Measurement of Nitric Oxide GE Analytical Instruments September 04, 2014 Imagination at work.
Nitric Oxide Synthase Argini inine ne + O 2 + NADPH H -> NO + Citru rull llin ine + NADP DP Type I (nNOS) – Neurotransmitter, intestinal motility, cerebral blood flow, LTP, pain. • Chromosome 12, 150 kb, 29 exons (160 kDa) Type II (iNOS) – Host defense, inflammation, apoptosis. • Chromosome 17, 37 kb, 26 exons (130 kDa) Type III (eNOS) – Regulates vascular tone, inhibits platelet aggregation, neutrophil infiltration, smooth muscle proliferation • Chromosome 7, 21-22 kb, 26 exons (130 kDa)
Type I (nNOS) Neurotransmitter, intestinal • motility, cerebral blood flow, LTP, pain Type II (iNOS) Host defense, inflammation, • apoptosis Type III (eNOS) Regulates vascular tone, • inhibits platelet aggregation, neutrophil infiltration, smooth muscle proliferation Nitric Oxide Analyzer
Nitric Oxide Analyzer
Reactions of NO
Reactions of NO NO (aq) <-> NO (g) K H ~20 Gas Phase NO + O 2 -> NO 2 + ½ O 2 k≈10 -38 cm 6 molecules -2 s -1
Consequences of NO Reactions • NO has a short half-life (seconds) and is present at low concentrations (nM). • NO is rapidly converted to nitrite, nitrate and RSNO. • Most researchers measure stable reaction products
Techniques for NO Measurement • Direct - Measure NO, NO 2 - , NO 3 - , RSNO. • Indirect -cGMP • Citrulline Assays • NOS Inhibitors • Bioassays
Direct Measurement Techniques • Chemiluminescence - Ozone, Luminol • Electrochemical - NO Electrodes, Fuel Cells • Spectrophotometric/Fluorometric - EPR, Griess Reaction, DAN, DAF-2 • Chromatographic - Capillary Electrophoresis, Ion Chromatography
Installation Vacuum pump NOA 280i Purge Vessel Hot Water Bath Restricted Exhaled Breath Kit Bag Kit
Theory of Operation
Chemiluminescence * + O 2 NO + O 3 -> NO 2 * -> NO 2 + light (>600 nm) NO 2
Nitric Oxide Analyzer Imagination at work.
NOA Applications – Liquids
Measurement Techniques • Gas-Phase NO • Solution NO • Solution Nitrite & NO • Total NO (NO, nitrite, nitrate and RSNO) • Nitrosothiols
Nitrite Measurement I - + NO 2 - +2H + NO + ½I 2 + H 2 O
Nitrite Injections
Nitrite Calibration • Prepare Stock Solution of 100 mM. • Make dilutions fresh each day. • Periodically inject standards to make sure response has not changed.
Nitrite Measurement Applications • Cell Culture Media • Serum/Plasma • Perfusates, Tissue Bath Fluid
Vaso-Active Peptides Gastric Smooth Muscle Cells
Nitrate Measurement 2NO 3 - + 3V +3 + 2H 2 O + + 4H + 2NO + 3VO 2 NaOH Trap
Nitrate Measurement Applications • Serum/Plasma, Urine, CS Fluid • BAL Fluid, Breath Condensate • Cell Culture Media • Perfusates, Tissue Bath Fluid • Tissue Homogenates
Serum Nitrate Measurement
Standard Addition Experiment Human Serum Human Plasma Direct 28 µM Direct 17 µM Std Add 30 µM Std Add 18 µM
Reduction of Nitroarginines by VCl 3 @ 90 ° C Nitro Group slowly converted to NO
Measurement of Nitrosothiols Three Techniques Cu(I)/cysteine – Low Molecular Weight RSNO’s 1. I-/I 2 /H + – Remove nitrite by chromatography or reaction 2. with Griess reagent Quinone/Hydroquinone 3. • Prepare RSNO by reacting nitrite and thiol • Use KCN/K 3 Fe(CN) 6 to remove Fe-bound NO
Measurement of Fe-Bound NO • Measure NO released with and without treatment with KCN/K 3 Fe(CN) 6 • Remove nitrite by chromatography • Gladwin, M.T., et al., PNAS 2000;97(21):11482-11487.
NOA Applications – Gas
NOA Gas Measurements • Exhaled Breath • NO and NO2 in Ventilator Circuits • Headspace Analysis • NO Production from other Organs
Calibration • Use gas sampling package to equalize humidity • Step 1: Measure signal KMnO4 when sampling gas that Carbon contains <1 ppb NO (Zero Air Filter) 45 ppm NO • Step 2: Measure signal when sampling gas of known NO concentration
Accuracy of ppb Measurements with ppm Calibration
NO in Exhaled Breath Controls Asthmatics Smokers Persson, M.G. et. al., Lancet 1994, 343, 146-147
Exhaled Nitric Oxide • NO is produced in the airways and paranasal sinuses • [NO] is increased during lung inflammatory states • Abnormal NO in Asthma, Infection, Transplant Rejection ( ); Kartagener's syndrome, HIV, CF ( ) • [NO] decreases with inhaled glucocortico- steroids • May be useful indicator of airway inflammation
Factors Influencing [NO] in Exhaled Breath • Expiratory Flow Rate • Nasal Contamination • Inspiratory [NO] • Inflammation • PEEP ( ), Hypercapnia ( ) • Hypoxia ( )
NO PLAT ppb vs. Expiratory Flow P. Silkoff, et al., Am. J. Respir. Crit. Care Med. 1997, 155, 260-267.
Nasal Contribution to Exhaled NO Kimberly, B.; et al.,Am. J. Respir. Crit Care Med. 1996 153, 829-836.
Effect of Inspired NO
Techniques for the Measurement of Exhaled Nitric Oxide • Restricted Exhaled Breath • Single Breath (Mylar Balloons) • Breath-by-Breath (Mouthpiece, Face Mask) • Nasal • Ventilator • Bronchoscope
Standard Techniques for Measurement of Exhaled NO • Adults and Children (<12 years old) • On-Line • Off-Line • Nasal • Instrument Requirements Am. J. Respir. Crit. Care Med. Vol 160, 1999.
On-Line Exhaled NO • Expiratory resistance (>5 cm H 2 O) to close vellum and eliminate nasal NO. • Monitor and Display exhalation pressure • Fixed flow rate 50 mL/s BTPS (always report flow)
Restricted Exhaled Breath Kit Restrictors Bacterial 30 mL/s and Viral Filter 50 mL/s 100 mL/s NO and 150 mL/s Pressure Ports 200 mL/s 250 mL/s
Thermal Mass Flowmeter • Permits direct measurement of exhaled flow rate Calibrate using • calibration syringe
On-Line Exhaled NO
Nitric Oxide Analyzer On-Line Exhaled NO Adults ults Chil ildren dren Durat atio ion 6 6 (sec) c) Plateau eau 3 2 (sec) c) 3-10% 10% Variatio iation 3-5% 5% 2-5% 5%
Changes in ENO 120 120 100 100 80 80 60 60 40 40 20 20 INHALED STEROID 0 VISIT IT 1 2 3 4 5 6 7 Silkoff, P. E. et al., J. Asthma 1998; 35 (6) 473-479
Off-Line Exhaled NO • Collect Exhaled Breath in Mylar Balloon • ATS Recommendations (Adults and Children) » Oral TLC inhalation of low NO air » Vital Capacity Exhalation at 350 mL/s ± 10% • Analyze balloons within 8-12 hours
Off-Line Exhaled NO • Advantages » Subject does not have to be near analyzer » Large number of samples can be collected for later analysis • Disadvantages » No real-time feed back on validity of data » Samples have limited lifetime
Bag Kits for Off-Line Collection • Vital Capacity » ATS 350 mL/s • Dead space Discard » 50 mL/s
Stability of NO in Mylar Bags Vital Capacity Dead space Discard
Measurement of Nasal Nitric Oxide • Transnasal Air Flow Rate 3 L/min » Vacuum Pump or Compress Air » Side-Stream Sampling to NOA • Vellum Closure by Oral Exhalation (>10 cm H 2 O) • Steady Plateau within 20 seconds • Use higher flows if necessary (3-6 L/min)
Nasal NO Measurement
Exhaled NO - Intubated Asthmatic
Models for Exhaled NO Toukias, N.M., George, S. C. J. Appl. Physiol. 1998: 85; 653-666. Silkoff,P.E., Sylvester, J.T., Zamel, N. Permutt, S. Am. J. Respir. Crit. Care Med. 2000: 161;1218-1228. Pietropauoli, A.P., Perillo, I.B., Torres, A., Perkins, P.T., Frasier, L.M., Utell, M.J., Frampton, M.W., and Hyde, R.W. J Appl Physiol 1999;87(4):1532-1542.
Using ENO Models • Measure On-Line Exhaled NO and Flow at Different Flow Rates • Use Non-linear Regression to Calculate Parameters from ENO vs. Flow • D NO and D NO C W higher in asthmatics vs. normal subjects and D NO unchanged with steroid treatment
Using ENO Models • Measure On-Line Exhaled NO and Flow at > 59 mL/s • Use linear Regression to Calculate Parameters from ENO* Flow vs. Flow
ENO Models Repeatability Comparison of Models CAlv Smoker (n=9)
Measurement of Exhaled Nitric Oxide in Animals Ventilated Animals (intubated or direct • connection to trachea) • Chamber Sampling Spontaneous Breathing • No Nasal NO (except primates) •
Breath-by-Breath (Rat)
LPS Treated Rat
Bronchoconstrictor (Guinea Pig) 16 14 12 10 [NO] (ppb) 8 6 4 2 0 20 30 40 50 60 70 80 90 Time (sec) 14 12 Pressure (torr) 10 8 6 4 2 0 20 30 40 50 60 70 80 90 Time (sec)
Exhaled NO - Horse 5.0 4.5 4.0 3.5 [NO] (ppb) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 10 20 30 40 50 60 Time (sec)
Future Applications of Exhaled NO Measurements Research Use Only • • Diagnosis (Asthma, Rejection, Infections) • Steroid Dosage Patient Compliance •
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