The impact of alcohol abuse on selected biochemical parameters in patients with chronic pancreatitis

INTRODUCTION

Chronic pancreatitis (CP) is a disease in which the prolonged inflammatory process leads to progressive impairment of the exo- and endocrine function of the pancreas, and its fibrosis thereafter. Mortality in CP is significantly higher compared to the general population and taking into account the unknown pathogenesis of the disease, it is an important problem of current medicine [1]. The consumption of ethyl alcohol is the most important factor contributing to CP development [2]. The disturbances of the connective tissue metabolism within the pancreas have not been finally clarified. However, pancreatic stellate cells (PSCs) seem to play a critical role in the pathogenesis of pancreatic fibrosis [3]. One hypothesis of pancreatic fibrosis assumes the insufficient modeling of extracellular proteins by matrix metalloproteinases (MMPs) [4]. MMPs are a large family of Zn-dependent endopeptidases that are able to digest various proteins such as collagens, elastin and laminin. In addition, MMPs are involved in the activation of biologically active compounds such as interleukin (IL)1β and chemokine CXCL-8 [5]. Extracellularly, the activity of MMPs is regulated by their four natural tissue inhibitors (tissue inhibitor of MMPs, TIMPs). Two metalloproteinases, MMP-2 and MMP-9, forming a group of gelatinases, play a particularly important role in the pathogenesis of numerous diseases, mainly cancer and inflammation [6]. TIMP-1 and TIMP-2 were found to be the strongest inhibitors of MMP-9 and MMP-2 respectively. The ratio of the enzyme to inhibitor concentrations was previously used as an indicator of MMPs’ activity in vivo [7, 8]. A previous study revealed a negative correlation between serum MMP-2 activity and the severity of cirrhosis suggesting the reduction of collagenolytic activity as a potential cause of liver fibrosis [9]. Hyaluronic acid (HA) is a natural biopolymer formed from repeating disaccharide units containing a D-glucuronic acid and N-acetyl-D-glucosamine. In the animals, HA is a constituent of many organs and tissues such as skin, joints, eyes, body fluids (especially synovial fluid, the vitreous body of the eye) and lung [10, 11]. Previous research indicated that the concentration of HA in the blood correlates closely with the degree of fibrosis and cirrhosis of the liver, regardless of the etiology of damage [12]. Some publications also suggest the possibility of using HA as a marker of fibrosis in renal [13] and lung failure [14]. The involvement of HA in fibrosis during CP has not been studied so far. The purpose of this study was to determine the concentration of MMP-2 and MMP-9, along with the concen­tration of their natural inhibitors TIMP-1 and TIMP-2 in patients with CP as well as the relationship of MMPs vs. HA levels.

MATERIAL AND METHODS

Fifty-seven patients with definite alcoholic CP diagnosed according to M-ANNHEIM criteria [15] were enrolled in the study. The basic criterion for inclusion in the study was the absence of clinical exacerbation of the disease during blood collection. The alcoholic etiology of the CP was established by the interview statement of intake of alcohol over 80 g/day (expressed in pure ethanol) for men and over 60 g/day for women, for at least 5 years. The characteristics of the patients are presented in Table 1.
The exclusion criteria of the study were cirrhosis in medium or serious degree on the Pugh-Child scale [16, 17] cancer, any autoimmunological diseases and the clinical or laboratory symptoms of an active inflammatory process (fever, elevated white blood cell count and C-reactive protein serum level). All these stages could affect the MMP-2 and MMP-9 activity in serum.
Twenty sex- and age-matched healthy individuals, without a history of gastrointestinal diseases with no evidence of active infection or other inflammatory process, were enrolled as the control group.
The study design was approved by the Bioethics Committee of the Medical University of Lublin (No. KE 0254/49/2010). All the patients gave their informed consent for participation in the study.
Venous blood was collected into a sterile tube without the addition of anticoagulant. After clot formation and centrifugation, the supernatant (serum) was divided into smaller portions, transferred to test tubes and kept at –70°C. Further procedures were performed after completion of the material from all patients.

EVALUATION OF MMP-2, MMP-9, TIMP-1, TIMP-2 AND HA IN SERUM

For determination of MMP-2, MMP-9, TIMP-1, TIMP-2 and hyaluronic acid in serum commercially available ELISA kits were applied (R&D Systems, USA). The procedures were performed according to the manufacturer’s instructions. All assays were performed in duplicate. The results were read using a Bio-Rad (USA) microplate reader at a wavelength of 450 nm (540 nm correction).

STATISTICAL ANALYSIS

The normality of distribution was determined using the Lilliefors test. In the case of a normal distribution the t-test was used for the comparison of values within the study and control groups and the results were expressed as the mean ± standard deviation (SD). Otherwise, the Mann-Whitney test was applied and the results were expressed as the median and lower/upper quartiles. The evaluation of the relationship between MMP/TIMP ratio vs. hyaluronic acid level was performed using Pearson correlation. The 2 test was applied for evaluation of differences in gender and smoking in the study and control groups. Statistical calculations were performed using InStat GraphPad (La Jolla, USA). Significance of differences was set at the level of less than 0.05.

RESULTS

A significant increase in TIMP-1 level and a decrease in MMP-2/TIMP-1 ratio were noted in patients with alcoholic CP compared to the control group. The difference between MMP-2, MMP-9, TIMP-2, MMP-9/TIMP-1, MMP-9/TIMP-2, MMP-2/TIMP-2 serum levels in the study and control group did not reach statistical significance.
Assuming that MMP level could indicate the fibrosis intensity within the pancreas, we compared the HA level (a potential marker of fibrosis within the pancreas) in serum of CP patients vs. controls and we estimated the correlation between combined MMP/TIMP ratio (MMP-2+MMP-9 levels/TIMP-1+TIMP-2 levels) and HA level. The observed higher HA level in CP patients in comparison to controls did not reach statistical significance. Similarly, the negative, weak correlation (r = –0.19) between combined MMP/TIMP vs. HA was not statistically significant (p > 0.05). The study results are presented in Table 2.

DISCUSSION

According to our knowledge, this study is the first report related to the concentration of gelatinases and their inhibitors in the serum of patients with alcoholic CP measured not under exacerbation of the disease.
The results showed no statistically significant differences between the levels of both MMP-2 and MMP-9 in sera of patients with CP compared to healthy volunteers. These results do not confirm the reports of other authors who have reported elevated levels of MMP-9 in patients with CP [4, 9, 18]. However, in our study, blood was collected during a scheduled follow-up visit in out-patient clinic and none of the study patients had exacerbation of the disease. Exacerbation of CP induces several pro-inflammatory cytokines that up-regulate mostly MMP-9 and TIMP-1 expression [4]. Therefore, we decided to collect the blood between the exacerbation periods to show the role of MMPs and TIMPs in the chronic phase of the disease. The relationship between MMP-2 or MMP-9 expression depending on the stage of the disease has previously been observed in other relapse-remitting diseases. For example, in multiple sclerosis (MS) the activity of MMP-9 increases dramatically during periods of relapse, returning to values comparable to the control group during remissions. Otherwise, MMP-2 increases during remission of RR-MS [19].
In this study, a statistically significant increase of TIMP-1 concentrations in patients with alcoholic CP was noted. Previous studies indicated TIMP-1 as a marker of liver fibrosis. In addition, TIMP-1 is one of the components of the panel of biomarkers (Enhanced Liver Fibrosis test – ELF), which together with hyaluronic acid and the amino terminal propeptide of type III procollagen (PIIINP) was used for the intermediate assessment of liver fibrosis [20]. Previous experiments using tissue homo­genates obtained during resection of the pancreas due to CP or cancer showed significantly lower levels of both TIMP-1 and TIMP-2 in CP as compared to cancer [21]. Unfortunately, the above results were not compared to normal pancreatic tissue.
MMP activity can be determined by a semi-quantitative method such as zymography or by calculating the ratio of MMP to TIMP. In the literature the ratios of MMP-9/TIMP-1, MMP-9/TIMP-2 and MMP-2/TIMP-2, MMP-2/TIMP-1 have been used [22-24].
The lack of elevated MMP-9 level during CP, together with a significant increase in the concentration of TIMP-1 (the strongest tissue inhibitor of MMP-9), suggests that the activity of this enzyme in CP is lowered in vivo. This event is partially confirmed by the reduced (on the border of statistical significance) value of the MMP-9/TIMP-1 ratio. These observations classify MMP-9 as an enzyme whose activity tends to decrease in the CP during the phase without exacerbation of symptoms.
The statistically significantly lower value of the MMP-2/TIMP-1 ratio in CP patients compared to controls indicates the decrease of gelatinolytic activity accompanying CP. The reduction in serum MMP-2 activity was previously observed in patients with cirrhosis and hepatic fibrosis [9]. The decrease in MMP-2 level may cause excessive growth of connective tissue within the pancreas resulting in progressive fibrosis that occurs during CP.
The increase in the concentration of hyaluronic acid in patients with CP was not found to be statistically significant. Previous studies have shown an increase of HA serum level in exacerbations of CP [25, 26]. Although the authors of these studies did not correlate the studied parameters with the results of biopsy of the pancreas, the obtained results were related to the degree of organ fibrosis, similarly to the role which HA plays in liver fibrosis.
The negative correlation coefficient between the integrated MMP/TIMP and the concentration of hyaluronic acid did not reach statistical significance; however, it appears to be interesting. Previous studies have indicated a reduction of gelatinolytic activity in patients with advanced liver fibrosis, but these observations were not studied in pancreatic disease [9].
The study has some limitations. Chronic exposure to ethanol in CP patients affects many biochemical processes inside the body and it may also interfere with the expression of gelatinases. Therefore, there is a possibility that the observed changes in the activity of MMP-2 and MMP-9 are the result of chronic alcohol abuse, but it is not directly related to the inflammatory process in the pancreas. However, the effects of ethanol on the activity of gelatinases are ambiguous. Previous studies revealed that ethanol exerts different effects on various tissues. It decreases the expression of MMP-2 and MMP-9 [27], increases the expression of MMP-2 in the kidney [28], but does not affect the activity of both gelatinases in the liver and in the aortic wall [29]. Reports on the activity of gelatinases in other chronic diseases with the etio­logy of alcohol also tend to present inconclusive results. Some studies showed an increase in the activity of MMP-9 in blood of patients with cirrhosis [30] while others reported a decrease in MMP-2 without an apparent effect on MMP-9 activity in cirrhosis [31]. Therefore, this issue requires further observation with the enrolment of an additional study group consisting of abusers without a diagnosis of pancreatitis.

CONCLUSIONS

The elevation of TIMP-1 and decrease in MMP-2/TIMP-1 ratio in serum of CP patients indicates reduced gelatinolytic activity in the progression of CP. This finding supports the hypothesis that insufficient remodeling of extracellular components accompanying CP could be one cause of the intense fibrosis within the pancreas.

DISCLOSURE

The authors report no conflict of interest.

References