electrochemical detection of nitric oxide in biological fluids
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Jaunal club 2 :Electrochemical Detection Of Nitric Oxide In Biological FluidsTRANSCRIPT
Electrochemical Detection of NitElectrochemical Detection of Nitric Oxide in Biological Fluidsric Oxide in Biological Fluids
METHODS IN ENZYMOLOGY, VOL. 396 ,2005
BARRY W. ALLEN, JIE LIU, and CLAUDE A. PIANTADOSI
Nitric Oxide in Blood
Name Description
Neuronal NOS (nNOS or NOS1)
Produces NO in neuronal tissue in both the central and peripheral nervous system.
Inducible NOS (iNOS or NOS2)
Can be found in the immune system used by macrophages in immune defence against pathogens.
Endothelial NOS (eNOS or NOS3 or Constitutive / cNOS)
Generates NO in blood vessels and is involved with regulating vascular function
Three isoforms of NO synthase (NOS)
Furchgott R, Zawadzki J (1980).
Nitric Oxide in Blood•NO-depentdent Vasodilator (Acetylcholine, Bradykinin)•Sheer stress•Inflamatory•hypoxia
Nitric Oxide in Blood
• NO has a half-life of about 4 s in biological fluids and is oxidized to nitrite and nitrate anions
Nitric Oxide in Blood
NO may be present in the blood in at least 2 active Forms
1. Aqueous form as a dissolved gas The half life of aqueous NO in red cell-free
plasma in vitro is around 1 min (Rassaf et al., 2002).
2. Nitrosothiols or RSNOs
Nitric Oxide in Blood
(oxyhaemoglobin) (methaemoglobin) (nitrate)
J. P. Wallis (2005)
(Adrian J. Hobbs ,2002)
Nitric Oxide in Blood
Why Detection of NO in Blood
Diseases or Conditions Associated with Abnormal NO Production and Bioavailability• Hypertension• Obesity• Dyslipidemias (particularly hypercholesterolemia and hyp
ertriglyceridemia)• Diabetes (both type I and II)• Heart failure• Atherosclerosis • Cigarette smoking• Septicemia • Etc.
Introduction
• Electrochemistry– fluids in real time and in situ
• NO electrodes can be made small eno ugh to be used in vivo
• NO in biological fluids that are maint ained in contact with a gaseous envir
onment,• R elease of NO from blood cells as the
y move between regions of high and l ow PO
2 levels
Materials and Methods
Helix Diameter 1.
85mm
100 µM MM MMMM MMMM , 3 .mm long
S uspended a20 µL
drop of rabbit aortic blood
Electrodes
•P 100latinum wires, µM MM MMMM MMMM
•M ultiwalled carbon nanotubes•CMMMMM MMMM MMMMMMMMM•C oated with Nafion
Electrode
Gas flow
•Air–CO2
M( 20% O2
, 5% CO2
, 75% N2
)
•CO2
–nitrogen mixture (5%CO
2 , 95% N
2)
MMM MMMM MMM MMMMMMMMMM MM MMMMMMMM MMMe
Blood Samples
• 3Rabbit aortic blood mL•C M MMMMM MM MMMM7
MMMMMMM MMMMMMM • 30keptonicef or up t o mi n bef or e use.
Chemical Reagents
•P 100repared µM MMMMMMMMM MM M eionized water
–ascorbate–M-MMMMMMM– -MMMMMMMMM-M -MMMMMMMM MMMM 2 ,3(DPG)
– sodi umni tri te– sodium nitrate
Electrochemical Methods
•Amperometry•BAS 100 B/W potentiostat equipped
•+ 675 mV (vs. Ag/AgCl,)•T he electrodes were activated e
lectrochemically by applying altern ating potentials of 200 and 800
- mV for 250 ms each at 500 ms in tervals for a total of 120 s
The data were not used
•the composite resistance of the electrochemical cell was m easured three times, final avera
ge was more than 1 0 % greater t hanthei ni t i al average,foul edor tha tthe bl ooddrophaddri ed,
•T he bl oodwas not fl ui d• thedropdi dnot fi l l the hel i x,
Results
• Selectivity of the Sensor for Nitric Oxide
1 0 0 µ M solutions in deionized water
Responses to Changing GMM M MMMMMMM
Change Gas mixed
350 s
NO oxidation
Control
Responses to Changing Gas Mixtures
• Responses to Changing Gas Mixtures– NO oxidation signals were first
detected from 200 to 400 s aft er the flowing gas was change
d– spike was followed by a continuou
s signal of 1–2 nA
Discussion
• The blood-drop preparation described here may represent a useful approach for further investigation of the response of NO levels
Discussion
• limiting the potential or by applying coatings to the electrode that exclude species that have certain characteristics of charge or size
• always useful to confirm anyexperimental result by using other nonelectrochemical methods
Conclusions•M MMMMMM MMMMMMMMM MMMMMMMMMMMMM
MM MMMMMM MMM MM MMMMMMMM MM MMMMM t nMconcentrati ons of NOacti vi t
y•F 200 400rom to s after a suspen
deddropof rabbi t arteri al bl ood was exposed to a decrease in ambie
MM MM2
•a nexperimentalcondition—hypoxia—inwhi chhypoxemi a coul dbe i nMMMMM MM MMM MMMMMMMM MMMMM MMMMM
Conclusions
•we did not measure the change in either ambient PO2 or blood-drop PO2 in this deliberately kept low in order to prevent drying of the blood drop, PO2 will change slowly.
• we cannot assign this release to a pa rticular source in the blood, for exam
ple, the red cells or the plasma