AN OVERVIEW OF FATTY ACID ETHYL ESTERS

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1 AN OVERVIEW OF FATTY ACID ETHYL ESTERS Michael Laposata, M.D., Ph.D. Director of Clinical Laboratories Massachusetts General Hospital Professor, Harvard Medical School

2 OUTLINE OF PRESENTATION Background - Toxicity of Synthesis Secretion Transport in Blood Degradation as Markers of Ethanol Intake

3 BACKGROUND

4 STRUCTURE OF FATTY ACID AND FATTY ACID ETHYL ESTER O CH -CH 3 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -C-OH FATTY ACID (FA) CH 3 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -C-O -CH 2 -CH 3 O FATTY ACID ETHYL ESTER ()

5 Pathological Significance of Fatty Acid Ethyl Esters Organs most frequently damaged by ethanol abuse include: Pancreas > Liver > Heart > Brain After ethanol intoxication, these organs have the highest levels of fatty acid ethyl esters and fatty acid ethyl ester synthase

6 OXIDATIVE ALCOHOL DEHYDROGENASE MICROSOMAL ETHANOL OXIDIZING SYSTEM ACETALDEHYDE ALDEHYDE DEHYDROGENASE OXIDATIVE AND NON-OXIDATIVE METABOLISM OF ETHANOL ACETATE ETHANOL SYNTHASE I, II & III NON-OXIDATIVE ETHANOL O-ACYLTRANSFERASE CARBOXYLESTERASE LIPOPROTEIN LIPASE PANCREATIC TRIGLYCERIDE LIPASE CHOLESTEROL ESTERASE FA FA CoA FA / TG-FA TG-FA TG-FA FA FATTY ACID ETHYL ESTER

7 METHOD: SYNTHESIS AND LDL RECONSTITUTION LDL ISOLATED LDL CORE RECONSTITUTED WITH RADIOLABELED TRIGLYCERIDE ELUTED WITH HEXANE.5 M KOH IN ETHANOL CORE LIPIDS REMOVED WITH HEPTANE RADIOLABELED PURIFIED BY SOLID PHASE EXTRACTION

8 Isolation and Quantitation of Fatty Acid Ethyl Esters ACETONE HEXANE GC-MS All samples were extracted with acetone / hexane and s were isolated by solid phase extraction s were then quantitated by gas chromatography-mass spectrometry (GC-MS)

9 TOXICITY

10 METHOD: ASSESSMENT OF CYTOXITY OF WITHIN LDL USING 3 H-THYMIDINE INCORPORATION AS AN INDEX CYTOTOXICITY HepG2 Cells Incubated with Delipidated Medium for 6 Hours to Increase Number of LDL Receptors 3 H-THYM 3 H-THYM 3 H-THYM 3H 3H 3 H-thymidine added for 5 hours Cell monolayers harvested and 3 H- thymidine incorporated into cells determined HepG2 cells incubated with rldl- or rldl-tg or rldl-ce or native LDL for 12 hours

12 UNLABELED CHOLESTEROL ESTER UNLABELED LDL RECONSTITUTED WITH CHOLESTEROL ESTER METHOD INTRA- ARTERIAL BOLUS AND INFUSION INTRA- ARTERIAL BOLUS AND INFUSION LDL RECONSTITUTED WITH ANIMALS SACRIFICED BLOOD AND PANCREAS REMOVED FOR ANALYSIS TOXICITY DETERMINED BY: HISTOLOGICAL EXAMINATION AND ELECTRON MICROSCOPY FOR CELLULAR ABNORMALITIES TRYPSINOGEN ACTIVATION PEPTIDE (TAP) MEASUREMENT FOR PANCREATIC CELL INJURY WET/DRY RATIO FOR EDEMA FORMATION

13 RATS RECEIVING SHOWED MARKEDLY HIGHER LEVELS OF TRYPSINOGEN ACTIVATION PEPTIDE IN THE PANCREAS COMPARED TO RATS RECEIVING EITHER CHOLESTEROL ESTER OR SALINE Human LDL reconstituted with either or CE administered to rats intra-arterially Rats sacrificed at 3, 6, and 12 hours Toxicity determined by measuring trypinsogen activation peptide (TAP) P<.1 P<.2 Nmol TAP/mg tissue weight P<.1 P<.1 CE Saline 3 HRS P<.1 P<.1 CE Saline CE Saline 6 HRS 12 HRS 75 Percentile Median 25 Percentile

14 RATIONALE FOR INHIBITOR STUDIES ETOH ADH 4-methlpyrazole MEOS Diallyl Sulfide Catalase Aminotriazole Acetaldehyde Organ Damage FA Synthase

15 THE INHIBITION OF THE OXIDATIVE PATHWAY OF ETHANOL METABOLISM LEADS TO AN INCREASED INFLUX THROUGH THE NON-OXIDATIVE PATHWAY IN VIVO A Bolus of Either Saline or Inhibitors Administered to Rats (nmol/gm wet wt.) Control Liver 4 MP 2.6 g/kg Ethanol Administered and Rats Sacrificed after 12 min Triple (nmol/gm wet wt.) Liver and Pancreas Harvested, Lipid Extracted with GC-MS to Quantitate Control Pancreas 4 MP Triple

16 Overview of Experiments: Hepatitis C Virus (HCV) and Ethanol Ethanol + HCV Ethanol Added in Increasing Concentration, 25, 5, 1, and 2 mm Daily to Cells HEPG2 Cells Incubate 7 Days Trypsinize Cells Real Time- PCR Assay Extract Lipids and Quantitate by GC-MS LDL Uptake Assay In Situ Hybridization Assay

17 Ethanol increases the viral load in HCV - infected cells dose dependently Ethanol Ethanol LDL Particle HCV LDL Receptor + HCV

18 Effect of Ethanol Treatment on HCV Viral Load in HepG2 Cells Viral Load geq x 1 4 /mg Cell Protein Ethanol Concentration (mm)

19 Hepatitis C virus infection decreases production in ethanol treated cells Ethanol Ethanol Fatty Acids LDL Particle HCV LDL Receptor HCV

20 Decreased Synthesis in in Ethanol Treated- HCV Infected HepG2 Cells (Pmol/mg Protein) ETOH ETOH+HCV HCV No Treatment

21 SYNTHESIS AND SECRETION OF FATTY ACID ETHYL ESTERS

22 METHOD 12 Hrs 37º C 1.25 µm 3 H HepG2 cells Cells rinsed and serum free medium containing, 5, or 1 mm ethanol added for 9 Hrs quantitated by scintilation counting CE TG FFA PL in cells and medium isolated by TLC Lipids extracted Medium and cell monolayers harvested

23 INCREASE IN THE ACCEPTOR CONCENTRATION WILL INCREASE PRODUCTION AND SECRETION Albumin Lipoprotein Fatty Acid Binding Protein Addition of carriers FABP FABP GOLGI GOLGI ER ER

24 CELLS RETAIN A FIXED AMOUNT OF, INDEPENDENT OF ACCEPTOR CONCENTRATION & TIME OF INCUBATION Albumin Lipoprotein Fatty Acid Binding Protein Addition of carriers FABP FABP GOLGI GOLGI ER ER

25 THE ADDITION OF ALBUMIN TO THE CULTURE MEDIUM MARKEDLY ENHANCED THE SECRETION OF 3 H-18:1 Added to HepG2 Cells for 12 hrs Cellular Ethyl Oleate (% of control) Cellular Ethyl Oleate Serum Free Medium Cells Rinsed & Serum Free Medium Containing 1 mm Ethanol and 1.5% BSA Added for 9 hrs BSA Cellular Ethyl Oleate (% of control) Cellular Ethyl Oleate Serum Free Medium Isolated & Quantitated by Scintillation Counting BSA

26 LIPOPROTEIN PARTICLES IN THE CULTURE MEDIUM STIMULATE SECRETION 3 H-18:1 added to HEP G2 cells for 12 hours Ethyl Oleate (dpm/mg Cell Protein Cells rinsed & serum medium containing 1 mm ethanol and 1 µg/ml lipoprotein added for 9 hours Serum Medium isolated & quantitated by scintilation counting VLDL LDL HDL Cellular Secreted

27 IN THE CYSTOSOLOF HEPG2 CELLS ARE BOUND PREDOMINANTLY TO A kda PROTEIN HEPG2 cells + 3 H-18:1 for 12 Hrs Protein Concentration (ug/ml) Serum free medium containing 1 mm ethanol added for 12 Hrs Cells harvested & fractionated by centrifugation FRACTION NUMBER Cytosol fractionated by gel filtration Ethyl Oleate (dpm)

28 TRANSPORT IN BLOOD

29 ALBUMIN A 66 kda molecular weight protein Primary functions include: - building and transport of fatty acids, bilirubin, calcium and many other small molecules - maintenance of osmotic pressure in the blood Fatty Acid Albumin

30 FREE FATTY ACIDS DISPLACE ETHYL [ 14 C] OLEATE FROM ALBUMIN Ethyl [14C] Oleate Recovered with Albumin as a Percent of Total :2 16: 18:1 18: 14: 2:4 Ratio of free fatty acids to ethyl oleate

31 CONCLUSIONS: BINDING TO LDL AND PHOSPHOLIPID VESSICLES LDL Phospholipid Vesicle Ethyl oleate binds to the core of lipoproteins align parallel to the fatty acid moieties of phospholipids in vesicles and are soluble up to 3 mole percent The transfer of ethyl oleate between phospholipid vesicles and LDL is a rapid bidirectional process

33 DEGRADATION

34 METHOD Blood collected at 5, 15, 3, 6, 9, and 12 minutes with sacrifice and harvest of organs Radiolabeled triglyceride.5 M KOH in ethanol Radiolabeled Delivered as oil directly into rat stomach CE TG FFA PL Labeled mixed in oil of purified unlabeled TLC to separate lipid classes

35 FATTY ACIDS FROM CAN BE ESTERIFIED INTO OTHER LIPIDS Fatty Acid + Ethanol Phospholipid (PL) Triglyceride (TG) Cholesterol Ester (CE)

36 AFTER ADMINISTRATION OF INTO THE STOMACH, ARE EXTENSIVELY HYDROLYZED TO FREE FATTY ACIDS IN THE DUODENUM % of Total Lipid DPM Portions of Harvested Organs Homogenized Stomach Lipids Extracted [ 3 H] Ethyl Oleate Metabolism TLC to Separate the Different Lipid Classes Duodenum Pancreas Liver Blood PL FFA TG CE % of Total Lipid DPM Stomach [ 3 H] Ethyl EPA Metabolism Duodenum Pancreas Liver Blood PL FFA TG CE

37 IS THERE DEGRADATION OF ORALLY INGESTED PRIOR TO ENCOUNTER WITH TARGET ORGAN? GI TRACT BLOOD TARGET ORGAN FA FA

38 METHOD Blood collected at: 15 sec, 3 sec, 1,2,3,4,5 min Radiolabeled triglyceride.5 M KOH in ethanol Injected into rat intra-arterially CE TG FFA PL LDL core reconstituted with TLC to separate lipid classes

39 THE DEGRADATION OF IN THE BLOOD IS EXTREMELY RAPID WITH A HALF-LIFE OF 58 SECONDS Human LDL reconstituted with [ 3 H] ethyl oleate administered to rats intra-arterially 1 Blood collected at: 15 sec, 3 sec, 1,2,3,4,5 min Lipids extracted and TLC performed to separate the different lipid classes % of DPM Remaining in Time (Seconds)

40 METHOD Radiolabeled triglyceride Platelet poor plasma Buffy coat Red blood Cells Platelet rich plasma Mixture incubated at 37º C for 2 hrs.5 M KOH in ethanol CE Radiolabeled TG FFA PL LDL core reconstituted with TLC to isolate

41 RBC, WBC AND PLATELETS ARE ALL CAPABLE OF HYDROLYZING Hydrolysis 8 % of Remaining Plasma RBC WBC Platelets 7.5 x x x 1 3

42 THE DEGRADATION OF IN THE BLOOD IS EXTREMELY RAPID WITH A HALF-LIFE OF 58 SECONDS 9 [ 3 H] Ethyl Oleate Metabolism 8 % of Total Lipid DPM sec 3 sec 6 sec 2 min 3 min 4 min 5 min 1 Phospholipid Fatty Free Acid Triglyceride Cholesterol ester

43 UPTAKE OF BY HUMAN PLATELETS IS MAXIMAL BY 6 SECONDS Isolation of platelets 1 Incubation with 3 H-E18:1 Measurement of radioactivity in platelets % of maximum uptake in experiment experiments, with n=2 / timepoint in each experiment Time (sec)

44 1 HYDROLYSIS OF INCORPORATED INTO PLATELETS Distribution of Total Lipid DPM (% In Each Fraction) experiments, with n=3 / lipid fraction in each experiment SEC 3 SEC 6 SEC 3 SEC 6 SEC Phospholipid Free Fatty Acid Triglyceride Fatty Acid Ethyl Ester Cholesteryl Ester FRACTIONS CONTAINING FATTY ACIDS DERIVED FROM

METHOD FOR ASSESSING EFFECT ON PLATELET AGGREGATION Add E 18:1 Add 5 µl Measure Aggregation To platelets Agonists By Light Transmission Turbid plasma absorbs light Clear plasma

45 METHOD FOR ASSESSING EFFECT ON PLATELET AGGREGATION Add E 18:1 Add 5 µl Measure Aggregation To platelets Agonists By Light Transmission Turbid plasma absorbs light Clear plasma absorbs little light Platelet rich plasma was incubated with 25 µm ethyl oleate for one minute At the end of the incubation, epinephrine (1 µm) was added to stimulate platelet aggregation

46 µm REVERSES THE EFFECT OF IBUPROFEN ON PLATELET AGGREGATION IN RESPONSE TO ARACHIDONATE Platelets obtained from 1 subject ingesting 4 mg ibuprofen % aggregation ACA + 5 µm E18:1 ACA + 1 µm E18:1 ACA + 25 µm E18:1 2 Archidonate (ACA)- No Time (sec)

47 Summary Platelets treated with have increased sensitivity to activation briefly after ethanol intake. At this point, platelets may be predisposed to aggregation. A loss of platelet granules in the brief activation may produce the hypofunctioning platelets reported to exist following ethanol intake. 3 sec to max uptake Activation Hydrolysis FFA Increased Ability to Aggregate Granules Loss Decreased Ability to Aggregate

48 SUMMARY Lipoprotein Albumin Lymphocytes Stomach? PLT RBC WBC ETOH T 1/2 = 1 min ETOH Duodenum FA FA FA E16: E18:1 Acetaldehyde FA Toxicity FA Toxicity FA + ETOH FA + ETOH PL TG CE LIVER PL TG CE PANCREAS

49 AS MARKERS OF ETHANOL INTAKE

50 STATE MARKERS Has there been recent intake of ethanol? Ethanol Fatty Acid Ethyl Esters () Is there evidence of chronic ethanol intake? Fatty Acid Ethyl Esters Carbohydrate Deficient Transferrin (CDT)

51 HAVE BEEN RECENTLY DISCOVERED IN SERUM AFTER ETHANOL INGESTION 2 Total (µm) > 1 ETOH (mg/dl)

52 STUDY DESIGN ETOH Ingestion Time (minutes) Blood Drawn -4 hr hr Low Fat Breakfast Low Fat Lunch Low Fat Supper

53 DEMOGRAPHICS: 7 STUDY PARTICIPANTS Physical Data Sex Height (feet/inches) Weight (pounds) % Body Fat - Skinfold % Body Fat - Bioelectric Basal Energy Expenditure Alcohol History Male Male Female Female Female Male Male # of beers per Month # of glasses of wine per month # of drinks of liquor per month <1 < <1 6 1 <1 8 < <1 12 Laboratory Data (Non Fasting) Cholesterol, mg/dl Triglyceride, mg/dl Subjective Sense of Sobriety Intoxication Intoxication Intoxication Intoxication Highly Intoxicated Highly Intoxicated No Signs of Intoxication

54 ARE DETECTABLE IN SERUM UP TO 24 HOURS AFTER ETHANOL INGESTION Serum ETOH (mg/l) at 24 hours Total (pmol / ml serum) at 24 hours Ethanol

56 REUTERS HEALTH INFORMATION SYSTEMS New Blood Test Confirms Alcohol Use New York (Reuters) - Fatty acid ethyl esters (s) are not a new rock group. They are highly sensitive biochemical markers for the presence of alcohol (ethanol) in the blood, which according to researchers can be used to confirm other blood tests for drunk driving.

57 CLINICAL SIGNIFICANCE OF 8: am: 11: am: Result: Train derailment Train conductor arrives at the hospital for evaluation and blood ethanol level determined Blood negative for ethanol Was the train conductor alcohol-free at the time of accident?

58 SUBJECTS (n = 8) AGE SOCIAL DRINKERS NO DIAGNOSED MEDICAL CONDITION WOMEN WERE NOT PREGNANT PROTOCOL STUDY BEGINS No alcohol consumption 5 days prior to study 9 minute ethanol consumption period STUDY ENDS No alcohol consumption Until 48 hour time point Blood Sampling: Base line Hours after start of ethanol intake

59 RBC ACCOUNT FOR APPROXIMATELY 7% OF WHOLE BLOOD % of Total Blood in RBC Hours After Start of Ethanol Intake

60 RED BLOOD CELL AND PLASMA CONCENTRATIONS AFTER ETHANOL INTAKE Blood Ethanol Concentration Blood Ethanol Concentration RBC (pmol / 1 9 Cells) RBC Ethanol Concentration (mg/dl) Plasma (pmol/ml) PLASMA Ethanol Concentration (mg/dl) Hours after Start of Ethanol Intake Hours after Start of Ethanol Intake Fatty acid ethyl esters remained detectable in plasma 48 hours post ethanol intake despite undetectable blood ethanol levels

61 Is the Fatty Acid Composition in Fatty Acid Ethyl Esters from Plasma and Red Blood Cells the Same? Plasma RBC? 16: 18: 18:1 Fatty Acid Composition 16: 18: 18:1 Fatty Acid Composition

62 THE FATTY ACID COMPOSITION OF RBC & PLASMA ARE DIFFERENT 1.75 Hours after start of ethanol intake (n = 8) Plasma 8 RBC 6 * 4 2 ** E16: E18: E18:1 E18:2 % of Total Species in Plasma and RBC 3.5 Hours after start of ethanol intake (n = 8) Plasma 8 RBC 6 * *** 4 2 * E16: E18: E18:1 E18:2 % of Total Species in Plasma and RBC 5.5 Hours after start of ethanol intake (n = 8) ** Plasma 8 RBC 6 4 *** 2 ** E16: E18: E18:1 E18:2 % of Total Species in Plasma and RBC 7.25 Hours after start of ethanol intake % of Total * Plasma (n = 8) RBC (n = 4) E16: E18: E18:1 E18:2 Species in Plasma and RBC

63 MEN HAVE SERUM LEVELS 2-FOLD HIGHER THAN WOMEN DESPITE COMPARABLE BLOOD ETHANOL LEVELS (nmol/l) ** * * * SERUM * * Men n-4 Women n-3 Blood Ethanol (mg/dl) * BLOOD ETHANOL ** * * * * Men n-4 Women n Time (min) Time (min) Time course for serum concentrations and blood ethanol concentrations for men and women consuming alcohol to legal limits of intoxication *indicates significant differences between sexes ( p<.5) for a given time point

64 TYPE OF ALCOHOLIC BEVERAGE INGESTED HAS NO IMPACT ON PLASMA LEVELS (nmol/l) Beer Vodka Alcoholic Beverage Ingested 15 min 12 min Plasma levels at two representative peak time points for blood ethanol in subjects who consumed equivalent amounts of ethanol adjusted for body weight

65 Detecting Chronic Alcoholism Never Drinks Social Drinking Light Drinking Light- Moderate Drinking Moderate Drinking Chronic Occasional Chronic Alcoholic / Binge Alcoholic Binge Never Stops Drinking The Test distinguishes between levels of drinking behavior Because ethanol intake is at most moderate, distinguishing between the different levels of drinking behavior is not contributory to evaluation Often clinically difficult to distinguish between chronic alcoholics and binge drinkers even with patient history, physical exam, and routine laboratory tests Because ethanol intake is so great, distinguishing between the different levels of drinking behavior is not contributory to evaluation

66 Ethyl Oleate as Percent of Total is Significantly Different in Chronic Alcoholics and Binge Drinkers Approximately 24 Hours after Ethanol Consumption was Discontinued 1 Sensitivity: 1 % and Specificity: 1 % p <.1 Ethyl 18:1 as % of Total Threshold for Chronic Alcoholism: Ethyl 18:1 > 52 % of Total Chronic Alcoholics n = 15 Mean ± SEM: 71 ± 2 Binge Drinkers n = 13 Mean ± SEM: 1 ± 5

67 Absolute Difference between Ethyl Stearate and Ethyl Oleate Both as Percent of Total is Significantly Different in Chronic Alcoholics and Binge Drinkers Approximately 24 Hours after Ethanol Consumption was Discontinued Absolute Difference between Ethyl 18: and Ethyl 18:1 Both as % of Total Sensitivity: 1 % and Specificity: 1 % p <.1 Threshold for Chronic Alcoholism: [Ethyl 18: % - Ethyl 18:1 %] > 45 Chronic Alcoholics n = 15 Mean ± SEM: 71 ± 2 Binge Drinkers n = 13 Mean ± SEM: 1 ± 3

68 THE HUMAN STUDY 31 cases from the Massachusetts Medical Examiner s Office and the Department of Pathology at the Massachusetts General Hospital were evaluated Postmortem intervals ranged from 5-29 hours (mean of 16 hours) In all cases blood ethanol concentrations were determined at autopsy Liver and adipose tissue fatty acid ethyl esters were extracted and quantitated

69 3 GROUPS OF SUBJECTS 1. Detectable blood ethanol at the time of autopsy 2. Negative blood ethanol at the time of autopsy with a history of chronic alcoholism 3. Negative blood ethanol at the time of autopsy with a history of social drinking or abstinence

70 LIVER FATTY ACID ETHYL ESTERS FOR THE 3 GROUPS OF SUBJECTS Total (pmol / g) ,521 1,235 Individuals with detectable blood ethanol at autopsy Chronic alcoholics with negative blood ethanol at autopsy Sensitivity=93% Specificity=1% 5,315 5,485 Social drinkers with negative blood ethanol at autopsy

71 OUTLINE OF PRESENTATION Background - Toxicity of Synthesis Secretion Transport in Blood Degradation as Markers of Ethanol Intake

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