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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 1  |  Page : 1-5

Sulfur-containing amino acids and oxidative stress in chronic pancreatitis patients


1 Department of Physiology, Amrita School of Medicine, Amrita Institute of Medical Sciences, Amrita Viswa Vidyapeetham, Kochi, Kerala, India
2 Department of Gastroenterology, Amrita School of Medicine, Amrita Institute of Medical Sciences, Amrita Viswa Vidyapeetham, Kochi, Kerala, India
3 Department of Biochemistry, Amrita School of Medicine, Amrita Institute of Medical Sciences, Amrita Viswa Vidyapeetham, Kochi, Kerala, India

Date of Submission23-Jan-2020
Date of Acceptance06-Mar-2020
Date of Web Publication07-Jul-2020

Correspondence Address:
Prof. Rajesh Gopalakrishna
Department of Gastroenterology, Amrita School of Medicine, Amrita Institute of Medical Sciences, Amrita Viswa Vidyapeetham, Kochi - 682 041, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/AMJM.AMJM_7_20

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  Abstract 


Background: Chronic pancreatitis (CP) patients are at high risk of malnutrition due to malabsorption. Sulfur amino acids (SAAs) being important antioxidant could affect pancreatic function. In this study, we have analyzed blood SAAs and its relation to antioxidant levels. Methods: One hundred and seventy-five CP patients and 113 healthy normal controls were prospectively studied. Disease characteristics and imaging features were recorded. Plasma SAAs were estimated using high-performance liquid chromatography. Erythrocyte reduced glutathione (GSH), GSH peroxidase, superoxide dismutase, plasma Vitamin C, erythrocyte thiobarbituric acid reactive substance (TBARS), urinary inorganic sulfate, and creatinine were estimated by spectrophotometry. Results: Plasma SAAs, urinary inorganic sulfate/creatinine ratio, and blood antioxidant levels were lower, whereas TBARS was higher in CP patients as compared to controls. Plasma methionine and TBARS were inversely correlated, whereas plasma cysteine and GSH level were directly correlated. Plasma cysteine and Vitamin C levels were lower, whereas TBARS was higher in CP patients with atrophy as compared to patients without atrophy. No statistical difference in these parameters between diabetic and nondiabetic CP patients was observed. Conclusion: Deficiency of SAAs appears to be associated with oxidative stress in CP patients. Possible benefit of supplementation of SAAs needs to be elucidated.

Keywords: Chronic pancreatitis, cysteine, inorganic sulfate/creatinine ratio, methionine, oxidative stress


How to cite this article:
Girish BN, Gopalakrishna R, Vaidyanathan K. Sulfur-containing amino acids and oxidative stress in chronic pancreatitis patients. Amrita J Med 2020;16:1-5

How to cite this URL:
Girish BN, Gopalakrishna R, Vaidyanathan K. Sulfur-containing amino acids and oxidative stress in chronic pancreatitis patients. Amrita J Med [serial online] 2020 [cited 2020 Aug 5];16:1-5. Available from: http://www.ajmonline.org.in/text.asp?2020/16/1/1/289142




  Introduction Top


Chronic pancreatitis (CP) is defined as a persistent inflammatory disease of the pancreas characterized by irreversible morphological changes typically causing pain and/or permanent loss of function which may be associated with exocrine and/or endocrine insufficiency.[1] Balaji et al. showed an enormous difference in incidence rates of CP in South India, especially Kerala state (125/100,000 population) as compared to the Western world (10–15/100,000 population).[2] In South India, nonalcoholic and nonhereditary form of CP was more common among children and young adults called tropical chronic pancreatitis (TCP). It is characterized by recurrent abdominal pain, pancreatic calculi, and diabetes with young age of onset, malnutrition, and severe pancreatic damage.[3] However, there are changing time trends in the natural history of tropical pancreatitis with improving socioeconomic status of a population. Alcoholic chronic pancreatitis (ACP) is emerging as a dominant etiology over recent years.[4] Exact pathologic mechanisms of CP are still unclear. Various hypotheses have been suggested, namely role of micronutrient deficiency, dietary toxins, and genetic influences.[4] The role of oxidative stress has received much attention. However, metabolic derangements and pathogenetic mechanisms remain a gray area. There is a paucity of literature in this regard.

Antioxidant potential of a sulfur amino acid (SAA) plays an important role in combating oxidative stress. Disturbed SAA metabolism was indicated in chronic relapsing pancreatitis by Martensson and Bolin.[5] Conversion of S-adenosyl methionine to S-adenosyl homocysteine plays a crucial role in methylation reaction which is essential for cell growth, differentiation, and survival, suggesting that deficiency of methyl donors could affect pancreatic function. Previously, we have reported diminished ability to remethylate homocysteine back to methionine in CP patients, probably due to folate deficiency.[6] Disturbed methyl metabolism may contribute to the pathogenesis of the pancreatic disease.[7]

The aim of the current study was to investigate the relationship between SAA level and antioxidant status in CP patients.


  Methods Top


A total of 175 consecutive CP patients were who had evidence of pancreatic calcification (ultrasonography/computed tomography) and/or parenchymal or ductal changes on imaging (computed tomography/endoscopic retrograde cholangiopancreatography/endoscopic ultrasonography) were recruited (84 ACP and 91 TCP) from the Pancreas clinic of the Department of Gastroenterology, Amrita Institute of Medical Sciences, Kerala, India. One hundred and thirteen healthy, nonalcoholic, nonsmoker controls were also recruited from the bystanders of patients. ACP was defined in CP patients with alcohol consumption of ≥80 g/d for at least 5 years. TCP was defined as per previously reported criteria.[1]

Diabetes mellitus was diagnosed if the fasting plasma glucose value was ≥126 mg/dL, confirmed on two occasions, and/or a plasma glucose value ≥200 mg/dL after a 2-h glucose load confirmed on two occasions, and/or there are requirements for insulin or oral hypoglycemic drugs. Patients with pancreatic cancer, those who had undergone pancreatic surgery, those with complications, such as a pseudocyst or common bile duct obstruction, and those consuming protein supplements were excluded from the study.

History of illness, including presenting complaints, duration of illness, pain, and diabetes mellitus and risk factors, such as alcohol and smoking, were recorded. Demographic parameters and anthropometric measurements were elicited, and a detailed physical examination was carried out. Body mass index (BMI) was calculated by the formula weight/height2 (kg/m2). A detailed dietary history was recorded.

Fasting blood samples were collected in ethylenediaminetetraacetic acid tubes and immediately placed in an icebox. Blood samples were centrifuged immediately at 1000 g for 10 min at 4°C. Cells were separated and stored at −20°C until assayed.

Plasma amino acids (methionine, cysteine) were derivatized with phenylisothiocyanate and separated and quantified by reverse-phase high-performance liquid chromatography[8] using a C18 column (150 mm × 4.6 mm ID, 3 μm) from norleucine (100 μM) as an internal standard. The biochemical tests were performed in the metabolic laboratory of the institute. Standard reactions were used to measure the levels of erythrocyte glutathione (GSH),[9] glutathione peroxidase (GPx),[10] superoxide dismutase (SOD),[11] thiobarbituric acid reactive substance (TBARS),[12] hemoglobin,[13] plasma Vitamin C,[14] urinary inorganic sulfate,[15] and creatinine[16] using a ultraviolet–visible double-beam spectrophotometer (Systronics 2201, Ahmedabad, India).

Institutional ethics committee clearance was obtained. Written informed consent was obtained from all patiens who participated in the study. The study protocol conforms to the ethical guidelines of the “World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects” adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964, as revised in Tokyo 2004.

Statistical analysis was done using SPSS version 11 software (SPSS Inc., Chicago, USA). Student's t-test was performed to compare means. Nonparametric Mann–Whitney U-test and Kruskal–Wallis test, as appropriate, were used to compare variables without a normal distribution. Linear correlation between the two groups was evaluated by calculating the Spearman rank correlation coefficient. Biochemical values were expressed as the mean ± standard error for comparison. Two-tailed P < 0.05 was considered statistically significant.


  Results Top


The demographic characteristics of the study population are given in [Table 1]. The mean age of CP patients was comparable with the age of controls. The mean BMI was lower in CP patients.
Table 1: Demographic representation of the study population

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Blood antioxidants such as GSH, GPx, SOD, and Vitamin C levels were significantly lower, and membrane lipid peroxidation product – TBARS – was higher in CP patients as compared to controls [Table 2]. Plasma levels of methionine and cysteine and urinary inorganic sulfate/creatinine ratio were significantly lower in CP patients as compared to controls [Table 2].
Table 2: Blood antioxidant levels, lipid peroxidation product, and sulfur amino acid level in chronic pancreatitis patients and controls (mean±standard error)

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Correlation studies between plasma SAA levels and antioxidants revealed significant correlation between erythrocyte GSH and plasma cysteine levels (r = 0.434, P < 0.001) [Figure 1]. Plasma methionine and erythrocyte TBARS showed a statistically significant negative correlation (r = −0.310, P < 0.001). Urinary inorganic sulfate/creatinine ratio and BMI were found to have significant positive correlation (r = 0.417, P < 0.001).
Figure 1: Correlation between erythrocyte glutathione and plasma cysteine level in chronic pancreatitis patients

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Plasma Vitamin C and cysteine levels were significantly lower, whereas TBARS was higher in CP patients with atrophy as compared to CP patients without atrophy [Table 3].
Table 3: Blood antioxidants, lipid peroxidation product, and sulfur amino acid level in chronic pancreatitis patients with and without atrophy (mean±standard error)

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We did not find any significant difference in these parameters between diabetic and nondiabetic CP patients [Table 4].
Table 4: Blood antioxidants, lipid peroxidation product, and sulfur amino acid level in chronic pancreatitis patients with and without diabetes mellitus (mean±standard error)

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  Discussion Top


No specific treatment exists for CP. Most of the treatment modalities targeted to reduce symptoms such as amelioration of exocrine insufficiency by pancreatic enzyme supplement, pain management, and/or surgical removal. Hence, more effective treatment is essential which can act more synergistically with current therapy. Antioxidant treatment was found to be promising since oxidative stress being the common denominator for several disorders.

We observed a significant decrease in blood antioxidant levels and increase in lipid peroxidation product TBARS indicating oxidative stress in CP. Oxidative stress plays a key role in the development of fibrosis and necrosis which is the hallmark of CP. One of the mechanisms by which oxidative stress can cause fibrosis is by inducing the release of proinflammatory cytokines that can stimulate pancreatic stellate cells and thereby increases collagen synthesis.[17]

Significant decrease in SAA level observed in our patients may be due to malnutrition and malabsorption which is associated with CP. SAAs plays a vital role in the functional integrity of the pancreas. Parsa et al. showed that methionine is an obligatory requirement for the differentiation of the pancreatic acinar cell in organ culture.[18] Methionine deficiency may have adverse effects on pancreatic growth, differentiation, and survival. Feeding choline (methyl donor)–deficient, ethionine (methionine antagonist)-supplemented diet has been successfully used as an animal model for pancreatitis.[19]

A significant negative correlation between plasma methionine and TBARS and positive relationship between plasma cysteine and erythrocyte GSH levels would highlight the antioxidant potential of SAA level. Methionine acts as an efficient oxidant scavenger. Methionine is readily oxidized to methionine sulfoxide by reactive oxygen species. Recycling of methionine by methionine sulfoxide reductase would allow the antioxidant system to function.[20] L-cysteine is the precursor of GSH, either synthesized from methionine by transmethylation pathway or it is derived from the diet. The supply of L-cysteine is rate-limiting step for GSH biosynthesis.[21] Studies conducted by Braganza et al. proposed that derangements in the transsulfuration pathway can cause oxidative stress in pancreatic acinar cells.[22] In our previous study, we reported that decreased plasma methionine and increased homocysteine plus the reduced level of folate in CP patients suggest a diminished ability to remethylate homocysteine.[6] Further studies required to test whether hyperhomocysteinemia has any pathologic consequences through possible vascular damage in the pancreas.

Meta-analysis of trials evaluating methionine containing antioxidants in CP patients showed a significant reduction in episodes of pain.[23] All these trials were carried out for a maximum of 12 months. Since CP is developed after many years of illness, it is essential to study the supplementation of methionine for a longer duration. Interestingly, Yang et al.[24] showed the administration of L-cysteine suppressed the proliferation and extracellular matrix production of pancreatic stellate cells in CP in rats.

Genetic predisposition is an important risk factor of pancreatitis. Recently, a study conducted in North India showed a significant association between Methylenetetrahydrofolate reductase (MTHFR) (C667T) polymorphism in the susceptibility of TCP.[25] Since MTHFR is involved in methylation, inflammation, and protection against oxidative stress, the process is especially important for pancreatic homeostasis.


  Conclusion Top


This study reports a significant negative relationship between SAA levels and oxidative stress in CP. Future clinical trials are needed to evaluate the potential benefits of supplementation with SAAs in boosting the status of antioxidants to improve the health of CP patients.

Financial support and sponsorship

The authors thank the Kerala State Council for Science Technology and Environment (KSCSTE), Government of Kerala, Kerala state, India, for the financial support.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Balakrishnan V, Unnikrishnan AG, Thomas V, Choudhuri G, Veeraraju P, Singh SP, et al. Chronic pancreatitis. A prospective nationwide study of 1,086 subjects from India. JOP 2008;9:593-600.  Back to cited text no. 1
    
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Balakrishnan V. Tropical chronic pancreatitis: A historical perspective. Gut 2011;60:1441.  Back to cited text no. 3
    
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Mårtensson J, Bolin T. Sulfur amino acid metabolism in chronic relapsing pancreatitis. Am J Gastroenterol 1986;81:1179-84.  Back to cited text no. 5
    
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Girish BN, Vaidyanathan K, Rao NA, Rajesh G, Reshmi S, Balakrishnan V. Chronic pancreatitis is associated with hyperhomocysteinemia and derangements in transsulfuration and transmethylation pathways. Pancreas 2010;39:e11-6.  Back to cited text no. 6
    
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Bonsnes RW, Taussky HH. Determination of creatinine in urine. In: Varley H, editor. Practical Clinical Biochemistry. 4th ed. London, UK: Heinemann; 1967:197-8.  Back to cited text no. 16
    
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Apte MV, Haber PS, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA, et al. Pancreatic stellate cells are activated by proinflammatory cytokines: Implications for pancreatic fibrogenesis. Gut 1999;44:534-41.  Back to cited text no. 17
    
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Parsa I, Marsh WH, Fitzgerald PJ. Pancreas acinar cell differentiation. 3. Importance of methionine in differentiation of pancreas anlage in organ culture. Am J Pathol 1970;59:1-22.  Back to cited text no. 18
    
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Lombardi B, Estes LW, Longnecker DS. Acute hemorrhagic pancreatitis (massive necrosis) with fat necrosis induced in mice by DL-ethionine fed with a choline-deficient diet. Am J Pathol 1975;79:465-80.  Back to cited text no. 19
    
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Levine RL, Mosoni L, Berlett BS, Stadtman ER. Methionine residues as endogenous antioxidants in proteins. Proc Natl Acad Sci U S A 1996;93:15036-40.  Back to cited text no. 20
    
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Taniguchi M, Hiramaya K, Yamaguchi N, Takeishi N, Suzuki M. Nutritional Aspects of glutathione metabolism and function. In: Dolphin, Poulson, Avramovic O (eds) Glutathione: Chemical, Biological and Mediacal Aspects, Part B. New York: Wiley; 1989. p. 645-727.  Back to cited text no. 21
    
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Braganza JM, Lee SH, McCloy RF, McMahon MJ. Chronic pancreatitis. Lancet 2011;377:1184-97.  Back to cited text no. 22
    
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Talukdar R, Murthy HV, Reddy DN. Role of methionine containing antioxidant combination in the management of pain in chronic pancreatitis: A systematic review and meta-analysis. Pancreatology 2015;15:136-44.  Back to cited text no. 23
    
24.
Yang L, Shen J, He S, Hu G, Shen J, Wang F, et al. L-cysteine administration attenuates pancreatic fibrosis induced by TNBS in rats by inhibiting the activation of pancreatic stellate cell. PLoS One 2012;7:e31807.  Back to cited text no. 24
    
25.
Singh S, Choudhuri G, Kumar R, Agarwal S. Association of 5, 10- methylenetetrahydrofolate reductase C677T polymorphism in susceptibility to tropical chronic pancreatitis in North Indian population. Cell Mol Biol (Noisy-le-grand) 2012;58:122-7.  Back to cited text no. 25
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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