PEth - Echelon Biosciences

Phosphatidylethanol (PEth) As A Long-Term Alcohol Biomarker
In Binge And Chronic Drinking
Paul Neilsen, Piotr Rzepecki, Jeff Johnson, Chad Testa Echelon Biosciences, Salt Lake City, Utah
Goals
1. Synthesize PEth analogs and generate specific monoclonal antibodies to PEth
2. Develop assays and protocols for detecting PEth in human clinical samples
3. Differentiate moderate and heavy social drinkers from non-drinkers
Drinking Category
n
Teetotaler
Light
Moderate
Heavy
3
3
4
2
Non-oxidative Ethanol Metabolism
Ethyl glucuronide
Urine - 3-5 days
OH
O
Phosphatidylethanol
Blood - 14 days
400
300
200
100
Ethyl palmitate
Blood - 1 day
Hair - 3 months
600
Lipid
0
10 - 3 10 - 2 10 - 1 10 0 10 1 10 2 10 3
Moderate
Moderate
8
C16:0/C20:4
40
C16:0/C18:2
46
46
20
23
Binge
Consistent
Heavy
Pattern
Inverse a-PEth IgG Correlation With EtOH Ingestion
800
a -PEth IgG (ng/mL)
Total PEth, (nM)
Light
Drinking Category
400
300
200
100
10
20
30
EPI, Previous
PEth (nM)
28-days
Med. (IQ range)
Binge
M
23.0
3.3
20
31 (25 to 63)
Consistent
M
26.8
4.9
23
207 (144 to 320)
400
Conclusions, Future, Acknowledgements
200
0
0
Gender BMI
Drinks/
Day
600
0
40
Expected PEth Index (%PEth/BMI), 28 Days
10
20
30
Expected PEth Index (%PEth/BMI), 28 Days
< 0.0001
Significant
P value
Deviation from zero?
0.0038
Significant
40
• Monoclonal antibodies and phosphatidylethanol
developed and used to detect anti-PEth IgG
(PEth)
analogs
• Autologous anti-PEth IgG was inversely (PEth was directly) correlated with
ingested ethanol and expected PEth
• Teetotalers & heavy consumers had different proportion of individual
PEth homologues compared to intermediate consumers
Four Homologues, Average 7 Time Points
O
O
PEth Species
O PO
-
100
O
O
NH
O
O
O
6.25 µg/mL
15
C16:0/C18:1
200
Non
500
0
C18:1/C18:1
0
Heavy
Positive PEth Correlation With EtOH Ingestion
P value
Deviation from zero?
Competitive Lipid (mM)
0 µg/mL
Light
12.5 µg/mL
25 µg/mL
50 µg/mL
+
N
F B N
F
100 µg/mL
9
6
80
% Total PEth
1
PE
PA
PC
PS
PG
SM
Cholesterol
PEth
Anti-PEth F(ab)2
• bound selectively to PEth when compared
with 7 abundant lipids
23
60
400
Red = Consistent
Anti-PEth FAB & Fluorescent PEth Analogs
19
20
Drinking Category
Ethanol Metabolism in Humans
• Most EtOH is eliminated by oxidation to acetaldehyde then CO2
• 1-3% converted non-oxidatively into ethyl glucuronide, ethyl sulfate, fatty acid ethyl esters (FAEE),
and phosphatidylethanol (PEth)
• PEth persists in blood ≥ 2 weeks making it an attractive biomarker for ethanol consumption
Anti-PEth Antibody Selective For PEth
PEth Species
100
0
Non
O
PEth Pilot: a-PEth IgG
800
0
2
Two Heavy Drinkers, Average 7 Points
Autologous Anti-PEth IgG
500
O
O
OD 450 nm
𝑔 𝐸𝑡𝑂𝐻 𝐶𝑜𝑛𝑠𝑢𝑚𝑒𝑑 × 𝐹𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑈𝑛𝑚𝑒𝑡𝑎𝑏𝑜𝑙𝑖𝑧𝑒𝑑 𝑃𝐸𝑡ℎ 𝑢𝑠𝑖𝑛𝑔 𝑡1/2 = 8 𝑑𝑎𝑦𝑠
𝐸𝑃𝐼 =
𝐵𝑜𝑑𝑦 𝑀𝑎𝑠𝑠 𝐼𝑛𝑑𝑒𝑥
PEth Pilot: PEth by MS
FAEE
Synthase
3
250 (240-270)
180 (160-250)
170 (150-260)
140 (110-170)
80
O
O
BODIPY-FL PEth Liposomes
• Multilamellar vesicles
(MLVs), incubated at
increasing PEth
concentrations with
human red blood cells
(RBC) stain successively
brighter by microscopy
and flow cytometry
0
0 4.3 (1.9-4.5)
0.46 (0.25-0.64) 2.5 (1.5-4.0) 5.6 (1.8-18)
1.2 (0.92-1.9) 9.0 (7.8-11) 20 (6.1-40)
4.1
(3.3-4.9) 23 (20-26) 65 (30-180)
Heavy Drinkers
• Had similar expected PEth indices (EPI), but vastly different PEth levels
• Noticed consistent versus binge drinking patterns
• No significant difference observed in proportion of PEth homologues
between binge and consistent volunteers
% Total PEth
or
OH
PLD
a-PEth IgG (mg/mL)
PEth (nM)
PEth by Mass Spectrometry
O
HO
O
S
O
Ethyl sulfate
Urine - 3-5 days
OH
OH
P
O
O
O
EPI*
O
HO
UTG or SULT
Drinks/Day
a -PEth IgG ( mg/mL)
O
∗
Total PEth, (nM)
HOOC
Drinking Patterns?
Pilot Study: Anti-PEth Levels Correlate Inversely with Drinking
16
23
21
18
21
11
60
C18:1/C18:1
C16:0/C20:4
C16:0/C18:1
C16:0/C18:2
70
40
48
46
46
• One binge-drinking volunteer had lower than expected levels of PEth, and
much lower levels compared to consistent-drinking volunteer
• Validate auto anti-PEth IgG correlation with larger cohort, establish cutoff
value for non versus heavy drinking, and
• Explore PEth levels in response to different drinking patterns
• Characterize PEth homologue differences with pattern and amount of
alcohol consumed
20
0
Teetotaller
n=3
Light
3
Moderate
4
Heavy
2
Four Individual PEth species
• Differences based on amount of alcohol consumed
• Abstainers had increased proportion of 16:0/18:1 with less 18:1/18:1 and 16:0/20:4
• Heavy drinkers had 30-40% greater relative levels of 16:0/18:2
• Ferrara and Weinmann groups recently reported differences in individual PEth homologues between alcoholic
and social drinkers
• We acknowledge Dr. Sean Davies and colleagues at Vanderbilt University
Medical Center for Mass Spectrometry analysis
• Supported by NIAAA, SBIR Contract, HHSN267200800020C
The content of this communication is
solely the responsibility of the authors
and does not necessarily represent the
official views of the National Institute
on Alcohol Abuse and Alcoholism nor
the National Institutes of Health.

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