Electrochemical Detection of Nitric Oxide in Biological Fluids METHODS IN ENZYMOLOGY, VOL. 396 ,2005 BARRY W. ALLEN, JIE LIU, and CLAUDE A. PIANTADOSI

Nitric Oxide in Blood Three isoforms of NO synthase (NOS) Furchgott R, Zawadzki J (1980). 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

Nitric Oxide in Blood NO-depentdent Vasodilator (Acetylcholine, Bradykinin) Sheer stress Inflamatory hypoxia

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

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 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

NO may be present in the blood in at least 2 active Forms

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)

Nitric Oxide in Blood (Adrian J. Hobbs ,2002)

Why Detection of NO in Blood Diseases or Conditions Associated with Abnormal NO Production and Bioavailability Hypertension Obesity Dyslipidemias (particularly hypercholesterolemia and hypertriglyceridemia) Diabetes (both type I and II) Heart failure Atherosclerosis Cigarette smoking Septicemia Etc.

Diseases or Conditions Associated with

Abnormal NO Production and Bioavailability

Hypertension

Obesity

Dyslipidemias (particularly hypercholesterolemia and hypertriglyceridemia)

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 enough to be used in vivo NO in biological fluids that are maintained in contact with a gaseous environment, R elease of NO from blood cells as they move between regions of high and low PO 2 levels

Electrochemistry

fluids in real time and in situ

NO electrodes can be made small enough to be used in vivo

NO in biological fluids that are maintained in contact with a gaseous environment,

R elease of NO from blood cells as they move between regions of high and low PO 2 levels

Materials and Methods Helix Diameter 1.85 mm 100 µ M in diameter , 3 mm long. S uspended a 20 µL drop of rabbit aortic blood

Electrodes P latinum wires, 100 µM in diameter M ultiwalled carbon nanotubes C oated with ruthenium C oated with Nafion

P latinum wires, 100 µM in diameter

M ultiwalled carbon nanotubes

C oated with ruthenium

C oated with Nafion

Electrode

Gas flow A ir–CO 2 mixture ( 20% O 2 , 5% CO 2 , 75% N 2 ) CO 2 –nitrogen mixture (5%CO 2 , 95% N 2 ) Gas flow was maintained at constant rat e

A ir–CO 2 mixture ( 20% O 2 , 5% CO 2 , 75% N 2 )

CO 2 –nitrogen mixture (5%CO 2 , 95% N 2 )

Gas flow was maintained at constant rat e

Blood Samples Rabbit aortic blood 3 m L C ontaining 7 units of lyophilized heparin kept on ice for up to 30 min before use.

Rabbit aortic blood 3 m L

C ontaining 7 units of lyophilized heparin

kept on ice for up to 30 min before use.

Chemical Reagents P repared 100 µ M solutions in deionized water ascorbate L-cystine 2,3-diphospho-D-glyceric acid (DPG) sodium nitrite sodium nitrate

P repared 100 µ M solutions in deionized water

ascorbate

L-cystine

2,3-diphospho-D-glyceric acid (DPG)

sodium nitrite

sodium nitrate

Electrochemical Methods A mperometry BAS 100 B/W potentiostat equipped + 675 mV (vs. Ag/AgCl,) T he electrodes were activated electrochemically by applying alternating potentials of 200 and 800 mV for 250 ms each at 500-ms intervals for a total of 120 s

A mperometry

BAS 100 B/W potentiostat equipped

+ 675 mV (vs. Ag/AgCl,)

T he electrodes were activated electrochemically by applying alternating potentials of 200 and 800 mV for 250 ms each at 500-ms intervals for a total of 120 s

T he data were not used the composite resistance of the electrochemical cell was m easured three times, final average was more than 10% greater than the initial average, fouled or that the blood drop had dried, T he bloodwas not fluid the drop did not fill the helix,

the composite resistance of the electrochemical cell was m easured three times, final average was more than 10% greater than the initial average, fouled or that the blood drop had dried,

T he bloodwas not fluid

the drop did not fill the helix,

Results Selectivity of the Sensor for Nitric Oxide 100 µ M solutions in deionized water

Selectivity of the Sensor for Nitric Oxide

Responses to Changing Gas Mixtures Control Change Gas mixed 350 s NO oxidation

Responses to Changing Gas Mixtures Responses to Changing Gas Mixtures NO oxidation signals were first detected from 200 to 400 s after the flowing gas was changed spike was followed by a continuous signal of 1–2 nA

Responses to Changing Gas Mixtures

NO oxidation signals were first detected from 200 to 400 s after the flowing gas was changed

spike was followed by a continuous 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

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

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 A highly sensitive electrochemical system can be designed to detect nM concentrations of NO activity F rom 200 to 400 s after a suspended drop of rabbit arterial blood was exposed to a decrease in ambient PO 2 a n experimental condition—hypoxia—in which hypoxemia could be induced in the captured blood drop.

A highly sensitive electrochemical system can be designed to detect nM concentrations of NO activity

F rom 200 to 400 s after a suspended drop of rabbit arterial blood was exposed to a decrease in ambient PO 2

a n experimental condition—hypoxia—in which hypoxemia could be induced in the captured blood drop.

Conclusions we did not measure the change in either ambient PO 2 or blood-drop PO 2 in this deliberately kept low in order to prevent drying of the blood drop, PO 2 will change slowly. we cannot assign this release to a particular source in the blood, for example, the red cells or the plasma

we did not measure the change in either ambient PO 2 or blood-drop PO 2 in this deliberately kept low in order to prevent drying of the blood drop, PO 2 will change slowly.

we cannot assign this release to a particular source in the blood, for example, the red cells or the plasma