BE1023199B1 - METHOD FOR ASSAYING VOLATILE SHORT CELL FATTY ACIDS - Google Patents

Abstract

Assay method comprising: - supplying a sample comprising volatile short-chain fatty acids and contaminants constituting the biological sample, - addition to said sample of an internal standard in a predetermined quantity with formation of a mixture, - homogenization of said mixture until a homogeneous mixture is obtained, - heating of at least part of said homogeneous mixture in an airtight container with volatilization of said fatty acids in a head space of said container, - injection, after said heating step, d 'at least part of said gas phase in a capillary column, and - measuring the quantity of said fatty acids and comparing said measured quantities of said fatty acids with predetermined values of quantities of corresponding fatty acids, and further comprising a step d adding to said biological sample a saturated saline solution to form said mixture.

Description

"METHOD FOR ASSAYING FATTY ACIDS WITH SHORT VOLATILE CHAINS"

The present invention relates to a volatile short-chain fatty acid (AGCCV) assay method from a human biological sample, preferably human stool. Generally, 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 β-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.

One of the beneficial effects of butyrate has been found to be the prevention and inhibition of colorectal cancer.

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.

In addition, butyrate has been observed to inhibit the proliferation of tumor cells, to act as a differentiating agent and to modulate anti-tumor immunity.

In addition, butyrate may have a prophylactic or therapeutic effect, particularly as an antidiarrheal, in the treatment of colitis diversion, ulcerative colitis, pouchitis, proctitis radicalitis. To date, the mechanism of butyrate is not yet well controlled.

More generally, 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.

It is therefore advantageous to be able to identify and assay the AGCCVs in order to be able to highlight a deficit or a surplus in AGCCV, which could lead to contributing to the diagnosis of numerous pathologies.

In order to do this, it is thus necessary to be able to isolate and correctly dose the AGCCV of interest from a biological sample from the human body such as blood, urine or faeces.

There are currently several methods for isolating AGCCVs. Current methods may use a series of chromatographic columns to attempt to isolate AGCCVs from their medium. It is essential to use a first chromatographic column to first separate the AGCCV from each other. Unfortunately, this first chromatographic step causes the AGCCV of interest but also many biological contaminants from the biological sample.

Then, it is necessary to use a second chromatographic column to separate each fatty acid of interest contaminants present in the biological sample. This second separation requires the use of various types of organic solvents to filter and thus attempt to separate the AGCCV said starting biological sample.

Unfortunately, the current methods are not sufficiently selective and require the use of several columns which make the separation complex and expensive. In addition, it has been observed that the use of so many steps does not allow to isolate efficiently and reliably AGCCV contaminants present in the biological sample. Indeed, the addition of solvent to separate each AGCCV contaminants from the biological sample leads to obtaining a mixture that includes both the AGCCV and contaminants contaminating the mixture. In addition, the use of as many steps and solvents can lead to a loss of fatty acid during treatment which is to be avoided since this affects the accuracy of the assay and the purification efficiency. The use of organic solvents also renders these types of processes unattractive on the market.

In addition, 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.

There is a real need to provide an easy and reliable assay method that can reproducibly and efficiently isolate and assay AGCCVs from a human biological sample, particularly human stool, while facilitating the implementation. of the process. 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.

To solve this problem, there is provided according to the invention a method which comprises the following steps: - supply of a human biological sample, in particular of human saddle, comprising volatile short-chain fatty acids and constitutive contaminants of the biological sample, - addition to said sample of an internal standard in a predetermined quantity with formation of a mixture, - homogenization of said mixture until a homogeneous mixture is obtained, - heating of at least a portion of said homogeneous mixture in an airtight container with volatilization of said gas phase volatile short-chain fatty acids in a headspace of said container to separate them from said constituents of the biological sample, - injecting, after said heating step, at least a portion of said gaseous phase comprising said volatile short-chain fatty acids and said standard i in a capillary column of a gas chromatograph, - measuring the amount of said volatile short-chain fatty acids in said at least a portion of said homogeneous mixture and comparing said measured amounts of said short-chain fatty acids with values predetermined amounts of corresponding short-chain fatty acids for assaying said short-chain fatty acids, characterized in that it further comprises a step of adding to said biological sample a saturated saline solution to form said mixture.

In the context of the present invention, it has been found that 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 of interest from said homogeneous mixture.

For this reason, the method according to the invention is made easy since it does not require additional and complex steps of pretreatments. Indeed, this homogeneous mixture can already be used to separate mainly AGCCV said homogeneous mixture by the application of the heating step.

Thus, 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.

Thus, the non-volatile species remain in the bottom of the container which allows to isolate AGCCV interests of said contaminants constituting the biological sample.

In this way, the passage of AGCCVs in the headspace in the gas phase in the hermetically sealed container makes it possible to isolate them efficiently and in a reproducible manner from the contaminants constituting the biological sample which are not or very little entrained in the phase. gas.

Thus, the process according to the invention is made easy because it provides a homogeneous mixture whose AGCCV can easily be separated from the constitutive contaminants of the human biological sample. The proposed method according to the invention is also selective in that it considerably reduces the presence of non-volatile species or contaminants in the gas phase obtained after the heating step.

When the gaseous phase is loaded with AGCCV and internal standard, part of this gaseous phase is removed and injected, for example by means of a syringe, directly into a chromatographic capillary column in order to separate each AGCCV according to their affinity. for the mobile phase.

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. 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.

Thus, the chromatogram obtained makes it easy to assay the AGCCV of interest by obtaining readable peaks which are sufficiently separated from each other.

It has thus been found that 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.

It has also surprisingly been found that the combination of the passage of mainly AGCCVs in the headspace of the hermetically sealed container and the capillary column enables the AGCCVs to be efficiently and quickly, reliably and reproducibly isolated and dosed. of a human biological sample by considerably reducing the risk of clogging of the capillary column following the unwanted passage of residual solid or non-volatile species inside thereof.

The method according to the invention thus proposes a method which involves chromatographic conditions particularly suitable for the assay of AGCCVs from a human biological sample, preferably of human saddle.

Advantageously, the process according to the invention comprises a step of adding to said biological sample a saturated saline solution to form said mixture. The addition of this saturated saline solution makes it possible to increase the volatility of the volatile species present in the homogeneous mixture, more particularly the AGCCVs.

Preferably, said saline solution is sodium hydrogen sulfate.

Preferably, 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.

Advantageously, said heating step is carried out in an oven, preferably an oven of a chromatography device.

According to a preferred embodiment, said heating step is carried out for a period of time of between 15-30 minutes, preferably 15-25 minutes.

Advantageously, 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.

Preferably, said internal standard is a solution of 2-ethyl-butyric acid.

Advantageously, 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.

Advantageously, 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.

Preferably, 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.

More preferably still, said column has a length of between 20 and 40 m, preferably between 25 and 35 m.

According to a preferred embodiment, said capillary column has a diameter of between 0.2-0.5 mm, preferably equal to 0.25 mm.

Advantageously, said capillary column has a film thickness of between 0.1-0.35 μm, preferably ranging from 0.20-0.25 μm.

Preferably, 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, iso-butyric acid, n-butyric acid, iso-valeric acid and n-valeric acid. Other embodiments of the process according to the invention are indicated in the appended claims. 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 . Other embodiments of the use according to the invention are indicated in the appended claims. Other features, details and advantages of the invention will become apparent from the description given below, without limitation and with reference to the accompanying drawings.

Thus, according to the method according to the present invention, 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.

This internal standard exhibits physicochemical behaviors similar to those of the AGCCVs to be assayed, which allows for a reliable and comparable measurement. Indeed, the disturbances experienced by the AGCCV will be also for the internal standard. Thus, each peak obtained on the chromatogram can be compared with 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.

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.

In the context of the present invention, the term "headspace" should be understood as the space created between the top of the sealed container and the surface of the mixture. This space can thus be loaded into analytes during a gas evolution.

Then, a heating step of this homogeneous mixture contained in the hermetically sealed container makes it possible to pass mainly the AGCCV in the head space and therefore in the gas phase.

This volatilization mainly targets AGCW, 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 risk of having impurities in the gaseous phase is considerably reduced and thus makes it possible to provide an efficient and inexpensive method by using a single and unique mixture without actually needing specific steps of pre-treatment of the sample. departure. 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 volatilization of AGCCVs.

The process according to the invention makes it possible to assay AGCCVs by using a gas chromatography technique coupled to a headspace.

In general, a gas chromatography device comprises a carrier gas forming the mobile phase, an injector, an oven, a chromatographic column, a detector, preferably a flame ionization detector, and a data processing device.

The principle of the chromatography being to be able to separate several analytes according to their affinity for the mobile phase. Each analyte will have a retention time of its own.

The principle is to add during the analysis an internal standard in the column. 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.

In addition, this internal standard is added in a known amount and thus makes it possible to measure by comparison the amount of each analyte.

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 height of the peak in order to compare this data with that of the internal standard.

Then, comparing these data with a calibration curve that includes predetermined values of AGCCV at different concentrations allows assay of each analyte.

In the context of the present invention, 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. As indicated above, 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 then consists in taking, with the aid of a needle, part of this AGCCV-loaded gas phase in the chromatographic capillary column in order to separate each AGCCV between them for the purpose of dosing them. The introduction of a part of the gas phase mainly loaded with AGCCV in the chromatographic capillary column makes it possible to selectively separate between each analyte to be assayed without risking contamination of the gas phase by non-volatile species or fouling of the gas phase. the capillary column, which would be detrimental to the dosage.

It has been found that the use of a capillary column makes it possible to facilitate the separation of AGCCV from interests in a reproducible manner.

Thus, it is possible to analyze a series of samples continuously without losing the quality of measurement and without risk of stopping the operation of the device to replace a plugged or fouled part.

Headspace chromatography is known under the trademark TurboMatrix from PerkinElmer or the Agilent "Headspace" brand from Agilent Technologies.

In the context of the present invention, the chromatographic capillary column may be an Elite-FFAP capillary column (0.25 mm × 30 × 0.25 μm).

Advantageously, 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.

Advantageously, 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.

Even more preferably, 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.

When a difference (deficit or excess of AGCCV) is found, it is possible to use the results obtained to highlight a pathology as explained in the preamble of the present invention. EXAMPLE 1 0.25 g of a biological sample of human saddle comprising acetic acid, propionic acid, isobutyric acid, n-butyric acid, iso-valeric acid and n-butyric acid. and constitutive contaminants of the human stool sample are mixed with 2.0 ml of 62% sodium hydrogen sulfate solution and with 30 μl of an aqueous solution of 2-ethyl-butyric acid (1.263 x10'3 mol / l), internal standard.

The mixture obtained is then vortexed for 10 minutes at 2500 rpm until a homogenized mixture is obtained.

The homogenized mixture is then transferred to a sealed bottle to create a head space above the mixture.

Heating the solution at a temperature of 85 ° C until the mixture reaches a temperature between 80 and 90 ° C to obtain a volatilization of AGCCV which will thus be separated from the constituent contaminants of the biological sample.

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. When transferring the gaseous phase, the temperature is 135 ° C.

The chromatographic capillary column is preferably an Elite-FFAP brand capillary column (0.25 μm, 0.25 μm).

In this way, each AGCCV having a certain affinity with the carrier gas will leave the capillary column with a retention time of its own.

At the end of the capillary column is a detector, preferably a flame ionization detector, which makes it possible to detect the analytes at the outlet of the capillary column.

Quantitative and qualitative analysis can then be performed to assay each AGCCV present in the starting human stool sample.

It is therefore necessary to compare the measured data with predetermined values of corresponding amount of AGCCV for assaying said AGCCVs.

It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made without departing from the scope of the appended claims.

Claims (14)

A method of assaying volatile short-chain fatty acids, said method comprising the steps of: providing a biological sample of human saddle, comprising volatile short-chain fatty acids and contaminants constituting the biological sample; adding to said sample an internal standard in a predetermined amount with the formation of a mixture, - homogenizing said mixture until a homogeneous mixture is obtained, - heating at least a portion of said homogeneous mixture in an airtight container with volatilization of said volatile gas phase short chain fatty acids in a headspace of said container for separating them from said contaminants constituting the biological sample, - injecting, after said heating step, at least a portion of said gaseous phase comprising said volatile short-chain fatty acids and said internal standard in a column capi a gas chromatograph device; - measuring the amount of said volatile short-chain fatty acids in said at least a portion of said homogeneous mixture and comparing said measured amounts of said short-chain fatty acids with predetermined values of amounts of said short-chain fatty acids; corresponding short-chain fatty acids for metering said short-chain fatty acids, and - comprising a step of adding to said biological sample a saturated saline solution to form said mixture, and - characterized in that it further comprises a step of adding to said biological sample a saturated saline solution to form said mixture. The method of claim 1, wherein said saline solution is a solution of sodium hydrogen sulphate. 3. A process according to any one of the preceding claims, wherein said step of heating said at least a portion of said homogeneous mixture is carried out at a temperature between 50 and 110 ° C, preferably between 70 and 95 ° C. 4. Method according to one of the preceding claims wherein said heating step is carried out for a period of time between 15-30 minutes, preferably 15-25 minutes. 5. Method according to one of the preceding claims, wherein 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 effect said volatilization. The process of any of the preceding claims, wherein said internal standard is a 2-ethyl-butyric acid solution. The method of claim 7, wherein said injecting step is performed at a first temperature of between 50 and 70 ° C, preferably between 55 and 65 ° C. The process according to claim 7, wherein said injecting step is carried out by progressively 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 180-195 ° C. 9. The method of claim 8, wherein said gradual increase in temperature is carried out in a period of time between 5 and 25 minutes, preferably between 10 and 20 minutes, more preferably between 10 and 17 minutes. The process according to any one of claims 6 to 8, wherein said capillary column has a length of between 20 and 40 m, preferably between 25 and 35 m. 11. A method according to any one of claims 6 to 9, wherein said capillary column has a diameter of between 0.2-0.5 mm, preferably equal to 0.25 mm. The method of claim 11, wherein said capillary column has a film thickness of 0.1-0.35 μm, preferably 0.20-0.25 μm. 13. A method according to one of the preceding claims, comprising the assay of short-chain fatty acids which include monocarboxylic acids selected from the group consisting of lactic acid, pyruvic acid, capric acid, lactic acid, and the like. caproic acid, caprylic acid, levulinic acid, lauric acid, acetic acid, propionic acid, isobutyric acid, n-butyric acid, iso-valeric acid and n-valeric acid. 14. Use of a headspace gas chromatograph device for assaying volatile short-chain fatty acids in the gaseous state from a human biological sample, in particular human saddle.