Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Abstract

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.

Figure 1

Fatty acids cause more cell death than their EEs. A and B: LDH leakage (A) and PI uptake (B) is increased compared with CONs in acini treated with unsaturated fatty acids LA and OA compared with LAEE and OAEE. C: LA and OA also cause decrease in ATP levels compared with their EEs. The saturated fatty acid PA does not cause any effect compared with its EE, PAEE. D: Western blot analysis for cytochrome c shows a leakage of cytochrome c from the M compartment to the C compartment in the presence of LA or OA. This intercompartment cytochrome c leakage is absent in LAEE and OAEE. Loading controls, α-tubulin for the C compartment and COX-IV for the M compartment, are equally present in all of the groups. Data are expressed as means ± SEM. ^∗P < 0.05 versus CONs; ^†P < 0.05 versus LA/OA. ATP, adenosine triphosphate; C, cytosolic compartment; CON, control; COX, cytochrome c oxidase; EE, ethyl ester; LA linoleic acid; LDH, lactate dehydrogenase; M, mitochondrial compartment; OA, oleic acid; PA, palmitic acid; PI, propidium iodide.

Figure 2

FAEEs affect mitochondrial membrane potential and cytosolic calcium levels less than FAs. A and B: All agents were added at zero second. LA and OA cause a significantly higher loss of mitochondrial membrane potential than their respective FAEEs, indicated by the increase in 530/590 emission ratio. C and D: Cytosolic calcium levels in Fura-2–loaded acini, measured by 340/380 emission ratio, show an increase in resting calcium by FAs, not FAEEs. Data are expressed as means ± SEM. n = 4 to 5 experiments. ^∗P < 0.05 versus the CON state. CON, control; EE, ethyl ester; FA, fatty acid; LA, linoleic acid; OA, oleic acid.

Figure 3

Intraductal injection of triolein causes higher rate of mortality and serum OA concentrations. A: Bar graphs of serum oleate level showing a significant increase in its concentrations with intraductal injection of triolein and OAEE. OAEE causes a significantly lesser increase compared with triolein. B: Kaplan-Meyer mortality curves show 70% mortality with the GTO group versus no mortality in the OAEE group. Data are expressed as means ± SEM. ^∗P < 0.05 versus the CON; ^†P < 0.05 versus triolein (GTO) group. CON, control; EE, ethyl ester; GTO, glyceryl tri-oleate; OA, oleic acid.

Figure 4

Triolein causes severe hemorrhagic necrotizing pancreatitis. A–E: Gross appearance (A–C) and hematoxylin and eosin-stained sections (D) of the pancreas in CONs (A and D), intraductal triolein (B and D), and OAEE (C and D). Intraductal injection of GTO causes significant hemorrhage and necrosis (B, D, and E) compared with the milder necrosis and no hemorrhage in OAEE (C, D, and E). F and G: OAEE caused a lesser increase in amylase and lipase than GTO. Data are expressed as means ± SEM. ^∗P < 0.05 compared with CONs; ^†P < 0.05 compared with GTO. Original magnification, ×10 (D). CON, control; GTO, glyceryl tri-oleate OAEE, oleic acid ethyl ester.

Figure 5

Triolein administration causes a worse cytokine response and inflammation compared with OAEE. A–D: Box plots depicting cytokines IL-1β (A), IL-18 (B), IL-6 (C), and KC-GRO (D) show significant increase in the group administered triolein intraductally compared with CONs. Administration of OAEE significantly reduces these cytokines. E: MPO⁺ cells are significantly increased in the GTO group versus the OAEE group. ^∗P < 0.05 compared with CONs; ^†P < 0.05 compared with GTO. Original magnification: ×20 (E); ×60 (insets). CON, control; KC-GRO, keratinocyte chemoattractant–growth-regulated oncogene; GTO, glyceryl tri-oleate; MPO, myeloperoxidase; OAEE, oleic acid ethyl ester; Sr., serum.

Figure 6

Triolein causes worse systemic injury compared with OAEE. A: Presence of TUNEL⁺ cells (inset, black arrows) in lungs are significantly higher in the GTO group than the OAEE group. B: Intraductal GTO also causes a significant increase in BUN and TUNEL⁺ cells in the kidney tubules. Representative TUNEL-stained image for each condition is shown to the right of the bar graphs (A and B). C–F: Peripheral blood mononuclear cells treated with OA show a significant decrease in live cells (C and D) and an increase in late apoptotic cells (C and E) and necrotic cells (C and F) compared with OAEE at equimolar amounts (10 μmol/L). ^∗P < 0.05 compared with CONs; ^†P < 0.05 compared with GTO. Original magnification: ×40 (A); ×20 (B); ×100 (insets). BUN, blood urea nitrogen; CON, control; EE, ethyl ester; FITC, fluorescein isothiocyanate; GTO, glyceryl tri-oleate; HPF, high power field; OA, oleic acid; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.