Non-invasive method for detecting respiratory gases comprising hydrogen sulfide

Abstract

The invention relates to a non-invasive method for detecting respiratory gases comprising hydrogen sulfide, in particular for use in the medicine, diagnosis, prediction, risk stratification, and therapy control for diseases, in particular intestinal diseases, of test subjects, wherein gases formed by bacteria are detected in the exhaled gas, and the hydrogen sulfide is not disadvantageously degraded.

Description

[0001] Non-invasive method for determining respiratory gases containing hydrogen sulfide

[0002] Description

[0003] The present invention relates to a non-invasive method for determining exhaled gases containing hydrogen sulfide, in particular for use in medicine, diagnosis, prediction, risk stratification and therapy control of diseases, especially intestinal diseases in test subjects, wherein gases formed by bacteria in the exhaled gas are determined, wherein the hydrogen sulfide is not adversely degraded.

[0004] In clinical practice, the determination of respiratory gases such as H2, CH4, CO2 or hydrogen sulfide (H2S) from exhalation samples is carried out either via gas chromatography or gas sensors. Corresponding tests are commercially available and described in the literature (Gasbarrini A, Corazza GR, Gasbarrini G, Montalto M, Di Stefano M, Basilisco G, Parodi A, Usai-Satta P, Vernia P, Anania C, Astegiano M, Barbara G, Benin! L, Bonazzi P, Capurso G, Certo M, Colecchia A, Cuoco L, Di Sario A, Fest! D, Lauritano C, Miceli E, Nardone G, Perri F, Portincasa P, Risicato R, Sorge M, Tursi A; 1st Rome H2-Breath Testing Consensus Conference Working Group. Methodology and indications of H2-breath testing in gastrointestinal diseases: the Rome Consensus Conference. Mar 30;29 Suppl 1:1-49.)

[0005] Breath gas analysis is a medical procedure in which the chemical composition of exhaled air is analyzed to obtain information about a person's health. This method can be used to diagnose diseases, monitor metabolic processes, particularly in sports medicine and geriatric medicine, analyze drug effects, and monitor therapy effectiveness.

[0006] The breath test is a non-invasive method and can be used, for example, to diagnose "Small Intestinal Bacterial Overgrowth" (SIBO) as follows:

[0007] Preparation: In the days leading up to the SIBO test, the subject is asked to avoid certain foods and medications that could affect the test result. These often include antibiotics, laxatives, prokinetics, and certain high-fiber foods.

[0008] Test substance: The subject receives a test substance, which is usually lactulose or glucose. These substances are primarily carbohydrates that are not normally digested in the small intestine but can be fermented by intestinal bacteria, provoking gas production.

[0009] Breath sample collection: The subject breathes into special breathing bags or tubes or sample containers that can collect respiratory gases.

[0010] Measurements / Determinations: After the test substance has been ingested, breath samples are taken at regular intervals, usually every 15-30 minutes over a period of several hours. These samples are then analyzed for their content of breath gases produced by intestinal bacteria as they ferment the test substance. The breath gases are detected, for example, by gas chromatography or mass spectrometry.

[0011] Interpretation of the results: An increase in respiratory gases indicates bacterial overgrowth in the small intestine. The patterns of exhaled gases can also help to differentiate between various types of SIBO. However, the success of the breath tests depends on the precise quantity and ratio of the respiratory gases.

[0012] Hydrogen sulfide (H₂S) is playing an increasingly important role in breath gas analysis, particularly in the diagnosis and investigation of gastrointestinal diseases; however, quantifying the analyte is challenging. H₂S is a gas produced by certain bacteria in the gut, especially during the breakdown of sulfur-containing amino acids. It can be detected in breath and provides valuable information about the state of the digestive tract and bacterial metabolism.

[0013] Surprisingly, it has now been found that hydrogen sulfide (H₂S), as a component of exhaled air, is unstable and degrades in the presence of atmospheric moisture. Consequently, the levels, quantity, concentration, or quantification of hydrogen sulfide can be distorted, especially if the exhaled air remains in a sample container after sampling.

[0014] Therefore, one object of the present invention is to provide a breath test so that test subjects obtain a reliable value of hydrogen sulfide and, particularly advantageously, the diagnostic value and determination are improved.

[0015] Therefore, the invention relates to a non-invasive method for determining breath gases containing hydrogen sulfide, wherein gases produced by bacteria, in particular intestinal bacteria, are determined in the exhaled gas of a subject, wherein i.) the moisture contained in the exhaled gas is removed by drying, and / or ii.) the hydrogen sulfide contained in the exhaled gas is passed through non-adsorbing inert materials and / or stored. Therefore, the invention relates to breath gases containing hydrogen sulfide for use a.) in medicine, in particular sports medicine or geriatric medicine, including the monitoring of metabolic processes, and / or b.) in the diagnosis or prediction or for risk stratification or therapy control of diseases in subjects, wherein gases produced by bacteria, in particular intestinal bacteria, are determined in the exhaled gas of a subject, wherein i.) the contained (d) e moisture of the exhaled gas is removed by drying, and / or ii.) the contained (d) e hydrogen sulfide of the exhaled gas is passed through non-adsorbing inert materials and / or stored.

[0016] Furthermore, in a further preferred embodiment, the invention relates to a aforementioned non-invasive method or use, wherein the drying is carried out by i.) cooling or by ii) a drying agent, in particular by means of calcium chloride.

[0017] Furthermore, in a further preferred embodiment, the invention relates to a non-invasive method or use, wherein drying is carried out by cooling using a cold trap. For example, the cold trap can be part of a sample tube or arranged in relation to the sample chamber, so that the humidity is extracted from the exhaled gas. This is usually done at temperatures below 0 degrees Celsius.

[0018] Furthermore, in a further preferred embodiment, the invention relates to non-adsorbing inert materials that do not adsorb hydrogen sulfide, thus preventing its removal from the breath gas sample. The non-adsorbing inert material can be a component of the sample container, particularly in the form of the (inner) wall of the sample container. Furthermore, conduits or tubes for guiding the breath gas, including the mouthpiece or receiving unit, can be made of such a non-adsorbing inert material. Such non-adsorbing inert materials are known to those skilled in the art.

[0019] Non-adsorbing inert materials are substances that do not interact significantly with other substances, especially not by adsorption, and are chemically unreactive.

[0020] "Inert" means that the material is chemically stable and does not readily combine with other substances.

[0021] "Non-adsorbing" means that the material does not hold particles from the environment to its surface.

[0022] In particular, silicon-containing materials can interact with hydrogen sulfide.

[0023] For example, ordinary glasses are not inert. They can release metal ions that can catalytically trigger degradation reactions of hydrogen sulfide. Similarly, surface defects can catalyze chemical reactions and serve as nucleation sites for crystallization. Siliconizing glasses or other materials can create the necessary inertness and prevent adsorption.

[0024] A silicone layer can be applied temporarily (film application) or permanently to a glass or other material surface by means of baked-on siliconization or coating processes such as chemical vapor deposition (CVD). The different manufacturing methods are known to those skilled in the art (Diss. T. Mundry, Humboldt University, Berlin, 1999).

[0025] The baked-on siliconization process typically yields a homogeneous, approximately 15–50 nm thin hydrophobic layer, covering the initial glass or other material surface and any existing defects (Reuter B., Petersen C., The siliconization of syringes, TechnoPharm2, No. 4, 238–244 (2012)). As a result, a reduced interaction between hydrogen sulfide and the siliconized material is achieved.

[0026] Suitable non-adsorbing inert materials also include hydrogenated amorphous silicon substrates (e.g. SilcoNert® 2000 (Bellefonte, USA) , as described in US 6,444,326 Bl), quartz glass, and coatings.

[0027] Furthermore, plastics with non-adsorbing properties can be used, such as polytetrafluoroethylene (PTFE), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), polyethylene terephthalate (PET), or polymethyl methacrylate (PMMA). Therefore, the invention also relates to the inventive method or use, wherein the hydrogen sulfide contained in the exhaled gas is passed through and / or stored in non-adsorbing inert materials.

[0028] The breathing gas can be guided through the mouthpiece or receiving unit into lines and tubes, either into the sample container, optionally with an integrated cooling trap, or into the detector. The breathing gas can be stored in a sample container. The sample container itself, particularly its wall, can be made of a non-adsorbing inert material. Furthermore, lines or tubes for guiding the breathing gas, including the mouthpiece or receiving unit, can also be made of such a non-adsorbing inert material. Such non-adsorbing inert materials are known to those skilled in the art.

[0029] Therefore, the invention also relates to the inventive method or use for determining respiratory gases containing hydrogen sulfide, wherein non-adsorbing inert materials are a component of the sample container, in particular in the form of the wall of the sample container and / or lines or tubes for guiding the respiratory gas, in particular into a detector, optionally including the mouthpiece or receiving unit, comprising such non-adsorbing inert materials.

[0030] A particular advantage is that the inventive method does not affect the content of other breathing gases. Consequently, a reliable calibration based on hydrogen sulfide can be carried out for the first time using the inventive method or application, preferably with the aid of a breathing gas containing hydrogen sulfide, which is obtained by the inventive method according to i.) and ii.).

[0031] In a further embodiment, the invention relates to a non-invasive method for determining respiratory gases containing hydrogen sulfide for use in medicine, in particular sports medicine or geriatric medicine, including monitoring of metabolic processes and / or diagnosis or prediction or for risk stratification or therapy control of diseases in test subjects.

[0032] For the purposes of this invention, diseases such as gastrointestinal diseases, in particular intestinal diseases, SIBO (Small Intestinal Bacterial Overgrowth), irritable bowel syndrome (IBS), lactose or fructose malabsorption, chronic inflammatory bowel diseases (IBD) (Guo FF, Yu TC, Hong J and Fang JY (2016) Emerging Roles of Hydrogen Sulfide in Inflammatory and Neoplastic Colonic Diseases. Front. Physiol. 7:156), in particular Crohn's disease and ulcerative colitis, celiac disease, or functional dyspepsia are included. In particular, intestinal diseases exhibiting malabsorption are included according to the invention. Furthermore, diseases selected from the group of cancers, in particular colon carcinoma (Du P, Tseng Y, Liu P, et al.), are included according to the invention.Role of exhaled hydrogen sulfide in the diagnosis of colorectal cancer, BMJ Open Gastroenterol 2024, 11), as well as cardiovascular diseases, in particular arteriosclerotic diseases (ASCVD), and skin diseases, in particular wound healing and psoriasis (Xiao Q, Xiong L, Tang J, Li L, Li L, Hydrogen Sulfide in Skin Diseases: A Novel Mediator and Therapeutic Target. Oxid Med Cell Longev. 2021 Apr 20; 2021: 6652086). Furthermore, the detection of halitosis (bad breath) within the meaning of this invention is also included. Other relevant diseases within the meaning of this invention are lung diseases and diseases of the respiratory tract, in particular asthma and COPD (Debraj Jash, Hydrogen sulphide as a biomarker in COPD and asthma, European Respiratory Journal 2014, 44), neurological diseases, in particular Alzheimer's and other forms of dementia.

[0033] The bacteria can, for example, influence the aforementioned diseases through infections or even be the cause of them. Furthermore, one or more of the aforementioned diseases may be affected.

[0034] The bacteria may be intestinal bacteria in particular, or they may be located at the site of colonization, such as the stomach, lungs, respiratory tract, etc.

[0035] Another object of the invention is also the therapy control by means of a prediction or diagnosis of diseases, in particular intestinal diseases, wherein a test subject is examined according to an inventive method or use, with the proviso that the test subject receives medication, in particular antibiotics / phytobiotics for the treatment of a disease, in particular intestinal disease, or adheres to a diet for the treatment of an intestinal disease, in particular for the treatment of malabsorption, so that a therapeutic success or improvement of therapy can take place.

[0036] "Diagnosis" or "prediction" within the meaning of this invention means the positive determination or prediction / prognosis or probability of the onset and occurrence of a disease, in particular an intestinal disease. The term diagnosis further includes medical diagnostics and related examinations, in particular in-vitro diagnostics and laboratory diagnostics.

[0037] The diagnosis according to the invention takes place outside (ex vivo) of the human body, since the respiratory gases, or rather the determination of the respiratory gases according to the invention, is carried out using gas chromatography or gas sensors. Furthermore, the presence of a physician is not required. Preferably, the exhaled gas of a test subject is provided in a sample container.

[0038] Furthermore, the invention relates to a method for stratification, in particular for risk stratification and / or therapy control of a patient.

[0039] “Stratification or therapy control” within the meaning of this invention means that the inventive method allows decisions regarding the treatment and therapy of the subject, be it hospitalization of the patient, use, effect and / or dosage of one or more drugs, a therapeutic measure or the monitoring of a disease course as well as the course of therapy or etiology or classification of diseases.

[0040] In another embodiment of the invention, the term “stratification” includes in particular risk stratification with the prediction of an “outcome” of an adverse health event, in particular that a disease is present.

[0041] Within the scope of this invention, "subject" is understood to mean any human being or mammal, in particular a patient. Patients who already exhibit symptoms such as bloating, abdominal pain, diarrhea, constipation, nutrient deficiency, and weight loss due to malabsorption are particularly preferred.

[0042] Furthermore, the invention relates to the use of a device for determining respiratory gases containing hydrogen sulfide, wherein gases produced by bacteria, in particular intestinal bacteria, are determined in the exhaled gas of a subject, wherein the device comprises means i.) that the moisture contained in the exhaled gas is removed by drying, and / or ii.) that the hydrogen sulfide contained in the exhaled gas is passed through and / or stored by non-adsorbing inert materials.

[0043] The device can include a mouthpiece or receiving unit, with the breathing gas being guided through lines and tubes, firstly into the sample container, optionally with an integrated cooling trap or via a desiccant, and optionally into a detector. The breathing gas is preferably stored in a sample container. The sample container for receiving the breathing gases, particularly its wall, is made of a non-adsorbing inert material. Furthermore, lines or tubes for guiding the breathing gas, particularly into a detector, optionally including the mouthpiece, can also be made of such a non-adsorbing inert material.

[0044] Such a device further consists of a suitable detection means for detecting the respiratory gases (e.g., a flame photometer detector (FPD) for use according to the invention). A gas chromatograph can also be used as a detector, preferably coupled with an FDD or a mass spectrometer.

[0045] An exemplary device is shown in Figure 1a, where drying is carried out using a desiccant.

[0046] It should be noted that features described in connection with an exemplary design or an exemplary object can be combined with any other exemplary design or with any other exemplary object.

[0047] When a term is referred to with an indefinite or definite article, such as "ein" in the singular, this also includes the term in the plural and vice versa, unless the context clearly states otherwise.

[0048] The expression "encompass" as used here does not only include the meaning of "contain", but can also mean "consist of" and "essentially consisting of".

[0049] The invention will now be described in more detail in the following examples and figures, without being limited thereto.

[0050] Examples:

[0051] Example 1:

[0052] Experimental setups

[0053] Instrumentation: Samples / standards were collected using desiccant tubes and headspace glass tubes. Measurements were performed on a flame photometer detector gas chromatograph (GC-FPD) equipped with an SH-U-Bond column (30 m x 0.32 mm and 10 pm film thickness) and an AOC-6000 Plus autosampler (Shimadzu, Duisburg, Germany). Hydrogen (99% purity) was generated using an HG BASIC 180 generator (LNI Swissgas GmbH, Kamen, Germany). The column temperature was maintained isothermally at 65°C. The FPD and injection temperatures were maintained at 200°C and 250°C, respectively. The flow rates of synthetic air (from Air Liquide, Regensburg, Germany) and hydrogen were 60 mL / min and 40 mL / min, respectively. The injection volume and the flow rate of the helium carrier gas (purity 99.9999%) were 500 pl and 4 ml / min, respectively.

[0054] Drying tube:

[0055] - The drying tube was made from a polyethylene tube by filling it with 20 g of CaC12 granules (Carl Roth GmbH+Co.KG, Karlsruhe, Germany) and connecting it with a polyethylene mouthpiece.

[0056] For the production of hydrogen sulfide standards:

[0057] - 1 Polypropylene Tedlar breathing bag from SKC Inc. (Cat. No.: 249-01-PP, Pennsylvania, USA) .

[0058] - 3 ppm hydrogen sulfide standard in N2 from All-In-Gas (part code: C020512, Starnberg, Germany) .

[0059] Coating of glass vials:

[0060] - 2 0 mL headspace vials (Carl Roth GmbH+Co.KG, Karlsruhe, Germany)

[0061] - SilcoNert® 2000 from SilcoTek GmbH (Bad Homburg, Germany). Figures:

[0062] Figure 1: a) Illustration of the setup of the breath drying tube with the exhaled air outlet into a coated sample tube. b) The exhaled breath flows through a CaC12 tube into an uncoated sample tube; then H2S is added (Cspiked = 143 ppb). Drying the exhaled air leads to an increase in the H2S recovery rate.

[0063] Figure 2:

[0064] A mixture of four gases (hydrogen, methane, carbon dioxide, and hydrogen sulfide) was prepared in an exhalation bag, and the concentration of each gas was measured from the bag (CO = 30 ppm H₂, 9 ppm CH₄, 3% CO₂, 350 ppb H₂S) and after passing through a tube filled with CaC₁₂. The drying method results in no loss of the aforementioned exhalation gases.

[0065] Figure 3:

[0066] 300 ppb H₂S in exhaled breath, collected in a breathing bag. The humidity in the bag is measured with a hygrometer. Then, three drying tubes containing 5, 10, and 20 g of CaC₁₂ are prepared. The gas is passed through the drying tubes and measured with a GC-FPD. The humidity after passing through the tubes is also measured. Drying the breath leads to an increase in the recovery of hydrogen sulfide to 100%.

[0067] Figure 4:

[0068] H₂S was processed from exhaled air and collected in coated and uncoated tubes, with and without drying tubes. The concentration was measured daily for up to 7 days. The results demonstrate an insufficient effect of the coating alone, but a strong effect of drying the breath on the stability of H₂S in breath samples.

[0069] Figure 5a and Figure 5b:

[0070] 1 ppm (Co) H2S in exhaled air was passed through drying tubes in three densely coated (opaque) and three less densely coated (transparent) tubes. Samples were measured on seven consecutive days (Ct), and the recapture was calculated as follows: (Ct / Co) * 100. The graphs shown are the average of three replicates for each day, with error bars.

Claims

Patent claims:

1. Non-invasive method for determining respiratory gases containing hydrogen sulfide, wherein gases produced by bacteria in the exhaled gas of a subject are determined, characterized in that i.) the moisture contained in the exhaled gas is removed by drying, and / or ii.) the hydrogen sulfide contained in the exhaled gas is passed through non-adsorbing inert materials and / or stored.

2. Non-invasive method for determining respiratory gases comprising hydrogen sulfide according to claim 1, wherein the drying is carried out by i.) cooling or by ii) a drying agent, in particular by means of calcium chloride .

3. Non-invasive method for determining respiratory gases comprising hydrogen sulfide according to claim 1, wherein drying is carried out by cooling using a cold trap.

4. Non-invasive method for determining respiratory gases comprising hydrogen sulfide according to claim 1, wherein non-adsorbing inert materials are selected from the group consisting of silicone coatings, plastics, hydrogenated amorphous silicon substrates and quartz glass.

5. Non-invasive method for determining respiratory gases containing hydrogen sulfide according to one of the above Claims, wherein non-adsorbing inert materials are a component of the sample container, in particular in the form of the wall of the sample container and / or lines or tubes for guiding the breathing gas, optionally including the mouthpiece or receiving unit, comprising such non-adsorbing inert materials.

6. Non-invasive method for determining respiratory gases comprising hydrogen sulfide according to one of the preceding claims for use in medicine, in particular sports medicine or geriatric medicine, including the monitoring of metabolic processes.

7. Non-invasive method for determining respiratory gases comprising hydrogen sulfide according to any of the preceding claims for use in the diagnosis or prediction or for risk stratification or therapy control of diseases of subjects.

8. A non-invasive method for determining breath gases comprising hydrogen sulfide according to any of the preceding claims for use in the diagnosis or prediction or for risk stratification or therapy control of diseases in subjects, wherein one or more diseases are selected from the group of gastrointestinal diseases, in particular intestinal diseases such as SIBO (small intestinal bacterial overgrowth), irritable bowel syndrome (IBS), lactose or fructose malabsorption, chronic inflammatory bowel diseases (IBD), in particular Crohn's disease and ulcerative colitis, celiac disease or functional dyspepsia; cancers, in particular colon carcinoma; vascular diseases, in particular arteriosclerotic (ASCVD); skin diseases, in particular wound healing and Psoriasis, bad breath (halitosis), lung diseases, especially asthma and COPD, neurological diseases, especially Alzheimer's and forms of dementia.

9. Non-invasive method for determining respiratory gases comprising hydrogen sulfide according to one of the preceding claims for use in therapy control, provided that the subject receives medication for the treatment of a disease or adheres to a diet for the treatment of a disease, in particular for the treatment of an intestinal disease or malabsorption.

10. Use of a device for determining respiratory gases containing hydrogen sulfide, wherein gases produced by bacteria in the exhaled gas of a subject are determined, the device comprising means characterized in that i.) the moisture contained in the exhaled gas is removed by drying, and / or ii.) the hydrogen sulfide contained in the exhaled gas is passed through and / or stored in non-adsorbing inert materials.

11. Respiratory gases containing hydrogen sulfide for use a.) in medicine, in particular sports medicine or geriatric medicine, including the monitoring of metabolic processes, and / or b.) in the diagnosis or prediction or for risk stratification or therapy control of diseases of subjects, wherein gases produced by bacteria, in particular intestinal bacteria, in the exhaled gas of a subject are determined are, wherein i. ) the moisture contained in the exhaled gas is removed by drying, and / or ii. ) the hydrogen sulfide contained in the exhaled gas is passed through non-adsorbing inert materials and / or stored.

12. Respiratory gases containing hydrogen sulfide for use according to claim 11, wherein one or more diseases are selected from the group consisting of gastrointestinal diseases, in particular intestinal diseases such as SIBO (Small Intestinal Bacterial Overgrowth), irritable bowel syndrome (IBS), lactose or fructose malabsorption, chronic inflammatory bowel diseases (IBD), in particular Crohn's disease and ulcerative colitis, celiac disease or functional dyspepsia; cancers, in particular colon carcinoma; vascular diseases, in particular arteriosclerotic (ASCVD); skin diseases, in particular wound healing and psoriasis; halitosis; lung diseases, in particular asthma and COPD; neurological diseases, in particular Alzheimer's and forms of dementia.

13. Respiratory gases comprising hydrogen sulfide for use according to claim 11, wherein the therapy control is provided that the subject receives medication to treat a disease or adheres to a diet to treat a disease, in particular to treat an intestinal disease or malabsorption.

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