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Article
Anders Isaksson, et al. High-Throughput LC-MS/MS Method for Determination of the Alcohol Use Biomarker
Phosphatidylethanol in Clinical Samples by Use of a Simple Automated Extraction Procedure—Preanalytical and Analytical Conditions. J Appl Lab Med 2017;2:880-92.
Guest
Dr. Anders Isaksson is assistant professor in the Department of Laboratory
Medicine in the Division of Clinical Chemistry at Lund University in Lund, Sweden.
Transcript
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Randye Kaye:
Hello, and welcome to this edition of “JALM Talk” from The Journal of Applied Laboratory Medicine, a publication of the American Association for Clinical Chemistry. I’m your host, Randye Kaye.
Alcohol use disorders are a major health problem worldwide
and alcohol biomarkers are an important tool for diagnosis,
follow-up, and treatment. Recent alcohol use can be
evaluated by measuring ethanol in serum, urine, or breath
or through the analysis of urine ethyl glucuronide or ethyl
sulfate. More long-term use has conventionally been
evaluated through measurement of carbohydrate deficient
transferrin or gamma glutamyl transferase. Recently,
phosphatidylethanol has emerged as the more sensitive and
specific marker of alcohol consumption, but a high capacity,
reliable, and cost effective method was lacking.
A high throughput LC-MS/MS method for the determination
of phosphatidylethanol (PEth) in clinical samples using a
simple automated extraction procedure was published in the
May 2018 issue of The Journal of Applied Laboratory
Medicine. This work describes an improved automated
approach to analysis of the alcohol marker
phosphatidylethanol that uses liquid chromatography mass
spectrometry instead of conventional HPLC.
The first author is Dr. Anders Isaksson. Dr. Isaksson is
assistant professor in the Department of Laboratory
Medicine in the Division of Clinical Chemistry at Lund
University in Lund, Sweden. He is our guest for today's
podcast. Welcome, Dr. Isaksson. What is
phosphatidylethanol (PEth) and how is it formed?
Anders Isaksson:
Well, phosphatidylethanol, in short PEth, is an abnormal
phospholipid only formed in the presence of ethanol. This
molecule was discovered in 1983 by Professor Christer
Alling, a former colleague of mine. He and his coworkers
were investigating the effects of ethanol on the composition
and concentration of phospholipids in different tissues and
to do that, they fed rats with ethanol. On a thin layer chromatography, they found an extra spot in lipid extract
formed tissues of ethanol-fed rats.
Later, they showed that this molecule was PEth.
Phosphatidylcholine is a precursor of PEth and that is
formed from its precursor through the action of the enzyme
phospholipase D. Phosphatidylcholine is an important
component of the cell membrane, so this conversion of the
phosphatidylcholine to PEth takes place within the cell
membrane.
Randye Kaye:
I see, thank you, and why is PEth a good alcohol marker?
Anders Isaksson:
Well because for one reason is that it can be measured in
blood as PEth is incorporated in the cell membrane of the
red blood cell. Interestingly, this applies only to humans.
No PEth has been found in erythrocytes from other
investigated species. Now, the reason is that it has high
clinical sensitivity and specificity since it is an alcohol
metabolite, PEth correlates with the alcohol consumption
level. It has a long window of detection, approximately the
same as carbohydrate-deficient transferrin, with a short
name CDT, which is a commonly used alcohol marker. The
long window of detection is because the half-life of PEth in
circulation is quite long, about one week.
Randye Kaye:
Thank you. Now, can you give me a short description, can
you describe your method for determination of PEth?
Anders Isaksson:
It’s simple. Blood is drawn in a tube with EDTA as
anticoagulant. The sample workup is largely robotized and
consists of protein precipitation with isopropanol. Off the
centrifugation, the supernatant is injected on to the column,
which is C18 reverse phase column. Therapist then
quantified where a liquid chromatographic-tandem mass
spectrometry method we use to direct in to this tandem.
The method has the high capacity and this is due to its short
turnaround time where a new sample is injected every 30
minutes. That, similar to its precursor, phosphatidylcholine,
contains two fatty acids. These can vary which means that
there are a large number of different molecular forms of
PEth. We determined a form that contains one palmitic and
one folic acid residue as it is usually found in the highest
concentration.
Randye Kaye:
So, how long has PEth has been used as an alcohol marker
and has the number of requests changed over time?
Anders Isaksson:
Our laboratory started with PEth as a clinical alcohol marker
in the beginning of 2006. It was however not until 2010
that other laboratories in Sweden began to perform PEth
analysis and now there are a lot of laboratories doing it. I think that PEth has also begun to be used in some other
Nordic countries and perhaps in some other countries in
continental Europe. The number of requests for PEth
analysis at our laboratory has increased almost
exponentially from 1,300 for the first year to over 60,000
last year. For comparison, we perform approximately four
times as many PEth analysis as CDT analysis.
Randye Kaye:
Right, very interesting, so my final question to you, doctor,
is how stable is PEth during storage or transport and are
there any other preanalytic factors to take into account?
Anders Isaksson:
It is stable for a week at room temperature and for at least
three weeks in refrigerator. At minus 80 degrees Celsius, it
is stable for at least two years. However, the PEth can be
raised in vitro in samples containing ethanol. This
information is low and clinically insignificant in EDTA
samples of the storage at the room temperature for up to
two days and that is even in the presence of high ethanol
concentrations.
PEth can also be formed in vitro below zero so samples
should not be stored in a regular freezer at minus 20
degrees Celsius. For long-term storage, PEth should be
stored at minus 80 degrees Celsius. At this temperature,
PEth is stable and there is no in vitro formation. Another
option would be to use dry blood spots as sample material.
Under this condition, PEth is stable up to a month at room
temperature and no formation of PEth can occur under these
conditions.
Randye Kaye:
Thank you very, very much. That was very interesting.
Thank you for joining me today.
Anders Isaksson:
Thank you for the opportunity.
Randye Kaye:
That was Dr. Anders Isaksson, assistant professor in the
Department of Laboratory Medicine at Lund University,
talking about a high throughput LC-MS/MS method for the
determination of phosphatidylethanol PEth in clinical
samples using a simple automated extraction from the May
2018 issue of JALM.
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