AOCS meeting April 2014, Huiles Bioavailability of Esters of 3-MCPD: in vitro and in vivo Studies F. JOFFRE, S. AMARA, F. CARRIERE, L. COUËDELO and C. VAYSSE French Institute for Fats and Oils (ITERG) Laboratory of Enzymologie Interfaciale et Physiologie de la Lipolyse - UMR7282 CNRS - Aix Marseille University Raffinage
What do we know about 3-MCPD esters? Formed during the refining of oil (especially deodorization) Mainly 3-MCPD diesters >>> 2-MCPD diesters and 3-MCPD monoesters High structural diversity of esters (FA composition, localization of Cl): a family of contaminant 3 indirect analytical methods (AOCS) H 2 C FA FA CH H 2 C Cl 3-MCPD diesters
What do we know about the toxicity of 3-MCPD esters? Free 3-MCPD is genotoxic (in vitro but not in vivo) and carcinogen (kidney and testis) 3-MCPD diesters: 3-MCPD DE have lower toxicity than their counterparts ME (Tee, 2011; Liu, 2011) High mortality related with tubular necrosis was observed in female rats fed with free 3-MCPD (higher dose) but not with 3-MCPD DE (Barocelli 2011, Onami, 2014) -30% of urinary metabolites after feeding 3-MCPD DE in comparison with free MCPD (Barocelli 2011) Renal and testicular injuries are similar in phenotype (tubular necrosis, apoptosis of the epididymis) but not in intensity after acute ingestion of 3- MCPD DE in comparison with free 3-MCPD (Barocelli, 2011; Liu, 2011; Onami, 2014) lowest toxicity of diesters may be explain by differences during digestion/absorption steps
Our hypothesis concerning the digestion/absorption of 3-MCPD esters Free 3-MCPD 3-MCPD DE Renal necrosis Carcinogenicity Death at high level Barocelli (2011), Onami (2014) Blood vessel Gastric lipolysis Free 3-MCPD 1 3-MCPD DE 3-MCPD ME Free 3-MCPD 3-MCPD ME Free 3-MCPD 3-MCPD-E are substrates for PPL (Seefelder et al., 2008) but hydrolysis of 3-MCPD DE is slower Ingested MCPD-esters may not be 100% hydrolyzed in the gut (Schilter, ILSI, 2009) Enterocyte 3-MCPD ME Tissue accumulation Milk (Zelinkova, 2008) Others? 3 Pancreatic lipolysis 3-MCPD DE Lymph compartment 2 3-MCPD DE
1st study: In vitro lipolysis of 1,2-dioleoyl-3-MCPD 1 ml of buffer, 10 mg of 3-MCPD DE + 2µg of enzyme, 1h, 37 C TAG Good rate of lipolysis Low rate of lipolysis FFA 1,3-DAG 1,2(2,3)-DAG MAG Pure 3-MCPD dic18:1 Control Without enzyme rdgl rhpl nppl rhplrp2 nceh PPE (2 µg) Gastric lipase PPE (20 µg) specific Non-specific Pancreatic Pancreatic lipases extract TAG 3-MCPD DE were hydrolyzed by gastric lipase (sn1me and sn2me) and pancreatic lipases (sn2me). This regioselectivity of lipases is usually demonstrated for TAG
1st study: In vitro two step model to evaluate the gastrointestinal lipolysis of 1,2-dioleoyl-3-MCPD + rdgl (ph 5) + PPE (ph 6) Gastric phase Duodenal phase 3-MCPD dioleate Decrease of 3-MCPD DE FFA 1,3-DAG 1,2(2,3)-DAG Increase of hydrolysis products: FFA and 3-MCPD ME MAG RS 0 15 29 35 40 45 60 90 min Loss of sn1-me (red arrow) generated during gastric phase indicated that pancreatic lipase produced free 3-MCPD. Sn2-ME (blue arrow) continously produced during duodenal phase may be absorbed by enterocyte.
Hydrolysis level (% of free FA) 1st study: In vitro two step model to evaluate the gastrointestinal lipolysis of 1,2-dioleoyl-3-MCPD 20 Gastric phase Duodenal phase 15 10 5 0 0 15 30 45 60 75 90 Time (min) Digestion rate of dioleate-3mcpd : 15% of total lipolysis after 90 min (gastric phase, 5% and duodenal phase, 10%) compared to 60-70% usually observed for TAG in the same conditions
2 nd study: In vivo absorption of 3-MCPD DE (lymphatic duct fistula model 0.3ml of oil spiked with 52 mg/kg of 3-MCPD dic18:1 + triisopalmitin 140 120 100 80 60 40 20 Content of eq. 3-MCPD (ng/ml lymphe) N=6 Surgically, mesenteric lymph duct was cannulated Wistar rats were placed in individual restraining cages and were intubated with spiked oil Collect of lymph during 24h (0-6h and 6-24h) Extraction of lipids (Folch) and quantification of 3- MCPD by indirect method (AOCS Cd29b) 0 0-6h 6-24h Constant absorption of 3-MCPD DE during 24 hours reflect slow absorption of ME produced during lipolysis and/or slow re-esterification in enterocyte or slow excretion in chylomicron Rate of absorption of 3-MCPD DE: 27%
3rd study: Tissue distribution of 3-MCPD after 4 weeks diet in female and male rats 30d of diet containing 10% of oil spiked with 52 mg/kg eq. 3-MCPD without 3-MCPD No effect on food intake, total weight gain and weight of tissue Rats were apparently healthy No anatomical abnormalities No accumulation of 3-MCPD in control rat Monitoring of food intake and weight gain Extraction of lipids from plasma, gonad, heart, liver, kidney, gut and adipose tissue Indirect determination of 3-MCPD (AOCS Cd 29b)
3rd study: Tissue distribution of 3-MCPD after 4 weeks diet in female and male rats 300,0 250,0 Content of eq. 3-MCPD (ng/ml plasma and ng/g of tissue) 200,0 150,0 plasma gonad heart liver kidney gut adipose tissue 100,0 50,0 0,0 female Tissue accumulation of 3-MCPD was higher in females than males Ovaries, kidney, heart and liver were good targets for 3-MCPD accumulation for both female and male rats male
Conclusion In vitro and in vivo studies confirmed the following points: In vitro, 3-MCPD diesters were hydrolyzed by gastric and pancreatic lipases. Nevertheless, the level of hydrolysis was lower than those obtained with TAG After ingestion, 3-MCPD diesters were absorbed since we found them in the lymph and they were accumulate throughout the body Nevertheless the level of absorption of 3-MCPD diesters in the body was lower than that of TAG The target organs that accumulated 3-MCPD diesters were the gonads and kidneys as described in the literature, liver (key-tissue for lipid metabolism) and more surprisingly the heart. The impact of these chlorinated contaminants on cardiac function is unknown.
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