DE4224901A1 - Determn. of nitrite formation in biochemical reactions - by using stable isotope nitrogen =15 as marker and determining its content in reaction prods. using mass spectroscopy - Google Patents
Determn. of nitrite formation in biochemical reactions - by using stable isotope nitrogen =15 as marker and determining its content in reaction prods. using mass spectroscopyInfo
- Publication number
- DE4224901A1 DE4224901A1 DE19924224901 DE4224901A DE4224901A1 DE 4224901 A1 DE4224901 A1 DE 4224901A1 DE 19924224901 DE19924224901 DE 19924224901 DE 4224901 A DE4224901 A DE 4224901A DE 4224901 A1 DE4224901 A1 DE 4224901A1
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- Germany
- Prior art keywords
- nitrogen
- isotope
- reaction
- labeled
- nitrite
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- 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.)
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Classifications
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- 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
Abstract
Description
Die Erfindung betrifft eine Anordnung und ein Verfahren zur Be stimmung des Nitrosierungspotentials in einem biochemischen Reak tionssystem durch Anwendung der 15N-Markierungstechnik.The invention relates to an arrangement and a method for loading tuning the nitrosation potential in a biochemical reac tion system using the 15N marking technique.
Mit der Erarbeitung isotopentechnischer Routineverfahren und der Entwicklung leistungsfähiger isotopenanalytischer Geräte hat die Tracertechnik mit stabilen Isotopen in zunehmendem Male Anwendun gen bei der Untersuchung biochemischer Reaktionssysteme gefunden.With the development of routine isotope technology and the The company has developed powerful isotope analytical devices Tracer technique with stable isotopes increasingly used gene found in the study of biochemical reaction systems.
Der Vorteil des Arbeitens mit stabilen Isotopen liegt besonders darin, daß keine Strahlenbelastungen für Experimentator und Umwelt auftreten und die Untersuchungen aus isotopen-analytischer Sicht zeitlich unbegrenzt sind. Die Markierung z. B. des Stickstoffs durch sein stabiles Isotop 15N (Markierung bedeutet hier die Erhö hung des Anteils des selteneren Isotops) gestattet es, das Schick sal des Stickstoffs bei biochemischen Reaktionsabläufen zu verfol gen bzw. endogenen von exogenem Stickstoff (z. B. Luftstickstoff) zu unterscheiden.The advantage of working with stable isotopes is particularly great in that no radiation exposure to the experimenter and the environment occur and the investigations from an isotope-analytical point of view are unlimited in time. The marking z. B. the nitrogen due to its stable isotope 15N (marking here means the increase The share of the rarer isotope) allows the chic Sal of nitrogen in biochemical reactions genes or endogenous of exogenous nitrogen (e.g. atmospheric nitrogen) to distinguish.
Das Ziel der Erfindung ist die störungsfreie bzw. nichtinvasive Bestimmung des Nitrosierungspotentials anhand kleinster endogen gebildeter Stickstoffmengen in einem biochemischen Reaktions system, die mit keiner anderen nichtisotopischen Methode meßbar sind.The aim of the invention is trouble-free or non-invasive Determination of the nitrosation potential using the smallest endogenous Amounts of nitrogen formed in a biochemical reaction system that cannot be measured with any other non-isotopic method are.
Der Erfindung liegt die Aufgabe zugrunde, ein Verfahren anzugeben, mit dem das Nitrosierungspotential eines biochemischen Reaktions systems über seine endogene Stickstoffproduktion quantitativ ge messen werden kann. Eine radioaktive Methode mit dem Stickstoff isotop 13N ist zwar prinzipiell denkbar, hat aber gegenüber der Stabilisotopen-Methode entscheidende Nachteile (Radioaktivität, Kurzlebigkeit des Isotops 13N [Halbwertszeit: 10 min.]).The invention has for its object to provide a method with which the nitrosation potential of a biochemical reaction systems quantitatively about its endogenous nitrogen production can be measured. A radioactive method with the nitrogen In principle, isotope 13N is conceivable, but compared to Stabilisotope method decisive disadvantages (radioactivity, Short life span of the isotope 13N [half-life: 10 min.]).
Das erfindungsgemäße Verfahren besteht darin, daß bei dem zu un tersuchenden biochemischen Reaktionssystem zunächst der im Reak tionsraum befindliche Luftstickstoff durch ein Spül- und Trägergas ausgetauscht wird.The method according to the invention is that at the un investigating biochemical reaction system first in the reac air nitrogen through a purge and carrier gas is exchanged.
Danach wird eine entsprechende stabilisotop 15N-markierte Verbin dung in das biochemische Reaktionssystem einmalig, in zeitlicher Staffelung oder kontinuierlich eingebracht und anschließend die 15N-Isotopenhäufigkeit des bei Vorhandensein reaktionsfähiger Verbindungen (salpetrige Säure aus Nitrit und aliphatische Amino verbindungen) sich bildenden endogenen gasförmigen Stickstoff s zeitabhängig massenspektrometrisch gemessen. Then a corresponding stable isotope 15N-labeled verb in the biochemical reaction system once, in time Graduated or continuously introduced and then the 15N isotope frequency of the more reactive in the presence Compounds (nitrous acid from nitrite and aliphatic amino compounds) forming endogenous gaseous nitrogen s measured time-dependent mass spectrometry.
Zwei Ausführungsbeispiele der Erfindung sind in der Zeichnung dargestellt und werden im folgenden näher beschrieben.Two embodiments of the invention are in the drawing are shown and are described in more detail below.
1. Mit Hilfe der stabilisotopen Tracertechnik soll bei der Umset zung von Harnstoff mit salpetriger Säure die endogene Stickstoff- Produktion quantitativ nach einem nichtvolumetrischen Verfahren bestimmt werden.1. With the help of the stable isotopic tracer technique should be implemented of urea with nitrous acid the endogenous nitrogen Production quantitatively using a non-volumetric process be determined.
Zunächst werden die Reaktionspartner (1 mmol Harnstoff und 2 mmol Natriumnitrat) sowie 0,01 mmol 15N-markierter Harnstoff (95 At.-%) in den Reaktionsraum 6 gebracht (Fig. 1). Danach wird der im Reaktionsraum befindliche Luftstickstoff durch ein Edelgas, z. B. Argon, verdrängt.First, the reactants (1 mmol of urea and 2 mmol of sodium nitrate) and 0.01 mmol of 15N-labeled urea (95 at%) are brought into the reaction space 6 ( FIG. 1). Then the atmospheric nitrogen in the reaction chamber is replaced by an inert gas, e.g. B. argon, displaced.
Die Umsetzung kann dann durch das Hinzufügen von 4 bis 5 mmol halbkonzentrierter Salzsäure in Gang gesetzt werden. Der entstehende Stickstoff wird mittels des Edelgases als Träger gas in einen Exetainer (= luftdicht verschließbares Probengefäß mit einem Septum) überführt. (Eine quantitative Überführung des Stickstoffs ist dabei nicht erforderlich!)The reaction can then be done by adding 4 to 5 mmol semi-concentrated hydrochloric acid. The nitrogen produced is used as a carrier by means of the noble gas gas into an Exetainer (= airtight sealable sample container with a septum). (A quantitative transfer of the Nitrogen is not required!)
Mit Hilfe einer massenspektrometrischen Messung wird die 15N- Häufigkeit des Stickstoffs aus den Exetainern bestimmt.With the help of a mass spectrometric measurement, the 15N Frequency of nitrogen determined from exetainers.
Aus der Isotopenverdünnung wird die endogen gebildete Stickstoff menge berechnet.The endogenously formed nitrogen becomes from the isotope dilution quantity calculated.
2. In einer biologischen Flüssigkeit (Speichel) soll der Nitrit gehalt mit Hilfe der Isotopenverdünnungstechnik bestimmt werden.2. In a biological fluid (saliva) the nitrite content can be determined using the isotope dilution technique.
Unter Verwendung der in Fig. 1 dargestellten Reaktionsapparatur werden zu 2 ml Speichel 1 µmol 15N-markiertes Natriumnitrit (10 At.-%) gegeben. (Reaktionspartner, z. B. aliphatische Verbindungen mit primären Aminogruppen sind ausreichend im Speichel vorhanden und brauchen deshalb nicht hinzugefügt werden.) Danach wird der unmarkierte Luftstickstoff mittels eines durch den Reaktionsraum geleiteten Argonstromes aus dem Reaktionsraum verdrängt. Die Reak tion wird durch Ansäuern mit 1 mmol halbkonzentrierter Salzsäure gestartet.Using the reaction apparatus shown in FIG. 1, 1 μmol of 15N-labeled sodium nitrite (10 at.%) Is added to 2 ml of saliva. (Reaction partners, for example aliphatic compounds with primary amino groups, are sufficiently present in the saliva and therefore do not need to be added.) The unlabeled atmospheric nitrogen is then displaced out of the reaction space by means of an argon stream passed through the reaction space. The reaction is started by acidification with 1 mmol of semi-concentrated hydrochloric acid.
Die nachfolgenden Arbeitsschritte erfolgen wie unter Anwendungs beispiel 1 beschrieben.The following steps are the same as under Application Example 1 described.
Claims (4)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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DE19924224901 DE4224901A1 (en) | 1992-07-28 | 1992-07-28 | Determn. of nitrite formation in biochemical reactions - by using stable isotope nitrogen =15 as marker and determining its content in reaction prods. using mass spectroscopy |
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DE19924224901 DE4224901A1 (en) | 1992-07-28 | 1992-07-28 | Determn. of nitrite formation in biochemical reactions - by using stable isotope nitrogen =15 as marker and determining its content in reaction prods. using mass spectroscopy |
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DE4224901A1 true DE4224901A1 (en) | 1994-02-03 |
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DE19924224901 Ceased DE4224901A1 (en) | 1992-07-28 | 1992-07-28 | Determn. of nitrite formation in biochemical reactions - by using stable isotope nitrogen =15 as marker and determining its content in reaction prods. using mass spectroscopy |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111983007A (en) * | 2020-08-27 | 2020-11-24 | 上海化工研究院有限公司 | For determining nitric acid or nitrate15Method and apparatus for N isotope abundance |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4015936A (en) * | 1975-08-26 | 1977-04-05 | Hiryokagaku Kenkyusho | Method for determination of total nitrogen and heavy nitrogen content |
DE2630419A1 (en) * | 1976-07-02 | 1978-01-05 | Schering Ag | (15,16)-Tritium labelled steroid derivs. - for radioimmunoassay of oral contraceptive blood levels |
DD205011A1 (en) * | 1982-04-12 | 1983-12-14 | Adw Der Ddr Zi Fuer Isotopen U | MARKING PLUG FOR STABLE ISOTOP N-15 |
DE3341806A1 (en) * | 1983-11-19 | 1985-05-30 | Glossmann, Hartmut, Prof.Dr.med., 6301 Heuchelheim | RADIOACTIVE WITH (UP ARROW) 1 (UP ARROW) (UP ARROW) 2 (UP ARROW) (UP ARROW) 5 (UP ARROW) I-LABELED 1,4-DIHYDROPYRIDINE DERIVATIVES AND 1,4-DIHYDROCHINOLINE DERIVATIVES, THEIR PRODUCTION AND YOUR PRODUCTION VERIFICATION OF MEDICINAL PRODUCTS |
DD260334A1 (en) * | 1987-05-08 | 1988-09-21 | Leipzig Duengungsforschung | METHOD FOR MEASURING RELEASED GASFOERMIGIC NITROGEN COMPOUNDS FROM BIOLOGICAL SUBSTRATES |
DE3814515A1 (en) * | 1988-04-29 | 1989-11-23 | Juergen Prof Dr Schrader | EVIDENCE OF REGIONAL MYOCARDICAEMIA |
-
1992
- 1992-07-28 DE DE19924224901 patent/DE4224901A1/en not_active Ceased
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4015936A (en) * | 1975-08-26 | 1977-04-05 | Hiryokagaku Kenkyusho | Method for determination of total nitrogen and heavy nitrogen content |
DE2630419A1 (en) * | 1976-07-02 | 1978-01-05 | Schering Ag | (15,16)-Tritium labelled steroid derivs. - for radioimmunoassay of oral contraceptive blood levels |
DD205011A1 (en) * | 1982-04-12 | 1983-12-14 | Adw Der Ddr Zi Fuer Isotopen U | MARKING PLUG FOR STABLE ISOTOP N-15 |
DE3341806A1 (en) * | 1983-11-19 | 1985-05-30 | Glossmann, Hartmut, Prof.Dr.med., 6301 Heuchelheim | RADIOACTIVE WITH (UP ARROW) 1 (UP ARROW) (UP ARROW) 2 (UP ARROW) (UP ARROW) 5 (UP ARROW) I-LABELED 1,4-DIHYDROPYRIDINE DERIVATIVES AND 1,4-DIHYDROCHINOLINE DERIVATIVES, THEIR PRODUCTION AND YOUR PRODUCTION VERIFICATION OF MEDICINAL PRODUCTS |
DD260334A1 (en) * | 1987-05-08 | 1988-09-21 | Leipzig Duengungsforschung | METHOD FOR MEASURING RELEASED GASFOERMIGIC NITROGEN COMPOUNDS FROM BIOLOGICAL SUBSTRATES |
DE3814515A1 (en) * | 1988-04-29 | 1989-11-23 | Juergen Prof Dr Schrader | EVIDENCE OF REGIONAL MYOCARDICAEMIA |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111983007A (en) * | 2020-08-27 | 2020-11-24 | 上海化工研究院有限公司 | For determining nitric acid or nitrate15Method and apparatus for N isotope abundance |
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