Patent Description:
Phosphatidylethanol is a substance that forms from cell membrane phospholipids during exposure to ethanol (i.e. alcohol drinking). Phospholipase D (PLD) is the enzyme responsible for the reaction and is caused by the feature of the enzyme to prefer ethanol as substrate over water when acting to form phosphatidic (PA) acid from phosphatidylcholines (PCs) as part of a regulatory system. When formed, PEth will be incorporated into the cell membrane and becomes accumulated over time. Repeated alcohol drinking will lead to elevated blood levels of PEth and it has been suggested as biomarker to detect riskful alcohol consumption from risk concluding limits. For this purpose, several assays have been suggested in for example <CIT>; <CIT>; <CIT> and <CIT>.

Sampled blood is an active biofluid that retains many biological activities after sampling. One such activity is the enzyme Phospholipase D (PLD), which is involved in regulation of the lipid content of the cell membrane. PLD has the unique feature to have activity also after cooling and freezing. Accordingly, PEth can be formed in vitro post sampling if ethanol is present in the blood at the time of sampling, which is not uncommon in real practice.

<NPL> discloses using dried blood spot (DBS) samples collected on filter paper by means of a volumetric DBS device for PEth measurement.

<NPL> discloses the development of standardized tests for measuring individual formation of PEth following alcohol consumption. The authors were aware of the problem with post-sampling formation of PEth and introduced the well-known PLD inhibitors halopemide and FIPI (<NUM>-fluoro-<NUM>-indolyl-deschlorohalopemide) to improve the quality of the PEth analysis in order to prevent in vitro formation of PEth in alcohol positive blood samples after blood sampling. However, halopemide and FIPI were considered unsuitable for clinical use due to low potency and high costs. For this reason, the problem with post-sampling PLD needs to be solved before feasible routine assays of PEth can be established.

<NPL> discloses assessing the alterations in serum urea and serum creatinine levels from a centrifuged mixture of serum, distilled water, NH2SO4 and a <NUM>% solution of sodium tungstate.

<NPL> describes a method for reducing ultra micro copper by using a method wherein sodium tungstate is used with copper sulphate to precipitate proteins from blood samples.

In addition, it is desirable to conform routine PEth assay to the economic and rapid screening technology admitted by DBS (Dried Blood Spot Sampling) where blood samples are conveniently collected and dried, as represented by the device disclosed by <CIT>. Although, it was estimated that dried blood spots may solve the problems with post-sampling formation of PEth, it was found that PLD remains active and that PEth forms also during the drying process and possibly also in the dried blood. There is an obvious need to find a solution to the possibility of post-sampling formation of PEth as it may lead to erroneous measurements and false interpretations.

The present invention therefore meets need of finding better strategies to develop a reliable, economic and convenient assay of PEth.

An object of the present invention is thus to provide a simple solution to a reliable, economical and convenient assay of PEth that avoids the problems with post-sampling formation of PEth.

In one aspect of the invention, there is provided a device configured for collection and subsequent testing for phosphatidylethanol (PEth) of a blood sample of less than <NUM>. The device is a test tube and includes at least one inhibitor of the enzyme phospholipase D (PLD) selected from at least one of a salt comprising a vanadium oxyanion and a salt comprising a tungsten oxyanion. The PLD inhibitor is immobilized in the test tube.

The inhibitor of the enzyme phospholipase D may be selected from at least one of NaVOs (sodium metavanadate) and Na<NUM>WO<NUM> (sodium tungstate).

The at least one salt of inhibitor of the enzyme phospholipase D may have a mass of less than <NUM>.

The at least one salt is present in amount that admits concentration of less than <NUM> in the blood sample.

The PLD inhibitor may be present in a gel attached to the walls of the test tube.

The walls of the test tube may be impregnated with the PLD inhibitor.

The walls of the test tube may have a coating, e.g. of less than <NUM> micrometers, comprising the at least one inhibitor of the enzyme phospholipase D.

The inner coating may be sprayed on the on the bottom and/or on the lid of the test tube.

In another aspect of the invention, a method is disclosed for preparing a sample for analysis of phosphatidylethanol (PEth) the method comprising adding a blood sample from a patient with a volume of less than <NUM> to a test tube (<NUM>), wherein the test tube includes at least one inhibitor of the enzyme phospholipase D (PLD) selected from at least one of a salt comprising a vanadium oxyanion and a salt comprising a tungsten oxyanion; contacting the blood sample with the PLD inhibotor; and admitting inhibition of the PLD in the blood sample with the at least one PLD inhibitor so formation of PEth is blocked.

The invention is now described, by way of example, with reference to the accompanying drawing, in which:
<FIG> shows a test tube device according to the invention.

In the following, a detailed description of embodiments of the invention is disclosed.

<FIG> shows a device <NUM> according to the present invention, comprising a blood collection test tube <NUM> that contains an PLD inhibitor enzyme. The blood collection test tube can be a conventional blood test tube made of glass or suitable polymers, such as Vacutainer®, specially labelled for PEth analysis. Similar to the blood collection test tubes with added components such as EDTA, heparin, etc. that are already known in the art, the test tube comprises an immobilized PLD inhibitor, as an added component.

In one alternative, the PLD inhibitor enzyme may be present in a gel attached to the walls of the test tube. Other means of immobilizing the PLD inhibitor enzyme are also contemplated.

It has been found that the salts of a transition metal belonging to column <NUM> or <NUM> of the periodic table, in particular, the salts of vanadium and tungsten are effective inhibitors of the enzyme phospholipase D. The PLD inhibitor is selected from at least one of a salt comprising a vanadium oxyanion and a salt comprising a tungsten oxyanion, preferably NaVOs (sodium metavanadate) or Na<NUM>WO<NUM> (sodium tungstate).

Other agents that may also be used as inhibitors of the enzyme D may include certain anti-cancer drugs, such as <NUM>-fluoro-<NUM>-indolyldes-chlorohalopemide (FIPI), or agents with certain ionic properties, such as phosphate analogues.

In further embodiments, the walls of the test tube may have been impregnated with a PLD inhibitor, or the walls of the test tube may have a coating comprising the PLD inhibitor. The PLD inhibiting agents can be included in thin film coatings of the container (see for example <CIT>) or added by other conventional ways to supply blood test tubes with additives.

An exemplary method of applying a coating of at least one inhibitor of the enzyme phospholipase D to a test tube is disclosed. The method comprises the steps of stabilizing the test in a substantially vertical position; inserting a spray nozzle inside the test tube, the spray nozzle being in fluid connection to a container holding a solution comprising at least one inhibitor of the enzyme phospholipase D; spraying the solution comprising at least one inhibitor of the enzyme phospholipase D inside the test tube; and allowing the solution comprising at least one inhibitor of the enzyme phospholipase D to dry.

In addition to the PLD inhibitor, the test tube may further include other conventional additives such as anticoagulants.

The test tube is used for blood collection. Approximately <NUM> -<NUM> of blood is added to the test tube. The test tube is then sealed/closed with a test tube cap and the now sealed test tub is gently rocked for approximately <NUM> minutes. Thereafter, the sealed test tube containing the blood sample is stored at either room temperature or +<NUM> pending further analysis.

The blood specimens used for the present investigation were deidentified surplus volumes of venous whole blood selected among those sent to the Department of Clinical Pharmacology, Karolinska University Laboratory (Stockholm) for routine analysis of PEth as an alcohol biomarker.

Additional blank specimens were collected from non-drinking healthy volunteers at the laboratory. The blood was collected in EDTA tubes and stored at <NUM> where PEth is reported to be stable for at least <NUM> weeks. Ethanol was not tested for. The procedures followed were approved by the ethics committee at the Karolinska University Hospital (No. <NUM>/<NUM>-<NUM>/<NUM>).

Routine analysis of PEth <NUM>:<NUM>/<NUM>:<NUM> in whole blood specimens was done essentially as previously described. <NUM>µL whole blood was mixed with <NUM>µL IS solution (PEth-d31; Avanti Polar Lipids, Alabaster, AL, USA), <NUM>µL acetonitrile, and <NUM>µL acetone. The mixture was gently shaken (<NUM> rpm) for <NUM> at room temperature and then centrifuged at <NUM> for <NUM>. The supernatant was transferred to a new vial and centrifuged again for another <NUM>. LC-MS/MS quantification of PEth <NUM>:<NUM>/<NUM>:<NUM> was done by comparison with a calibration curve covering <NUM>-<NUM>µmol/L prepared similarly in blank (i.e. PEth negative) blood specimens spiked with known amounts of PEth <NUM>:<NUM>/<NUM>:<NUM> (Avanti Polar Lipids) [<NUM>]. Two quality control samples (low and high PEth level) were prepared in the same way. The detection limit (LOD) and lower quantification limit (LLOQ) of the method were <NUM>µmol/L and <NUM>µmol/L, respectively.

For method evaluation and validation, DBS samples were prepared by pipetting <NUM>µL of the venous blood specimen onto the filter paper (Whatman <NUM> Protein Saver Card; GE Healthcare Ltd. , Cardiff, UK), which was then allowed to dry at room temperature for at least <NUM> hours in horizontal position with air on both sides. For the analysis, <NUM> filter paper punches (<NUM> diameter) were taken from each DBS and placed in a test tube and added with <NUM>µL IS solution and <NUM>µL methanol. The tube was covered and gently shaken (<NUM> rpm) for <NUM> hour, followed by centrifugation at <NUM> for <NUM>. The liquid phase was transferred to an autosampler vial and centrifuged for another <NUM>, prior to analysis. LC-MS/MS quantification of PEth <NUM>:<NUM>/<NUM>/<NUM> in the sample was done by comparison with a DBS calibration curve prepared as described for venous blood.

The value of volumetric DBS measurement of PEth was examined using a disposable prototype DBS device (provided by Capitainer AB, Stockholm, Sweden) [<NUM>] constructed with an inlet cavity for applying <NUM>-<NUM>µL (i.e. about one drop) of blood. After the application of blood, a capillary channel is automatically filled with <NUM>µL of the sample which is eventually emptied onto a Whatman <NUM> filter paper disc (<NUM> diameter). The paper disc is impregnated with, for example, <NUM> of salt. For this study, <NUM>µL venous whole blood was applied to the inlet cavity using a pipette.

After drying at room temperature for at least <NUM> hours, the volumetric filter disc was transferred to a glass test tube and extracted with <NUM>µL isopropanol containing IS (<NUM>µmol/L PEth-d5; Chiron AS, Trondheim, Norway). The test tube was gently shaken (<NUM> rpm) for <NUM> at room temperature and <NUM>µL of the liquid phase transferred to an autosampler vial. A <NUM>-µL aliquot was injected on the column and the PEth <NUM>:<NUM>/<NUM>:<NUM> concentration was measured by LC-MS/MS as described above. The standards were prepared by fortifying DBS spots from blank blood with reference PEth <NUM>:<NUM>/<NUM>:<NUM> in isopropanol. The method was calibrated between <NUM> and <NUM>µmol/L PEth <NUM>:<NUM>/<NUM>:<NUM>. Uncertainty in quantification was documented both for intra- and interassay imprecision and accuracy, using authentic blood quality controls with assigned PEth concentrations.

The results demonstrate that with the inhibitor present during drying additional artificial formation of PEth does not take place.

Claim 1:
A device (<NUM>) configured for collection and subsequent testing for phosphatidylethanol (PEth) of a blood sample of less than <NUM>, wherein the device is a test tube (<NUM>) and includes at least one inhibitor of the enzyme phospholipase D (PLD) selected from at least one of a salt comprising a vanadium oxyanion and a salt comprising a tungsten oxyanion, characterized in that the PLD inhibitor is immobilized in the test tube.