exchange (Q) columns and ProteinChips were purchased from Ciphergen Biosystems (Fremont, CA). Sequencing grade trypsin was obtained from Roche Diagnostics (Indianapolis, IN). Except where noted, all other chemicals were obtained from Sigma Chemical Company (St Louis, MO) and were of the highest purity available. Human sera from normal, diabetic, and pancreatic cancer patients were obtained from Clinomics BioSciences, Inc. (Pittsfield, MA). Diabetic samples came from a mixture of type I and type II diabetics, and all pancreatic cancer samples were obtained from patients with stage 4 pancreatic adenocarcinoma. Human sera samples from patients diagnosed with pancreatitis or vasculitis were obtained from the University of Michigan in a collaborative agreement in Institutional Review Board–approved studies.
In Vivo Studies
Use of the animals in the following studies was reviewed and approved by the Pfizer Institutional Animal Care and Use Committee. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International.
Rat studies.
Male Sprague-Dawley IGS rats between 6 and 8 weeks of age were housed in a controlled environment with 12-h light/dark cycle and were provided with water and standard lab chow ad libitum. The animals were fasted 14–16 h prior to dosing. For animals in all studies, three sections of pancreas from different regions (head, body, and tail) were collected and fixed in formalin for histopathological examination and the remainder of the pancreas was frozen for proteomic analyses.
Study A. Caerulein-induced pancreatitis in rats. To induce pancreatitis, caerulein is generally administered at doses from 5 to 50 µg/kg by infusion or multiple hourly injections over a period up to 6 h (Jaworek et al., 2004; Neuschwander-Tetri et al., 1992; Robert et al., 1989). We chose a moderate dose of a saline suspension of caerulein (15 µg/kg) which was administered in three doses, 1 h apart, by intraperitoneal injection. Eight control animals received intraperitoneal injection of saline. At 8-h postdose, blood was collected from the tail vein of animals anesthetized with CO2. At the scheduled euthanasia at 24-h postdosing, additional samples of serum were collected from the tail vein and inferior vena cava of animals anesthetized with CO2.
Study B. CHB dose-response study. In groups of eight animals each, animals were administered a single dose of saline or a saline suspension of CHB (50, 100, 150, or 200 mg/kg) subcutaneously. At 8-h postdose, blood was collected from the tail vein of animals anesthetized with CO2. At the scheduled euthanasia at 24-h postdosing, additional samples of serum were collected from the tail vein and inferior vena cava of animals anesthetized with CO2.
Mouse study.
Male Swiss Webster mice between 10 and 12 weeks of age were housed in a controlled environment with 12-h light/dark cycle and were provided with water and standard lab chow ad libitum. The animals were fasted 14–16 h prior to dosing. In groups of 10 animals, mice were administered a single dose of saline or a saline suspension of CHB (50 or 150 mg/kg) subcutaneously. For all animals, three sections of pancreas from different regions (head, body, and tail) were collected and fixed in formalin for histopathological examination and the remainder of the pancreas was frozen for proteomic analyses. At 8-h postdose, blood was collected from the retro-orbital venous plexus of animals anesthetized with CO2. At the scheduled euthanasia at 24-h postdosing, additional samples of serum were collected from the inferior vena cava of animals anesthetized with CO2.
Serum Chemistry
Serum was analyzed on a Hitachi 912 serum chemistry analyzer, using standard clinical chemistry procedures. Sera were analyzed for multiple serum enzymes and metabolic parameters, including total protein, blood urea nitrogen, creatinine, albumin, pancreatic amylase, and lipase levels.
Global Serum Digestion and Analysis by Surface-Enhanced Laser Desorption-Ionization-Time of Flight Mass Spectrometry
Sera collected from rats or mice 8- to 24-h posttreatment with vehicle control, 15 µg/kg caerulein, or 150 mg/kg CHB, or sera from human patients, were normalized to 1 mg/ml by dilution with distilled water. Human sera samples were randomly numbered so as to be blinded upon initial surface-enhanced laser desorption-ionization-time of flight (SELDI) analysis. Ten micrograms of each serum sample was then combined with 2 µg of sequencing grade trypsin (Roche) in 25mM ammonium bicarbonate pH 8.0 (100 µl total reaction volume). The sera were digested overnight at 37°C and the resulting digests were then analyzed by SELDI mass spectrometry on NP20 and IMAC3 chip surfaces. The NP20 chip surface captures hydrophilic proteins, while the IMAC3 chip surfaces isolate metal-binding proteins.
For NP20 ProteinChip analysis, the chip surface was prewashed with distilled water, and 1 µl of sample was loaded with 1 µl of distilled water and incubated for 30 min in a humidity chamber. The chip surface was then washed twice with 3 µl of distilled water and allowed to air dry. 0.5 µl of 0.83% Alpha-cyano-4-hydroxycinnamic acid (CHCA) was added to each spot and after drying a second application of CHCA was applied. Human sera samples were not amenable to analysis on NP20 ProteinChips; the intensity of the 1623 m/z peptide, which is the peptide in human serum digests that corresponds to the same region of albumin as the 1609 m/z peptide in rat samples, was weak and not easily quantified. To evaluate the 1623 m/z peptide in human sera, therefore, we analyzed the diluted sera on a gold chip surface, diluting the sample 1:3 with 0.83% CHCA.
For IMAC3 ProteinChip analysis, the chip spots were loaded with 100mM copper sulfate twice for 15-min incubations each, and then rinsed with distilled water. The chip surfaces were then rinsed twice with 50mM sodium acetate, pH 4. Two microliters of samples digest was then loaded into 4 µl of 0.1% Triton in phosphate-buffered saline (PBS) and the chip was incubated in a humidity chamber at room temperature for 1 h. Following incubation the chip surfaces were washed three times with 5 µl 0.1% Triton X-100 in PBS followed by two washes with 5 µl of distilled water. Two applications of 0.5 µl of 0.83% CHCA were applied to each surface and allowed to dry completely.
Digestion of HSA with Trypsin and Carboxypeptidase A
A 1 mg/ml solution of human serum albumin (HSA) (Sigma Chemical Co.) was prepared in 25mM ammonium bicarbonate, pH 8. In one reaction, 10 µl of HSA solution was combined with 10 µl of 200 ng/µl trypsin and 65 µl of 25mM ammonium bicarbonate pH 8. In a second reaction, 10 µl of HSA solution was combined with 10 µl of 200 ng/µl trypsin and 65 µl of a 5 U/ml solution of carboxypeptidase A (Sigma Chemical Co.). HSA was digested overnight at 37°C. Resulting digests were analyzed by SELDI mass spectrometry on a gold chip surface using 0.83% CHCA in a ratio of 3:1 with digest.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Histopathology of Caerulein-Treated Rats
In order to determine if the peptide biomarkers could detect pancreatic lesions induced by other toxicants, we treated rats with caerulein, which is commonly used for animal models of pancreatitis. Pancreas sections obtained from rats 24 h past the initial dose of caerulein (Study A) were examined. The most remarkable observation at this time point was acinar degeneration, as well as mild acinar cell apoptosis and inflammation and rare acinar cell necrosis. Unlike CHB-induced pancreatic injury where zymogen depletion would be extensive at this time point, this event likely occurred at a much earlier time point in the caerulein-treated rats. The pancreatic degeneration seen in caerulein-treated rats was multifocal and characterized by shrinkage of acini with loss of zymogen and increased interstitial space and cellularity.
Analysis of Peptide Markers in Sera from Caerulein-Treated Rats
Serum collected from rats 8 and 24 h after treatment with caerulein or vehicle control was digested with trypsin and analyzed on NP20 and IMAC-Cu chip surfaces. The prominent albumin fragment at 1609 m/z was abundant in control samples and was found to be significantly reduced in sera from all caerulein-treated animals (Table 1). The 2864 m/z peak was detected on the IMAC-Cu chip surface in sera from those animals with caerulein-induced pancreatic damage. The changes in these peptide markers generally agreed with the elevations in amylase and lipase levels detected at 8 h. At 24 h, however, lipase and amylase values had returned to a normal range while 1609 m/z remained significantly decreased and 2864 m/z was still significantly elevated (Fig. 1), corresponding to the acinar degeneration seen in pancreatic tissue from caerulein-treated animals at 24 h (histopathology results).
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Histopathology of CHB-Treated Rats in Dose-Response Study
To evaluate sensitivity of the pancreas biomarkers to pancreatic injury, a dose-response study with CHB (50, 100, 150, and 200 mg/kg) in rats was completed. Pancreas sections obtained from rats 24 h following CHB treatment (Study B) were examined for evidence of dose-response relationships. Mild acinar cell apoptosis in a few of the animals, and mild diffuse zymogen depletion occurred in all but one animal treated at the lowest dose (50 mg/kg) of CHB. Enhanced pancreatic injury was very evident in rats treated with 100 mg/kg CHB, where all animals were noted to have acinar cell apoptosis and diffuse zymogen depletion (Fig. 2B–2E). Mild oncotic acinar cell necrosis was also seen in all animals, as well as an increased incidence of interstitial edema and inflammation in the pancreas (Fig. 2B). In rats dosed with 150 mg/kg CHB, the pancreatic injury was very similar to those animals dosed with 100 mg/kg, except that the incidence of oncotic acinar cell necrosis was decreased (Fig. 2C) and coagulative acinar necrosis was noted in two of the animals. Rats administered the highest dose of CHB (200 mg/kg) in this study also had similar pancreatic injury as those animals dosed at 150 mg/kg CHB with an even higher incidence of coagulative acinar necrosis (all but one animal) (Fig. 2D and 2E). In one animal dosed with 200 mg/kg CHB, acinar cell apoptosis, zymogen depletion, edema, and coagulative acinar necrosis were noted as severe (histopathology score = 5). Mean histopathology scores for each group of animals in this study are shown in Figure 3A.
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and inset); (C) 150 mg/kg group, acinar zymogen depletion and scattered apoptosis (
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Analysis of Peptide Markers in CHB Rat Dose-Response Study
Histopathology and serum chemistry results indicated that while there was an increase in apoptosis, and a significant increase in zymogen depletion, even in rats treated with the lowest dose (50 mg/kg) of CHB, lipase levels did not reflect this pathology (Fig. 3A and 3B). Even as robust pathologic changes were evident at the higher dose levels (
100 mg/kg CHB), only very mild elevations were noted in lipase levels in half of the rats treated with 100 mg/kg CHB. It was of interest then to compare the pathology and serum chemistry results to serum peptide marker levels in these samples. Sera from rats treated with the various doses (0–200 mg/kg) of CHB were digested with trypsin and analyzed on NP20 and IMAC-Cu chip surfaces. A "positive" test for pancreatitis from the biomarkers analyzed was defined as a test value outside of the range of the average for the control samples ± 2 SDs. The mean increase in the 2864 m/z marker was significantly elevated in the 150 and 200 mg/kg CHB groups (Fig. 3C), however, two animals in the 100 mg/kg group did test positive for this marker at 24-h postdose. While the mean decrease in the 1609 m/z peptide marker was significant in samples from groups treated with 100 mg/kg CHB (Fig. 3C), decreases in the 1609 m/z peptide marker turned out to be quite sensitive at detecting even minor pancreatic damage, as even three animals in the 50 mg/kg CHB-treated group (one at 8 h and two at 24 h) were identified as having significant reductions in this serum albumin fragment. The 1609 m/z marker also detected rats with pancreatic damage in the 100 mg/kg CHB-treated group which were not identified by changes in lipase levels. Overall the 1609 m/z marker improved the sensitivity for selecting out animals with pancreatic lesions 18% over using lipase levels (sensitivity at 8-h posttreatment—lipase, 60%; RA1609, 78%; RT2864, 72%) (Fig. 4).
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Analysis of Peptide Markers in Sera from CHB-Treated Mice
In order to determine whether similar peptide fragments would be useful as biomarkers of pancreatic damage in multiple species, mice were treated acutely with CHB. Serum collected from mice 24 h after treatment with CHB or vehicle control was digested with trypsin and analyzed on NP20 and IMAC-Cu chip surfaces. Sera from the 8-h time point were not analyzed. As anticipated from the minor changes seen both by histopathological examination and by lipase measurement, sera from only three mice showed changes in the peptide markers. The two animals (M03 and M05) with the most pancreatic damage (diffuse microvesiculation) also had substantial changes in the peptide markers (Table 2). Interestingly, these changes, particularly the increases in the 2864 m/z peak, were comparable to changes in the peptide markers seen in rats treated with caerulein or CHB.
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Digestion of HSA with Trypsin and Carboxypeptidase A
In order to determine if the 1609 m/z peptide marker would be useful in detecting pancreatic damage in species other than rodents, we compared the sequence of the rodent trypsin albumin fragment in a series of other species, including pig, horse, cat, dog, and human. A similar trypsin fragment of albumin should be present in serum digests from these other species. Only the HSA sequence contains two amino acid differences from the other species, which would produce a slightly larger fragment (approximately 1623 m/z). We then digested HSA with trypsin in the presence and absence of carboxypeptidase A to determine if the presence of carboxypeptidase A would produce the decrease in the 1623 m/z albumin fragment as is seen with rodent serum albumin. We had found previously through experiments with rat sera incubated with combinations of trypsin and various specific protease inhibitors that carboxypeptidase A appeared to be responsible for producing the peptide fragments (Walgren, Mitchell, Whiteley, and Thompson, manuscript in review). The 1623 m/z peak was in fact prominent in the spectrum from trypsin-digested HSA, and this peak was dramatically reduced in the presence of carboxypeptidase A (Fig. 5).
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Analysis of Peptide Markers in Human Sera Samples
Serum samples were obtained from 98 individuals (Table 3). The samples included sera from 20 normal, 33 pancreatitis, 10 diabetes, 10 pancreatic cancer, and 25 vasculitis patients. Prior to sample dilution (standardized to 1 mg/ml) and analysis of peptide biomarkers, serum amylase, lipase, albumin, and total protein were analyzed in all samples. Serum albumin was increased in 15% (5) of the pancreatitis patient samples and 32% (8) of the vasculitis patient samples; total protein was increased in 33% (11) of pancreatitis and 52% (13) of vasculitis samples. Total protein values for pancreatitis and vasculitis patients with significantly elevated total protein were 7.09 and 7.5 g/dl, respectively, versus a mean total protein of 4.5 g/dl in normal samples. The mean albumin concentration was 4.28 and 4.34 g/dl in those pancreatitis and vasculitis patient samples, respectively, that displayed a significant increase in albumin, versus the mean albumin concentration of 2.75 g/dl in normal samples. Serum amylase was elevated significantly in all pancreatitis patients (mean 502.3 U/l) and in 72% (18) of vasculitis patient samples (the mean value in vasculitis patients with elevated amylase was 48.3 vs. 15.8 U/l mean in normal samples). Serum lipase was also elevated in all pancreatitis patient samples (mean 1244.9 U/l), one normal subject sample (44.5 U/l), and in 40% of vasculitis patient samples (the mean value in vasculitis patients with elevated lipase was 80.8 vs. 23.9 U/l mean in normal samples).
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Samples were normalized relative to protein concentration (diluted to 1 mg/ml) and then analyzed for both the RT2864 fragment and the RA1609 fragment, which in human samples corresponds to a fragment at 1623 Da. As previously mentioned, the 1623-Da fragment was evaluated on a gold chip surface for the human samples. The sequence of the human peptide contains a methionine residue that is easily oxidized and generates a peak at approximately 1641 Da. The sum of the heights of these two peaks was used for all analyses. Again, a "positive" test for pancreatitis from the biomarkers analyzed was defined as a test value outside of the range of the mean for normal human sera ± 2 SDs.
Both the 2864 peptide fragment and the 1623/1641 peptide fragments identified samples from pancreatitis patients (2864 fragment sensitivity = 85%, specificity = 94%; 1623/1641 fragments sensitivity = 100%, specificity = 88%) (Fig. 6, representative spectra). The only group of patient samples that overlapped to a small degree with the pancreatitis group was the vasculitis group. Information on the location of the inflammation or the extent of vasculitis in patients was not provided. In all but one case, the samples which showed a "positive" result for the 1623/1641 marker were vasculitis patients that displayed either elevated amylase or lipase, or both. Of the vasculitis samples which gave a "positive" result with the 2864 marker, all had elevated lipase, and three of the four were the vasculitis samples displaying the greatest elevations in lipase and amylase.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent biomarker studies in our laboratory led to the identification of two peptides, RA1609 and RT2864, whose levels changed in rats in response to exocrine pancreatic injury induced by CHB. Through in vitro studies in acinar cells these peptide biomarkers were shown to be pancreas-specific and were the result of the release of pancreatic enzymes and cleavage of albumin and trypsin in the serum sample digests. As these peptide markers appeared to be sensitive for detection of pancreatic injury, we desired to determine if these biomarkers would be more broadly applicable to injury induced by other toxicants as well as in predicting pancreatic damage in other species.
Caerulein induces an acute, edematous pancreatitis that is said to resemble the early phase of this disease in humans. Following an acute dosing phase of caerulein in rats, apoptosis is induced in acinar cells, accompanied by edema in the lobules, moderate infiltration, cell vacuolization and partial degranulation. Inflammatory infiltrate persists for several days but in general regeneration of the acinar cells occurs by one week postdosing (Reid and Walker, 1999). Caerulein has therefore been used for many years as a standard animal model of acute pancreatitis (Gorelick et al., 1993). In the current study we administered caerulein by intraperitoneal injection in three doses of 15 µg/kg, 1 h apart. Histopathologic changes at 24-h postdose corresponded with the expected response of acinar cell apoptosis, degeneration, and pancreatic inflammation. Sera were collected at two time points, 8- and 24-h postdose. A significant decrease in the 1609 m/z peak and a significant increase in 2864 m/z peak were evident at both time points, despite a return of the amylase and lipase levels to baseline by 24 h (Table 1 and Fig. 1). These results indicate that these peptide biomarkers track with the degeneration of the acini beyond the initial degranulation.
To determine the sensitivity and specificity of the peptide biomarkers in our experimental animal models of pancreatitis, we also ran a dose-response study with CHB in rats. In this study rats were dosed with 50, 100, 150, or 200 mg/kg CHB once subcutaneously and were euthanized at 24-h postdose, with blood collections at 8- and 24-h postdose. A range of histological response was seen, from mild apoptosis and zymogen degranulation at 50 mg/kg to moderate to severe acinar cell apoptosis, zymogen depletion, edema, and coagulative acinar necrosis in animals dosed at 200 mg/kg (see Figs. 2 and 3A). Lipase levels, which as a laboratory diagnostic test is thought to be more specific to and indicative of pancreas injury, did not reflect the zymogen depletion seen with histopathology at the lower doses (50 and 100 mg/kg) of CHB. While the peptide biomarkers did not on average significantly change in animals with acinar necrosis or zymogen depletion at 50 mg/kg CHB, three of the animals (one at 8 h and two at 24 h) showed a "positive" test for pancreatic injury based on the 1609 m/z biomarker. RA1609 also proved to be the best biomarker for detecting damage in the 100 mg/kg CHB-treated rats, identifying rats with pancreatic damage that were not identified by changes in lipase levels. The most impressive result was the ability of the peptide markers to improve sensitivity of detecting pancreas injury in the rats at the earlier (8 h) time point; sensitivity of lipase at 8-h postdose was 60%, while sensitivity was 78% for RA1609 and 72% for RT2864. All markers were specific for pancreatic injury (no false positives in vehicle control animals).
To determine if we could detect pancreatic injury in other species using the peptide markers, we analyzed sera both from mice treated with 50 or 150 mg/kg CHB, and from human subjects in various disease states. Sequence similarity and the results from the HSA experiment gave us confidence that we should be able to detect changes in these peptide biomarkers in sera from other species following pancreatic damage where increased levels of serum enzymes (carboxypeptidase A) would be expected. Mice are much less sensitive to CHB, so the lesions that were noted were very mild and consisted of microvesiculation of acinar epithelium with mild and variable decrease in the prominence of zymogen granules; only two animals had more diffuse microvesiculation. Based on values of the biomarkers in control mice in this study, the two animals with more diffuse vacuolation were identified by lipase levels, RA1609, and RT2864. In addition, the RT2864 peptide identified one of the mice with milder multifocal microvesiculation that was not identified by any other biomarker.
The 1623/1641 m/z (equivalent to RA1609) and 2864 m/z (RT2864) peptides were measured in sera from normal human subjects, and patients with diabetes, pancreatic adenocarcinoma, pancreatitis, and vasculitis. The peptide biomarkers successfully identified sera from patients with pancreatitis. These biomarkers differentiated sera from pancreatitis patients from patients with other diseases involving the pancreas, such as type I and II diabetes and pancreatic cancer. As the pancreatitis patients were identified based on significant increases in amylase and lipase, both of these markers had 100% sensitivity in identifying pancreatitis samples. However, the 1623/1641 peptide biomarker also showed 100% sensitivity (the 2864 biomarker had 85% sensitivity), and both peptide biomarkers improved specificity of detection. Lipase and amylase displayed 85% and 74% specificity, respectively, while the 1623/1641 and 2864 peptide biomarkers had 88% and 94% specificity, respectively, for pancreatitis samples. Interestingly, the only group of sera with some overlap in signal was the sera from vasculitis patients. This is not unexpected, as the patient samples we received were from patients with active episodes of systemic vasculitis, which could involve inflammation in the gastrointestinal (GI) tract. Additional information regarding the type of vasculitis was not available; however, all but one of the vasculitis samples which were positive for the peptide biomarkers had elevated amylase or lipase, or both. Interestingly, in the case of the vasculitis samples which gave a positive test for the peptide biomarker RA1609 (1623/1641), four out of seven were from patients under the age of 50, and three of the four which showed a positive test result for RT2864 were males under the age of 50. These patients are more likely to have a form of vasculitis which could involve the GI tract (i.e., microscopic polyangitis, polyarteritis nodosa) rather than the most common form of vasculitis which is found almost exclusively in patients over the age of 50 (Giant Cell arteritis/temporal vasculitis) (Langford, 2003).
In conclusion, the peptide biomarkers RA1609 and RT2864 are sensitive and specific biomarkers to exocrine pancreatic injury induced by different toxicants, and in at least three species (rat, mouse, and human). As these peptide biomarkers are able to detect pancreatic damage just hours after toxicant administration in animal models of pancreatitis, and continue to track with acinar cell injury even after the initial degranulation of zymogen granules, these markers could be valuable tools in drug development research to detect pancreatic injury in potentially several preclinical species. In addition to being useful for in vivo studies, these peptide markers were also applicable to detecting pancreatic toxicants in vitro in acinar cell cultures (Walgren, Mitchell, Whiteley, and Thompson, manuscript in review), and could therefore be used to predict the potential of a compound to cause pancreatic injury.
Corresponding peptides are also specific in identifying serum samples from patients with pancreatitis. Further clinical studies could determine if these peptide biomarkers could be sensitive at a very early stage of pancreas injury. Also, as changes in these peptides tracked fairly well with the severity of the pancreatic lesion in animal models in our studies, it would be interesting to determine if they would be useful in establishing the extent of pancreas injury in humans, as current biomarkers generally are not useful in this regard.
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ACKNOWLEDGMENTS |
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The authors would like to thank the Drug Safety Research and Development (DSRD) histology and necropsy group and the DSRD Biomarkers group for analyses of serum amylase and lipase.
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REFERENCES |
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