Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
  • A61B10/0038—Devices for taking faeces samples; Faecal examination devices
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/22—Devices for withdrawing samples in the gaseous state
  • G01N1/2226—Sampling from a closed space, e.g. food package, head space
  • G01N2001/2229—Headspace sampling, i.e. vapour over liquid
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/04—Preparation or injection of sample to be analysed
  • G01N30/06—Preparation
  • G01N30/12—Preparation by evaporation
  • G01N2030/126—Preparation by evaporation evaporating sample
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
  • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
  • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/06—Gastro-intestinal diseases

Definitions

• the present invention relates to a volatile short-chain fatty acid (AGCCV) assay method from a human biological sample, preferably human stool.
• ATCV volatile short-chain fatty acid
• AGCCVs can group C1 to C6 monocarboxylic acids. They constitute the bulk of organic anions present in the colon and are absorbed by the colonic mucosa. Butyrate, the conjugated base of butyric acid (C4), is the main energy source of colonocytes located on the wall of the transverse colon.
• Colonocytes are the epithelial cells of the colon and actually absorb this source of butyrate by a chemical reaction of oxidation of butyrate. For this reason, butyrate is the main contributor of energy to colonocytes.
• the metabolism of butyrate by colonocytes takes place in the mitochondrial compartment corresponding to an energy factory where reactions are essential for the proper functioning of the body.
• the butyrate is therefore degraded by the route commonly known as ⁇ -oxidation which produces butyryl-CoA which will then join the last steps of the aforesaid route in order to finally produce acetyl-CoA.
• the latter is used in the Krebs cycle or in the ketogenic pathway.
• the Krebs cycle provides energy in the form of ATP to cells throughout the body.
• the ketogenic pathway also consists of using acetyl-CoA. This pathway occurs when the fatty acids can not be converted to citrate and thus can not enter the Krebs cycle but will be used as a substrate in the ketogenic pathway.
• the ketone body formation is also an energy supply that can be used throughout the body. The energy intake then used by the cells of the whole body depends strongly on this source of butyrate.
• Butyrate may also be involved in the pathology of Crohn's syndrome. Indeed, it modulates the expression of HLA-DR and HLA class I molecules on colonic epithelial cells which makes it possible to increase antigens contained in the colonic lumen.
• butyrate has been observed to inhibit the proliferation of tumor cells, to act as a differentiating agent and to modulate anti-tumor immunity.
• butyrate may have a prophylactic or therapeutic effect, particularly as an antidiarrheal, in the treatment of colitis diversion, ulcerative colitis, pouchitis, proctitis radicalitis.
• AGCCVs act as signaling factors in various known metabolic pathways, for example, the regulation of cholesterol and glucose synthesis.
• Deficiencies in AGCCV have been observed in colorectal cancer, obesity, allergies or type II diabetes.
• Garner's article "volatile organic compounds from feces and their potential diagnosis of gastrointestinal disease" (vol.21, June 2007 - The FASEB Journal) aims to identify volatile organic compounds in a human stool sample. .
• the author describes the use of two coupled systems each comprising a gas chromatography device coupled to a mass spectrometer.
• This technique requires the use of a solid phase micro-extraction fiber using polydimethylsiloxane / carboxene, which makes the use of such a system expensive.
• this method is impractical because it is necessary to leave the fiber in the container for a relatively long time for the volatile organic compounds to adsorb to the fiber. It is then necessary to desorb them in order to dose them, which makes this type of analysis more complex.
• the current assay methods require the use of several pre-treatments of the sample whose objective is to avoid as much as possible the presence of contaminants that disturb the measurement or foul the device used.
• the accumulation of residues in a metering device is a real limitation for the user who is forced to stop dosing in order to replace the defective part or to clean the dirty or clogged part. This is not acceptable when the device must assay a series of continuous human samples.
• the aim of the invention is to overcome the drawbacks of the state of the art by providing a simpler and more economical method of assay which makes it possible to selectively, efficiently and reproducibly and reliably separate the AGCCVs present in a biological sample, in particular of human saddle.
• a human biological sample in particular of human stool, comprising volatile short-chain fatty acids and contaminants constituting the biological sample, adding to said sample an internal standard in a predetermined quantity with formation of a mixture,
• the assay method provided makes it possible to effectively separate the AGCCV from the contaminants originating from the starting human biological sample, which contributes to facilitating the assay of each AGCCV.
• the assay method proposed in the present invention requires the formation of a homogeneous mixture which contains, preferably in aqueous medium, both the AGCCV of interest and the constitutive contaminants of the biological sample as well as the internal standard.
• the mixture thus obtained is ready to be heated to separate the AGCCV from the interests of said homogeneous mixture.
• this homogeneous mixture can already be used to separate mainly AGCCV said homogeneous mixture by the application of the heating step.
• the homogeneous mixture is contained in a hermetically sealed container which is heated to a predetermined temperature, which makes it possible to volatilize mainly the AGCCVs of interest in the gas phase in a headspace of the container.
• Two phases are thus formed, a solid-liquid phase containing contaminants constituting the human biological sample and optionally solid residues and a gas phase comprising the volatilized AGCCVs as well as the internal standard in a predetermined amount.
• non-volatile species remain in the bottom of the container which allows to isolate AGCCV interests of said contaminants constituting the biological sample.
• the method according to the invention is made easy because it provides a homogeneous mixture whose AGCCV can easily be separated from the constituent contaminants of the human biological sample.
• the method proposed according to the invention is also selective in that it makes it possible to considerably reduce the presence of non-volatile species or at best to eliminate said non-volatile species.
• pressurization is performed in the head space of the container in order to create an overpressure.
• the pressurization step generates a pressure in the headspace of the container which is between 2 and 10 psi, preferably 3 and 6 psi, more preferably equal to 5 psi. This applied pressure is greater than that of the injector of the gas chromatograph.
• This pressurization is carried out simultaneously or in a delayed manner with the aid of a mobile phase comprising an inert gas, preferably helium.
• the injection step after the heating step (thermostatization), consists of a transfer step that takes place by stopping the application of the overpressure in the head space of the container and the opening a valve at the injection system which allows the vapor phase of the head space to be driven to the injector of the gas chromatograph, said overpressure being greater than the pressure of the chromatographic column of the gas chromatography device.
• This overpressure can be achieved for a short period of time which can be between 0.5 and 2 minutes, preferably equal to 1 minute.
• stopping the pressurization step during a period of time which may be between 0.05 and 0.2 minutes, preferably equal to 0.1 minutes, makes it possible to carry out the injection step.
• the method according to the invention therefore uses a gas chromatography device provided with a headspace for assaying the AGCCVs from a human biological sample.
• a gas chromatography device provided with a headspace for assaying the AGCCVs from a human biological sample.
• the coupling between a gas chromatography device and a "head space" type injector makes it possible to extract and inject a part of the gas phase comprising mainly and essentially the AGCCVs of interest to be assayed, as well as the standard internal.
• the chromatogram obtained makes it easy to assay the AGCCVs of interest by obtaining readable peaks which are sufficiently separated from each other.
• the process according to the invention makes it possible to reduce the presence of contaminants in the part of the homogeneous mixture to be assayed while not requiring a substantial pretreatment before assaying. Indeed, it is no longer necessary to condition the sample before the assay or to carry out several pretreatments in order to assay the AGCCV of interest, which makes the process simple, practical, effective, attractive and inexpensive. The formation of this mixture no longer requires having to separate the AGCCV from each other beforehand.
• the process according to the invention thus proposes a method which involves particularly chromatographic conditions. suitable for assaying AGCCVs from a human biological sample, preferably human saddle.
• the process according to the invention comprises a step of adding to said biological sample a saturated saline solution to form said mixture.
• said saline solution is sodium hydrogen sulfate.
• said step of heating said at least a portion of said homogeneous mixture is carried out at a temperature of between 50 and 110 ° C, preferably between 70 and 95 ° C.
• said heating step is carried out in an oven, preferably an oven of a chromatography device.
• said heating step is carried out for a period of time of between 15-30 minutes, preferably 15-25 minutes.
• said heating step is carried out until said homogeneous mixture reaches a temperature between 50 and 110 ° C, preferably between 70 and 95 ° C, more preferably still between 75 and 86 ° C to achieve said volatilization.
• said internal standard is a solution of 2-ethyl-butyric acid.
• the injection step is carried out at a first temperature of between 50 and 70 ° C., preferably between 55 and 65 ° C.
• the injection step is advantageously carried out using a needle or a syringe provided for this purpose.
• said injection step is carried out by gradually increasing said first temperature between 50 and 70 ° C, preferably between 55 and 65 ° C to a second temperature between 170-210 ° C, preferably between 180 and 170 ° C. - 195 ° C.
• said gradual increase in temperature is carried out in a space of time between 5 and 25 minutes, preferably between 10 and 20 minutes, more preferably between 10 and 17 minutes.
• said column has a length of between 20 and 40 m, preferably between 25 and 35 m.
• said capillary column has a diameter of between 0.2-0.5 mm, preferably equal to 0.25 mm.
• said capillary column has a film thickness of between 0.1-0.35 ⁇ m, preferably ranging from
• the process according to the invention comprises the determination of short chain fatty acids which include the monocarboxylic acids chosen from the group consisting of lactic acid, pyruvic acid, capric acid, caprylic acid, caprylic acid, levulinic acid, lauric acid, acetic acid, propionic acid, isobutyric acid, n-butyric acid, iso-valeric acid and n-valeric acid.
• monocarboxylic acids chosen from the group consisting of lactic acid, pyruvic acid, capric acid, caprylic acid, caprylic acid, levulinic acid, lauric acid, acetic acid, propionic acid, isobutyric acid, n-butyric acid, iso-valeric acid and n-valeric acid.
• the subject of the invention is also a use of a headspace gas chromatograph device for assaying volatile short-chain fatty acids in the gaseous state and coming from a human biological sample, in particular from human saddle .
• a human biological sample preferably of human saddle, which comprises AGCCVs such as acetic acid, propionic acid, isobutyric acid, n-butyric acid iso-valeric acid and n-valeric acid, as well as constitutive contaminants of the biological sample is mixed with an internal standard. It will be preferentially these AGCCVs which will be determined in the context of the present patent application.
• This internal standard exhibits physicochemical behaviors similar to those of the AGCCVs to be assayed, which enables a reliable and comparable measurement to be made. Indeed, the disturbances experienced by the AGCCV will be also for the internal standard. Thus, each peak obtained on the chromatogram can be compared to the peak corresponding to the internal standard which will have undergone the same disturbances during the assay.
• the assay of the AGCCVs from the human biological sample can be made easy by adding to said starting human biological sample also containing the internal standard a saturated saline solution to form a mixture whose AGCCV will pass more easily into phase gas during the heating step.
• the mixture When the mixture is obtained, it is homogenized so as to obtain a homogeneous mixture. Homogenization can be obtained by vortexing the mixture.
• the homogeneous mixture is introduced into a hermetically sealed container so as to create a headspace.
• headspace should be understood as the space created between the top of the container tightly closed and the surface of the mixture. This space can thus be loaded in analts during a gas evolution.
• This volatilization mainly targets AGCVV, in particular lactic acid, pyruvic acid, capric acid, caproic acid, caprylic acid, levulinic acid, lauric acid, acetic acid, propionic acid, iso-butyric acid, n-butyric acid, iso-valeric acid and n-valeric acid.
• the heating step makes it possible to pass mainly the AGCCVs, species of interest, in the gas phase, which has the effect of separating them in a reliable and reproducible manner from said constitutive contaminants of the human biological sample.
• the AGCCVs of interest are preferably chosen from the group consisting of acetic acid, propionic acid, isobutyric acid, n-butyric acid, iso-valeric acid and n-valeric acid.
• the AGCCS of interest to be determined in the context of the present invention are the following: acetic acid, propionic acid, iso-butyric acid, n-butyric acid, iso-valeric acid and n-valeric acid.
• the aforementioned heating step may be carried out in an oven having a temperature of between 50 and 110 ° C, preferably 70 and 95 ° C. It has been found that it is necessary to heat the mixture until the latter reaches a temperature of between 50 and 110.degree. C., preferably between 70 and 95.degree. C., more preferably between 75 and 86.degree. to obtain the volatilization of AGCCV in the head space of the container.
• the process according to the invention makes it possible to assay AGCCVs by using a gas chromatography technique coupled to a headspace.
• a gas chromatography device comprises a carrier gas forming the mobile phase, an injector, a furnace, a chromatographic column with a stationary phase lining the wall of the column, a detector, preferably an ionization detector. flame, and a data processing device.
• the gas chromatography device is advantageously coupled to a flame ionization detector.
• the principle of the chromatography being to be able to separate several analytes according to their affinity for the stationary phase. Each analyte will have a retention time of its own.
• the principle is to add during the analysis an internal standard in the sample.
• This standard exhibits physicochemical behavior similar to the analytes to be assayed. In this way, all the perturbations observed for the internal standard will be also for the analytes of interest which makes it possible to obtain a reliable measurement.
• this internal standard is added in a known quantity to said sample to be assayed and thus makes it possible to correct the concentration calculated for the different AGCCVs of interest.
• This correction makes it possible to cancel variations related to the injection volume of the sample in the chromatographic column and variations related to the extraction step (vortex).
• Such a device ultimately makes it possible to obtain a chromatogram which comprises several peaks where each peak corresponds to a response of a particular analyte, in this case each AGCCV.
• the analysis consists in measuring the area under the peak or the height of the peak so as to compare this datum with a calibration line.
• a headspace gas chromatograph is used.
• This device comprises the aforementioned elements but is characterized by a specific injection system in that it provides for recovering the mixture containing the analytes to be dosed in a headspace of a hermetically sealed container.
• a specific injection system in that it provides for recovering the mixture containing the analytes to be dosed in a headspace of a hermetically sealed container.
• heating to a predetermined temperature makes it possible to obtain volatilization of the AGCCVs in the gas phase, in this case in the headspace.
• the constituent contaminants of the biological sample being heavier than the AGCCV, they are maintained in the container at the bottom of the mixture with the non-volatile species.
• the injection consists in taking, with the aid of a needle, part of this gas phase loaded with AGCCV and injecting into the chromatographic capillary column in order to separate each AGCCV between them for the purpose of dosing them.
• the container containing the homogeneous mixture which contains the sample, the saline solution and the internal standard is placed on a turntable which has a thermostatically controlled oven which is arranged to support between 1 and 110 containers.
• the mixture can thus be heated to a temperature between 50 and 110 ° C, preferably between 70 and 95 ° C, more preferably between 75 and 86 ° C.
• This heating step (temperature control) of the mixture can last between 10 and 30 minutes, preferably for 20 minutes.
• a mechanical arm holds the container to be dosed and a syringe pierces the top of the container and enters the gaseous phase located in the head space thereof.
• the introduction of the syringe into the head space makes it possible to inject a mobile phase, preferably helium, and to create a pressurization in the container.
• This pressurization makes it possible to create an overpressure in the head space which is greater than that of the injector of the chromatographic column.
• Pressurization creates a pressure in the container of between 2 and 10 psi, preferably 3 and 6 psi, more preferably 5 psi, or 34473.8 Pa, which is higher than the pressure of the chromatograph injector. in the gas phase.
• the pressurization time is between 0.5 and 2 minutes, more preferably for a minute.
• the pressurization is stopped for a short period which can be between 0.05 and 0.2 minute, preferably equal to 0.1 minute.
• the AGCCVs present in the gaseous phase of the container and the mobile phase are transferred by means of a valve system to a line of transfer which precedes the injector of the chromatographic column.
• This transfer line consists of a column which has an internal diameter similar or identical to that of the chromatographic column and comprises an inert stationary phase, unlike the chromatographic column.
• the transfer line has a higher temperature than the mixture present in the container.
• the temperature at the transfer line is in the range of 100 to 180 ° C, preferably 120 to 145 ° C, or 135 ° C.
• the mobile phase and the AGCCV contained in the gas phase are introduced into the chromatographic capillary column to perform the assay.
• the transfer line and the chromatographic column are connected by a piece called "zero dilution liner".
• Headspace chromatography is known as the PerkinElmer brand TurboMatrix or the agilent brand "Headspace” of Agitent Technologies.
• the chromatographic capillary column may be an Elite-FFAP capillary column (0.25 mm ⁇ 30 ⁇ m ⁇ 0.25 ⁇ m).
• the oven has a temperature between 60 and 100 ° C.
• the transfer temperature is preferably greater than the oven temperature in order to avoid premature condensation of the analytes to be assayed.
• the oven temperature is in the range of 100 to 180 ° C, preferably 120 to 145 ° C, or 135 ° C.
• the needle used for injecting the gas phase into the capillary column has a temperature of between 80 and 120 ° C., preferably equal to 105 ° C.
• the duration of the analysis is between 10 and 30 minutes, preferably equal to 20 minutes.
• the method according to the invention thus makes it possible to assay AGCCVs in a biological sample, in particular of human saddle.
• 0.25 g of a biological sample of human saddle comprising acetic acid, propionic acid, iso-butyric acid, n-butyric acid, iso-valeric acid and n-valeric acid and constitutive contaminants from the human stool sample are mixed with 2.0 ml of a 62% sodium hydrogen sulfate solution and with 20 ⁇ of an aqueous solution of 2-ethyl-butyric acid (0.1 mol / l), internal standard.
• the mixture obtained is then vortexed for 10 minutes at 2500 rpm until a homogenized mixture is obtained.
• the homogenized blend is then transferred to a sealed bottle to create a head space above the mixture.
• This volatilization makes it possible to pass the analytes of interest, in this case acetic acid, propionic acid, iso-butyric acid, n-butyric acid, iso-valeric acid and acid.
• n-valeric gas phase in the head space of the hermetically sealed container.
• Example 2 The homogenized mixture as described in Example 1 is obtained and introduced into a furnace of a headspace gas chromatograph.
• the oven has a temperature of 85 ° C.
• the needle of the chromatography device then extracts a portion of the gas phase containing the AGCCVs and injects it into a capillary chromatography column.
• the needle has a temperature of 105 ° C.
• the temperature is 135 ° C.
• the chromatographic capillary column is preferably an Elite-FFAP brand capillary column (0.15 mm ⁇ 30 ⁇ m, 0.25 ⁇ m).
• each AGCCV having a certain affinity with the stationary phase will leave the capillary column with a retention time of its own.
• a detector preferably a flame ionization detector, which makes it possible to detect the analytes at the outlet of the capillary column.
• a quantitative and qualitative analysis can then be performed to assay each AGCCV present in the starting human stool sample,

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• Physics & Mathematics (AREA)
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• Analytical Chemistry (AREA)
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• General Health & Medical Sciences (AREA)
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• Immunology (AREA)
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• Investigating Or Analysing Biological Materials (AREA)
• Sampling And Sample Adjustment (AREA)
• Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

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• 2015
  • 2015-09-21 BE BE2015/5584A patent/BE1023199B1/fr not_active IP Right Cessation
• 2016
  • 2016-09-21 WO PCT/EP2016/072413 patent/WO2017050819A1/fr active Application Filing
  • 2016-09-21 EP EP16775542.0A patent/EP3353541A1/de not_active Withdrawn
  • 2016-09-21 MA MA042927A patent/MA42927A/fr unknown

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