Author Affiliations: Department of Endocrinology (Yang N) and Department of Gastroenterology (Zhang DL and Hao JY), Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China

Corresponding Author: Jian-Yu Hao, MD, Department of Gastroenterology, Beijing Chaoyang Hospital, Capital Medical University, No. 8 Gongren Tiyuchang Nanlu, Chaoyang District, Beijing 100020, China (Tel: +86- 10-85231714; Fax: +86-10-85231520; Email: haojianyucyyy@sina.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60376-9

Published online May 21, 2015.

 

Contributors: HJY proposed the study. YN and ZDL performed research, collected and analyzed the data and wrote the first draft, YN and ZDL contributed equally to this article. All authors contributed to the design and interpretation of the study and to further drafts. HJY is the guarantor.

Funding: None.

Ethical approval: The study protocol was designed according to Declaration of Helsinki guidelines and approved by the hospital Institutional Ethics Committee.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

BACKGROUND: Coagulopathy and its association with disease severity in hyperlipidemia (HL)- and non-hyperlipidemia (NHL)-induced acute pancreatitis (AP) are not clear. The present study was to evaluate the relationship between coagulation homeostasis and AP.

 

METHODS: This study included 106 AP patients admitted to our hospital between October 2011 and January 2013. Stratified by disease severity, the patients were divided into two groups: a mild AP (MAP) group (n=69); and a moderately severe AP (MSAP) group (n=37). Based on disease etiology, there were 31 HL-induced AP (HLP) cases and 75 NHL-induced AP (NHLP) cases. The HLP and NHLP groups were compared for parameters of coagulation homeostasis, lipid metabolism, and disease severity. Correlations between disease severity and levels of D-dimer and protein C were investigated, and the prognostic potential of D-dimer was evaluated.

 

RESULTS: Compared with MAP patients, MSAP patients showed higher levels of D-dimer and lower levels of protein C. HLP patients had higher protein C levels than NHLP patients. Both D-dimer and protein C levels were significantly associated with the disease severity, not the disease etiology. D-dimer levels correlated positively with low density lipoprotein cholesterol levels and performed well as a sensitive and specific predictor of disease severity in AP patients, especially in HLP patients.

 

CONCLUSIONS: The coagulation homeostasis is different between HLP and NHLP patients, and HL may be a contributing factor for thrombosis and fibrinolysis in HLP. D-dimer may be a robust marker of disease severity in HLP.

 

(Hepatobiliary Pancreat Dis Int 2015;14:633-641)

 

KEY WORDS: hyperlipidemia-induced acute pancreatitis; coagulation homeostasis; D-dimer; protein C; disease severity

Acute pancreatitis (AP) is a protean pancreatic disease with a wide spectrum symptoms, which may range from mild and self-limiting to severe and life-threatening.^{[1, 2]} While patients with mild AP (MAP) may require minimal medical intervention, those with moderately severe AP (MSAP) often experience transient organ failure or local complications and can progress to severe AP (SAP). SAP can cause systemic inflammation, sepsis, multiple organ failure (MOF), and even death.^[3-6] The mortality rate for SAP can be as high as 10%-30%. Therefore, it is important to characterize the pathological alterations at an early phase of AP and identify risk factors that are associated with disease severity and therefore, to prevent the patients with MSAP from progressing to SAP.

 

During the early phase of AP, trypsinogen produced in situ is abnormally activated in the pancreas, leading to pancreatic tissue self-destruction. This damage induces a strong systemic inflammatory response mediated by the release of proinflammatory molecules such as interleukins, chemokines, tumor necrosis factor, and platelet activation factor into the circulation.^[7] These proinflammatory cytokines then activate neutrophils, monocytes and endothelial cells, which, along with cells in the diseased pancreas, produce large quantities of tissue factors to initiate the coagulation cascade.^{[8, 9]} Therefore, AP is often accompanied by coagulopathy, which is characterized by increased thrombosis and fibrinolysis.

 

In 1952, Klatskin and Gordon^[10] reported that relapsing pancreatitis had hyperlipidemia. The authors proposed that alterations in serum lipids might be the underlying cause of AP recurrence. Since then, numerous efforts have been made to elucidate the association between hyperlipidemia and AP. These studies found that 12%-50% of AP patients have dyslipidemia, and that hyperlipidemia is the third major cause of AP after gallstones and alcohol abuse and accounts for 1%-12% of all AP cases.^[11-14] Hyperlipidemia promotes the conversion of triglycerides (TG) to free fatty acids (FFAs). Higher circulating levels of FFAs damage the pancreatic acinar and endothelial cells. hyperlipidemia also activates trypsinogen and impairs microcirculation.^[15-17] Compared with non-hyperlipidemia AP (NHLP) patients, hyperlipidemia-induced AP (HLP) patients have higher risk of developing acute respiratory distress syndrome (ARDS) and MOF and therefore HLP needs longer and more intensive clinical care.^[18] Since the serum amylase level, the current gold standard diagnostic marker for AP, is often normal or only minimally elevated in patients with HLP and therefore, it is difficult to identify HLP patients in the early phase of the disease. The association between hyperlipidemia and abnormal coagulation homeostasis has been well documented. We hypothesized that hyperlipidemia-associated changes in coagulation homeostasis may be present in patients with HLP, and such changes could be used to predict AP severity in HLP patients.

 

The association between coagulopathy and disease severity or outcome in AP patients has been well documented.^{[19, 20]} It is known that the levels of D-dimer, a small protein fragment produced during blood clot degradation by fibrinolysis and a sensitive marker of coagulation and fibrinolysis, are positively correlated with clinical complications and therefore negatively correlated with overall survival in SAP patients.^[21] Additionally, the levels of protein C, another well-characterized anticoagulant precursor, were lower in SAP patients before the development of MOF.^[22] Activated protein C, an efficient anticoagulant, has been reported to significantly reduce mortality in patients with severe sepsis and at a high risk of death.^{[23, 24]} However, no study has examined the impact of hyperlipidemia on the coagulation homeostasis, and especially on the levels of protein C and D-dimer in AP patients. The present study was to evaluate the predictive effect of D-dimer on AP severity in HLP and NHLP patients at an early phase of the disease.

 

 

Patients with AP were enrolled between October 2011 and January 2013 in our hospital. Among 137 patients, 20 with SAP were excluded; the other 11 patients were excluded because they had previously taken anti-coagulation medications or/and had a concomitant acute cardiovascular disease. Our cohort included 106 patients that had abdominal pain characteristic of AP and elevated serum amylase levels. All patients in this study were admitted to the hospital within the first 24 hours of disease onset, and their AP diagnosis was confirmed by contrast-enhanced computed tomography (CECT) within 48 hours of disease onset. The study protocol was approved by the hospital Institutional Ethics Committee and conformed to the Declaration of Helsinki. A written informed consent was obtained from all patients.

 

AP severity was determined according to the 2012 version of the Atlanta classification of AP.^[25] MAP: no organ failure or local or systemic complications; MSAP: transient organ failure (<48 hours) and/or local or systemic complications without persistent organ failure. We used this classification to divide the study cohort into two groups: MAP group (69 patients) and MSAP group (37 patients).

 

HLP patients were defined as those who had clinical presentations of AP, elevated TG (>11.30 mmol/L in the absence of lactescent serum, or 5.65-11.30 mmol/L with lactescent serum), and no other known causes for AP. Using this definition, the study cohort was divided into an HLP group (31 patients) and an NHLP group (75 patients). Among the 75 patients with NHLP, 30 had biliary pancreatitis and 45 had AP resulted from alcohol abuse.

 

A fasting blood sample (10 mL) was collected from a peripheral vein of each patient within the first 24 hours after admission and subjected to a routine analysis including white blood cell (WBC) count, hematocrit (HCT), and platelet (PLT) count. Additional tests were made to measure albumin (ALB), creatinine (Cr), calcium (Ca), glucose (Glu), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and TG. Coagulation parameters measured included prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen level (Fbg), and thrombin time (TT).

 

WBC, HCT and PLT were measured using a Sysmex XE-2100 autoanalyzer (Sysmex, Kobe, Japan). Reference intervals for WBC, HCT and PLT were 3.97-9.15×10⁹/L, 0.380%-0.508% and 100-300×10⁹/L, respectively. PT, APTT, Fbg and TT were determined using a Sysmex CA6000 automated analyzer (Sysmex, Milton Keynes, UK) with the coagulation detector. Reference intervals for PT, APTT, Fbg and TT were 9.6-13.0 seconds, 21-34 seconds, 170-400 mg/dL and 14-21 seconds, respectively. ALB, Cr, Ca, Glu, TC, HDL-C, LDL-C and TG levels were quantified using a Dade Behring Dimension RXL Autoanalyzer (Dade Behring Diagnostics, Marburg, Germany). Reference intervals for ALB, Cr, Ca, Glu, TC, HDL-C, LDL-C and TG were 32-55 g/L, 53-115 µmol/L, 2.1-2.6 mmol/L, 3.3-6.1 mmol/L, 3.62-5.70 mmol/L, 1.03-1.55 mmol/L, 1.81-3.36 mmol/L and 0.56-2.26 mmol/L, respectively.

 

A portion of each blood sample was mixed with sodium citrate (final concentration 10.9 mmol/L) and centrifuged at 2000 g for 10 minutes. The isolated plasma was then stored at -80?�� for later analyses of D-dimer, protein C, protein S, tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1). The levels of D-dimer, tPA and PAI-1 were measured using enzyme-linked immunosorbent assay (ELISA) kits purchased from Sun Biotech (Shanghai, China) according to the manufacturer's instructions. Reference intervals for D-dimer, tPA and PAI-1 were 0-0.5 mg/L, 1-12 µg/L and 5-45 µg/L, respectively. The levels of protein C and protein S were measured using a Sysmex CA6000 automated analyzer with a Berichrom Protein C kit and protein S Ac kit (Siemens Healthcare Diagnostics, Marburg, Germany), respectively. Reference intervals for protein C and protein S were 70%-130% and 55%-130% of the normal values, respectively.

 

Variables used for calculating acute physiology and chronic health evaluation II (APACHE II) and Ranson scores were collected at the time of admission and again at 24 and 48 hours after admission. CECT results were used to calculate the Balthazar CT severity index (CTSI).

 

Statistical analyses were performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data that follow a normal distribution are presented as mean±SD; data that follow a skewed distribution are presented as median (Q1, Q3). Two-group comparisons were done using the Mann-Whitney U test and Student's t test. Multiple-group comparisons were done by one-way ANOVA and least-significant difference analyses. Count variables were analyzed by the Chi-square test or continuity correction analysis. Correlations between two variables were evaluated by Pearson's or Spearman's correlation coefficient analysis. A P value <0.05 was considered statistically significant.

 

 

The MAP and MSAP groups were in similar age and gender, and had comparable rates of concurrent diabetes, fatty liver and cholecystectomy. Compared with the MAP group, the MSAP group had higher WBC and PLT counts, elevated levels of Fbg and D-dimer, lower levels of protein C, increased Cr and decreased Ca. Additionally, the MSAP group showed significantly lower ALB, higher Glu, and higher LDL-C levels than the MAP group (Table 1).

 

Patients with HLP had higher levels of Glu, TC, HDL-C and LDL-C than patients with NHLP, and almost all patients with HLP had a fatty liver. The HLP and NHLP groups showed no difference in gender, history of cholecystectomy, or disease severity scores; however, patients with HLP were significantly younger and had a greater incidence of diabetes. The HLP group also had higher levels of HCT, PLT, Fbg, protein C and protein S, and lower levels Cr and Ca. Additionally, the PT and TT were significantly shorter in HLP patients than in NHLP patients (Table 2).

 

We subdivided the patients of the HLP and NHLP groups into four groups (HL-MAP, HL-MSAP, NHL-MAP and NHL-MSAP) based on disease severity, and compared the groups for parameters of coagulation and lipid metabolism. The results showed no differences in gender or history of cholecystectomy among the four groups. While the average age in the two HL-groups was lower than that in the two NHL-groups; among the NHL patients, the MSAP subgroup was significantly older than the MAP subgroup. Additionally, the HL-MSAP subgroup had the highest rate of diabetes among the four subgroups.

 

It is noteworthy that among patients with the same hyperlipidemia status, the levels of D-dimer in the two MSAP subgroups were significantly higher than those in the corresponding MAP subgroups; also, the HL-MSAP subgroup had the highest levels of D-dimer. While NHLP patients had the lower levels of protein C than HLP patients; when comparing patients within groups stratified by hyperlipidemia status, the MSAP subgroups had significantly lower protein C levels than the corresponding MAP subgroups. Moreover, Fbg levels, PLT counts, APTT, Ca level and severity scores were all found different among groups with different hyperlipidemia status and severity classification (Table 3). Interestingly, among patients with the same hyperlipidemia status, the LDL-C levels were always significantly higher in the MSAP subgroups than those in the MAP subgroups (Table 3).

 

Regardless of hyperlipidemia status, the higher levels of D-dimer always significantly correlated with higher disease severity scores, even when the scores were calculated by different systems (Ranson, APACHE II and CTSI). In contrast, protein C levels were negatively correlated with disease severity scores. Following stratification of patients by hyperlipidemia status, D-dimer levels were still positively correlated with disease severity in most cases, except when severity was measured by the CTSI score in both groups or the APACHE II in the HLP group. And there was a significant negative correlation between protein C levels and disease severity (Table 4).

 

Regardless of hyperlipidemia status, higher D-dimer levels were significantly correlated with higher LDL-C levels (Table 4 and Fig. 1). Additionally, in all patients, HDL-C levels were negatively correlated with levels of D-dimer. However, protein C levels were positively correlated with TC, HDL-C and TG in all patients. Following hyperlipidemia stratification, only the correlation between D-dimer and HDL-C levels was found to be significant in patients with NHLP (Table 4).

 

Finally, we attempted to identify indicators of AP severity in patients with HLP and NHLP. A backward stepwise elimination process was used in a logistic regression analysis of AP severity and various parameters associated with a significant difference between the MAP and MSAP groups. We found that both D-dimer levels and WBC were indicators for MSAP (OR=2.637 and 1.202, P=0.041 and 0.029, respectively), whereas protein C was a protective factor (OR=0.959, P=0.004). We then performed a receiver-operating characteristics curve analysis using D-dimer as an identifier for MSAP in all NHLP and HLP patients, respectively. At the cut-off of 0.91 mg/L, D-dimer levels provided a more reliable indication of disease severity in patients with HLP than in all patients or NHLP patients, with the sensitivity, specificity, and negative predictive value all >90% (Table 5 and Fig. 2).

 

 

AP is a reversible inflammatory disease. During this disease process, cytokines and tissue factors such as prostaglandins and activated complements are released to activate the coagulation cascade and inhibit fibrinolysis, leading to blood hypercoagulability.^[26] Abnormal homeostasis of coagulation, fibrinolysis and lipid metabolism are present in AP, and these abnormalities are associated with the severity of AP.^{[19, 20]} Especially in patients with SAP, markers related to disseminated intravascular coagulation, such as D-dimer and AT-III levels, may correlate with disease progression.^[27] hyperlipidemia has been identified as a cause of both abnormal coagulation and AP, and can modulate coagulation homeostasis. Changes in coagulation homeostasis can further induce pancreatic tissue damage. Thus, patients with HLP have a higher risk of developing MSAP and SAP symptoms, such as ARDS, acute renal failure and MOF. Intravenous infusions of insulin and heparin, which activate lipoprotein lipase, have been reported to decrease TG levels in HLP patients.^[28-31] Patients who do not respond to such treatment can be treated using plasma exchange to reduce serum lipid levels.^[32-36] However, robust markers for identifying early stage HLP are still lacking, and it is important to identify such biomarkers so that patients with HLP can receive tailored clinical management to prevent disease progression and achieve an optimal outcome.^[18]

 

D-dimer is a small protein fragment produced during fibrinolysis and has been widely used in the diagnosis of the disseminated intravascular coagulation and to exclude the possibility of thromboembolic disease where the risk is low.^[37] Consistent with previous study,^[21] we observed a positive correlation between levels of D-dimer and disease severity. To our knowledge, this is the first time to demonstrate that patients with HLP had higher D-dimer levels than those with NHLP, even when the disease severity scores for the HLP and NHLP groups were similar. In addition, HL-MSAP patients had the highest D-dimer levels among all of our enrolled AP patients. Therefore, D-dimer may be particularly useful for identifying HL-MSAP patients. This observation is in agreement with the previous finding that HLP patients often have abnormal thrombosis and fibrinolysis processes, and suggests that hyperlipidemia may partially contribute to coagulation abnormalities.^{[38, 39]} In our study, fibrinolytic activity was higher and anti-coagulation activity was lower in the HLP patients compared with those in the NHLP patients.

 

Our study is the first to describe the correlation between D-dimer and LDL-C levels in AP patients. Because patients with hyperlipidemia are characterized by an LDL-C abnormality,^[38-40] and D-dimer levels correlate with LDL-C levels, we believe that the significant elevation of D-dimer may be due to both over-activation of inflammation-induced coagulation and increases in serum LDL-C in the early phase of AP, especially in patients with HLP. Although the positive correlation between D-dimer levels and AP severity was not limited to patients with HLP, the correlation was stronger in patients with HLP than in those with NHLP. These observations confirmed that hypercoagulability is associated with AP severity, and indicated that hyperlipidemia may further activate the coagulation pathway in AP patients. Therefore, D-dimer level may be a reliable marker for disease severity in HLP.

 

Protein C is zymogenic protein that is activated by thrombin and the thrombomodulin complex. Activated protein C, an important anticoagulant involved in inflammation, blood clot formation, and blood vessel permeability, exerts its effect by inactivating PAI-1, coagulation factors Va and VIIIa, and also by regulating thrombomodulin and endothelial cell protein C receptor.^[41-43] In our study, protein C levels were lower in the MSAP patients than those in the MAP patients, regardless of hyperlipidemia status. This difference may be due to decreased protein C synthesis in the liver and excessive protein C consumption in patients with MSAP.^[44] Interestingly, the patients with HLP had significantly higher levels of protein C than those with NHLP, and levels of protein C in HLP patients appeared to be normal or only slightly decreased. However, given that protein C levels in HL-MSAP patients were significantly lower than those in HL-MAP patients, and the HL-MSAP subgroup had the highest D-dimer levels, we reasoned that HL-MSAP patients could still have abnormal coagulation. In fact, PLT, PT and Fbg were all increased in HL-MSAP patients, indicating abnormal coagulation homeostasis in this population. Recombinant activated protein C has been developed as a therapeutic agent, and was found to be able to significantly reduce mortality in patients with severe sepsis and at high risk of death.^{[23, 24, 45-47]} However, activated protein C associated hemorrhage is a major safety concern of the drug, especially in patients with a low mortality risk.^[43] And a recent study found that activated protein C administration had no benefit in patients with AP.^[48-51] Patients with sepsis and those with AP have many clinical and pathophysiologic similarities.^[1] Based on our finding that protein C levels in patients with HLP were higher than those in NHLP, we speculated that only patients with NHLP, and a subgroup of HL-MSAP patients who have low protein C levels, may demonstrate a better response to activated protein C treatment. Further investigations are needed to confirm our findings in a larger patient population.

 

In conclusion, patients with HLP have higher levels of protein C, and the levels of D-dimer and protein C in HLP patients are correlated with disease severity in positive and negative manners, respectively. D-dimer is correlated with LDL-C in AP, especially in HLP patients. D-dimer levels could predict disease severity with a high sensitivity and specificity. These findings indicate that D-dimer may serve as a prognostic marker in patients with HLP.

 

 

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