DE19719098C1 - Diagnostic determination of labelled nitric oxide in breath - Google Patents

Diagnostic determination of labelled nitric oxide in breath

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Publication number
DE19719098C1
DE19719098C1 DE1997119098 DE19719098A DE19719098C1 DE 19719098 C1 DE19719098 C1 DE 19719098C1 DE 1997119098 DE1997119098 DE 1997119098 DE 19719098 A DE19719098 A DE 19719098A DE 19719098 C1 DE19719098 C1 DE 19719098C1
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exhalation
production
diagnosis
therapy
breath
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Expired - Fee Related
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DE1997119098
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German (de)
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Christian Dr Med Plath
Klaus-Dieter Dr Rer Nat Wutzke
Rolf Dr Rer Nat Russow
Inken Dipl Chem Sich
Bismarck Philip Dr Med Von
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WUTZKE KLAUS DIETER DR RER NAT
Helmholtz Zentrum fuer Umweltforschung GmbH UFZ
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WUTZKE KLAUS DIETER DR RER NAT
Helmholtz Zentrum fuer Umweltforschung GmbH UFZ
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1206—Administration of radioactive gases, aerosols or breath tests
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/08—Detecting, measuring or recording devices for evaluating the respiratory organs
    • A61B5/0813—Measurement of pulmonary parameters by tracers, e.g. radioactive tracers
    • 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

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  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
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Abstract

Method of measuring <15>NO exhalation for the diagnosis and therapy of human diseases comprises administering a tracer comprising "universally <15>N-labelled proteins from yeasts and algae, preferably L-guanidino-<15>N2arginine monohydrochloride" to a subject and measuring the <15>N content of NO in the subject's breath by cryogenic NO enrichment and a combination of gas chromatography and quadrupole mass spectrometry.

Description

Die Erfindung betrifft ein Verfahren zur Bestimmung der 15NO-Exhalation aus L-[guanidino-15N2]Argininmonohydrochlorid zur Diagnostik und Therapie beim Menschen.The invention relates to a method for determining the 15 NO exhalation from L- [guanidino- 15 N 2 ] arginine monohydrochloride for diagnosis and therapy in humans.

NO wird durch die NO-synthase in den Endothelzellen der pulmonalen Arterien gebildet, diffundiert in die glatte Gefäßmuskulatur und wird an der pro­ sthetischen Gruppe der löslichen Guanylylzyklase gebunden. Dieser Prozeß führt über die Freisetzung von zyclischem Guanosinmonophosphat und Senkung des verfügbaren intrazellulären Kalziums unmittelbar zur NO-induzierten Vasorelaxa­ tion. Nach neuesten Erkenntnissen gilt NO als bedeutender Regulator des vasku­ laren Tonus und stimuliert die Vasodilation.NO is produced by the NO synthase in the pulmonary endothelial cells Arteries are formed, diffuses into the smooth vascular muscles and is attached to the pro aesthetic group of soluble guanylyl cyclase bound. This process leads on the release of cyclic guanosine monophosphate and lowering the available intracellular calcium immediately to the NO-induced vasorelaxa tion. According to the latest knowledge, NO is an important regulator of vasku laren tone and stimulates vasodilation.

Das terminale Guanidino-Stickstoffatom von L-Arginin gilt als Präcursor von NO und wird bei Säugetieren zu Nitrit und Nitrat oxidiert und anschließend renal ausgeschieden.The terminal guanidino nitrogen atom of L-arginine is considered a precursor of NO and is oxidized to nitrite and nitrate in mammals and then renally excreted.

Bisher konnte der Nachweis der endogenen NO-Bildung lediglich indirekt über die Messung von NO3 - im Serum und Harn geführt werden (Castillo et al. Pediatr Res 1995; 38: 17-24).So far, the detection of endogenous NO formation could only be carried out indirectly by measuring N O3 - in serum and urine (Castillo et al. Pediatr Res 1995; 38: 17-24).

Ein direkter Nachweis der Bildung von NO aus L-Arginin in der Exhalati­ onsluft konnte bisher nicht erbracht werden.Direct evidence of the formation of NO from L-arginine in the exhalati Onsluft could not be provided so far.

Die Isotopenverhältnis-Massenspektrometrie (IRMS) gehört zu den eta­ blierten Standardmethoden bei der Messung von 15N-Häufigkeiten in festen und flüssigen Proben.Isotope ratio mass spectrometry (IRMS) is one of the established standard methods for measuring 15 N frequencies in solid and liquid samples.

Die Messung von 15N-Anreicherungen in den seltenen Atemgasen N2O und NO ist aufgrund der sehr niedrigen Konzentrationen dieser Gase in der Atemluft mit diesem Verfahren nicht möglich.The measurement of 15 N enrichments in the rare breathing gases N 2 O and NO is not possible with this method due to the very low concentrations of these gases in the breathing air.

Der im Patentanspruch angegebenen Erfindung liegt das Problem zugrun­ de, ein massenspektometrisches Verfahren zur nicht-invasiven Messung des N- Transfers von L-[guanidino-15N2]Argininmonohydrochlorid und universell 15N- markierten Proteinen aus Hefe und Algen auf 15NO in der Exhalationsluft zu entwickeln, um den klinische Verlauf von Erkrankungen (beispielsweise der persistierenden pulmonalen bzw. arteriellen Hypertension), den Einfluß von Medikamenten, die Wirkung der oralen und intravenösen Zufuhr des NO-Präcur­ sors Arginin als Agens zur Vasodilation, die Wirkung von NO-Beimengungen zu Beatmungszwecken sowie den Einfluß der Ernährung bei Menschen und Säuge­ tieren untersuchen zu können. The problem specified in the invention is based on the problem, a mass spectrometric method for the non-invasive measurement of the N transfer of L- [guanidino- 15 N 2 ] arginine monohydrochloride and universally 15 N-labeled proteins from yeast and algae to 15 NO in the To develop exhalation air to determine the clinical course of diseases (e.g. persistent pulmonary or arterial hypertension), the influence of medication, the effect of oral and intravenous administration of the NO precursor arginine as an agent for vasodilation, the effect of NO admixtures to be able to investigate for ventilation purposes and the influence of nutrition in humans and mammals.

Dieses Problem wird durch die im Patentanspruch aufgeführten Merkmale gelöst.This problem is solved by the features listed in the claim solved.

Das Wesen der Erfindung liegt in der Nutzung des NO-Pathways des L- Arginins und der nicht-invasiven Messung von 15NO in der Exhalationsluft mit­ tels eines Gaschromatographie-Quadrupolmassenspektrometrie-Kopplungssystems (GC-QMS).The essence of the invention lies in the use of the NO pathway of L-arginine and the non-invasive measurement of 15 NO in the exhalation air by means of a gas chromatography-quadrupole mass spectrometry coupling system (GC-QMS).

Bei diesem modifizierten Verfahren dient eine mit flüssigem Stickstoff gekühlte Edelstahlkapillare als Kühlfalle zur Aufkonzentrierung von NO (Kryo­ trapping). Durch thermische Desorption wird das NO punktuell auf die GC-Säule aufgebracht, gaschromatographisch getrennt und im QMS massenspektrometrisch gemessen.This modified process uses one with liquid nitrogen cooled stainless steel capillary as a cold trap for concentrating NO (cryo trapping). The NO is selectively applied to the GC column by thermal desorption applied, separated by gas chromatography and mass spectrometry in the QMS measured.

Es wurde gefunden, daß die Verabreichung von 7,5 mg/kg L-[guanidino- 15N2]Argininmonohydrochlorid (95 Atom-%) an 3 Erwachsene nach 30 bis 45 Minuten zu einer maximalen 15NO-Markierung von 4,4 bis 6,1 Atom-%-exzess in der Atemluft führte.It was found that the administration of 7.5 mg / kg L- [guanidino- 15 N 2 ] arginine monohydrochloride (95 atomic%) to 3 adults after 30 to 45 minutes to a maximum 15 NO label of 4.4 to 6.1 atomic% excess resulted in the air we breathe.

Der Vorteil des erfindungsgemäßen Verfahrens liegt in der Möglichkeit auf nicht-invasivem Wege mittels in vivo Bestimmungen der endogenen NO-Produk­ tion den klinischen Verlauf von Erkrankungen (beispielsweise der persistierenden pulmonalen bzw. arteriellen Hypertension), den Einfluß von Medikamenten, die Wirkung der oralen und intravenösen Zufuhr des NO-Präcursors Arginin als Vasodilationsagens, die Wirkung von NO-Beimengungen zu Beatmungszwecken sowie den Einfluß der Ernährung bei Menschen und Säugetieren untersuchen zu können.The advantage of the method according to the invention lies in the possibility non-invasive way by means of in vivo determinations of the endogenous NO product tion the clinical course of diseases (for example the persistent pulmonary or arterial hypertension), the influence of drugs that Effect of oral and intravenous administration of the NO precursor arginine as Vasodilation agent, the effect of NO admixtures for ventilation purposes as well as investigating the influence of nutrition in humans and mammals can.

Ein Ausführungsbeispiel wird im folgenden näher beschrieben.An exemplary embodiment is described in more detail below.

Gesunden Erwachsenen im Alter von 28 bis 56 Jahren mit einem Körper­ gewicht von 54 bis 84 kg werden nach dem Frühstück um 08.00 Uhr 7,5 mg/kg L-[guanidino-15N2]Argininmonohydrochlorid (95 Atom-%) oral verabreicht.Healthy adults aged 28 to 56 years with a body weight of 54 to 84 kg are orally administered after breakfast at 8:00 a.m. 7.5 mg / kg L- [guanidino- 15 N 2 ] arginine monohydrochloride (95 atom%).

Die Ausatemluft wird im 30- bzw. 60-Minuten Rhythmus in 2-Liter Atemgasbeuteln über 12 Stunden fraktioniert gesammelt.The exhaled air becomes 2-liter every 30 or 60 minutes Breathing gas bags collected fractionally over 12 hours.

Zusätzlich wird der Harn zur Bestimmung der 15 N-Häufigkeiten im Ge­ samtstickstoff, Harnstoff, Ammoniak, Nitrit und Nitrat im 2- bzw. 4-Stunden Rhythmus über 24 Stunden quantitativ gesammelt.In addition, the urine is used to determine the 15 N frequencies in the Ge Velvet nitrogen, urea, ammonia, nitrite and nitrate in 2 or 4 hours Rhythm collected quantitatively over 24 hours.

Die 15N-Häufigkeiten im NO, N2O, NO2- und NO3 werden mittels GC- QMS gemessen (QP 2000, Shimadzu, Japan), während die 15N-Häufigkeiten in den Harnfraktionen Gesamt-N, Harnstoff und Ammoniak mittels IRMS (Europa Scientific, Crewe, U. K.) ermittelt werden.The 15 N frequencies in NO, N 2 O, NO 2 - and NO 3 are measured using GC-QMS (QP 2000, Shimadzu, Japan), while the 15 N frequencies in the urine fractions total N, urea and ammonia using IRMS (Europa Scientific, Crewe, UK) can be determined.

Die orale Verabreichung des 15N-markierten Arginins führt nach 30 bis 45 Minuten in der Atemluft zu maximalen 15NO-Markierungen von 4, 4 bis 6,1 Atom-%-exzess während die 15N-Markierungen im N2O erst nach 45 bis 90 Minuten mit 0,78 bis 1,43 Atom-%-exzess ihr Maximum erreichen. Die kumula­ tive 15NO-Exhalation liegt im Mittel bei 0,09 Promille während die renalen Gesamt-15N-Exkretionen zwischen 24 und 36% beträgt.Oral administration of the 15 N-labeled arginine leads to a maximum of 15 NO-markings of 4.4 to 6.1 atom% excess in the breathing air after 30 to 45 minutes, while the 15 N-markings in the N 2 O only after 45 up to 90 minutes with 0.78 to 1.43 atomic% excess. The cumulative 15 NO exhalation is on average 0.09 per thousand, while the total renal 15 N excretion is between 24 and 36%.

Claims (2)

1. Verfahren zur Bestimmung der [15N]Stickstoffmonoxid-Exhalation zur Diagnostik und Therapie bei Erkrankungen des Menschen dadurch gekenn­ zeichnet, daß
  • 1. 1.1. als Tracersubstanzen universell 15N-markierte Proteine aus Hefe und Algen, vorzugsweise L-[guanidino-15N2]Argininmonohydrochlorid (95 Atom-%), ver­ wendet werden,
  • 2. 1.2. die Messung der 15N-Häufigkeiten des NO im Atemgas mit einer Gaschro­ matographie-Quadrupolmassenspektrometrie Kopplung mit kryogener NO-Anrei­ cherung erfolgt.
1. A method for determining the [ 15 N] nitrogen monoxide exhalation for diagnosis and therapy in human diseases is characterized in that
  • 1.1.1. 15 N-labeled proteins from yeast and algae, preferably L- [guanidino- 15 N 2 ] arginine monohydrochloride (95 atom%), are used universally as tracer substances,
  • 2. 1.2. the 15 N frequencies of the NO in the breathing gas are measured using a gas chromatography-quadrupole mass spectrometry coupling with cryogenic NO enrichment.
2. Verfahren zur Bestimmung der [15N]Stickstoffmonoxid-Exhalation zur Diagnostik und Therapie bei Erkrankungen des Menschen nach Anspruch 1, dadurch gekennzeichnet, daß
  • 1. 2.1. mittels in vivo Bestimmungen der endogenen 15NO-Produktion auf nicht- invasivem Weg der klinische Verlauf von Erkrankungen vorzugsweise der persi­ stierenden pulmonalen bzw. arteriellen Hypertension beurteilt wird,
  • 2. 2.2. der Einfluß von Medikamenten auf die 15NO-Produktion ermittelt wird,
  • 3. 2.3. die Wirkung der oralen und intravenösen Zufuhr des NO-Präcursors Argi­ nin als Vasodilationsagens bestimmt wird,
  • 4. 2.4. die Wirkung von NO-Beimengungen zu Beatmungszwecken gemessen wird und
  • 5. 2.5. der Einfluß der Ernährung bei Menschen und Säugetieren auf die endogene 15NO-Produktion untersucht wird.
2. A method for determining the [ 15 N] nitrogen monoxide exhalation for diagnosis and therapy in human diseases according to claim 1, characterized in that
  • 1. 2.1. the clinical course of diseases, preferably persistent pulmonary or arterial hypertension, is assessed by means of in vivo determinations of endogenous 15 NO production in a non-invasive way,
  • 2. 2.2. the influence of drugs on 15 is determined NO production,
  • 3. 2.3. the effect of oral and intravenous administration of the NO precursor arginin is determined as a vasodilation agent,
  • 4. 2.4. the effect of NO additions for ventilation purposes is measured and
  • 5. 2.5. the influence of diet in humans and mammals on endogenous 15 NO production is investigated.
DE1997119098 1997-05-06 1997-05-06 Diagnostic determination of labelled nitric oxide in breath Expired - Fee Related DE19719098C1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001031334A1 (en) * 1999-10-26 2001-05-03 Diabact Ab Method and means for detecting inflammatory processes
EP1380838A2 (en) * 2002-07-11 2004-01-14 Ralph Gäbler Method of gas analysis for determining the reaction of a biological system
DE102004035916A1 (en) * 2004-07-23 2006-03-16 Prof. Dr. Karl-Heinz Gericke Technische Universität Braunschweig Institut für Physikalische und Theoretische Chemie Device, useful for isotopically determining nitrogen monoxide concentration in e.g. breathing air, comprises a UV source of light (emits a light of wavelength and electronically energizes isotope of nitrogen monoxide) and a light detector

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Castillo et al. Pediatr. Res. 1995, 38 S.17-24 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001031334A1 (en) * 1999-10-26 2001-05-03 Diabact Ab Method and means for detecting inflammatory processes
EP1380838A2 (en) * 2002-07-11 2004-01-14 Ralph Gäbler Method of gas analysis for determining the reaction of a biological system
EP1380838A3 (en) * 2002-07-11 2004-09-08 Ralph Gäbler Method of gas analysis for determining the reaction of a biological system
DE102004035916A1 (en) * 2004-07-23 2006-03-16 Prof. Dr. Karl-Heinz Gericke Technische Universität Braunschweig Institut für Physikalische und Theoretische Chemie Device, useful for isotopically determining nitrogen monoxide concentration in e.g. breathing air, comprises a UV source of light (emits a light of wavelength and electronically energizes isotope of nitrogen monoxide) and a light detector
DE102004035916B4 (en) * 2004-07-23 2007-10-18 Prof. Dr. Karl-Heinz Gericke Technische Universität Braunschweig Institut für Physikalische und Theoretische Chemie Method for the isotope-selective determination of nitrogen monoxide concentrations

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