Differences in Plasma Fatty Acid Composition Related to Chronic Pancreatitis: A Pilot Study.

Healthy control subjects (n=22) and subjects with CP (n=47) who were consecutively enrolled in an observational biobanking study at the Ohio State University Wexner Medical Center Pancreas Clinic from 2015 to 2016 were selected for this analysis under the approval of the OSU Institutional Review Board. Control subjects included individuals with a family history of PDAC and no clinical or imaging evidence of CP or other diseases of the exocrine pancreas. Clinical data were obtained by reviewing medical records and recorded on a standardized form. Extracted information included disease history, past medical history, and social history. Smoking status and alcohol use patterns were self-reported prospectively using a standardized questionnaire. Excessive alcohol use was defined as consuming ≥14 alcoholic drinks per week for ≥5 years and tobacco use as smoking ≥100 cigarettes during the subject’s lifetime. EPD was defined as the presence of a supporting clinical diagnosis (based on either overt steatorrhea or abnormal indirect pancreatic function testing) and/or the use of pancreatic enzyme replacement therapy. Non-fasting blood samples were collected from all study subjects at the time of enrollment in the study. Aliquots of plasma were frozen at −80°C until the time of sample analysis. Research personnel conducting the analyses were blinded to the subjects’ group assignment. We measured FAs composition as previously described. 16 In brief, total lipids were extracted from formerly frozen plasma samples using the classic 2:1 chloroform:methanol method followed by 0.88% potassium chloride wash. 16 This method has the advantages of low variability and is comparable to other studies in the field. 17 , 18 Total esterified and non-esterified lipids were methylated using 5% hydrochloric acid in methanol at 76°C. 19 , 20 FA methyl esters (FAMEs) were analyzed using gas chromatography on a 30-m Omegawax TM 320 fused silica capillary column (Supelco Inc, Bellefonte, PA, USA), with the oven temperature and carrier gas flow rate settings as previously described. 17 Sample retention times were compared to standards for each FA (Matreya, LLC, Pleasant Gap, PA; Nu-Check Prep Inc, Elysian, MN). The data were presented as the quantity of each FAME as a percentage of all identified FAMEs (% area) indicating the relative abundance of each FAME, as recommended by experts in the field. 18 The percent coefficient of variance (%CV) for the FAME measurements are provided for inter-sample variability calculation ( Supplemental Table 1 )). A de novo lipogenesis index was calculated using a ratio of palmitic acid to linoleic acid. 21 Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc, San Diego, CA). Frequencies were reported for categorical variables and were compared between groups using a chi-squared test of independence or Fisher’s exact test, as appropriate. Means and standard deviations were reported by study group for the continuous variables of age and BMI and compared using an independent samples t-test and the non-parametric Mann-Whitney U test, respectively. Differences in FA composition between groups were compared using 2-way ANOVA with a Fisher’s LSD post-hoc test for pairwise comparisons. A non-parametric Mann-Whitney U test was used for FAs whose distribution was non-normal. P-values were adjusted for multiple testing using a Bonferroni correction. Spearman’s rho correlation coefficients were calculated to assess associations between FA composition and age or BMI. Fisher’s z transformation compared differences in correlation between control and CP groups. Propensity matching was performed using an outcome of FA composition with covariates of sex, age, BMI, and smoking history. Diabetes and EPD were excluded from propensity matching since these are common complications of CP and would cause a bias.

A total of 69 subjects were included in the study population: 22 controls and 47 subjects with CP. On average, the CP group was older, had a higher proportion of males, and had a lower BMI ( Table 1 ). The CP group had a higher prevalence of diabetes and excessive alcohol use compared with the control group, but no difference was observed for smoking history. Most CP subjects in this study had either an idiopathic or substance use etiology of disease ( Table 1 ). Plasma FA composition analysis revealed palmitic acid (% of plasma FAs) was higher (p < 0.0001) and linoleic acid was lower (p < 0.0001) in CP compared to controls ( Figure 1 and Supplemental Table 2 ). Similar trends were observed in FAs after propensity score matching to the control group ( Supplemental Table 3 ). Within the control group, palmitic, oleic and linoleic acids were different between males and females; however, these trends disappeared in the CP group ( Supplemental Figure 1A and B , respectively ). Since the control and CP groups differed by age and BMI characteristics and those characteristics can influence FA metabolism, we used correlations between age or BMI and FAs to explore the differences in FA composition. 22 – 24 We found linoleic acid was negatively correlated with age and dihomo-γ-linolenic acid was negatively correlated with age in the control group; however, these correlations were lost in the CP group (p < 0.05 for each) ( Table 2 ). Palmitic, palmitoleic and dihomo-γ-linolenic acids were strongly positively correlated with BMI in the control group; however, these correlations were not observed in the CP group (p < 0.01, p < 0.05, and p < 0.001, respectively) ( Table 3 ). Overall, these data suggest correlations between plasma FAs normally seen in healthy individuals during aging or across the BMI spectrum are lost in CP. Diabetes and EPD are common complications in CP that influence fat soluble nutrient absorption and metabolism. 25 , 26 We dichotomized subjects with CP according to the presence or absence of diabetes. We found that oleic acid was decreased (p < 0.01) and linoleic acid increased (p < 0.05) in subjects with CP and diabetes when compared to subjects with CP but without diabetes ( Figure 2A ). Although EPD affects nutrient absorption, we did not detect any significant differences in any FAs in subjects with CP and EPD compared to subjects with CP without EPD ( Figure 2B ). Calcific CP is often considered as “severe CP” and is a common morphologic feature of late-stage CP. Therefore, we compared FA composition within CP between calcific and non-calcific CP. There was an increase in oleic acid (p < 0.01) and a decrease in linoleic acid in calcific CP (p < 0.0001, Figure 2C ). Excessive tobacco and alcohol use are common risk factors for CP. 27 Therefore, we evaluated whether differences in plasma FA composition were associated with substance use and CP. Linoleic acid was decreased (p < 0.0001) in subjects with CP who had a history of alcohol use compared to subjects with CP without a history of alcohol use ( Figure 3A ). There was an increase in oleic acid in subjects with CP and a history of smoking compared to those without a history (p < 0.05). Linoleic acid was also decreased in subjects with CP who had a history of smoking (p < 0.01, Figure 3B ). There was also a trend for a decreased in arachidonic acid in subjects with CP who had a history of smoking (unadjusted p = 0.0558). These data suggest that in patients with CP complications and risk factors for CP could influence the relative abundance of oleic and linoleic acids. Dysregulation of de novo lipogenesis is often associated with metabolic dysregulation in multiple pathologic conditions. 28 Since CP is also associated with multiple metabolic disorders, we used a ratio of palmitic acid (a product of de novo lipogenesis) and linoleic acid (an essential FA that can only be acquired through diet) to create a de novo lipogenesis index. 21 Subjects with CP had an elevated de novo lipogenesis index compared to controls (p<0.001, Figure 4A ). Propensity score matching to the control group also supported this elevation in CP subjects (p < 0.04, Supplemental Table 3 ). Within the CP population, there was an increase in de novo lipogenesis in subjects with calcific CP compared to those with non-calcific CP (p < 0.05, Figure 4B ).

Pancreatitis often results in deficiencies in lipids that have been implicated in disease progression; however, many studies group all CP patients together even though it is a heterogenous disease. 8 , 15 , 29 In this study, we now highlight that linoleic acid and oleic acid are altered within CP subjects when dichotomizing by CP complications including pancreatic calcification and diabetes, or a history of substance use (alcohol and smoking). We show CP and calcific CP is associated with an increase in markers of de novo lipogenesis of saturated and monounsaturated FAs. Finally, we show that expected associations between plasma FAs and age or BMI are altered in CP. These results suggest the specific complications and risk factors of CP can lead to unique metabolic alterations. Hyperlipidemia, an elevation of lipids in the blood, is an etiology of pancreatitis and can drive inflammation and acinar cell necrosis through increased levels of palmitic acid. 12 , 30 , 31 Palmitic acid, which we observed elevated in CP compared to controls, is elevated in severe AP compared to controls and is a marker of de novo lipogeneisis. 15 Palmitic acid can induce inflammation by stimulating the secretion of pro-inflammatory cytokines like interleukin 6, tumor necrosis factor α, interleukin 8, and interleukin 1β, inducing insulin resistance and promoting macrophage M1 polarization. 12 , 32 , 33 Diets high in palmitic and myristic acids have been associated with lipotoxicity related to non-alcoholic steatohepatitis. 34 To counteract this, omega-3 FAs, such as those in fish oils, have been recommended as nutritional supplementation to reduce inflammation in pancreatitis, particularly in acute pancreatitis. 35 , 36 The long chain omega-3 FAs, eicosapentaenoic and docosahexaenoic acids were not different between controls and CP and within CP. Although there may be promise to using omega-3 FAs in treating fibrotic CP in mice using omega-3 FAs, 37 reducing de novo lipogenesis of saturated FAs or increasing linoleic acid in the diet may also help reduce fibrosis and inflammation in CP. Increased age generally correlates with changes in FA composition 38 – 40 and can worsen pancreatitis. 41 Additionally, older patients and those with CP often have increased visceral adipose tissue and peripancreatic fat compared with individuals without CP. 42 , 43 Fatty replacement of pancreatic acinar cells and inflammation from increased adipokine levels can lead to exocrine dysfunction. 44 Although visceral adiposity is increased in CP and aging, 42 , 43 the correlations observed between age or BMI and the relative abundance of several FAs in the control group did not persist in the CP group. To our knowledge, this is the first study to identify a loss of correlation between plasma FA composition and age or BMI in CP compared to a control group. However, a larger sample size is needed to verify this observation using controls that better matched the disease group. Nevertheless, these data further illustrate how pancreatic diseases can dysregulate metabolism and alter nutrient absorption beyond changes caused by differences in body composition or age. We observed differences in oleic acid and linoleic acid in CP compared to controls and in the CP subgroup with diabetes compared to without diabetes. Oleic acid supplementation can improve insulin sensitivity of adipocytes by modulating PI3K signaling. 45 Additionally, high linoleic acid levels are also associated with improved glycemic control and a reduced incidence of diabetes 46 and reduces diabetes in preclinical models 10 and clinical trials. 47 Linoleic acid supplementation of less than one serving of oil per day increases plasma linoleic acid and tetralinoleoyl cardiolipin (important in mitochondrial function) in peripheral blood mononuclear cells of healthy subjects . 48 Our unexpected finding of higher linoleic acid and lower oleic acid in CP with diabetes may be related to an increased use in diabetic drugs modulating FA uptake and metabolism or that the combined endocrine and exocrine dysfunction may modify these FAs within CP in a previously unreported fashion. Therefore, further assessment of FAs and their metabolism within CP and diabetes will be needed to understand the complex regulation of FAs. Substance abuse is another important etiologic risk factor for developing CP. Several studies have shown polyunsaturated FAs (PUFA) like linoleic acid are decreased and monounsaturated FAs like oleic acid are increased in patients with CP who had a history of excessive alcohol consumption compared to healthy controls; 7 , 49 , 50 however, many of these studies do not address how smoking affect FAs in patients who already have CP. In our study, we confirm that linoleic acid decreased, and oleic acid increased with alcohol use in patients with CP. However, we found only linoleic acid was decreased with a history of smoking in patients with CP compared to patients with CP who did not have a history of smoking. Linoleic acid as well as other PUFAs and monounsaturated fatty acids (MUFAs) and their metabolites are altered in people who smoke without a history of pancreatitis. 51 , 52 Therefore, increasing linoleic acid or modifying linoleic acid metabolism in patients with a history of substance abuse may be a beneficial target for therapy in this subgroup of CP patients. There are limitations to this pilot study that should be considered. First, our findings, particularly those related to subgroup analyses (even with the proper statistical comparison correction), should be considered preliminary because of the small sample size and low proportion of males in the control group, limiting the power of our analysis and potentially contributing to the overall differences observed between the control and CP groups. Additionally, blood samples were obtained while patients were non-fasting, and nutritional intake was not recorded, which may have contributed to variances in our data, particularly in linoleic acid. 7 , 8 , 49 However, our data aligns with previously published studies suggesting there is a dysregulation of linoleic acid metabolism, indicated by increased metabolites in CP, 53 or a reduction in linoleic acid absorption, and an increased de novo lipogenesis in CP and changes of FA composition, including linoleic acid, in diabetes and CP. 6 , 8 Unexpectedly, we did not observe any differences in FA composition in CP with and without EPD, often seen in patients with cystic fibrosis-associated pancreatitis. 54 Although this study did not have subjects with cystic fibrosis or hyperlipidemia, a future study including these additional forms of pancreatic disease would further determine if our findings can also apply to these subsets of patients. Despite the stated limitations, the results of this pilot study highlight the intrinsic heterogeneity within CP and reveal oleic and linoleic acid to be consistently altered in CP subgroups. Therefore, this pilot study provides the basis for more extensive and mechanistic studies to evaluate the role of de novo lipogenesis and linoleic acid metabolism in the pathogenesis of CP.

Pancreatitis is a debilitating inflammatory disease of the pancreas, which can occur as an acute or chronic disease. Risk factors for chronic pancreatitis (CP) include excessive alcohol consumption, smoking, genetic predisposition, autoimmune disease, and ductal obstructions. 1 CP is characterized by inflammation of increasing severity, resulting in declining pancreatic function and irreversible morphologic changes, including calcification and fibrosis. 2 The unremitting abdominal pain that accompanies this fibro-inflammatory process is linked to reduced quality of life, increased disability, and increased utilization of healthcare services. 3 In addition, CP is a recognized risk factor for pancreatic ductal adenocarcinoma (PDAC), a highly aggressive malignancy that is difficult to detect at an early stage. 2 , 4 , 5 Therefore, identifying targetable molecules and pathways that contribute to the pathogenesis of CP will be valuable to improve care of CP patients and help reduce the incidence of PDAC. Nutritional complications are some of the hallmarks of CP, often due to diabetes and exocrine pancreatic dysfunction (EPD), including impaired fat absorption secondary to pancreatic lipase deficiency. These sequelae lead to direct and indirect consequences, including altered dietary patterns that lead to changes in lipid and lipid-soluble nutrient metabolism. In prior studies from Europe and Japan, patients with CP exhibited differences in fatty acid (FA) composition compared with healthy individuals. 6 , 7 Diabetes, when combined with CP, also influences the composition of plasma FAs. 8 Furthermore, changes in FA composition have been associated with increased adiposity, a key driver of diabetes 9 – 11 FA products of de novo lipogenesis, a mechanism of converting excess carbohydrates into saturated and monounsaturated FAs, have been implicated in both pancreatic damage and insulin resistance. 12 – 14 Furthermore, there is an increase in the circulating levels of several markers of de novo lipogenesis including palmitic acid, palmitoleic and, and oleic acid in severe acute pancreatitis compared to controls. 15 However, the collective impact of CP and complications like EPD and diabetes, or obesity on plasma FA composition has not been assessed. In this pilot study, we aimed to compare differences in plasma FA composition not only between CP and healthy individuals, but also within CP subgroups based on etiology or complications of the disease. We further explored whether the differences observed in FA composition could be related to de novo lipogenesis.