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Determination of the formation rate of phosphatidylethanol by phospholipase D (PLD) in blood and test of two selective PLD inhibitors
Accepted Manuscript Determination of the formation rate of phosphatidylethanol by phospholipase D (PLD) in blood and test of two selective PLD inhibitors Alexandra Schröck, Anna Henzi, Peter Bütikofer, Stefan König, Wolfgang Weinmann PII:
S0741-8329(18)30001-6
DOI:
10.1016/j.alcohol.2018.03.003
Reference:
ALC 6784
To appear in:
Alcohol
Received Date: 3 January 2018 Revised Date:
8 March 2018
Accepted Date: 13 March 2018
Please cite this article as: Schröck A., Henzi A., Bütikofer P., König S. & Weinmann W., Determination of the formation rate of phosphatidylethanol by phospholipase D (PLD) in blood and test of two selective PLD inhibitors, Alcohol (2018), doi: 10.1016/j.alcohol.2018.03.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Determination
of
the
formation
rate
of
phosphatidylethanol
by
phospholipase D (PLD) in blood and test of two selective PLD inhibitors Authors:
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Alexandra Schröcka,c, Anna Henzia, Peter Bütikoferb, Stefan Königa, Wolfgang Weinmanna
Affiliations: a
Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern
Bühlstrasse 20
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3012 Bern
b
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Switzerland
Institute of Biochemistry and Molecular Medicine, University of Bern
Bühlstrasse 28 3012 Bern Switzerland
Graduate School for Cellular and Biomedical Sciences (GCB), University of Bern
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c
c/o Theodor Kocher Institute Freiestrasse 1
Switzerland
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3012 Bern
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Corresponding author: Wolfgang Weinmann
Institute of Forensic Medicine, University of Bern Bühlstrasse 20
CH-3012 Bern
Tel. +41 (0)31 631 5668 E-Mail: [email protected]
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Abstract Phosphatidylethanol (PEth) is an alcohol biomarker formed from phosphatidylcholine (PC) by the enzyme phospholipase D (PLD) in the presence of ethanol. A drinking study revealed individual differences in maximum PEth levels after drinking up to a targeted blood alcohol concentration (BAC) of 1 ‰. This seemed to be due to different PLD activities in the tested
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persons. Furthermore, post-sampling formation of PEth occurred in blood samples, still containing alcohol. Therefore, a standardized in-vitro test for measuring individual PEth formation rates was developed. Two PLD inhibitors were tested for their potency to inhibit post-sampling PEth formation.
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PEth-negative blood samples were collected from a volunteer. Ethanol was added in different concentrations (0.1 – 3 ‰ BAC) directly after blood sampling. The specimens were incubated at 37 °C. Aliquots were taken at the start of the incubation, and every hour until 8 hours after
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start of incubation, and one sample was taken on subsequent days over one week. PEth 16:0/18:1 and PEth 16:0/18:2 were determined by online-SPE-LC-MS/MS. Furthermore, this test system was applied to blood samples of 12 volunteers.
For the inhibition tests, fresh blood (spiked with 1 ‰ ethanol) was spiked with 30, 300, 3000, 30000 nM of either halopemide or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), and
and once on day 2 and 3.
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incubated at 37 °C. PEth concentrations were determined hourly over 5 hours on the first day
PEth formation was linear in the first 7 hours of incubation and dependent on the alcohol concentration. The formation rates of PEth 16:0/18:1 were 0.002 µmol·L-1·h-1 (0.1 ‰ BAC),
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0.016 µmol·L-1·h-1 (1 ‰ BAC), 0.025 µmol·L-1·h-1 (2 ‰ BAC) and 0.029 µmol·L-1·h-1 (3 ‰ BAC). For PEth 16:0/18:2, the formation rates were 0.002 µmol·L-1·h-1 (0.1 ‰ BAC), 0.019
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µmol·L-1·h-1 (1 ‰ BAC), 0.025 µmol·L-1·h-1 (2 ‰ BAC) and 0.030 µmol·L-1·h-1 (3 ‰ BAC). Maximum concentrations reached 431 ng/mL (PEth 16:0/18:1) and 496 ng/mL (PEth 16:0/18:2) at 3 ‰ BAC after 3 days. Maximum velocity (vmax) was not reached under these conditions. PEth formation in blood of the 12 volunteers ranged between 0.011 – 0.025 µmol·L-1·h-1 for PEth 16:0/18:1 and between 0.014 – 0.021 µmol·L-1·h-1 for PEth 16:0/18:2. PEth formation in human blood was inhibited by halopemide in a concentration-dependent manner. However, a complete inhibition was not achieved by the applied maximum concentration of 30000 nM. FIPI showed a better inhibition of PEth formation. A complete inhibition could be achieved by a concentration of 30000 nM for the first 24 h (for PEth 16:0/18:1) and for 48 h (for PEth 16:0/18:2).
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ACCEPTED MANUSCRIPT Formation of PEth was found to be dependent on the BAC. As a consequence, it is essential to inhibit PLD activity after blood collection to avoid post-sampling formation of PEth in blood samples with a positive BAC. Inhibition of PEth formation was more effective using FIPI compared to halopemide.
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Key words Phosphatidylethanol (PEth); alcohol biomarker; online-SPE-LC-MS/MS; Phospholipase D
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(PLD) activity, PEth formation rate, PLD inhibitors
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Introduction As alcohol abuse is a major global factor contributing to disease, injury and death, the development of methods for the early detection of risky alcohol consumption by means of alcohol biomarkers is important [1]. A promising direct alcohol biomarker with high
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specificity and sensitivity is phosphatidylethanol (PEth) [2]. The term PEth represents a group of phospholipid analogues. 48 analogues have been identified in human blood [3], with PEth 16:0/18:1 and PEth 16:0/18:2 as the most abundant analogues [4, 5]. In vivo, PEth is produced from ethanol (after alcohol intake and as long as ethanol is present [6, 7]) and
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phosphatidylcholine (PC) in a reaction catalyzed by phospholipase D (PLD) [8].
PLD is a membrane-associated enzyme found in most eukaryotes and also in prokaryotes. There are two isoforms of mammalian PLD, termed PLD1 and PLD2 [9]. Both isoforms have
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similar regulatory and catalytic domains, but show differences in activation and have different biological functions [10]. Unstimulated PLD1 has a low activity and is regulated by protein kinase C, Arf and Rho GTPases [11]. In contrast, PLD2 shows a high basal activity and is involved in several protein interactions [12, 13].
Normally PLD catalyzes the hydrolysis of its principal substrate PC into phosphatidic acid
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(PA) and choline as shown in figure 1. PA is an important lipid second messenger involved in several signaling pathways, affecting cellular metabolism, cell cycle progression and cell growth [14]. From a therapeutic point of view, PLD or rather its aberrant function and/or overexpression is for example implicated to play a role in cancer [15, 16] and metastasis [13],
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cell motility and viral infection [18].
However, in the presence of primary alcohols, PLD catalyzes a transphosphatidylation
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reaction using alcohol as substrate instead of water to produce phosphatidylalcohol. In previous studies on the function of PLD, formation of phosphatidylalcohols (e.g. phosphatidylbutanol) is often used to assay PLD activity in vitro, as phosphatidylalcohols are normally not produced under physiological conditions [19]. Adding high amounts of alcohol can completely divert PLD away from producing PA and was thus used to inhibit PLDmediated pathways, when no suitable inhibitors were available [13, 19]. PEth, the product of the transphosphatidylation reaction with ethanol (figure 1), is suitable as alcohol biomarker [6, 20-23]. Due to an elimination half-life of approximately four days [24], which can vary widely between individuals [7], PEth gets accumulated in the body after repeated drinking. A number of drinking studies with moderate alcohol consumption have
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ACCEPTED MANUSCRIPT shown that measured PEth concentrations correlate with the consumed amounts of alcohol [7, 25]. O R1 O
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O H 2O
R2
O
O
R1
H
N
R2
+
H 3C H 3C
O
O O
-
Phosphatidylcholine (PC)
O
EtOH
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H 3C
O
R1
Phospholipase D (PLD)
P
-
Phosphatidic acid (PA)
O O
P
O
O
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CH 3
O
O
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O
O
O
R2
O O
O
O
P O
-
Phosphatidylethanol (PEth)
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Figure 1. Enzymatic reactions catalyzed by phospholipase D
Such a drinking study with 16 volunteers revealed individual differences in maximum PEth
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concentrations after a single alcohol ingestion up to a targeted blood alcohol concentration (BAC) of 1 ‰ [26]. This seemed to be due to different PLD activities in the tested persons. Additionally, post-sampling formation of PEth was shown to occur at several storage conditions in blood samples, which still contained alcohol [27]. Further investigations have long been impaired by the lack of specific inhibitors of this enzyme. Several compounds including diethylstilbestron, honokiol, raloxifen [13] and ceramide [19] as well as the antibiotic neomycin [28] are known to inhibit PLD activity indirectly. In 2007, halopemide was identified as a PLD inhibitor by high-throughput screening [29]. Halopemide was first described as an antipsychotic drug with dopamine-blocking properties and structural analogy to neuroleptics of butyrophenone type [30]. The structural formula of 5
ACCEPTED MANUSCRIPT halopemide is depicted in figure 2A. Halopemide and its congeners were identified to be mostly dual and slightly PLD1 preferring inhibitors [13]. Halopemide inhibited PLD1 with an IC50 of 220 nM and PLD2 with an IC50 of 310 nM. Since then, numerous isoform selective PLD inhibitors have been developed using halopemide as a lead compound [10, 13, 31], for example the very potent 5-fluoro-2-indolyl-deschlorohalopemide (FIPI) which inhibits PLD1
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and PLD2 at sub-nM concentrations [32] (figure 2B). By using the N-terminally truncated enzyme PLD1.d311, a constitutively highly active form of PLD, Scott et al. could confirm that the compounds inhibit PLD in a direct manner and not due to interference with myr-Arf-1 GTPase stimulation, which is commonly used in other experiments for testing PLD activity
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[13].
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B
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A
Figure 2. Structure of halopemide (A) and 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) (B)
The aims of this study were to develop a standardized test system for measurement of individual PEth formation rates, and to apply this test to different individuals. Furthermore, we investigated the dual PLD1/2 inhibitors halopemide and FIPI for their ability to inhibit PEth formation by PLD in human blood after adding ethanol. The introduction of such 6
ACCEPTED MANUSCRIPT inhibitors may have the potential to further improve the quality of PEth analysis by preventing additional in-vitro formation of PEth in ethanol positive blood samples after blood sampling.
Material and methods
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Chemicals and materials PEth 16:0/18:1, PEth 16:0/18:2, PC 16:0/18:1 and PC 16:0/18:2 were obtained from Avanti Polar Lipids (Alabaster, USA). Phospholipase D (PLD) (from cabbage), halopemide, FIPI and formic acid (HCOOH) were purchased from Sigma Aldrich (Buchs, Switzerland).
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Ammonium acetate, diethyl ether, dimethylsulfoxide (DMSO), calcium chloride and disodium-hydrogen-phosphate were provided by Merck (Darmstadt, Germany). Acetonitrile was supplied by Agros Organics (New Jersey, USA), and 2-propanol was
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obtained from Fisher Scientific (Loughborough, UK). HPLC solvents were of gradient grade; all other solvents were of analytical grade. Deionized water was produced in-house by a MilliQ water system from Millipore (Billerica, USA). Deuterated standards were synthesized from PC 16:0/18:1 or PC 16:0/18:2 and D6-ethanol using PLD [23]. Heparin S-Monovettes (volume 9 mL) were obtained from Sarstedt (Nümbrecht, Germany). Lithium heparin whole
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blood, which was used as blank blood, was obtained from 8 volunteers (5 females, 3 males) who were abstinent for at least 4 weeks.
Determination of PEth formation rates
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Fresh PEth-negative lithium-heparinized blood samples were collected from a volunteer who had been abstinent for at least four weeks. Ethanol was added to 5 mL aliquots of this sample
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to reach different blood alcohol concentrations (BAC: 0.1 ‰, 0.5 ‰, 1.0 ‰, 1.5 ‰, 2.0 ‰, 2.5 ‰ and 3 ‰) directly after blood sampling. The seven 5 mL aliquots were incubated at 37 °C to obtain the same temperature as in the human body. For determination of PEth formation rate, 200 µL from each sample were taken over 7 hours on the first day and once daily on seven subsequent days.
To study the applicability of this test, the experiment was repeated with fresh blood samples from twelve additional volunteers (4 females, 8 males). For the first four volunteers (2 females and 2 males), the blood samples were spiked to a BAC of 1 ‰ to investigate possible inter-individual differences in PEth formation rates. 7
ACCEPTED MANUSCRIPT The blood samples of the next eight volunteers (2 females and 6 males) were spiked with ethanol to reach a BAC of 0.8 ‰. Samples were taken every hour for five hours to determine in-vitro PEth formation as described above. Drinking study design for in-vivo PEth formation rates To determine in-vivo PEth formation rates, the above mentioned eight volunteers (2 female
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and 6 male) ingested an individual single dose of alcohol (vodka mixed with a soft drink), which should lead to the targeted BAC of 0.8 ‰. Blank blood samples were obtained from all subjects before starting the experiment. Further samples were collected after 2, 4 and 6 h after the alcohol intake. The collected samples were stored at 4 °C prior to analysis to prevent post-
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sampling formation of PEth in the ethanol containing blood samples and were analyzed
PLD inhibition experiments
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promptly after sampling to ensure the overall PEth stability [27, 33, 34].
Preparation of halopemide and FIPI solutions
Halopemide was dissolved in DMSO to prepare a stock solution of 1 mg/mL. To prepare halopemide concentrations of 30 nM, 300 nM and 3000 nM in the blood samples, the stock solution was diluted further with water to 100 µg/mL and 10 µg/mL. Solutions with
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halopemide concentrations >3000 nM could not be tested because of poor solubility of the compound in DMSO:water (1:4; v/v).
FIPI was diluted in DMSO and water in the same way as halopemide (1 mg/mL and 5
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mg/mL, respectively, in DMSO, then diluted with water), and the same concentrations as for halopemide were used. In contrast to halopemide, FIPI was soluble at a concentration of
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30000 nM in DMSO:water (1:4, v/v).
PLD inhibition experiments For the inhibition experiments, preliminary experiments were performed in diethyl ether/water solution (1:1.5, v/v) to test the activity of PLD (from cabbage) and afterwards to test the inhibition potential of halopemide. These experiments were performed like the synthesis of the internal standards D5-PEth 16:0/18:1 and D5-PEth 16:0/18:2 described in detail by Schröck et al. [23], but instead of D6-ethanol, ethanol was used. Production of PEth 16:0/18:1 and PEth 16:0/18:2 was used to monitor PLD activity. Inhibition of PLD was determined using halopemide concentrations of 300 nM, 600 nM and 3000 nM. The IC50 values for halopemide have been reported to be approximately 300 nM for inhibition of PLD2 8
ACCEPTED MANUSCRIPT [10, 13, 29]. Fresh blood samples of 9 mL each (Heparin S-Monovettes) were taken from a healthy abstinent volunteer. Halopemide was added to final concentrations of 30 nM, 300 nM, and 3000 nM. Prior to adding ethanol to obtain a BAC of 1 ‰ and to start the reacti on, the halopemide containing blood samples were incubated for 30 min at 37 °C. The blood samples were kept at this temperature for the rest of the experiment. Aliquots of blood were sampled at
of PLD, was tested under identical conditions.
Determination of PEth
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0, 1, 2, 3, 4, 5, 24, 48 and 72 h after the start of the experiment. FIPI, another potent inhibitor
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PEth analysis was determined by online-SPE-LC-MS/MS with a QTrap 3200 mass spectrometer with a TurboIon source (Sciex, Toronto, Canada) and column-switching. PEth 16:0/18:1 and PEth 16:0/18:2 were analyzed in 200 µL of whole blood with D5-PEth
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16:0/18:1 and D5-PEth 16:0/18:2 as internal standards. The two analogues were trapped on a Polar-RP column, 20 mm x 2 mm, 5 µm particle size (Phenomenex, Brechbühler, Schlieren, Switzerland) and then chromatographically separated on a Luna RP-C5 column, 50 mm x 2 mm, 5 µm particle size (Phenomenex, Brechbühler, Schlieren, Switzerland). Multiple-reaction monitoring (MRM) was used for the detection of analytes and deuterated internal standards. A
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more detailed procedure has been published before [26].
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Results Monitoring of PEth formation rates
3.0 ‰ 2.5 ‰ 2.0 ‰ 1.5 ‰ 1.0 ‰
2.0 ‰ 1.5 ‰ 1.0 ‰
0.5 ‰
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0.5‰
3.0 ‰ 2.5 ‰
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resulted in concentration-dependent formation of PEth (figure 3).
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Incubation of human blood at 37 °C in the presence of different concentrations of ethanol
0.1 ‰
0.1 ‰
Figure 3. Formation of PEth 16:0/18:1 (left) and PEth 16:0/18:2 (right) over one
‰, 2.5 ‰ and 3 ‰)
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week dependent on alcohol concentrations (BAC: 0.1 ‰, 0.5 ‰, 1.0 ‰, 1.5 ‰, 2.0
Addition of PC 16:0/18:1 and PC 16:0/18:1 produced PEth 16:0/18:1 and PEth 16:0/18:2,
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respectively. Formation of PEth was linear over 7 h of incubation and occurred at the following rates: at BACs of 0.1 ‰, 1 ‰, 2 ‰ and 3 ‰, PEth 16:0/18:1 was formed at 0.002
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µmol·L-1·h-1, 0.016 µmol·L-1·h-1, 0.025 µmol·L-1·h-1 and 0.029 µmol·L-1·h-1, respectively. Similar rates were also observed when PC 16:0/18:2 was used as substrate, i.e. 0.002 µmol·L1
·h-1 (at BAC 0.1 ‰), 0.019 µmol·L-1·h-1 (at BAC 1 ‰), 0.025 µmol· L-1·h-1 (at BAC 2 ‰)
and 0.030 µmol·L-1·h-1 (at BAC 3 ‰). Maximum concentrations of 431 ng/mL (PEth 16:0/18:1) and 496 ng/mL (PEth 16:0/18:2) were detected at 3 ‰ (figure 3 and 4) on day three.
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ACCEPTED MANUSCRIPT 0.035
y = -0.0031x2 + 0.0191x + 0.0003 R² = 0.9858
0.025 0.02
y = -0.0028x2 + 0.0178x + 0.0002 R² = 0.998
0.015
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V0 (µmol·L-1·h-1)
0.03
0.01
0 0
0.5
1
1.5
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0.005
2
3
3.5
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BAC (‰)
2.5
PEth 16:0/18:1
PEth 16:0/18:2
Figure 4. In-vitro PEth formation rates depending on the alcohol concentration in the incubated blood samples
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PEth formation rates with spiked blood samples (in-vitro)
The determination of the PEth formation rates (in-vitro) in blood samples obtained from 12 persons (4 female and 8 male) showed that the developed test system also worked with a shortened incubation time of 5 hours, and differences in PEth formation rates between those
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12 volunteers were found.
For the first four volunteers (2 female and 2 male), the blood samples were spiked to a BAC
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of 1 ‰ and the PEth 16:0/18:1 the formation rates ranged between 0.016 – 0.021 µmol·L-1·h-1 (mean: 0.018 ± 0.002 µmol·L-1·h-1), and for PEth 16:0/18:2 between 0.015 – 0.020 µmol·L1
·h-1 (mean: 0.017 ± 0.002 µmol·L-1·h-1).
The blood samples of the next eight volunteers (2 female and 6 male) were spiked with ethanol up to a BAC of 0.8 ‰. The in-vitro formation rates for PEth 16:0/18:1 ranged between 0.011 – 0.025 µmol·L-1·h-1 (mean: 0.020 ± 0.004 µmol·L-1·h-1), and for PEth 16:0/18:2 between 0.014 – 0.021 µmol·L-1·h-1 (mean: 0.017 ± 0.003 µmol·L-1·h-1) (figure 5).
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ACCEPTED MANUSCRIPT Formation rate (µmol·mL-1·h-1)
0.030 0.025 0.020
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0.015 0.010
0.000 1
2
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Test persons
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0.005
In-vitro PEth 16:0/18:1 formation rate
In-vitro PEth 16:0/18:2 formation rate
Figure 5. Comparison of in-vitro formation rates of PEth 16:0/18:1 and PEth 16:0/18:2 in 12 blood samples from different test persons at two different BACs (1-4: BAC 1 ‰; 5-12: BAC 0.8 ‰)
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In a preliminary experiment, which was performed under the same conditions as described above (BAC 0.8 ‰, 37 °C, 5 hours), we tried to compare these in-vitro formation rates of PEth to the in-vivo formation rates from a drinking study. The corresponding in-vivo formation rates ranged for PEth 16:0/18:1 between 0.007 – 0.011 µmol·L-1·h-1 (mean: 0.010 ±
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0.002 µmol·L-1·h-1), and for PEth 16:0/18:2 between 0.006 – 0.013 µmol·L-1·h-1 (mean: 0.010 ± 0.002 µmol·L-1·h-1), showing that the in-vivo formation rates were approximately factor 2
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smaller.
PLD inhibition experiments As shown above, incubation of PC 16:0/18:1 in the presence of ethanol resulted in the formation of PEth during the first hour of incubation; accordingly, the amount of PC decreased. Interestingly, in the presence of the PLD inhibitor, halopemide, formation of PEth was not affected in the experiments with PLD from cabbage. In contrast, as expected no PEth was formed in the absence of ethanol. It has previously been shown that pre-incubation of the halopemide analogue FIPI for 15 min prior to addition of the primary alcohol to start the reaction was sufficient to completely 12
ACCEPTED MANUSCRIPT inhibit PLD activity [32]. In contrast, we found no inhibition of PLD activity even after a 30 min pre-incubation of halopemide with PLD. Since it is not known if halopemide also inhibits plant (cabbage) PLDs, we repeated the experiments using human PLD from blood. Interestingly, PEth formation in blood was inhibited by halopemide in a concentrationdependent way. However, complete inhibition could not be achieved by the applied maximum
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concentration of 3 µM. The rates for 16:0/18:1 and 16:0/18:2 PEth production were determined from the values obtained during the first 5 hours of the experiment (figure 6 A).
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A
Figure 6 Formation rate of PEth 16:0/18:1 and 16:0/18:2 as a function of halopemide concentration (A) and FIPI concentration (B) 13
ACCEPTED MANUSCRIPT We found that FIPI was a better inhibitor than halopemide of PEth formation in human blood. Complete inhibition of PEth formation was observed at a concentration of 30 µM during the first 24 h (for PEth 16:0/18:1) or 48 h (for PEth 16:0/18:2) (figure 6 B). The rates of PEth
Discussion
Standardized measurement of PEth formation rates
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16:0/18:1 and PEth 16:0/18:2 formation were determined by linear regression.
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Our results demonstrate linear PEth formation in blood during the first 7 h of incubation in the presence of ethanol. The rate and extent of PEth formation was dependent on the alcohol
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concentration, following the trend that increasing amounts of alcohol spiked into the samples, led to a higher PEth formation. PEth 16:0/18:1 and PEth 16:0/18:2 were formed nearly to the same extent.
The rates of PEth formation were determined by linear regression during the first 7 h of incubation at 37 °C, the slopes representing the respective velocities of PEth 16:0/18:1 and PEth 16:0/18:1 formation. Under our experimental conditions, the PLD in the collected blood
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samples formed PEth for three days, or at this point of time PEth degradation had also started in a higher rate than PEth formation itself.
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Application of the standardized measurement of PEth formation rates For a standardized test involving fresh “PEth negative” blood samples from 12 test persons, the time of incubation at 37 °C was shortened to 5 hours. The results showed small
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differences in the rates of PEth formation. Similarly, no major differences in PEth formation were seen when the blood samples were spiked with 0.8 ‰ or 1 ‰ ethanol (figure 5). By comparison of the in-vitro formation rates of PEth to the in-vivo formation rates from a drinking study, the in-vivo formation rates were found to be approximately factor 2 smaller. The in-vivo formation rate could be influenced by the distribution of the formed lipophilic compound PEth in the body tissues; this would leave less detectable PEth in the blood. Another factor could be the elimination kinetics of ethanol leading to metabolic decrease of the blood alcohol concentration in the human body. Mainly, alcohol is converted to acetaldehyde in the liver by the enzyme alcohol dehydrogenase (ADH) to acetaldehyde. Acetaldehyde is then rapidly metabolized to acetate mainly by the enzyme aldehyde 14
ACCEPTED MANUSCRIPT dehydrogenase 2 (ALDH2) [35]. These processes leave less alcohol in the blood to form PEth via PLD; in contrast, the ethanol concentration is not significantly declining in the sample during the in vitro-experiment. Nevertheless, this in-vitro test system enables us to compare the PLD activities in blood samples of different test persons. However, PEth formation is not only dependent on the
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substrate ethanol; it may also depend on the amounts of different individual concentrations of
further investigations seem to be necessary.
PLD inhibition experiments
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the PC homologues in human blood, which could be affected by nutrition [36]. Therefore,
Halopemide and its homologue FIPI have been described to be potent inhibitors of mammalian PLD in several experiments [10, 13, 29, 32].
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In the preliminary experiments with PC 16:0/18:1 dissolved in diethyl ether and PLD from cabbage, halopemide did not prevent formation of PEth at any of the applied concentrations. Halopemide might not be able to inhibit the PLD obtained from plants (cabbage) which we used in this preliminary experiment. In previous studies, experiments were performed with mammalian PLD and some variants of bacterial PLD only, which all share a conserved HKD
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amino acid domain (histidine, lysine, aspartate) that is thought to be the catalytic site [37]. In our experiments with human blood, in other words with human PLD, the formation of PEth analogues could be partially inhibited by halopemide and FIPI, but with a much lower potency than previously described. A complete inhibition of PEth formation could only be
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achieved by FIPI at a concentration of 30000 nM. Su et al. [32] performed a pre-incubation of the halopemide analogue FIPI prior to addition of the primary alcohol to start the reaction.
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They found that 15 min was sufficient to achieve a complete inhibition of PLD. In our experiment however, 30 min pre-incubation of halopemide with PLD did not change the results, either.
Although the PLD inhibitors halopemide and FIPI do inhibit PEth formation in human blood samples, due to their low potency they are not useful to guarantee stability in human blood samples. It would not be feasible for clinical use as well as from an economical point of view (due to high costs of halopemide and FIPI) to add these substances at the high concentrations needed for a measurable effect and to avoid formation of PEth in blood samples which contain ethanol.
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ACCEPTED MANUSCRIPT Possible reasons for the low potency of halopemide and FIPI in our experimental setting are that PEth could also be formed in alternative pathways, or the PLD in human blood is not targeted as well as in other tissues by halopemide and/or FIPI. So far the easiest and the most robust way for PEth determination in blood is the analysis of PEth from dried blood spots (DBS) as PEth analysis in DBS in comparison to analysis of
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PEth in whole blood shows no post-sampling formation of PEth [38]. By drying the blood on a filter paper, the PLD activity is inhibited and no post-sampling formation takes place; and DBS sampling inhibits PEth degradation as well.
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Conclusion
In the in-vivo drinking study we found inter-individual differences in PEth formation rates.
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In-vitro experiments with freshly sampled blood revealed that the formation rate of PEth in blood is dependent on BAC. The in-vitro PEth formation during the first 7 h in the presence of alcohol proceeds in a zero order kinetic, leading to a standardized test to determine the individual PEth formation rates in human blood specimens via linear regression. Although we found high formation rates of PEth in blood, which might be responsible for a high amount of PEth production in the human body, we do not have knowledge about PEth
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formation in liver or other organs, which are mainly responsible for metabolism of xenobiotics.
With the application of the PLD inhibitors halopemide and FIPI, there was inhibition of PEth formation, but only at rather high inhibitor concentrations. However, if PEth is used as
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solved.
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alcohol marker in routine analysis the problem with post-sampling PLD activity needs to be
Funding
This study was supported by the Swiss Foundation of Alcohol Research (Grant 254/2014: Studies on phosphatidylethanol – a promising biomarker for the detection of harmful ethanol consumption – and its possible use for abstinence monitoring).
Ethical Approval This study has been approved by the Cantonal Ethics Commission Bern (064/13) on December 10, 2015.
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References
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W.H.O. (WHO), Global Status Report on Alcohol and Health 2014. http://www.who.int/substance_abuse/publications/global_alcohol_report/en/. Accessed 04 Dezember 2015 2014. A. Schröck, et al., Progress in monitoring alcohol consumption and alcohol abuse by phosphatidylethanol. Bioanalysis, 2014. 6(17): p. 2285-94. H. Gnann, et al., Identification of 48 homologues of phosphatidylethanol in blood by LC-ESIMS/MS. Anal Bioanal Chem, 2010. 396(7): p. 2415-23. A. Helander and Y. Zheng, Molecular species of the alcohol biomarker phosphatidylethanol in human blood measured by LC-MS. Clin Chem, 2009. 55(7): p. 1395-405. Y. Zheng, O. Beck, and A. Helander, Method development for routine liquid chromatography–mass spectrometry measurement of the alcohol biomarker phosphatidylethanol (PEth) in blood. Clin Chim Acta, 2011. 412(15–16): p. 1428-1435. H. Gnann, et al., Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC-ESI-MS/MS. J Mass Spectrom, 2009. 44(9): p. 1293-9. H. Gnann, W. Weinmann, and A. Thierauf, Formation of phosphatidylethanol and its subsequent elimination during an extensive drinking experiment over 5 days. Alcohol Clin Exp Res, 2012. 36(9): p. 1507-11. M. Kobayashi and J.N. Kanfer, Phosphatidylethanol formation via transphosphatidylation by rat brain synaptosomal phospholipase D. J Neurochem, 1987. 48(5): p. 1597-603. H.A. Brown, et al., Biochemical analysis of phospholipase D. Methods Enzymol, 2007. 434: p. 49-87. J.A. Lewis, et al., Design and synthesis of isoform-selective phospholipase D (PLD) inhibitors. Part I: Impact of alternative halogenated privileged structures for PLD1 specificity. Bioorg Med Chem Lett, 2009. 19(7): p. 1916-20. J.H. Exton, Regulation of phospholipase D. FEBS Lett, 2002. 531(1): p. 58-61. J.F. Hancock, PA promoted to manager. Nat Cell Biol, 2007. 9(6): p. 615-7. S.A. Scott, et al., Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness. Nat Chem Biol, 2009. 5(2): p. 108-17. N.A. Babenko and V.S. Kharchenko, Modulation of Insulin Sensitivity of Hepatocytes by the Pharmacological Downregulation of Phospholipase D. Int J Endocrinol, 2015. 2015: p. 794838. D.Y. Noh, et al., Overexpression of phospholipase D1 in human breast cancer tissues. Cancer Lett, 2000. 161(2): p. 207-14. Y. Yamada, et al., Association of a polymorphism of the phospholipase D2 gene with the prevalence of colorectal cancer. J Mol Med (Berl), 2003. 81(2): p. 126-31. J.A. Aguirre Ghiso, et al., A phospholipase D and protein kinase C inhibitor blocks the spreading of murine mammary adenocarcinoma cells altering f-actin and beta1-integrin point contact distribution. Int J Cancer, 1997. 71(5): p. 881-90. T.H. Oguin, 3rd, et al., Phospholipase D facilitates efficient entry of influenza virus, allowing escape from innate immune inhibition. J Biol Chem, 2014. 289(37): p. 25405-17. W. Su, Q. Chen, and M.A. Frohman, Targeting phospholipase D with small-molecule inhibitors as a potential therapeutic approach for cancer metastasis. Future Oncol, 2009. 5(9): p. 147786. S. Aradottir, et al., PHosphatidylethanol (PEth) concentrations in blood are correlated to reported alcohol intake in alcohol-dependent patients. Alcohol Alcohol, 2006. 41(4): p. 431-7. G. Viel, et al., Phosphatidylethanol in blood as a marker of chronic alcohol use: a systematic review and meta-analysis. Int J Mol Sci, 2012. 13(11): p. 14788-812.
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M. Winkler, et al., Comparison of direct and indirect alcohol markers with PEth in blood and urine in alcohol dependent inpatients during detoxication. Int J Legal Med, 2013. 127(4): p. 761-8. A. Schröck, et al., Phosphatidylethanol (PEth) in blood samples from "driving under the influence" cases as indicator for prolonged excessive alcohol consumption. Int J Legal Med, 2016. 130(2): p. 393-400. A. Varga, et al., Normalization rate and cellular localization of phosphatidylethanol in whole blood from chronic alcoholics. Clin Chim Acta, 2000. 299(1-2): p. 141-50. H. Gnann, Phosphatidylethanol - Ein Alkoholkonsummarker auf dem Weg in die klinischforensische Routinediagnostik. Dissertation, University of Freiburg, Germany. http://www.freidok.uni-freiburg.de/volltexte/8428/pdf/Diss_Heike_Gnann.pdf. Accessed 15 April 2014, 2011: p. 101. A. Schröck, et al., Phosphatidylethanol (PEth) detected in blood for 3 to 12 days after single consumption of alcohol-a drinking study with 16 volunteers. Int J Legal Med, 2016. S. Aradóttir, et al., Phosphatidylethanol in Human Organs and Blood: A Study on Autopsy Material and Influences by Storage Conditions. Alcohol Clin Exp Res, 2004. 28(11): p. 17181723. R.C. Bruntz, C.W. Lindsley, and H.A. Brown, Phospholipase D signaling pathways and phosphatidic acid as therapeutic targets in cancer. Pharmacol Rev, 2014. 66(4): p. 1033-79. L. Monovich, et al., Optimization of halopemide for phospholipase D2 inhibition. Bioorg Med Chem Lett, 2007. 17(8): p. 2310-1. A.J. Loonen and W. Soudijn, Halopemide, a new psychotropic agent. Cerebral distribution and receptor interactions. Pharm Weekbl Sci, 1985. 7(1): p. 1-9. R. Lavieri, et al., Design and synthesis of isoform-selective phospholipase D (PLD) inhibitors. Part II. Identification of the 1,3,8-triazaspiro[4,5]decan-4-one privileged structure that engenders PLD2 selectivity. Bioorg Med Chem Lett, 2009. 19(8): p. 2240-3. W. Su, et al., 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), a phospholipase D pharmacological inhibitor that alters cell spreading and inhibits chemotaxis. Mol Pharmacol, 2009. 75(3): p. 437-46. S. Aradottir and B.L. Olsson, Methodological modifications on quantification of phosphatidylethanol in blood from humans abusing alcohol, using high-performance liquid chromatography and evaporative light scattering detection. BMC Biochem, 2005. 6: p. 18. A. Faller, et al., Stability of phosphatidylethanol species in spiked and authentic whole blood and matching dried blood spots. Int J Legal Med, 2013. 127(3): p. 603-10. S. Zakhari, Overview: how is alcohol metabolized by the body? Alcohol Res Health, 2006. 29(4): p. 245-54. W. Weinmann, A. Schröck, and F.M. Wurst, Commentary on the Paper of Walther L. et al.: Phosphatidylethanol is Superior to CDT and GGT as an Alcohol Marker and Is a Reliable Estimate of Alcohol Consumption Level. Alcohol Clin Exp Res, 2016. 40(2): p. 260-2. Z. Xie, W.T. Ho, and J.H. Exton, Association of N- and C-terminal domains of phospholipase D is required for catalytic activity. J Biol Chem, 1998. 273(52): p. 34679-82. N. Kummer, et al., Quantification of phosphatidylethanol 16:0/18:1, 18:1/18:1, and 16:0/16:0 in venous blood and venous and capillary dried blood spots from patients in alcohol withdrawal and control volunteers. Anal Bioanal Chem, 2016. 408(3): p. 825-38.
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Highlights
The formation rate of PEth in blood is dependent on blood alcohol concentration.
•
Measurement of individual PEth formation rates in fresh blood samples is standardized.
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5-fluoro-2-indolyl-deschlorohalopemide is a more effective inhibitor than halopemide.
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S0741-8329(18)30001-6
DOI:
10.1016/j.alcohol.2018.03.003
Reference:
ALC 6784
To appear in:
Alcohol
Received Date: 3 January 2018 Revised Date:
8 March 2018
Accepted Date: 13 March 2018
Please cite this article as: Schröck A., Henzi A., Bütikofer P., König S. & Weinmann W., Determination of the formation rate of phosphatidylethanol by phospholipase D (PLD) in blood and test of two selective PLD inhibitors, Alcohol (2018), doi: 10.1016/j.alcohol.2018.03.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Determination
of
the
formation
rate
of
phosphatidylethanol
by
phospholipase D (PLD) in blood and test of two selective PLD inhibitors Authors:
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Alexandra Schröcka,c, Anna Henzia, Peter Bütikoferb, Stefan Königa, Wolfgang Weinmanna
Affiliations: a
Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern
Bühlstrasse 20
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3012 Bern
b
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Switzerland
Institute of Biochemistry and Molecular Medicine, University of Bern
Bühlstrasse 28 3012 Bern Switzerland
Graduate School for Cellular and Biomedical Sciences (GCB), University of Bern
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c
c/o Theodor Kocher Institute Freiestrasse 1
Switzerland
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3012 Bern
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Corresponding author: Wolfgang Weinmann
Institute of Forensic Medicine, University of Bern Bühlstrasse 20
CH-3012 Bern
Tel. +41 (0)31 631 5668 E-Mail: [email protected]
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Abstract Phosphatidylethanol (PEth) is an alcohol biomarker formed from phosphatidylcholine (PC) by the enzyme phospholipase D (PLD) in the presence of ethanol. A drinking study revealed individual differences in maximum PEth levels after drinking up to a targeted blood alcohol concentration (BAC) of 1 ‰. This seemed to be due to different PLD activities in the tested
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persons. Furthermore, post-sampling formation of PEth occurred in blood samples, still containing alcohol. Therefore, a standardized in-vitro test for measuring individual PEth formation rates was developed. Two PLD inhibitors were tested for their potency to inhibit post-sampling PEth formation.
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PEth-negative blood samples were collected from a volunteer. Ethanol was added in different concentrations (0.1 – 3 ‰ BAC) directly after blood sampling. The specimens were incubated at 37 °C. Aliquots were taken at the start of the incubation, and every hour until 8 hours after
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start of incubation, and one sample was taken on subsequent days over one week. PEth 16:0/18:1 and PEth 16:0/18:2 were determined by online-SPE-LC-MS/MS. Furthermore, this test system was applied to blood samples of 12 volunteers.
For the inhibition tests, fresh blood (spiked with 1 ‰ ethanol) was spiked with 30, 300, 3000, 30000 nM of either halopemide or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), and
and once on day 2 and 3.
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incubated at 37 °C. PEth concentrations were determined hourly over 5 hours on the first day
PEth formation was linear in the first 7 hours of incubation and dependent on the alcohol concentration. The formation rates of PEth 16:0/18:1 were 0.002 µmol·L-1·h-1 (0.1 ‰ BAC),
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0.016 µmol·L-1·h-1 (1 ‰ BAC), 0.025 µmol·L-1·h-1 (2 ‰ BAC) and 0.029 µmol·L-1·h-1 (3 ‰ BAC). For PEth 16:0/18:2, the formation rates were 0.002 µmol·L-1·h-1 (0.1 ‰ BAC), 0.019
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µmol·L-1·h-1 (1 ‰ BAC), 0.025 µmol·L-1·h-1 (2 ‰ BAC) and 0.030 µmol·L-1·h-1 (3 ‰ BAC). Maximum concentrations reached 431 ng/mL (PEth 16:0/18:1) and 496 ng/mL (PEth 16:0/18:2) at 3 ‰ BAC after 3 days. Maximum velocity (vmax) was not reached under these conditions. PEth formation in blood of the 12 volunteers ranged between 0.011 – 0.025 µmol·L-1·h-1 for PEth 16:0/18:1 and between 0.014 – 0.021 µmol·L-1·h-1 for PEth 16:0/18:2. PEth formation in human blood was inhibited by halopemide in a concentration-dependent manner. However, a complete inhibition was not achieved by the applied maximum concentration of 30000 nM. FIPI showed a better inhibition of PEth formation. A complete inhibition could be achieved by a concentration of 30000 nM for the first 24 h (for PEth 16:0/18:1) and for 48 h (for PEth 16:0/18:2).
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ACCEPTED MANUSCRIPT Formation of PEth was found to be dependent on the BAC. As a consequence, it is essential to inhibit PLD activity after blood collection to avoid post-sampling formation of PEth in blood samples with a positive BAC. Inhibition of PEth formation was more effective using FIPI compared to halopemide.
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Key words Phosphatidylethanol (PEth); alcohol biomarker; online-SPE-LC-MS/MS; Phospholipase D
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(PLD) activity, PEth formation rate, PLD inhibitors
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Introduction As alcohol abuse is a major global factor contributing to disease, injury and death, the development of methods for the early detection of risky alcohol consumption by means of alcohol biomarkers is important [1]. A promising direct alcohol biomarker with high
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specificity and sensitivity is phosphatidylethanol (PEth) [2]. The term PEth represents a group of phospholipid analogues. 48 analogues have been identified in human blood [3], with PEth 16:0/18:1 and PEth 16:0/18:2 as the most abundant analogues [4, 5]. In vivo, PEth is produced from ethanol (after alcohol intake and as long as ethanol is present [6, 7]) and
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phosphatidylcholine (PC) in a reaction catalyzed by phospholipase D (PLD) [8].
PLD is a membrane-associated enzyme found in most eukaryotes and also in prokaryotes. There are two isoforms of mammalian PLD, termed PLD1 and PLD2 [9]. Both isoforms have
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similar regulatory and catalytic domains, but show differences in activation and have different biological functions [10]. Unstimulated PLD1 has a low activity and is regulated by protein kinase C, Arf and Rho GTPases [11]. In contrast, PLD2 shows a high basal activity and is involved in several protein interactions [12, 13].
Normally PLD catalyzes the hydrolysis of its principal substrate PC into phosphatidic acid
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(PA) and choline as shown in figure 1. PA is an important lipid second messenger involved in several signaling pathways, affecting cellular metabolism, cell cycle progression and cell growth [14]. From a therapeutic point of view, PLD or rather its aberrant function and/or overexpression is for example implicated to play a role in cancer [15, 16] and metastasis [13],
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cell motility and viral infection [18].
However, in the presence of primary alcohols, PLD catalyzes a transphosphatidylation
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reaction using alcohol as substrate instead of water to produce phosphatidylalcohol. In previous studies on the function of PLD, formation of phosphatidylalcohols (e.g. phosphatidylbutanol) is often used to assay PLD activity in vitro, as phosphatidylalcohols are normally not produced under physiological conditions [19]. Adding high amounts of alcohol can completely divert PLD away from producing PA and was thus used to inhibit PLDmediated pathways, when no suitable inhibitors were available [13, 19]. PEth, the product of the transphosphatidylation reaction with ethanol (figure 1), is suitable as alcohol biomarker [6, 20-23]. Due to an elimination half-life of approximately four days [24], which can vary widely between individuals [7], PEth gets accumulated in the body after repeated drinking. A number of drinking studies with moderate alcohol consumption have
4
ACCEPTED MANUSCRIPT shown that measured PEth concentrations correlate with the consumed amounts of alcohol [7, 25]. O R1 O
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O H 2O
R2
O
O
R1
H
N
R2
+
H 3C H 3C
O
O O
-
Phosphatidylcholine (PC)
O
EtOH
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H 3C
O
R1
Phospholipase D (PLD)
P
-
Phosphatidic acid (PA)
O O
P
O
O
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CH 3
O
O
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O
O
O
R2
O O
O
O
P O
-
Phosphatidylethanol (PEth)
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Figure 1. Enzymatic reactions catalyzed by phospholipase D
Such a drinking study with 16 volunteers revealed individual differences in maximum PEth
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concentrations after a single alcohol ingestion up to a targeted blood alcohol concentration (BAC) of 1 ‰ [26]. This seemed to be due to different PLD activities in the tested persons. Additionally, post-sampling formation of PEth was shown to occur at several storage conditions in blood samples, which still contained alcohol [27]. Further investigations have long been impaired by the lack of specific inhibitors of this enzyme. Several compounds including diethylstilbestron, honokiol, raloxifen [13] and ceramide [19] as well as the antibiotic neomycin [28] are known to inhibit PLD activity indirectly. In 2007, halopemide was identified as a PLD inhibitor by high-throughput screening [29]. Halopemide was first described as an antipsychotic drug with dopamine-blocking properties and structural analogy to neuroleptics of butyrophenone type [30]. The structural formula of 5
ACCEPTED MANUSCRIPT halopemide is depicted in figure 2A. Halopemide and its congeners were identified to be mostly dual and slightly PLD1 preferring inhibitors [13]. Halopemide inhibited PLD1 with an IC50 of 220 nM and PLD2 with an IC50 of 310 nM. Since then, numerous isoform selective PLD inhibitors have been developed using halopemide as a lead compound [10, 13, 31], for example the very potent 5-fluoro-2-indolyl-deschlorohalopemide (FIPI) which inhibits PLD1
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and PLD2 at sub-nM concentrations [32] (figure 2B). By using the N-terminally truncated enzyme PLD1.d311, a constitutively highly active form of PLD, Scott et al. could confirm that the compounds inhibit PLD in a direct manner and not due to interference with myr-Arf-1 GTPase stimulation, which is commonly used in other experiments for testing PLD activity
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[13].
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B
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A
Figure 2. Structure of halopemide (A) and 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) (B)
The aims of this study were to develop a standardized test system for measurement of individual PEth formation rates, and to apply this test to different individuals. Furthermore, we investigated the dual PLD1/2 inhibitors halopemide and FIPI for their ability to inhibit PEth formation by PLD in human blood after adding ethanol. The introduction of such 6
ACCEPTED MANUSCRIPT inhibitors may have the potential to further improve the quality of PEth analysis by preventing additional in-vitro formation of PEth in ethanol positive blood samples after blood sampling.
Material and methods
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Chemicals and materials PEth 16:0/18:1, PEth 16:0/18:2, PC 16:0/18:1 and PC 16:0/18:2 were obtained from Avanti Polar Lipids (Alabaster, USA). Phospholipase D (PLD) (from cabbage), halopemide, FIPI and formic acid (HCOOH) were purchased from Sigma Aldrich (Buchs, Switzerland).
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Ammonium acetate, diethyl ether, dimethylsulfoxide (DMSO), calcium chloride and disodium-hydrogen-phosphate were provided by Merck (Darmstadt, Germany). Acetonitrile was supplied by Agros Organics (New Jersey, USA), and 2-propanol was
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obtained from Fisher Scientific (Loughborough, UK). HPLC solvents were of gradient grade; all other solvents were of analytical grade. Deionized water was produced in-house by a MilliQ water system from Millipore (Billerica, USA). Deuterated standards were synthesized from PC 16:0/18:1 or PC 16:0/18:2 and D6-ethanol using PLD [23]. Heparin S-Monovettes (volume 9 mL) were obtained from Sarstedt (Nümbrecht, Germany). Lithium heparin whole
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blood, which was used as blank blood, was obtained from 8 volunteers (5 females, 3 males) who were abstinent for at least 4 weeks.
Determination of PEth formation rates
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Fresh PEth-negative lithium-heparinized blood samples were collected from a volunteer who had been abstinent for at least four weeks. Ethanol was added to 5 mL aliquots of this sample
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to reach different blood alcohol concentrations (BAC: 0.1 ‰, 0.5 ‰, 1.0 ‰, 1.5 ‰, 2.0 ‰, 2.5 ‰ and 3 ‰) directly after blood sampling. The seven 5 mL aliquots were incubated at 37 °C to obtain the same temperature as in the human body. For determination of PEth formation rate, 200 µL from each sample were taken over 7 hours on the first day and once daily on seven subsequent days.
To study the applicability of this test, the experiment was repeated with fresh blood samples from twelve additional volunteers (4 females, 8 males). For the first four volunteers (2 females and 2 males), the blood samples were spiked to a BAC of 1 ‰ to investigate possible inter-individual differences in PEth formation rates. 7
ACCEPTED MANUSCRIPT The blood samples of the next eight volunteers (2 females and 6 males) were spiked with ethanol to reach a BAC of 0.8 ‰. Samples were taken every hour for five hours to determine in-vitro PEth formation as described above. Drinking study design for in-vivo PEth formation rates To determine in-vivo PEth formation rates, the above mentioned eight volunteers (2 female
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and 6 male) ingested an individual single dose of alcohol (vodka mixed with a soft drink), which should lead to the targeted BAC of 0.8 ‰. Blank blood samples were obtained from all subjects before starting the experiment. Further samples were collected after 2, 4 and 6 h after the alcohol intake. The collected samples were stored at 4 °C prior to analysis to prevent post-
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sampling formation of PEth in the ethanol containing blood samples and were analyzed
PLD inhibition experiments
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promptly after sampling to ensure the overall PEth stability [27, 33, 34].
Preparation of halopemide and FIPI solutions
Halopemide was dissolved in DMSO to prepare a stock solution of 1 mg/mL. To prepare halopemide concentrations of 30 nM, 300 nM and 3000 nM in the blood samples, the stock solution was diluted further with water to 100 µg/mL and 10 µg/mL. Solutions with
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halopemide concentrations >3000 nM could not be tested because of poor solubility of the compound in DMSO:water (1:4; v/v).
FIPI was diluted in DMSO and water in the same way as halopemide (1 mg/mL and 5
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mg/mL, respectively, in DMSO, then diluted with water), and the same concentrations as for halopemide were used. In contrast to halopemide, FIPI was soluble at a concentration of
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30000 nM in DMSO:water (1:4, v/v).
PLD inhibition experiments For the inhibition experiments, preliminary experiments were performed in diethyl ether/water solution (1:1.5, v/v) to test the activity of PLD (from cabbage) and afterwards to test the inhibition potential of halopemide. These experiments were performed like the synthesis of the internal standards D5-PEth 16:0/18:1 and D5-PEth 16:0/18:2 described in detail by Schröck et al. [23], but instead of D6-ethanol, ethanol was used. Production of PEth 16:0/18:1 and PEth 16:0/18:2 was used to monitor PLD activity. Inhibition of PLD was determined using halopemide concentrations of 300 nM, 600 nM and 3000 nM. The IC50 values for halopemide have been reported to be approximately 300 nM for inhibition of PLD2 8
ACCEPTED MANUSCRIPT [10, 13, 29]. Fresh blood samples of 9 mL each (Heparin S-Monovettes) were taken from a healthy abstinent volunteer. Halopemide was added to final concentrations of 30 nM, 300 nM, and 3000 nM. Prior to adding ethanol to obtain a BAC of 1 ‰ and to start the reacti on, the halopemide containing blood samples were incubated for 30 min at 37 °C. The blood samples were kept at this temperature for the rest of the experiment. Aliquots of blood were sampled at
of PLD, was tested under identical conditions.
Determination of PEth
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0, 1, 2, 3, 4, 5, 24, 48 and 72 h after the start of the experiment. FIPI, another potent inhibitor
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PEth analysis was determined by online-SPE-LC-MS/MS with a QTrap 3200 mass spectrometer with a TurboIon source (Sciex, Toronto, Canada) and column-switching. PEth 16:0/18:1 and PEth 16:0/18:2 were analyzed in 200 µL of whole blood with D5-PEth
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16:0/18:1 and D5-PEth 16:0/18:2 as internal standards. The two analogues were trapped on a Polar-RP column, 20 mm x 2 mm, 5 µm particle size (Phenomenex, Brechbühler, Schlieren, Switzerland) and then chromatographically separated on a Luna RP-C5 column, 50 mm x 2 mm, 5 µm particle size (Phenomenex, Brechbühler, Schlieren, Switzerland). Multiple-reaction monitoring (MRM) was used for the detection of analytes and deuterated internal standards. A
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more detailed procedure has been published before [26].
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Results Monitoring of PEth formation rates
3.0 ‰ 2.5 ‰ 2.0 ‰ 1.5 ‰ 1.0 ‰
2.0 ‰ 1.5 ‰ 1.0 ‰
0.5 ‰
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0.5‰
3.0 ‰ 2.5 ‰
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resulted in concentration-dependent formation of PEth (figure 3).
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Incubation of human blood at 37 °C in the presence of different concentrations of ethanol
0.1 ‰
0.1 ‰
Figure 3. Formation of PEth 16:0/18:1 (left) and PEth 16:0/18:2 (right) over one
‰, 2.5 ‰ and 3 ‰)
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week dependent on alcohol concentrations (BAC: 0.1 ‰, 0.5 ‰, 1.0 ‰, 1.5 ‰, 2.0
Addition of PC 16:0/18:1 and PC 16:0/18:1 produced PEth 16:0/18:1 and PEth 16:0/18:2,
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respectively. Formation of PEth was linear over 7 h of incubation and occurred at the following rates: at BACs of 0.1 ‰, 1 ‰, 2 ‰ and 3 ‰, PEth 16:0/18:1 was formed at 0.002
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µmol·L-1·h-1, 0.016 µmol·L-1·h-1, 0.025 µmol·L-1·h-1 and 0.029 µmol·L-1·h-1, respectively. Similar rates were also observed when PC 16:0/18:2 was used as substrate, i.e. 0.002 µmol·L1
·h-1 (at BAC 0.1 ‰), 0.019 µmol·L-1·h-1 (at BAC 1 ‰), 0.025 µmol· L-1·h-1 (at BAC 2 ‰)
and 0.030 µmol·L-1·h-1 (at BAC 3 ‰). Maximum concentrations of 431 ng/mL (PEth 16:0/18:1) and 496 ng/mL (PEth 16:0/18:2) were detected at 3 ‰ (figure 3 and 4) on day three.
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ACCEPTED MANUSCRIPT 0.035
y = -0.0031x2 + 0.0191x + 0.0003 R² = 0.9858
0.025 0.02
y = -0.0028x2 + 0.0178x + 0.0002 R² = 0.998
0.015
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V0 (µmol·L-1·h-1)
0.03
0.01
0 0
0.5
1
1.5
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0.005
2
3
3.5
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BAC (‰)
2.5
PEth 16:0/18:1
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Figure 4. In-vitro PEth formation rates depending on the alcohol concentration in the incubated blood samples
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PEth formation rates with spiked blood samples (in-vitro)
The determination of the PEth formation rates (in-vitro) in blood samples obtained from 12 persons (4 female and 8 male) showed that the developed test system also worked with a shortened incubation time of 5 hours, and differences in PEth formation rates between those
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12 volunteers were found.
For the first four volunteers (2 female and 2 male), the blood samples were spiked to a BAC
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of 1 ‰ and the PEth 16:0/18:1 the formation rates ranged between 0.016 – 0.021 µmol·L-1·h-1 (mean: 0.018 ± 0.002 µmol·L-1·h-1), and for PEth 16:0/18:2 between 0.015 – 0.020 µmol·L1
·h-1 (mean: 0.017 ± 0.002 µmol·L-1·h-1).
The blood samples of the next eight volunteers (2 female and 6 male) were spiked with ethanol up to a BAC of 0.8 ‰. The in-vitro formation rates for PEth 16:0/18:1 ranged between 0.011 – 0.025 µmol·L-1·h-1 (mean: 0.020 ± 0.004 µmol·L-1·h-1), and for PEth 16:0/18:2 between 0.014 – 0.021 µmol·L-1·h-1 (mean: 0.017 ± 0.003 µmol·L-1·h-1) (figure 5).
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ACCEPTED MANUSCRIPT Formation rate (µmol·mL-1·h-1)
0.030 0.025 0.020
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Test persons
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In-vitro PEth 16:0/18:1 formation rate
In-vitro PEth 16:0/18:2 formation rate
Figure 5. Comparison of in-vitro formation rates of PEth 16:0/18:1 and PEth 16:0/18:2 in 12 blood samples from different test persons at two different BACs (1-4: BAC 1 ‰; 5-12: BAC 0.8 ‰)
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In a preliminary experiment, which was performed under the same conditions as described above (BAC 0.8 ‰, 37 °C, 5 hours), we tried to compare these in-vitro formation rates of PEth to the in-vivo formation rates from a drinking study. The corresponding in-vivo formation rates ranged for PEth 16:0/18:1 between 0.007 – 0.011 µmol·L-1·h-1 (mean: 0.010 ±
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0.002 µmol·L-1·h-1), and for PEth 16:0/18:2 between 0.006 – 0.013 µmol·L-1·h-1 (mean: 0.010 ± 0.002 µmol·L-1·h-1), showing that the in-vivo formation rates were approximately factor 2
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smaller.
PLD inhibition experiments As shown above, incubation of PC 16:0/18:1 in the presence of ethanol resulted in the formation of PEth during the first hour of incubation; accordingly, the amount of PC decreased. Interestingly, in the presence of the PLD inhibitor, halopemide, formation of PEth was not affected in the experiments with PLD from cabbage. In contrast, as expected no PEth was formed in the absence of ethanol. It has previously been shown that pre-incubation of the halopemide analogue FIPI for 15 min prior to addition of the primary alcohol to start the reaction was sufficient to completely 12
ACCEPTED MANUSCRIPT inhibit PLD activity [32]. In contrast, we found no inhibition of PLD activity even after a 30 min pre-incubation of halopemide with PLD. Since it is not known if halopemide also inhibits plant (cabbage) PLDs, we repeated the experiments using human PLD from blood. Interestingly, PEth formation in blood was inhibited by halopemide in a concentrationdependent way. However, complete inhibition could not be achieved by the applied maximum
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concentration of 3 µM. The rates for 16:0/18:1 and 16:0/18:2 PEth production were determined from the values obtained during the first 5 hours of the experiment (figure 6 A).
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Figure 6 Formation rate of PEth 16:0/18:1 and 16:0/18:2 as a function of halopemide concentration (A) and FIPI concentration (B) 13
ACCEPTED MANUSCRIPT We found that FIPI was a better inhibitor than halopemide of PEth formation in human blood. Complete inhibition of PEth formation was observed at a concentration of 30 µM during the first 24 h (for PEth 16:0/18:1) or 48 h (for PEth 16:0/18:2) (figure 6 B). The rates of PEth
Discussion
Standardized measurement of PEth formation rates
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16:0/18:1 and PEth 16:0/18:2 formation were determined by linear regression.
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Our results demonstrate linear PEth formation in blood during the first 7 h of incubation in the presence of ethanol. The rate and extent of PEth formation was dependent on the alcohol
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concentration, following the trend that increasing amounts of alcohol spiked into the samples, led to a higher PEth formation. PEth 16:0/18:1 and PEth 16:0/18:2 were formed nearly to the same extent.
The rates of PEth formation were determined by linear regression during the first 7 h of incubation at 37 °C, the slopes representing the respective velocities of PEth 16:0/18:1 and PEth 16:0/18:1 formation. Under our experimental conditions, the PLD in the collected blood
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samples formed PEth for three days, or at this point of time PEth degradation had also started in a higher rate than PEth formation itself.
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Application of the standardized measurement of PEth formation rates For a standardized test involving fresh “PEth negative” blood samples from 12 test persons, the time of incubation at 37 °C was shortened to 5 hours. The results showed small
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differences in the rates of PEth formation. Similarly, no major differences in PEth formation were seen when the blood samples were spiked with 0.8 ‰ or 1 ‰ ethanol (figure 5). By comparison of the in-vitro formation rates of PEth to the in-vivo formation rates from a drinking study, the in-vivo formation rates were found to be approximately factor 2 smaller. The in-vivo formation rate could be influenced by the distribution of the formed lipophilic compound PEth in the body tissues; this would leave less detectable PEth in the blood. Another factor could be the elimination kinetics of ethanol leading to metabolic decrease of the blood alcohol concentration in the human body. Mainly, alcohol is converted to acetaldehyde in the liver by the enzyme alcohol dehydrogenase (ADH) to acetaldehyde. Acetaldehyde is then rapidly metabolized to acetate mainly by the enzyme aldehyde 14
ACCEPTED MANUSCRIPT dehydrogenase 2 (ALDH2) [35]. These processes leave less alcohol in the blood to form PEth via PLD; in contrast, the ethanol concentration is not significantly declining in the sample during the in vitro-experiment. Nevertheless, this in-vitro test system enables us to compare the PLD activities in blood samples of different test persons. However, PEth formation is not only dependent on the
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substrate ethanol; it may also depend on the amounts of different individual concentrations of
further investigations seem to be necessary.
PLD inhibition experiments
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the PC homologues in human blood, which could be affected by nutrition [36]. Therefore,
Halopemide and its homologue FIPI have been described to be potent inhibitors of mammalian PLD in several experiments [10, 13, 29, 32].
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In the preliminary experiments with PC 16:0/18:1 dissolved in diethyl ether and PLD from cabbage, halopemide did not prevent formation of PEth at any of the applied concentrations. Halopemide might not be able to inhibit the PLD obtained from plants (cabbage) which we used in this preliminary experiment. In previous studies, experiments were performed with mammalian PLD and some variants of bacterial PLD only, which all share a conserved HKD
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amino acid domain (histidine, lysine, aspartate) that is thought to be the catalytic site [37]. In our experiments with human blood, in other words with human PLD, the formation of PEth analogues could be partially inhibited by halopemide and FIPI, but with a much lower potency than previously described. A complete inhibition of PEth formation could only be
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achieved by FIPI at a concentration of 30000 nM. Su et al. [32] performed a pre-incubation of the halopemide analogue FIPI prior to addition of the primary alcohol to start the reaction.
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They found that 15 min was sufficient to achieve a complete inhibition of PLD. In our experiment however, 30 min pre-incubation of halopemide with PLD did not change the results, either.
Although the PLD inhibitors halopemide and FIPI do inhibit PEth formation in human blood samples, due to their low potency they are not useful to guarantee stability in human blood samples. It would not be feasible for clinical use as well as from an economical point of view (due to high costs of halopemide and FIPI) to add these substances at the high concentrations needed for a measurable effect and to avoid formation of PEth in blood samples which contain ethanol.
15
ACCEPTED MANUSCRIPT Possible reasons for the low potency of halopemide and FIPI in our experimental setting are that PEth could also be formed in alternative pathways, or the PLD in human blood is not targeted as well as in other tissues by halopemide and/or FIPI. So far the easiest and the most robust way for PEth determination in blood is the analysis of PEth from dried blood spots (DBS) as PEth analysis in DBS in comparison to analysis of
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PEth in whole blood shows no post-sampling formation of PEth [38]. By drying the blood on a filter paper, the PLD activity is inhibited and no post-sampling formation takes place; and DBS sampling inhibits PEth degradation as well.
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Conclusion
In the in-vivo drinking study we found inter-individual differences in PEth formation rates.
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In-vitro experiments with freshly sampled blood revealed that the formation rate of PEth in blood is dependent on BAC. The in-vitro PEth formation during the first 7 h in the presence of alcohol proceeds in a zero order kinetic, leading to a standardized test to determine the individual PEth formation rates in human blood specimens via linear regression. Although we found high formation rates of PEth in blood, which might be responsible for a high amount of PEth production in the human body, we do not have knowledge about PEth
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formation in liver or other organs, which are mainly responsible for metabolism of xenobiotics.
With the application of the PLD inhibitors halopemide and FIPI, there was inhibition of PEth formation, but only at rather high inhibitor concentrations. However, if PEth is used as
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solved.
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alcohol marker in routine analysis the problem with post-sampling PLD activity needs to be
Funding
This study was supported by the Swiss Foundation of Alcohol Research (Grant 254/2014: Studies on phosphatidylethanol – a promising biomarker for the detection of harmful ethanol consumption – and its possible use for abstinence monitoring).
Ethical Approval This study has been approved by the Cantonal Ethics Commission Bern (064/13) on December 10, 2015.
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Highlights
The formation rate of PEth in blood is dependent on blood alcohol concentration.
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Measurement of individual PEth formation rates in fresh blood samples is standardized.
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5-fluoro-2-indolyl-deschlorohalopemide is a more effective inhibitor than halopemide.
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