Fatty acid ethyl esters: Non-oxidative metabolites of ethanol

PROSTAGLANDINSLEUKOTRIENES AND ESSENTIALFATTYACIDS Prostaglmldins Leukotrienes and Essential Fatty Acids (1995) 52, 87-91 © Pearson Professional Ltd 1995

Fatty Acid Ethyl Esters: Non-oxidative Metabolites of Ethanol M. Laposata, Z. M. Szczepiorkowski and J. E. Brown

Department of Pathology, Division of Clinical Laboratories, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA (Reprint requests to ML, Director of Clinical Laboratories, Room 235 Gray Building, Massachusetts General Hospital, Fruit St, Boston, MA 02114, USA) A B S T R A C T. Fatty acid ethyl esters are esterification products of fatty acids and ethanol. These compounds have been detected in the serum and cells of individuals following ethanol ingestion. Fatty acid ethyl esters can be quantitated by gas chromatography-mass spectroscopy (GC-MS) in the serum following ethanol ingestion and have been found in concentrations up to 42 gM. Fatty acid ethyl esters have also been isolated from adipose tissue of subjects ingesting fatty acid ethyl ester capsules as well as from subjects ingesting ethanol. HepG2 cells, a human hepatoblastoma cell line, have also been shown to generate fatty acid ethyl esters when incubated with 1.25 ~tM fatty acid and 0.17 M ethanol. Fatty acid ethyl esters were found to be toxic to HepG2 ceils when presented to the cells in the core of low density lipoprotein particles.

acetaldehyde is not found in the pancreas following ethanol intake, and negligible amounts are found in the circulation (1). Thus, it is difficult to explain how oxidative products of ethanol could be responsible for all of the organ damage seen with ethanol abuse. Direct evidence of fatty acid ethyl ester induced cytotoxicity for intact cells has been lacking, despite the strong circumstantial evidence. In a number of studies which have been performed to assess cytotoxicity, fatty acid ethyl esters in a non-physiologic vehicle, such as an emulsion, have been incubated with isolated organelles and were shown to have a variety of detrimental effects (6, 7). To address the question of whether fatty acid ethyl esters in a physiologic particle could be toxic for intact cells, we first performed a study to determine how fatty acid ethyl esters were transported in the blood following ethanol ingestion in order to identify physiologic carriers for ethyl esters (8). Using gas chromatographymass spectrometry with single ion monitoring, we demonstrated the presence of fatty acid ethyl esters in the blood and observed that they were bound both to lipoproteins (primarily LDL), and a plasma protein with a molecular weight identical to albumin.

INTRODUCTION It has been known for some time that ethanol can be oxidized by alcohol dehydrogenase and the microsomal ethanol oxidizing system to generate acetaldehyde (1). Acetaldehyde can then be further oxidized to acetate through the action of aldehyde dehydrogenase. Ethanol can also be metabolized in a non-oxidative fashion and esterified with a fatty acid to form fatty acid ethyl esters (2). This reaction is catalyzed by the action of the enzyme, fatty acid ethyl ester synthase. The enzyme fatty acid ethyl ester synthase has been purified and is found to have at least three isoforms originally separated as three peaks of activity in different organ homogenates by DEAE cellulose chromatography (2). It has been reported that free fatty acid (3) as well as fatty acyl CoA (4) may serve as fatty acid substrates for the enzyme. Interestingly, the organs most frequently damaged by ethanol abuse have been shown to contain the highest levels of fatty acid ethyl ester synthase activity and, after acute intoxication, the highest level of fatty acid ethyl esters (5). Following acute intoxication, fatty acid ethyl esters were in highest quantities in the pancreas with the next highest amount in the liver followed by smaller quantities in the heart and brain. The presence of fatty acid ethyl esters following ethanol ingestion, as well as the enzyme responsible for their synthesis, specifically in the organs damaged by ethanol abuse provides circumstantial evidence that fatty acid ethyl esters are toxic metabolites which may, at least in part, account for ethanolinduced organ damage. Unlike fatty acid ethyl esters,

MATERIALS AND METHODS Fatty acid ethyl ester isolation and quantitation Patient specimens, lipoprotein isolation, and fatty acid ethyl ester extraction from plasma and cells, using a 87

88 ProstaglandinsLeukotrienesand EssentialFattyAcids modified Folch method, have all been previously described (8). An internal standard of 500 pmol ethyl heptadecanoate (E17:0) (Nu Chek Prep, Elysian, MN) was added to each sample. Fatty acid ethyl esters were isolated by thin layer chromatography (TLC) using a petroleum ether: diethyl ether (75:5 v/v) solvent system and Silica gel 60 plates (EM Science, Gibbstown, NJ). Isolated fatty acid ethyl esters were scraped from TLC plates in a nitrogen atmosphere and eluted from the gel with acetone. Fatty acid ethyl esters in the eluate were concentrated by drying the sample under nitrogen. A 1 gl sample was then injected into a Hewlett Packard 5890 Series II gas chromatograph equipped with a Supelcowax 10 capillary column (Supelco, Inc., Bellefonte, PA), coupled to a HP5971 mass spectrometer. The injector and detector were maintained at 260°C and 280°C, respectively. The oven program was initially maintained at 150°C for 2 rain, then ramped to 160°C at 10°C/rain, ramped again at 2°C/rain to 180°C and held for 7 min, and finally ramped to 230°C at 15°C/rain and held for 3 min. Carrier gas flow rate was maintained at a constant 0.75 ml/min throughout. Single ion monitoring was performed, quantitating base ions 55, 88, and 101 for ethyl 16:0 (E16:0), ethyl 16:1 (E16:I), ethyl 17:0 (El7:0), ethyl 18:0 (E18:0), and ethyl 18:1 (E18:1); ions 55, 67, and 69 for ethyl 18:2 (E18:2); and ions 79 and 91 for ethyl 20:4 (E20:4).

HepG2 cell production of fatty acid ethyl esters Confluent monolayers of HepG2 cells were incubated overnight with medium containing 1.25 ~tM free fatty acids (16:0, 18:1 or 18:2). The following day, media was aspirated, mixed with ethanol (0.17 M) and returned to the cells for 7.5 h. An 0.17 M concentration of ethanol was maintained throughout the incubation period. At the end of the incubation period, the medium was again removed, the cells washed three times with warm phosphate "buffered saline solution and then harvested by scraping in cold phosphate buffered saline. Fatty acid ethyl esters were extracted, isolated and quantitated as described above.

Assessment of fatty acid ethyl ester cytotoxicity in HepG2 cells LDL was isolated from human plasma and reconstituted with fatty acid ethyl esters as previously described (8). HepG2 cells were first incubated with delipidated medium for 6 h to increase the number of LDL receptors. These cells were then incubated with LDL reconstituted with fatty acid ethyl esters or with LDL reconstituted with triglyceride and/or cholesteryl esters to serve as controls. For further comparison, HepG2 cells were also incubated for 12h with native LDL that was not delipidated and reconstituted with ethyl esters. [Methyl3H] thymidine (Dupont-New England Nuclear, Boston, MA) was then added to each test combination for 5 h.

Cell monolayers were harvested and 3H-thymidine incorporation into the cells determined by liquid scintillation counting. Similarly, L-[35S] methionine incorporation into newly synthesized protein by HepG2 cells was performed according to the method of Harlow et al (9). In these studies, HepG2 cells were preincubated for 12 h with LDL reconstituted with fatty acid ethyl esters or with native LDL. 35S-methionine was then added for 5 h and the cell monolayers rinsed and harvested to determine 35S-methionine incorporation into newly synthesized protein precipitated by trichloroacetic acid.

RESULTS Figure 1 (A) shows a total ion scan of all ions generated by gas chromatography-mass spectroscopy following injection of an extract of alcoholic serum into the instrument. Using single ion monitoring, small peaks of fatty acid ethyl esters in the midst of peaks unrelated to ethyl esters can be transformed into a spectrum (B) with large fatty acid ethyl ester peaks independent from non-ethyl ester peaks. The peaks can be conclusively identified as fatty acid ethyl esters by matching the electron impact spectra of these compounds with those of authentic standards in the mass spectrometry library. Figure 2 shows a typical GC-MS profile by single ion monitoring of fatty acid ethyl esters found in serum and cells. It is noted that the prominent fatty acid ethyl esters are comparable to the prominent fatty acids in cells and tissues, with relatively large amounts of ethyl palmitate, ethyl stearate, and ethyl oleate. The electron impact spectra, generated by GC-MS for the two most abundant fatty acid ethyl esters in serum are shown in Figure 3. It is noted that the molecular ion, as well as the base ions, can be used to identify the compounds. Spectra of these fatty acid ethyl esters match extremely closely to spectra of authentic standards in the instrument's library of spectra. The tracing in Figure 4 shows the pattern of serum fatty acid ethyl esters by single ion monitoring from a subject whose blood alcohol level was 0.2 g%. It can be seen that this individual had large amounts of ethyl palmitate and ethyl oleate in his serum with detectable amounts of four other fatty acid ethyl esters. We have previously demonstrated that fatty acid ethyl esters are present in the blood bound to plasma proteins and lipoproteins, of which low density lipoprotein (LDL) is the major carrier (8). With the knowledge that fatty acid ethyl esters are present in LDL following ethanol ingestion, we assessed whether toxicity could be produced by incubating HepG2 cells (a human hepatoblastoma cell line) in culture with LDL containing fatty acid ethyl esters. To carry out these experiments, LDL was isolated from human plasma and the cholesterol ester and triglyceride removed from its core and substituted with fatty acid ethyl esters. The LDL reconstituted with fatty acid was not oxidized, was highly soluble in aqueous medium,

Fatty Acid Ethyl Esters: Non-oxidative Metabolites of Ethanol 89

B

A

25I

~.b~ndance

1300000 l 1200000

04

150000]

20:4

~.4

140000 14,20

13000o] 1100000

17:0

17. ~,'7

11.36

120000

1000000

16:0

f26.0 6.

900000

34,

16:0

i

!

5

800000 700000

:2

~00000

23."

18;1

2

500000 40000

17:0

18:1 le.6o

1 .3798 14.02

l';.g

20.5,1

.5720.54

'

30000 200000 100000 "t~e--> °

looo0

'S.'00

,

,

lO.00

. . . . .

,

. . . .

15.OO

i

20,00

. . . .

,

25,00

,

,

rtm~- ->

o

' 5.'00 . . . .

10'. 00' ' ' ~.5~,O0'

,

,

20 '.00'

.

.

'25 ~00'

Fig. 1 Detection and qnantitation of fatty acid ethyl esters in serum by single ion monitoring. Panel A shows a scan of all ions (50-500 AMU) and Panel B shows a scan monitoring single ions of the identical sample.

Abundance

5oooc

.26

24 .93

14.06 17.77 E18:0

4000(

E20:4

E16:0

3000G 1"35 20.31

2000G

E18:

E18:2

IO00E

Time--~

"J,.b'0"

" ' ' " ' "~.bb'

"" '""

"1"2'.66"

"'"

' 'i6'.66"

" ' "" "2"o'.66"

" '"

" "2"d.66"

" '""

Fig. 2 Typical GC-MS profile of fatty acid ethyl esters found in serum and cells. The designation of El6:0 refers to ethyl 16:0• Other fatty acid ethyl esters are identified using similar terminology.

and was bound to cell surface receptors on HepG2 cells for LDL. W e demonstrated that fatty acid ethyl esters could decrease 3H-thymidine incorporation in the HepG2 cells (30% by 600 g M fatty acid ethyl esters in the medium) as well as protein synthesis (40% by 400 g M fatty acid ethyl esters in the medium). The amount of fatty acid ethyl esters found in the cells in these experiments was an amount very similar to that found in hepatocytes following acute intoxication (5).

DISCUSSION The identification of the toxic ethanol metabolite(s) responsible for organ damage has been elusive for decades. W e have demonstrated that fatty acid ethyl esters appear in the blood following ethanol ingestion within lipoproteins (primarily LDL) and bound to a plasma protein with molecular weight identical to albumin (8). Ethyl esters can be carefully quantitated and

90 Prostaglandins Leukotrienes and Essential Fatty Acids

A ETHYL 16:0 Abundance

88

100000

BASE IONS: 55,88,101

80000 43 60000

l

101

lJl,

40000

55

20000

1 7

,o,.:.

MOLECULAR

. . . . . . [1 . . k, .... L . . .129 . . . . ! 43" ~..171 185 199213227241 255 284 "'" "rb' ' ""oh'" i66'" i k r " i46"" i~6 '~ igd~' ' ~ i ' ' ) k 6 ' ' ~,~6"' ~gd'" ~6"'

m/z--> 0

B Abundance

115

ETHYL 18:1 55

160000i

BASE IONS: 55,88,101

1400001 120000i 100000i 800001 60000! 400001

200001 m~-->0 :4b--¸

69 88

L,iI,I

MOLECULAR

97

123137 155.~180 22223r.,... - 264 ,811L.,,Itl ,,11,, ,dl~.,J,l~.'.~.~ J.. 193 _. J. .vZ4b II 310 i -i6 0 "i~,6 i 4 i f "i~ 6 "i~O :~06 2~'5 2,~6 2t~6 2 8 0 :~6ff'":3~,0

Fig. 3 The electron impact spectra generated by GC-MS for selected fatty acid ethyl esters; (A) ethyl 16:0, (B) ethyl 18:1.

conclusively identified using gas chromatography-mass spectroscopy. Toxicity studies indicate that fatty acid ethyl esters, when bound to LDL, can be incorporated into cells and reduce their proliferative capacity and their capability to synthesize new proteins. Using the GC-MS for fatty acid ethyl ester quantitation, we have been able to detect as little as 1020 pmol of fatty acid ethyl esters in cells and tissues. In studies involving GC without MS, even for those instruments equipped with capillary columns, sensitivity for fatty acid ethyl ester detection is much less, and these results are always open to possibility that the peaks identified as fatty acid ethyl esters may instead be fatty acid methyl esters. Previous work with fatty acid ethyl ester-induced toxicity has largely avoided the use of physiologic carriers of fatty acid ethyl esters (6, 7). In most experiments, fatty acid ethyl esters are solubilized in emulsions which are not normally in the circulation and are targeted for isolated organelles rather than intact cells. It has been demonstrated that fatty acid ethyl esters in emulsions can uncouple oxidative phosphorylation in

isolated mitochondria (6). It has also been shown that fatty acid ethyl esters decrease the stability of isolated lysosomes (7). Our preliminary studies indicate that fatty acid ethyl esters are present in the circulation following ethanol ingestion (8). In addition, they can be incorporated into cultured HepG2 cells in amounts similar to those found in autopsy studies following ethanol ingestion. Further, fatty acid ethyl esters produce acute toxic effects on HepG2 cells as demonstrated by their decreased capacity for proliferation and decreased protein synthesis following fatty acid ethyl ester uptake.

Acknowledgements This work was supported by Grants DK37454 and DK43159 from the National Institutes of Health.

References 1. Lieber C S. Metabolism and metabolic effects of alcohol. Semin Hematol 1980; 17: 85-99.

Fatty Acid Ethyl Esters: Non-oxidative Metabolites of Ethanol ~bundance

5500000

, r,o E 16:o

5000000

4500000

4000000 3500000

3000000

2500000 E17:010,91 9.84. ,Fp,,1,B:I

2000000

18:1 1o

\

1500000

f

~5 L.29

~8:2 ]111.90

1000000

500000

Time-->

0

"

I

4.00

. . . .

I

6.00

. . . .

I

8,00

" "*"v'"'"l'-"

I0.00

12, O0

14.00

16.00

Fig. 4 The GC-MS tracing from a serum sample of a subject with a blood ethanol level of 0.2 g%. Concentrations of FAEE are as follows: El6:0, 2.0 p.M; El6:1, 0.6 gM; El8:0, 0.5 gM; El8:1, 4.7 gM; E18:2, 1.0 gM; E20:4, 0.5 gM.

2. Lange L G. Mechanism of fatty acid ethyl ester formation and biological significance. Ann NY Acad Sci 1991; 625: 802-806. 3. Mogelson S, Pieper S J, Lange L G. Thermodynamic bases for fatty acid ethyl ester synthase catalyzed esterification of free fatty acid with ethanol and accumulation of fatty acid ethyl esters. Biochemistry 1984; 23: 4082-4087. 4. Grigor M R, Bell Jr I C. Synthesis of fatty acid esters of short-chain alcohols by an acyltransferase in rat liver microsomes. Biochim Biophys Acta 1973; 306: 26-30. 5. Laposata E A, Lange L G. Presence of non-oxidative

6.

7.

8.

9.

ethanol metabolism in human organs commonly damaged by ethanol abuse. Science 1986; 231: 497-499. Lange L G, Sobel B E. Mitochondrial dysfunction induced by fatty acid ethyl esters, myocardial metabolites of ethanol. J Clin Invest 1983; 72: 724-731. Haber P S, Wilson J S, Apte M V, Pirola R C. Fatty acid ethyl esters increase rat pancreatic lysosomal fragility. J Lab Clin Med 1993; 121: 759-764. Doyle K M, Bird D A, AI-Salihi Set al. Fatty acid ethyl esters are present in human serum after ethanol ingestion. J Lipid Res 1994; 35: 428-437. Harlow E, Lane D. Antibodies. A laboratory manual. New York: Cold Spring Harbor Laboratory, 1988.

91