CA2312808C - A device for examining respiratory diseases, and diagnostic agents - Google Patents
A device for examining respiratory diseases, and diagnostic agents Download PDFInfo
- Publication number
- CA2312808C CA2312808C CA002312808A CA2312808A CA2312808C CA 2312808 C CA2312808 C CA 2312808C CA 002312808 A CA002312808 A CA 002312808A CA 2312808 A CA2312808 A CA 2312808A CA 2312808 C CA2312808 C CA 2312808C
- Authority
- CA
- Canada
- Prior art keywords
- air
- respiratory
- inlet valve
- diagnostic agent
- flow channel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/497—Physical analysis of biological material of gaseous biological material, e.g. breath
Abstract
The invention relates to a device which can be used in conjunction with a conventional analysis system for diagnosing the metabolic capacity of the respiratory tracts. The device enables specific quantities of suitable diagnostic agents to be added to the inspired air and after a certain time span, can be used to collect metabolites of the diagnostic agents present in the expired air and/or the diagnostic agent itself if still present. Conventional analysis systems are then used to identify the substances which have been expired and collected and determine the quantities present (e.g. using mass spectrometry, gas chromatography, GC-MS-MS, HPL-MS, isotope analysis). Conclusions can then be drawn as to the state of health of the respiratory tract organs. The invention also relates to diagnostic agents which can be used for determining the metabolic capacity of the respiratory tract epithelium, and which are used especially advantageously in conjunction with the inventive device, and to a n analytical chemical method for determining the metabolic capacity of the bronchial epithelium.
Description
A Device for Examining Respiratory Diseases, and Diagnostic Agents Specification The invention relates to a device which, in associa-tion with conventional analytical systems, can be used in diagnosing the metabolic performance of the respiratory or-gans. Using this device, it is possible to dose suitable diagnostic agents in specific quantities into the inhalation air and, following a defined period of time, collect metabo-lites of the diagnostic agent and/or the remaining diagnostic agent itself from the expired air. Using conventional analytical systems (e.g., mass spectrometry, gas chromatography, GC-MS-MS, HPLC-MS, isotope analysis), these expired and collected substances can be identified and determined quantitatively, thereby permitting conclusions as to the health condition of the respiratory organs.
The invention is also directed to diagnostic agents which can be used to determine the metabolic performance of the respiratory tract epithelium - with particular advantage also in association with the device according to the inven-tion, and to an appropriate method for the chemical-analyti-cal determination of the metabolic performance of the bron-chial epithelium.
Referring to the prior art, it has been familiar for a long time to collect volatile substances from expired air by passing the air over a sorbent, adsorbing the substances and determining them. In the mid-seventies, the so-called Tenax, a polymer based on 2,6-diphenyl-p-phenylene oxide, has been developed as a sorbent particularly suited for this purpose (Krotoszynski et al., J. Chromatogr. Sci. 15, 239-244, 1977; Krotoszynski et al., J. Analyt. Toxicol. 3, 225-234, 1979).
More recent studies by Wallace et al., Environmental Health Perspectives, Vol. 104, Supplement 5, pp. 861-869, 1996, deal with the utility of breath analysis in the deter-mination of volatile organic substances in respiratory air after exposing test persons to certain chemicals in their environment (automobile exhaust gases, gas stations, swimming pools, exposition to benzene and styrene during active smoking).
Various methods and devices for collecting both volatile and non-volatile substances from respiratory air have become familiar.
Thus, WO 91/05255 Al describes a method wherein non-volatile biopolymers such as proteins from the broncho-alveolar boundary fluid are detected in respiratory air. To this end, a test person breathes forced exhalations on a sample carrier plate 1 cm2 in size and cooled to -196 C. The sample then is freeze-dried on a cryo-table by applying an ultra-high vacuum and subsequently analyzed.
DE 195 05 504 Al describes a method and a device for collecting expired breath condensate. Therein, expired air flows through a sample collector tube, being cooled to a subfreezing temperature of below 0 C where liquid and soluble components undergo condensation, freezing to the interior wall of the sample collector tube which has a lower temperature than the respiratory condensate.
In Applied Cardiopulmonary Pathophysiology 5, pp.
215-219, 1995, Gunther Becher et al. describe the determina-tion of leukotriene B4 (LTB4) in respiratory condensate - a ~
The invention is also directed to diagnostic agents which can be used to determine the metabolic performance of the respiratory tract epithelium - with particular advantage also in association with the device according to the inven-tion, and to an appropriate method for the chemical-analyti-cal determination of the metabolic performance of the bron-chial epithelium.
Referring to the prior art, it has been familiar for a long time to collect volatile substances from expired air by passing the air over a sorbent, adsorbing the substances and determining them. In the mid-seventies, the so-called Tenax, a polymer based on 2,6-diphenyl-p-phenylene oxide, has been developed as a sorbent particularly suited for this purpose (Krotoszynski et al., J. Chromatogr. Sci. 15, 239-244, 1977; Krotoszynski et al., J. Analyt. Toxicol. 3, 225-234, 1979).
More recent studies by Wallace et al., Environmental Health Perspectives, Vol. 104, Supplement 5, pp. 861-869, 1996, deal with the utility of breath analysis in the deter-mination of volatile organic substances in respiratory air after exposing test persons to certain chemicals in their environment (automobile exhaust gases, gas stations, swimming pools, exposition to benzene and styrene during active smoking).
Various methods and devices for collecting both volatile and non-volatile substances from respiratory air have become familiar.
Thus, WO 91/05255 Al describes a method wherein non-volatile biopolymers such as proteins from the broncho-alveolar boundary fluid are detected in respiratory air. To this end, a test person breathes forced exhalations on a sample carrier plate 1 cm2 in size and cooled to -196 C. The sample then is freeze-dried on a cryo-table by applying an ultra-high vacuum and subsequently analyzed.
DE 195 05 504 Al describes a method and a device for collecting expired breath condensate. Therein, expired air flows through a sample collector tube, being cooled to a subfreezing temperature of below 0 C where liquid and soluble components undergo condensation, freezing to the interior wall of the sample collector tube which has a lower temperature than the respiratory condensate.
In Applied Cardiopulmonary Pathophysiology 5, pp.
215-219, 1995, Gunther Becher et al. describe the determina-tion of leukotriene B4 (LTB4) in respiratory condensate - a ~
mediator which, in the event of mucosa inflammation, is re-leased from the inflammatory cells into the respiratory tract. It has been shown that the amount of expired LTB4 correlates with the clinical stage of bronchial asthma. This written document concludes that collection of the respiratory condensate and biochemical analysis thereof is well-suited for diagnosing inflammatory respiratory diseases.
Being based on the measurement of a biochemical en-dogenous component, the determination of LTB4 is the most direct method at present. However, this method does not offer any option for true quantification of the stage of disease via standardizable reference values. On the other hand, practical application in routine diagnostics is limited by the necessity of accumulating and measuring exceedingly low amounts of material, and these amounts are subject to intra- and inter-individual variations which, in their normal range, may be greater than e.g. the effect of bronchial asthma.
Also, the problem is that in most of the cases one does not know in which way the mediators present in the ex-pired air, such as proteins, peptides, amino acids,. and phospholipides, correlate with the stage of disease in the respiratory tract or the lungs, so that diagnosis on this basis is not possible as yet. Similarly, WO 91/05255 wherein a method of diagnosing the health condition of lungs and respiratory tract is claimed, merely demonstrates that non-volatile biopolymers can be analyzed in human respiratory air. WO 91/05255 does not reveal any conclusions as to the health condition of the respiratory tract and, in particular, the metabolic performance of the respiratory epithelium.
The deficiency of all the previous solutions is their incapability of reliably characterizing the physiological-chemical condition and the metabolic capacity of the bronchial epithelium via the respiratory air.
It was therefore the object of the invention to develop a method of determining the health condition of the respiratory tract and, in particular, the metabolic activity of cells on the boundary surface of the respiratory tract, as well as the surface condition of the bronchial mucosa in a reproducible, reliable, standarized and direct fashion using chemical-analytical means, and to provide a simple device for this purpose.
According to one aspect the invention provides a device for examining respiratory diseases by dosing diagnostic agents into the respiratory air, which agents are capable of forming metabolites in the respiratory organs, and collecting substances present in the expired air, the device comprising a flow channel having a front end and an opposite end and having a mouthpiece at its front end, an inlet valve, an inhalation or injection unit for the diagnostic agent, a cold trap to separate substances present in the expired air, which is arranged at an angle relative to the flow channel, and an adsorption vessel arranged downstream of the cold trap at the opposite end of the flow channel.
According to another aspect the invention provides a method of determining the metabolic performance of the bronchial epithelium, wherein diagnostic agents capable of forming metabolites in the respiratory organs are dosed into the respiratory air of a test person using the novel device, the substances present in the expired air are collected subsequently, and the metabolites and/or remaining amount of diagnostic agent are subjected to analysis.
Being based on the measurement of a biochemical en-dogenous component, the determination of LTB4 is the most direct method at present. However, this method does not offer any option for true quantification of the stage of disease via standardizable reference values. On the other hand, practical application in routine diagnostics is limited by the necessity of accumulating and measuring exceedingly low amounts of material, and these amounts are subject to intra- and inter-individual variations which, in their normal range, may be greater than e.g. the effect of bronchial asthma.
Also, the problem is that in most of the cases one does not know in which way the mediators present in the ex-pired air, such as proteins, peptides, amino acids,. and phospholipides, correlate with the stage of disease in the respiratory tract or the lungs, so that diagnosis on this basis is not possible as yet. Similarly, WO 91/05255 wherein a method of diagnosing the health condition of lungs and respiratory tract is claimed, merely demonstrates that non-volatile biopolymers can be analyzed in human respiratory air. WO 91/05255 does not reveal any conclusions as to the health condition of the respiratory tract and, in particular, the metabolic performance of the respiratory epithelium.
The deficiency of all the previous solutions is their incapability of reliably characterizing the physiological-chemical condition and the metabolic capacity of the bronchial epithelium via the respiratory air.
It was therefore the object of the invention to develop a method of determining the health condition of the respiratory tract and, in particular, the metabolic activity of cells on the boundary surface of the respiratory tract, as well as the surface condition of the bronchial mucosa in a reproducible, reliable, standarized and direct fashion using chemical-analytical means, and to provide a simple device for this purpose.
According to one aspect the invention provides a device for examining respiratory diseases by dosing diagnostic agents into the respiratory air, which agents are capable of forming metabolites in the respiratory organs, and collecting substances present in the expired air, the device comprising a flow channel having a front end and an opposite end and having a mouthpiece at its front end, an inlet valve, an inhalation or injection unit for the diagnostic agent, a cold trap to separate substances present in the expired air, which is arranged at an angle relative to the flow channel, and an adsorption vessel arranged downstream of the cold trap at the opposite end of the flow channel.
According to another aspect the invention provides a method of determining the metabolic performance of the bronchial epithelium, wherein diagnostic agents capable of forming metabolites in the respiratory organs are dosed into the respiratory air of a test person using the novel device, the substances present in the expired air are collected subsequently, and the metabolites and/or remaining amount of diagnostic agent are subjected to analysis.
According to the invention, a device is provided by means of which a small, precisely defined amount of a suitable diagnostic agent is inhaled during inspiration, and the amount of said agent and/or its metabolites streaming back during expiration is collected by successive, well-defined cooling and adsorption and determined accurately.
A certain quantity of the diagnostic agent is absorbed by the bronchial epithelium and optionally converted in a biochemical reaction (metabolized) to give well-defined resultant products. The level of absorption and conversion depends on the health condition of the epithelium tissue and thus, on the present metabolic performance thereof. Consequently, it is possible to determine the epithelium condition in a direct biochemical way by measuring the quantity of expired diagnostic agent relative to the inhaled amount and/or the corresponding quantity of its defined metabolites.
The invention will now be described with reference to the accompanying drawings, in which:
Fig. 1 shows a device according to the invention having a fresh air valve 3 and a simple inhalation or injection unit 4.
Fig. 2 shows a device according to the invention having a fresh air valve 3 and an aerosol inhalation or injection unit 4.
Fig. 3 shows a device according to the invention having an antistatic reservoir bag.
Fig. 4 shows a part of the device according to the invention including suction device 11, analytical unit 12, cold trap 5, and adsorption section 6.
- 5a -Fig. 5 shows a reverse cold trap 5.
Referring to Figure 1, a device for examining respiratory diseases consists of a flow channel 1 having a mouthpiece 2 at its front end, an inlet valve 3, an inhalation or injection unit 4 for the diagnostic agent, a cold trap 5 to separate soluble substances present in the expired air, which is arranged at an angle relative to the flow channel 1, and an adsorption vessel 6 arranged down-stream of the cold trap 5 at the opposite end of the flow channel 1.
In a preferred embodiment of the invention, the longitudinal axis of the cold trap is arranged at an angle of about 450 relative to the flow channel, thereby achieving an approximately uniform flow through the cold trap.
The inhalation or injection unit 4 for dosing the diagnostic agent into the inspiration air can be designed in various ways, but using per se known equipment or parts of equipment. Thus, dosing can be effected using e.g. a dosing pump or a piston syringe, or by simple suction of the diagnostic agent in an appropriate dilution (cf., Fig. 1).
Previous nebulization or atomization of the diagnostic agent is also possible. Preferably, computer-controlled aerosol generation using a pressurized, nozzle or ultrasonic nebulizer or a centrifugal atomizer may also be employed (cf., Fig. 2). In these cases, fresh air is sucked in via inlet valve 3. For a person skilled in the art, however, it would be no problem to provide other suitable inhalation or injection units. Thus, for example, a previously prepared air/diagnostic agent mixture can be provided in a reservoir bag and inspired or dosed via inlet valve 3 (cf., Fig. 3).
According to the device of the invention, the expired air to be analyzed, in order to collect the - 5b -metabolites of the diagnostic agent present therein and/or the remaining diagnostic agent itself, initially flows through the cold trap 5 where the water present in the expired air is condensed together with non-gaseous substances that are present. To achieve optimum cooling performance, the cold trap 5 can be a reverse cold trap, for example, as illustrated in Fig. 5. However, any other design is also possible, and the only point is that the adjustable temperature range for cooling performance spans at least from -5 C to -25 C. This can be done in a very simple way by using a cooling jacket tube where water having a coolant additive is passed through. When using a reverse cold trap in accordance with Fig. 5, it may be connected to a refrigerating aggregate. The cold trap 5 may be arranged in a fashion enabling its removal from the device, so that the respiratory condensate can be collected in frozen condition.
With a computer-controlled design of the deVice, however, it is also possible to heat the cold trap 5 after collection of the exhaled material is completed and suck the water containing the substances to be determined directly into the analytical unit 12 (cf., Fig. 4). According to the invention, the device may also include multiple cold traps arranged in series.
Downstream of cold trap 5, the gaseous components and remaining residues of exhaled material pass through an adsorption vessel 6. The adsorption vessel 6 includes a sorbent commonly used to collect volatile substances in ex-pired air, e.g. an organic polymer or active charcoal. Basi-cally, the respiratory condensate will deposit on the interior wall of cold trap 5.
As can easily be seen by someone skilled in the art, the device is operable in two different ways, either by means of mechanical valves or by using computer-controlled valve switching. The passive flow control using a mechanical valve 3 permits a purely mechanical separation of the inspiratory and expiratory flow paths. The easy-running valve 3 opens even at slightly reduced. pressure (inspiration) within the mouthpiece, so that the influx of the inspired volume invariably takes place via this inlet.
As soon as the vacuum declines or excess pressure develops at the mouthpiece (no breath or expired air), the valve closes automatically. The cooling and adsorption section is remarkable for its system-inherent resistance, excluding influx during inspiration. In case of small flow differences (e.g., in measurements on children), an additional passive expi_ration valve is employed. When expiring, the only flow path for the expired air is through the cooling and adsorption section.
As an alternative to the passive variant, opening of the cooling and adsorption section may be effected depending on expiration volume and/or flow rate when using computer-controlled valves.
Hence, by sucking off the frozen and re-thawed sub-stances, the collected respiratory condensate can be fed directly, i.e., on-line, into the analytical unit 12, e.g. a usual combination of chromatograph and mass spectrometer. In the event of a cold trap 5 having a removable design, the respiratory condensate is removed manually from the interior wall of the cold trap by thawing and subjected to analysis or stored in a deep-frozen state until measurement.
In a particularly preferred embodiment of the inven-tion, the patient is subjected to a cold air provocation between two examinations, which is well-known in the diagno-sis of respiratory diseases, so that a differential measure-ment for type and amount of expired chemical substances prior to and subsequent to mucosa irritation can be performed. To effect cold air provocation, a per se kiiown air cooling device is arranged upstream of inlet valve 3, e.g. the RHES cold air provocation instrument by the company Jager, Wurzburg (Germany) . In the device according to the invention, this air cooling device may also be used to cool the inner tube of cold trap 5. Owing to this differential measurement, a substantial improvement in the reliability of the analytical results is achieved.
Depending on the diagnostic agent employed, the in-ventive analysis of diagnostic agent still present in the expired material or metabolites thereof is performed using conventional analytical methods, preferably mass-spectromet-ric and chromatographic investigations.
In order to ensure good tolerability of the diagnostic agent, formulations including endogenous or related substances are preferably used, e.g. amino acids or higher alcohols in high dilution. These compounds were found to be suitable diagnostic agents for the present method of investigation.
To ensure high specificity of diagnosis and optimize the sensitivity of determination, these substances are administered in a stable isotope labelled (i.e., non-radioactive) form. Only in this way it is possible to identify the expired diagnostic agent or a specific conversion product thereof as part of the inspired diagnostic agent.
Thus, the quantity of the stable isotope labelled diagnostic agent (or a defined resultant product thereof) recovered within a specific period of time is a reliable measure for the actual absorptive and metabolic performance of the epithelium.
The invention is also directed to a novel use of pharmaceutically tolerable higher alcohols or amino acids in a stable isotope labelled form in the diagnosis of respiratory diseases and to a method for the chemical-analytical determination of the metabolic performance of the bronchial epithelium using the device according to the invention. The test persons inhale diagnostic agents capable of forming metabolites in the respiratory organs and subsequently, the expired air is collected and the metabolites and/or remaining amount of diagnostic agent are determined after being recovered by freezing and adsorption.
A certain quantity of the diagnostic agent is absorbed by the bronchial epithelium and optionally converted in a biochemical reaction (metabolized) to give well-defined resultant products. The level of absorption and conversion depends on the health condition of the epithelium tissue and thus, on the present metabolic performance thereof. Consequently, it is possible to determine the epithelium condition in a direct biochemical way by measuring the quantity of expired diagnostic agent relative to the inhaled amount and/or the corresponding quantity of its defined metabolites.
The invention will now be described with reference to the accompanying drawings, in which:
Fig. 1 shows a device according to the invention having a fresh air valve 3 and a simple inhalation or injection unit 4.
Fig. 2 shows a device according to the invention having a fresh air valve 3 and an aerosol inhalation or injection unit 4.
Fig. 3 shows a device according to the invention having an antistatic reservoir bag.
Fig. 4 shows a part of the device according to the invention including suction device 11, analytical unit 12, cold trap 5, and adsorption section 6.
- 5a -Fig. 5 shows a reverse cold trap 5.
Referring to Figure 1, a device for examining respiratory diseases consists of a flow channel 1 having a mouthpiece 2 at its front end, an inlet valve 3, an inhalation or injection unit 4 for the diagnostic agent, a cold trap 5 to separate soluble substances present in the expired air, which is arranged at an angle relative to the flow channel 1, and an adsorption vessel 6 arranged down-stream of the cold trap 5 at the opposite end of the flow channel 1.
In a preferred embodiment of the invention, the longitudinal axis of the cold trap is arranged at an angle of about 450 relative to the flow channel, thereby achieving an approximately uniform flow through the cold trap.
The inhalation or injection unit 4 for dosing the diagnostic agent into the inspiration air can be designed in various ways, but using per se known equipment or parts of equipment. Thus, dosing can be effected using e.g. a dosing pump or a piston syringe, or by simple suction of the diagnostic agent in an appropriate dilution (cf., Fig. 1).
Previous nebulization or atomization of the diagnostic agent is also possible. Preferably, computer-controlled aerosol generation using a pressurized, nozzle or ultrasonic nebulizer or a centrifugal atomizer may also be employed (cf., Fig. 2). In these cases, fresh air is sucked in via inlet valve 3. For a person skilled in the art, however, it would be no problem to provide other suitable inhalation or injection units. Thus, for example, a previously prepared air/diagnostic agent mixture can be provided in a reservoir bag and inspired or dosed via inlet valve 3 (cf., Fig. 3).
According to the device of the invention, the expired air to be analyzed, in order to collect the - 5b -metabolites of the diagnostic agent present therein and/or the remaining diagnostic agent itself, initially flows through the cold trap 5 where the water present in the expired air is condensed together with non-gaseous substances that are present. To achieve optimum cooling performance, the cold trap 5 can be a reverse cold trap, for example, as illustrated in Fig. 5. However, any other design is also possible, and the only point is that the adjustable temperature range for cooling performance spans at least from -5 C to -25 C. This can be done in a very simple way by using a cooling jacket tube where water having a coolant additive is passed through. When using a reverse cold trap in accordance with Fig. 5, it may be connected to a refrigerating aggregate. The cold trap 5 may be arranged in a fashion enabling its removal from the device, so that the respiratory condensate can be collected in frozen condition.
With a computer-controlled design of the deVice, however, it is also possible to heat the cold trap 5 after collection of the exhaled material is completed and suck the water containing the substances to be determined directly into the analytical unit 12 (cf., Fig. 4). According to the invention, the device may also include multiple cold traps arranged in series.
Downstream of cold trap 5, the gaseous components and remaining residues of exhaled material pass through an adsorption vessel 6. The adsorption vessel 6 includes a sorbent commonly used to collect volatile substances in ex-pired air, e.g. an organic polymer or active charcoal. Basi-cally, the respiratory condensate will deposit on the interior wall of cold trap 5.
As can easily be seen by someone skilled in the art, the device is operable in two different ways, either by means of mechanical valves or by using computer-controlled valve switching. The passive flow control using a mechanical valve 3 permits a purely mechanical separation of the inspiratory and expiratory flow paths. The easy-running valve 3 opens even at slightly reduced. pressure (inspiration) within the mouthpiece, so that the influx of the inspired volume invariably takes place via this inlet.
As soon as the vacuum declines or excess pressure develops at the mouthpiece (no breath or expired air), the valve closes automatically. The cooling and adsorption section is remarkable for its system-inherent resistance, excluding influx during inspiration. In case of small flow differences (e.g., in measurements on children), an additional passive expi_ration valve is employed. When expiring, the only flow path for the expired air is through the cooling and adsorption section.
As an alternative to the passive variant, opening of the cooling and adsorption section may be effected depending on expiration volume and/or flow rate when using computer-controlled valves.
Hence, by sucking off the frozen and re-thawed sub-stances, the collected respiratory condensate can be fed directly, i.e., on-line, into the analytical unit 12, e.g. a usual combination of chromatograph and mass spectrometer. In the event of a cold trap 5 having a removable design, the respiratory condensate is removed manually from the interior wall of the cold trap by thawing and subjected to analysis or stored in a deep-frozen state until measurement.
In a particularly preferred embodiment of the inven-tion, the patient is subjected to a cold air provocation between two examinations, which is well-known in the diagno-sis of respiratory diseases, so that a differential measure-ment for type and amount of expired chemical substances prior to and subsequent to mucosa irritation can be performed. To effect cold air provocation, a per se kiiown air cooling device is arranged upstream of inlet valve 3, e.g. the RHES cold air provocation instrument by the company Jager, Wurzburg (Germany) . In the device according to the invention, this air cooling device may also be used to cool the inner tube of cold trap 5. Owing to this differential measurement, a substantial improvement in the reliability of the analytical results is achieved.
Depending on the diagnostic agent employed, the in-ventive analysis of diagnostic agent still present in the expired material or metabolites thereof is performed using conventional analytical methods, preferably mass-spectromet-ric and chromatographic investigations.
In order to ensure good tolerability of the diagnostic agent, formulations including endogenous or related substances are preferably used, e.g. amino acids or higher alcohols in high dilution. These compounds were found to be suitable diagnostic agents for the present method of investigation.
To ensure high specificity of diagnosis and optimize the sensitivity of determination, these substances are administered in a stable isotope labelled (i.e., non-radioactive) form. Only in this way it is possible to identify the expired diagnostic agent or a specific conversion product thereof as part of the inspired diagnostic agent.
Thus, the quantity of the stable isotope labelled diagnostic agent (or a defined resultant product thereof) recovered within a specific period of time is a reliable measure for the actual absorptive and metabolic performance of the epithelium.
The invention is also directed to a novel use of pharmaceutically tolerable higher alcohols or amino acids in a stable isotope labelled form in the diagnosis of respiratory diseases and to a method for the chemical-analytical determination of the metabolic performance of the bronchial epithelium using the device according to the invention. The test persons inhale diagnostic agents capable of forming metabolites in the respiratory organs and subsequently, the expired air is collected and the metabolites and/or remaining amount of diagnostic agent are determined after being recovered by freezing and adsorption.
In a preferred variant, a test person inhales a stable isotope labelled, pharmaceutically tolerable higher alcohol as diagnostic agent. Following an exposure period of about 1 to 3 hours, preferably 1 to 2 hours, the expired air is collected and the expired diagnostic agent or metabolites thereof are frozen out. Subsequently, the amount and/or the isotope content of the substances are determined and compared to the isotope basic value in the respiratory air zero sample of the test person.
Preferred diagnostic agents are higher alcohols, preferably C8 alcohols and more preferably having at least 13 C atoms, e.g. hexadecanol-1, which preferably is 13C-labelled.
In another preferred variant, stable isotope labelled amino acids are used as diagnostic agents. The expired air is collected over a time period of at least 30 minutes from the first expiration event on and analyzed specifically for gaseous metabolites of the diagnostic agent or N-labelled nitrous gases using per se known methods. A preferred diagnostic agent is a 15N-labelled amino acid, and particularly preferred is the amino acid L-arginine.
In another preferred variant, a cold air provocation on the test person is carried out after collecting the expired air, and the diagnostic agent is administered once more as an inhalation. The determination of the expired air is repeated, and these measured values are correlated with the values measured prior to provocation, thereby making it possible to determine the sensitivity for the bronchial surface and establish the severity of an inflammation.
Preferred diagnostic agents are higher alcohols, preferably C8 alcohols and more preferably having at least 13 C atoms, e.g. hexadecanol-1, which preferably is 13C-labelled.
In another preferred variant, stable isotope labelled amino acids are used as diagnostic agents. The expired air is collected over a time period of at least 30 minutes from the first expiration event on and analyzed specifically for gaseous metabolites of the diagnostic agent or N-labelled nitrous gases using per se known methods. A preferred diagnostic agent is a 15N-labelled amino acid, and particularly preferred is the amino acid L-arginine.
In another preferred variant, a cold air provocation on the test person is carried out after collecting the expired air, and the diagnostic agent is administered once more as an inhalation. The determination of the expired air is repeated, and these measured values are correlated with the values measured prior to provocation, thereby making it possible to determine the sensitivity for the bronchial surface and establish the severity of an inflammation.
Example 1 liters of air in a sealed vessel is mixed with defined quantities of between 1 and 100 mg of hexadecanol-1 labelled with the stable isotope 13C (and containing more 5 than 95 atom-% of 13C in the CH2OH group) . According to the invention, [1-13C] -hexadecanol serves as diagnostic agent for measuring the permeability of the bronchial epithelium.
Prior to applying the 13C-hexadecanol-1 formulation, a respiratory air sample is collected from the individual to be examined ("zero sample") in order to measure the actual natural 13C basic value of expired carbon dioxide of said person. The basic value is related to the so-called PDB
standard value, which is 1.1112328 atom-% 13C. From the second digit behind the decimal point on, this value is subject to deviation in each of the individuals, and this deviation can be measured with high precision. To this end, a mass spectrometer or a special respiratory gas 13C
measuring instrument is used in a per se known fashion (e.g., the FANci2 instrument supplied by the company Fischer Analysen Instrumente, Leipzig (Germany)).
Breathing ten times, the prepared air/hexadecanol mixture then is inspired completely using the array of Fig. 3 according to the invention. After a waiting period of 1 and 2 hours, respectively, the expired air of 50 exhalations is collected each time, using the array of the invention. Expired [1-13C]-hexadecanol and carbon dioxide are separated by fractionated freezing. Amount and 13C
content of both substances are determined.
Now, the 13C value of carbon dioxide in the expired air exhibits a characteristic deviation from the one of the zero sample: The more [1-13C]-hexadecanol has permeated the bronchial epithelium, the higher the increase of this value will be -(e.g. from 1.11127 to 1.2467... atom-% 13C) ; because, only after passing this barrier, the hexadecanol can enter the blood circulation to be degraded to carbon dioxide in the liver. Accordingly, the amount of 13C isotope derived from [1-13C] -hexadecanol and recovered in the COz of the expired air is a reliable measure for the permeability of the bronchial epithelium.
Example 2 The apparatus is used as in Example 1. In addition, however, a cold air provocation is performed after collecting the expired air, wherein a per se known RHES air cooling device by the company Jager, Wurzburg, is used which may be arranged upstream of valve 3 in the array according to the invention. 20 minutes after the cold air provocation, the steps of admixing, inspiring, expiring, and measuring illustrated in Example 1 are repeated in the same fashion once again. The 13C carbon dioxide values measured after cold air provocation are correlated with the values measured prior to provocation. In this way, an intra-individually standardized and thus, inter-individually comparable measure for the sensitivity of the bronchial surface is obtained.
Example 3 Between 1 and 100 mg of a 15N-labelled amino acid, preferably L- [guanino-15N2] arginine, dissolved in a physiological sodium chloride solution, is mixed into the storage air container. This commercially available 'SN-labelled arginine contains e.g. 95 atom-% 15N in its guanino group. Breathing up to 20 times, the prepared air/aerosol mixture is inspired completely using the inventive array of Fig. 3, and, according to the invention, the expired air from the first expiration event on is passed via valve 8 across the cold trap and adsorber section, namely, over a total time of 60 minutes, with cold trap and adsorber being exchanged after 30 minutes. After this time, the contents of the cold traps and adsorber sections is subjected to GC-MS
analysis. The substances frozen out together with expired moisture and the substances that underwent adsorption were specifically examined for gaseous metabolites of [15Nz] arginine, i.e., [15N] ammonia and 15N-labelled nitrous gases. In those cases where virtually no labelled ammonia or nitric oxide was found, the metabolic activity of the bronchial epithelium was normal, i.e., relatively low. In a condition of stress or inflammation, however, the metabolic activity is massively increased. This is seen in a comparatively large amount of 15N-labelled gaseous substances in the expiration condensate, which can only be derived from '5N-labelled arginine. Consequently, the amount of 15N isotope recovered in the condensate is a reliable measure for the metabolic activity and the inflammation level of the bronchial epithelium. In addition, urine may also be examined for metabolites containing 15N.
Example 4 The apparatus is used as in Example 3. After completing the one-hour collection of expiration condensate and 15N-labelled gases, cold air provocation in a per se known manner is performed as in Example 2. In this way, it is possible for the first time to perform a biochemical measurement of the bronchial epithelium sensitivity to cold air provocation - and indeed, a,non-invasive and in vivo one in the form of a reduced or increased amino acid metabolism.
Also, the severity of an inflammation can be determined from the ratio of the amount of 15N in the expired gases derived from inspired arginine prior to and subsequent to cold air provocation.
Prior to applying the 13C-hexadecanol-1 formulation, a respiratory air sample is collected from the individual to be examined ("zero sample") in order to measure the actual natural 13C basic value of expired carbon dioxide of said person. The basic value is related to the so-called PDB
standard value, which is 1.1112328 atom-% 13C. From the second digit behind the decimal point on, this value is subject to deviation in each of the individuals, and this deviation can be measured with high precision. To this end, a mass spectrometer or a special respiratory gas 13C
measuring instrument is used in a per se known fashion (e.g., the FANci2 instrument supplied by the company Fischer Analysen Instrumente, Leipzig (Germany)).
Breathing ten times, the prepared air/hexadecanol mixture then is inspired completely using the array of Fig. 3 according to the invention. After a waiting period of 1 and 2 hours, respectively, the expired air of 50 exhalations is collected each time, using the array of the invention. Expired [1-13C]-hexadecanol and carbon dioxide are separated by fractionated freezing. Amount and 13C
content of both substances are determined.
Now, the 13C value of carbon dioxide in the expired air exhibits a characteristic deviation from the one of the zero sample: The more [1-13C]-hexadecanol has permeated the bronchial epithelium, the higher the increase of this value will be -(e.g. from 1.11127 to 1.2467... atom-% 13C) ; because, only after passing this barrier, the hexadecanol can enter the blood circulation to be degraded to carbon dioxide in the liver. Accordingly, the amount of 13C isotope derived from [1-13C] -hexadecanol and recovered in the COz of the expired air is a reliable measure for the permeability of the bronchial epithelium.
Example 2 The apparatus is used as in Example 1. In addition, however, a cold air provocation is performed after collecting the expired air, wherein a per se known RHES air cooling device by the company Jager, Wurzburg, is used which may be arranged upstream of valve 3 in the array according to the invention. 20 minutes after the cold air provocation, the steps of admixing, inspiring, expiring, and measuring illustrated in Example 1 are repeated in the same fashion once again. The 13C carbon dioxide values measured after cold air provocation are correlated with the values measured prior to provocation. In this way, an intra-individually standardized and thus, inter-individually comparable measure for the sensitivity of the bronchial surface is obtained.
Example 3 Between 1 and 100 mg of a 15N-labelled amino acid, preferably L- [guanino-15N2] arginine, dissolved in a physiological sodium chloride solution, is mixed into the storage air container. This commercially available 'SN-labelled arginine contains e.g. 95 atom-% 15N in its guanino group. Breathing up to 20 times, the prepared air/aerosol mixture is inspired completely using the inventive array of Fig. 3, and, according to the invention, the expired air from the first expiration event on is passed via valve 8 across the cold trap and adsorber section, namely, over a total time of 60 minutes, with cold trap and adsorber being exchanged after 30 minutes. After this time, the contents of the cold traps and adsorber sections is subjected to GC-MS
analysis. The substances frozen out together with expired moisture and the substances that underwent adsorption were specifically examined for gaseous metabolites of [15Nz] arginine, i.e., [15N] ammonia and 15N-labelled nitrous gases. In those cases where virtually no labelled ammonia or nitric oxide was found, the metabolic activity of the bronchial epithelium was normal, i.e., relatively low. In a condition of stress or inflammation, however, the metabolic activity is massively increased. This is seen in a comparatively large amount of 15N-labelled gaseous substances in the expiration condensate, which can only be derived from '5N-labelled arginine. Consequently, the amount of 15N isotope recovered in the condensate is a reliable measure for the metabolic activity and the inflammation level of the bronchial epithelium. In addition, urine may also be examined for metabolites containing 15N.
Example 4 The apparatus is used as in Example 3. After completing the one-hour collection of expiration condensate and 15N-labelled gases, cold air provocation in a per se known manner is performed as in Example 2. In this way, it is possible for the first time to perform a biochemical measurement of the bronchial epithelium sensitivity to cold air provocation - and indeed, a,non-invasive and in vivo one in the form of a reduced or increased amino acid metabolism.
Also, the severity of an inflammation can be determined from the ratio of the amount of 15N in the expired gases derived from inspired arginine prior to and subsequent to cold air provocation.
Reference list Fig. 1:
1 Flow channel 2 Mouthpiece 3 Inlet valve for fresh air 4 Inhalation or injection unit 4a Supply pipe 4b Vessel for diagnostic agent 4c Valve Cold trap 6 Adsorption vessel 7 Flow meter 8 Valve Computer control Fig. 2:
1 Flow channel 2 Mouthpiece 3 Inlet valve for fresh air 4 Inhalation or injection unit 4a Supply pipe 4b Vessel for diagnostic agent 4c Valve 4d Device for nebulizing diagnostic agent 5 Cold trap 6 Adsorption vessel 7 Flow meter 8 Valve 10 Computer control 11 Suction device 12 Analytical unit Fig. 3..
1 Flow channel 2 Mouthpiece 3 Inlet valve for air/diagnostic agent mixture 4 Reservoir bag including air/diagnostic agent mixture =5 Cold trap 6 Adsorption vessel 7 Flow meter 8 Valve Computer control 11 Suction device 12 Analytical unit Fig. 4:
1 Flow channel 5 Cold trap 6 Adsorption vessel 11 Suction device 12 Analytical unit Fig. 5:
1 Flow channel 5 Cold trap 13 Internal tube 14 External body with cooling fins
1 Flow channel 2 Mouthpiece 3 Inlet valve for fresh air 4 Inhalation or injection unit 4a Supply pipe 4b Vessel for diagnostic agent 4c Valve Cold trap 6 Adsorption vessel 7 Flow meter 8 Valve Computer control Fig. 2:
1 Flow channel 2 Mouthpiece 3 Inlet valve for fresh air 4 Inhalation or injection unit 4a Supply pipe 4b Vessel for diagnostic agent 4c Valve 4d Device for nebulizing diagnostic agent 5 Cold trap 6 Adsorption vessel 7 Flow meter 8 Valve 10 Computer control 11 Suction device 12 Analytical unit Fig. 3..
1 Flow channel 2 Mouthpiece 3 Inlet valve for air/diagnostic agent mixture 4 Reservoir bag including air/diagnostic agent mixture =5 Cold trap 6 Adsorption vessel 7 Flow meter 8 Valve Computer control 11 Suction device 12 Analytical unit Fig. 4:
1 Flow channel 5 Cold trap 6 Adsorption vessel 11 Suction device 12 Analytical unit Fig. 5:
1 Flow channel 5 Cold trap 13 Internal tube 14 External body with cooling fins
Claims (10)
1. A device for examining respiratory diseases by dosing diagnostic agents into the respiratory air, which agents are capable of forming metabolites in the respiratory organs, and collecting substances present in the expired air, the device comprising a flow channel having a front end and an opposite end and having a mouthpiece at its front end, an inlet valve, an inhalation or injection unit for the diagnostic agent, a cold trap to separate substances present in the expired air, which is arranged at an angle relative to the flow channel, and an adsorption vessel arranged downstream of the cold trap at the opposite end of the flow channel.
2. The device according to claim 1, wherein the inhalation or injection unit comprises a supply pipe, a vessel for the diagnostic agent and a valve, and the inlet valve is an inlet valve for fresh air.
3. The device according to claim 1, wherein the inhalation or injection unit includes a nebulizer, and the inlet valve is an inlet valve for fresh air.
4. The device according to claim 1, wherein the inhalation or injection unit is a reservoir bag which includes a prepared air/diagnostic agent mixture and is connected with the flow channel via the inlet valve.
5. The device according to any one of claims 1 to 3, wherein a cold air provocation apparatus is arranged upstream of the inlet valve.
6. The device according to any one of claims 1 to 5, wherein the device is computer-controlled.
7. The device according to any one of claims 1 to 6, wherein a flow meter is arranged in the flow channel.
8. The device according to claim 6 or 7, wherein a suction device is arranged upstream of the cold trap in the flow channel.
9. The device according to claim 8, wherein the suction device is connected to an analytical unit.
10. A method of determining the metabolic performance of the bronchial epithelium, wherein diagnostic agents capable of forming metabolites in the respiratory organs are dosed into the respiratory air of a test person using the device according to any one of claims 1 to 9, the substances present in the expired air are collected subsequently, and the metabolites and/or remaining amount of diagnostic agent are subjected to analysis.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19755471A DE19755471A1 (en) | 1997-12-03 | 1997-12-03 | Device for examining respiratory diseases and diagnostic means |
| DE19755471.7 | 1997-12-03 | ||
| PCT/EP1998/007549 WO1999028743A1 (en) | 1997-12-03 | 1998-11-19 | Device for investigating respiratory tract diseases and diagnostic agents |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2312808A1 CA2312808A1 (en) | 1999-06-10 |
| CA2312808C true CA2312808C (en) | 2008-01-15 |
Family
ID=7851811
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002312808A Expired - Fee Related CA2312808C (en) | 1997-12-03 | 1998-11-19 | A device for examining respiratory diseases, and diagnostic agents |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1036322B1 (en) |
| AT (1) | ATE237805T1 (en) |
| CA (1) | CA2312808C (en) |
| DE (2) | DE19755471A1 (en) |
| WO (1) | WO1999028743A1 (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10125415A1 (en) * | 2001-05-25 | 2002-11-28 | Fischer Analysen Instr Gmbh | Apparatus for the measurement of respiratory gases converts exhaled air into the correct composition of breathing air using a non-dispersive infra red spectroscope |
| DE10137566B4 (en) * | 2001-07-30 | 2006-11-30 | Filt Lungen- Und Thoraxdiagnostik Gmbh | Method for quantifying the exchange processes in the respiratory tract |
| DE10137564A1 (en) * | 2001-07-30 | 2003-02-27 | Filt Lungen Und Thoraxdiagnost | Screening for drugs in a human or animal, comprises taking the condensation from exhaled breath, to be prepared for quantitative and qualitative analysis on site or to give a sample for a laboratory |
| DE10232849A1 (en) * | 2002-07-19 | 2004-02-12 | Abb Patent Gmbh | Gas analyzer, e.g. for field detection of odorless, invisible toxic agents and pollutants, has self-contained sampling and detector, and sample is transferred to detector under action of force field |
| DE10232850A1 (en) * | 2002-07-19 | 2004-02-12 | Abb Patent Gmbh | Mobile analysis facility |
| GB2451249A (en) | 2007-07-24 | 2009-01-28 | Equine Healthcare Ltd | A breath condensate collector |
| DE102009048644A1 (en) | 2009-09-30 | 2011-04-07 | Filt Lungen- Und Thoraxdiagnostik Gmbh | Device for fractioned extraction of substance of exhalation air, has collecting device aligned downstream-side to nozzle and accommodating fluid and/or frozen particles that are carried along exhalation air stream |
| WO2011038726A1 (en) | 2009-09-30 | 2011-04-07 | Mti Medtech Innovation Gmbh | Device and method for fractionally collecting contents of exhaled air |
| DE202009013577U1 (en) | 2009-09-30 | 2010-02-11 | Filt Lungen- Und Thoraxdiagnostik Gmbh | Apparatus for the fractionated recovery of ingredients of the exhaled air |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4803977A (en) * | 1982-03-25 | 1989-02-14 | Mallinckrodt, Inc. | Method and apparatus for the diagnosis of respiratory diseases and allergies |
| US4622976A (en) * | 1985-04-15 | 1986-11-18 | Enhancer R & D | Method and apparatus for conducting xenon inhalation studies |
| DE3932784C3 (en) * | 1989-09-30 | 2002-08-01 | Nmi Univ Tuebingen | Process for the extraction of non-volatile substances as analytical sample material from the human breath |
| US5386832A (en) * | 1993-06-11 | 1995-02-07 | New England Deaconess Hospital | Liver function breath test using aromatic amino acids |
| DE19505504A1 (en) * | 1994-05-13 | 1995-11-16 | Filt Forschungsgesellschaft Fu | Collection of exhaled breath sample |
| DE19528158A1 (en) * | 1995-07-24 | 1997-01-30 | Medium Sensor Gmbh | Device for extracting the contents of the exhaled air |
| DE19606470C2 (en) * | 1996-02-21 | 2001-03-15 | Mpo Ges Fuer Medizintechnische | Procedure for determining the functional residual capacity (FRC) |
-
1997
- 1997-12-03 DE DE19755471A patent/DE19755471A1/en not_active Ceased
-
1998
- 1998-11-19 DE DE59807995T patent/DE59807995D1/en not_active Expired - Fee Related
- 1998-11-19 CA CA002312808A patent/CA2312808C/en not_active Expired - Fee Related
- 1998-11-19 WO PCT/EP1998/007549 patent/WO1999028743A1/en active IP Right Grant
- 1998-11-19 EP EP98963501A patent/EP1036322B1/en not_active Expired - Lifetime
- 1998-11-19 AT AT98963501T patent/ATE237805T1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| DE19755471A1 (en) | 1999-06-17 |
| EP1036322A1 (en) | 2000-09-20 |
| WO1999028743A1 (en) | 1999-06-10 |
| ATE237805T1 (en) | 2003-05-15 |
| EP1036322B1 (en) | 2003-04-16 |
| DE59807995D1 (en) | 2003-05-22 |
| CA2312808A1 (en) | 1999-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6033368A (en) | Condensate colorimetric nitrogen oxide analyzer | |
| Horváth et al. | Exhaled breath condensate: methodological recommendations and unresolved questions | |
| Pietropaoli et al. | Simultaneous measurement of nitric oxide production by conducting and alveolar airways of humans | |
| Schmidt et al. | Ammonia in breath and emitted from skin | |
| Kharitonov et al. | Biomarkers of some pulmonary diseases in exhaled breath | |
| Lärstad et al. | Determination of ethane, pentane and isoprene in exhaled air–effects of breath‐holding, flow rate and purified air | |
| US5042501A (en) | Apparatus and method for analysis of expired breath | |
| Jones | Quantitative measurements of the alcohol concentration and the temperature of breath during a prolonged exhalation | |
| Dubowski | Breath analysis as a technique in clinical chemistry | |
| JP2002541429A (en) | Apparatus and method for monitoring asthma | |
| CA2312808C (en) | A device for examining respiratory diseases, and diagnostic agents | |
| Silkoff et al. | A method for the standardized offline collection of exhaled nitric oxide | |
| Jones | Role of rebreathing in determination of the blood-breath ratio of expired ethanol | |
| Ohlsson et al. | Accurate measurement of blood alcohol concentration with isothermal rebreathing. | |
| JP4452783B2 (en) | Cirrhosis test method and apparatus using breath analysis apparatus | |
| Lemaitre et al. | Effect of air diving exposure generally encountered by recreational divers: oxidative stress? | |
| Sällsten et al. | Determination of trace amounts of mercury vapour in humid air: performance of gold traps in an atomic fluorescence system | |
| EP1731095A2 (en) | Measurement and analysis of components of exhaled respiratory gas | |
| Španˇel et al. | Recent SIFT-MS studies of volatile compounds in physiology, medicine and cell biology | |
| Lindström et al. | A simple pharmacokinetic method to evaluate the pulmonary dose in clinical practice—analyses of inhaled sodium cromoglycate | |
| Periago et al. | Design and evaluation of an exhaled breath sampler for biological monitoring of organic solvents | |
| Schubert et al. | In vivo evaluation of a new method for chemical analysis of volatile components in the respiratory gas of mechanically ventilated patients | |
| Graham et al. | Implementing the three-equation method of measuring single breath carbon monoxide diffusing capacity | |
| Muskiet et al. | Mass-fragmentographic determination of catecholamine metabolites in amniotic fluid and its possible clinical usefulness. | |
| KR20190088662A (en) | Standard Sample and Method for Analysis of Exhaled Breath Gas |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |