Early changes in the urine proteome in a diethyldithiocarbamate-induced chronic pancreatitis rat model.

Without homeostasis mechanisms, urine accumulates early changes in biomarkers and can be a better and earlier biomarker source than blood, especially for chronic diseases. This study tests whether early changes can be detected in a rat model of chronic pancreatitis induced by intraperitoneal injection of diethyldithiocarbamate. Urinary proteins from three rats were profiled by liquid chromatography coupled with tandem mass spectrometry. Compared with before injection, fifty differential proteins that had human orthologs were significantly changed in the chronic pancreatitis rats. At week 2, fifteen differential proteins were identified when no obvious pathological changes had yet appeared. Among them, twelve proteins were altered at the other two time points, five had previously been associated with chronic pancreatitis. Inflammatory cytokines infiltration, acinar disruption and fibrosis were detected at week 3 and week 4, at which points fourteen identified differential proteins had been reported to be differentially expressed in the serum or pancreatic tissue of chronic pancreatitis patients and other animal model studies. These include proteins that are significant in acute-phase response (FETUA, F2, FINC and REG3G), extracellular matrix organization (COMP, FINC) and tissue remodeling (CSPG4, GAS6). REG3G has been described abnormally expressed in several human digestive system diseases such as chronic pancreatitis. SIGNIFICANCE: This study demonstrate that changes caused by chronic pancreatitis can be reflected early in urinary proteins. New clues for the early diagnosis of chronic pancreatitis can be found even with only the small number of model animals used.

Enteric hyperoxaluria in chronic pancreatitis.

Chronic pancreatitis may lead to steatorrhea, enteric hyperoxaluria, and kidney damage. However, the prevalence and determinants of hyperoxaluria in chronic pancreatitis patients as well as its association with renal function decline have not been investigated.We performed an observational study. Urine oxalate to creatinine ratio was assessed on 2 independent random urine samples in consecutive adult patients with chronic pancreatitis followed at the outpatient clinic from March 1 to October 31, 2012. Baseline characteristics and annual estimated glomerular filtration rate (eGFR) change during follow-up were compared between patients with hyper- and normo-oxaluria.A total of 48 patients with chronic pancreatitis were included. The etiology of the disease was toxic (52%), idiopathic (27%), obstructive (11%), autoimmune (6%), or genetic (4%). Hyperoxaluria (defined as urine oxalate to creatinine ratio >32 mg/g) was found in 23% of patients. Multivariate regression analysis identified clinical steatorrhea, high fecal acid steatocrit, and pancreatic atrophy as independent predictors of hyperoxaluria. Taken together, a combination of clinical steatorrhea, steatocrit level >31%, and pancreatic atrophy was associated with a positive predictive value of 100% for hyperoxaluria. On the contrary, none of the patients with a fecal elastase-1 level >100 µg/g had hyperoxaluria. Longitudinal evolution of eGFR was available in 71% of the patients, with a mean follow-up of 904 days. After adjustment for established determinants of renal function decline (gender, diabetes, bicarbonate level, baseline eGFR, and proteinuria), a urine oxalate to creatinine ratio >32 mg/g was associated with a higher risk of eGFR decline.Hyperoxaluria is highly prevalent in patients with chronic pancreatitis and associated with faster decline in renal function. A high urine oxalate to creatinine ratio in patients with chronic pancreatitis is best predicted by clinical steatorrhea, a high acid steatocrit, and pancreatic atrophy. Further studies will need to investigate the mechanisms of renal damage in chronic pancreatitis and the potential benefits of therapies reducing oxaluria.

Neutrophil Gelatinase-Associated Lipocalin (NGAL) concentration in urine is superior to CA19-9 and Ca 125 in differentiation of pancreatic mass: Preliminary report.

BACKGROUND: Currently pancreatic cancer is the fourth leading cause of cancer-related death worldwide. Because of its late manifestation and consequent dismal prognosis, there is an urgent need to develop highly sensitive and specific marker. Neutrophil Gelatinase-Associated Lipocalin (NGAL) recently emerged as a protein playing an important role in carcinogenesis of various neoplasms. OBJECTIVE: Our aim was to assess the potential of urine and bile concentration of NGAL in differentiating pancreatic adenocarcinoma from chronic pancreatitis. METHODS: Forty-two patients operated on due to pancreatobiliary lesions were enrolled in this study. All enrolled patients had eGFR within reference range. Levels of CEA, CA 125 and Ca19-9 were assessed using standard laboratory protocols. A sample of urine was collected prior to the surgery. Intraoperatively a 5 ml sample of bile was collected directly from the common bile duct. Bile and urine levels of NGAL were measured using a ELISA kit. After standard pathological examination of specimens obtained during surgery, patients were divided into 2 groups: 21 patients with pancreatic adenocarcinoma and 15 patients with focal chronic pancreatitis. RESULTS: NGAL concentration in bile in patients with PDAC vs CP was 75.72 ± 16.05 ng/mL vs 62.62 ± 18.6 ng/mL respectively (p= 0,011). NGAL concentration in urine was 43.26 ± 21.21 ng/mL vs 17.96 ± 14.58 ng/mL (p= 0.002) respectively. In order to compare these markers with routinely used ones, ROC curve was built for Ca125 to establish cutoff point and in case of CA19-9 clinically used cutoff (≥ 37U/mL) was applied. Sensitivity and specificity for NGALurine with cutoff value of 27 ng/mL was 80.95% and 80% respectively, while these values for NGALbile were 71.43% and 80% respectively. Ca19-9 measured in plasma with clinically used cutoff value had sensitivity of 71.43% and specificity of 73.33%. Sensitivity and specificity for Ca 125 measured in plasma with cutoff value of 13 U/mL were 85.71% and 66.67% respectively. CONCLUSIONS: In conclusion, NGAL in urine and bile are remarkably accurate in differentiating pancreatic mass due to chronic pancreatitis from pancreatic adenocarcinoma. Therefore, NGAL concentrations in bile and urine should be further investigated in order to assess their usefulness in early pancreatic adenocarcinoma diagnosis.

Urinary Peptide Analysis Differentiates Pancreatic Cancer From Chronic Pancreatitis.

OBJECTIVES: Differentiation of pancreatic cancer (PCA) from chronic pancreatitis (CP) is challenging. We searched for peptide markers in urine to develop a diagnostic peptide marker model. METHODS: Capillary electrophoresis-mass spectrometry was used to search for peptides in urine of patients with PCA (n = 39) or CP (n = 41). Statistical different peptides were included in a peptide multimarker model. Peptide markers were sequence identified and validated by immunoassay and immunohistochemistry (IHC). RESULTS: Applied to a validation cohort of 54 patients with PCA and 52 patients with CP, the peptide model correctly classified 47 patients with PCA and 44 patients with CP (area under the curve, 0.93; 87% sensitivity; 85% specificity). All 5 patients with PCA with concomitant CP were classified positive. Urine proteome analysis outperformed carbohydrate antigen 19-9 (area under the curve, 0.84) by a 15% increase in sensitivity at the same specificity. From 99 healthy subjects, only four were misclassified. Fetuin-A was the most prominent peptide marker source for PCA as verified by immunoassay and IHC. In silico protease mapping of the peptide markers' terminal sequences pointed to increased meprin-A activity in PCA, which in IHC was associated with neoangiogenesis. CONCLUSIONS: Urinary proteome analysis differentiates PCA from CP and may serve as PCA screening tool.

Urinary 1H-NMR metabolomics can distinguish pancreatitis patients from healthy controls.

CONTEXT: The characterization of the urinary metabolome may yield biomarkers indicative of pancreatitis. OBJECTIVES: We establish a non-invasive technique to compare urinary metabolic profiles in patients with acute and chronic pancreatitis to healthy controls. METHODS: Urine was obtained from healthy controls (HC, n=5), inpatients with mild acute pancreatitis (AP, n=5), and outpatients with chronic pancreatitis (CP, n=5). Proton nuclear magnetic resonance spectra were obtained for each sample. Metabolites were identified and quantified in each spectrum; resulting concentrations were normalized to account for differences in dilution among samples. Kruskal-Wallis test, post-hoc Mann-Whitney U tests, and principal component analysis were performed to identify metabolites that discriminate healthy controls, acute pancreatitis, and chronic pancreatitis. RESULTS: Sixty metabolites were identified and quantified; five were found to differ significantly (P<0.05) among the three groups. Of these, citrate and adenosine remained significant after validation by random permutation. Principal component analysis demonstrated that healthy control urine samples can be differentiated from patients with chronic pancreatitis or acute pancreatitis; chronic pancreatitis patients could not be distinguished from acute pancreatitis patients. CONCLUSIONS: This metabolomic investigation demonstrates that this non-invasive technique offers insight into the metabolic states of pancreatitis. Although the identified metabolites cannot conclusively be defined as biomarkers of disease, future studies will validate our findings in larger patient cohorts.

Elevated urinary levels of urokinase-type plasminogen activator receptor (uPAR) in pancreatic ductal adenocarcinoma identify a clinically high-risk group.

BACKGROUND: The urokinase plasminogen activator receptor is highly expressed and its gene is amplified in about 50% of pancreatic ductal adenocarcinomas; this last feature is associated with worse prognosis. It is unknown whether the level of its soluble form (suPAR) in urine may be a diagnostic-prognostic marker in these patients. METHODS: The urinary level of suPAR was measured in 146 patients, 94 pancreatic ductal adenocarcinoma and 52 chronic pancreatitis. Urine from 104 healthy subjects with similar age and gender distribution served as controls. suPAR levels were normalized with creatinine levels (suPAR/creatinine, ng/mg) to remove urine dilution effect. RESULTS: Urinary suPAR/creatinine values of pancreatic ductal adenocarcinoma patients were significantly higher (median 9.8; 25th-75th percentiles 5.3-20.7) than those of either healthy donors (median 0; 0-0.5) or chronic pancreatitis patients (median 2.7; 0.9-4.7). The distribution of values among cancer patients was widespread and asymmetric, 53% subjects having values beyond the 95th percentile of healthy donors. The values of suPAR/creatinine did not correlate with tumour stage, Ca19-9 or CEA levels. Higher values correlated with poor prognosis among non-resected patients at univariate analysis; multivariate Cox regression identified high urinary suPAR/creatinine as an independent predictor of poor survival among all cancer patients (odds ratio 2.10, p = 0.0023), together with tumour stage (stage III odds ratio 2.65, p = 0.0017; stage IV odds ratio 4.61, p < 0.0001) and female gender (odds ratio 1.85, p = 0.01). CONCLUSIONS: A high urinary suPAR/creatinine ratio represents a useful marker for the identification of a subset of patients with poorer outcome.

Insights into the development of alcoholic chronic pancreatitis at Soweto, South Africa: a controlled cross-sectional study.

OBJECTIVE: We asked why so few working-class Africans of Soweto have chronic pancreatitis (CP) when alcoholism is the norm. METHODS: Twenty-one alcoholics with acute psychosis but normal pancreas were investigated for lifestyle, micronutrient status, electrophilic stress, and iron overload. RESULTS: Alcoholics consumed more ethanol daily than did 14 previously studied patients with CP (P = 0.003); cigarette usage was similar; both groups had even poorer vitamin C status than 14 healthy controls, and no participant had iron overload. The CP group had higher scores for exposure to occupational xenobiotics than did alcoholics (P < 0.05), with lower plasma glutathione (P = 0.047) and urinary inorganic sulfate (P = 0.009). Further analysis identified hyperhomocysteinemia in the alcoholic set, with lower vitamin B12 (P < 0.001), higher folic acid (P = 0.003), and similar vitamin B6 levels compared with controls. CONCLUSIONS: The transition from alcoholism to CP in Soweto is associated with occupational exposure to xenobiotics. Among detoxification systems that are strained thereby, glutathione and inorganic sulfate depend on methionine intake, which is ample in Sowetans, whereas vitamin C, which exerts a glutathione-sparing effect, is deficient. Hence, a daily tablet of vitamin C may enable community prophylaxis against the disease--but homocysteine status would need monitoring.

Chronic pancreatitis is associated with hyperhomocysteinemia and derangements in transsulfuration and transmethylation pathways.

OBJECTIVES: Homocysteine has been implicated in vascular dysfunction and thrombosis, as well as inflammatory conditions. This study was aimed to find out whether chronic pancreatitis (CP) is associated with hyperhomocysteinemia and derangements of transmethylation and transsulfuration pathways. METHODS: We estimated homocysteine and its metabolites in 45 alcoholic CP patients, 45 tropical CP patients, and 48 healthy controls. RESULTS: Significant increases in plasma total homocysteine and decreases in red blood cell folate, reduced glutathione, plasma methionine, cysteine, and urinary inorganic sulfate/creatinine ratio were observed in both alcoholic and tropical CP patients in comparison with healthy controls. Red blood cell glutathione and plasma cysteine levels were significantly lower in alcoholic than in tropical CP patients. However, plasma vitamin B12 levels were comparable between CP patients and controls. No significant differences in these parameters were observed between diabetic patients and nondiabetic patients. Multivariate regression analysis showed a significant negative correlation between homocysteine and folate (r = -0.415, P = 0.001) and a positive correlation between glutathione and cysteine levels (r = 0.37, P = 0.003). CONCLUSIONS: Chronic pancreatitis is associated with hyperhomocysteinemia and derangements in transmethylation and transsulfuration pathways. Low folate levels observed in these patients seem to have a key role in this derangement.

Application of proteomic technology in identifying pancreatic secretory trypsin inhibitor variants in urine of patients with pancreatitis.

BACKGROUND: Although the analysis of genetic variability has traditionally been performed with molecular genetic techniques, the development of proteomic technology has raised the possibility of analyzing genetic variants at the protein level. This method provides additional information about posttranslational modifications and differences in expression. We used mass spectrometry to characterize 3 variants of the peptide encoded by the serine protease inhibitor Kazal type 1 (SPINK1) gene, pancreatic secretory trypsin inhibitor (PSTI). A genetic variant of PSTI, N34S, is associated with the development of pancreatitis. METHODS: We used a quadrupole/time-of-flight hybrid mass spectrometer equipped with an electrospray ionization source to analyze the molecular identity of PSTI purified from the urine of 12 patients with pancreatitis and from 3 controls. We also developed a rapid small-scale capture procedure to isolate and analyze PSTI from small volumes of urine. RESULTS: The mutations responsible for mass shifts of different PSTI variants could be verified. We observed differences in the expression of different variants as well as a novel proteolytic fragment of PSTI. Small-scale magnetic bead-mediated immunoaffinity chromatography PSTI enabled easy and rapid purification from small urine volumes, facilitating mass spectrometric analysis with adequate sensitivity. CONCLUSIONS: Pancreatitis-related PSTI variants occurring at nanomolar concentrations in urine can be detected and quantified by immunoaffinity purification and mass spectrometry. In addition, the N34S variant occurs at higher concentrations than the wild type. This finding casts new light on the possible role of PSTI as a cause of hereditary pancreatitis.