measurement of nitric oxide - university of...
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Imagination at work.
GE Analytical Instruments
September 04, 2014
Measurement of Nitric Oxide
Nitric Oxide Synthase
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)
Arginine + O2 + NADPH -> NO + Citrulline + NADP
Nitric Oxide Analyzer
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
Reactions of NO
Reactions of NO
NO(aq) <-> NO(g)
KH~20
Gas Phase
NO + O2 -> NO2 + ½ O2
k≈10-38 cm6 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, NO2-, NO3
-, 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
NO + O3 -> NO2* + O2
NO2* -> NO2 + light (>600 nm)
Imagination at work.
Nitric Oxide Analyzer
NOA Applications – Liquids
Measurement Techniques
• Gas-Phase NO
• Solution NO
• Solution Nitrite & NO
• Total NO (NO, nitrite, nitrate and RSNO)
• Nitrosothiols
Nitrite Measurement
I- + NO2- +2H+
NO + ½I2 + H2O
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
2NO3- + 3V+3 + 2H2O
2NO + 3VO2+ + 4H+
Nitrate Measurement
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
Human Serum
Direct 28 µM
Std Add 30 µM
Human Plasma
Direct 17 µM
Std Add 18 µM
Standard Addition Experiment
Nitro Group slowly converted to NO
Reduction of Nitroarginines by VCl3 @ 90 °C
Measurement of Nitrosothiols
Three Techniques 1. Cu(I)/cysteine – Low Molecular Weight RSNO’s
2. I-/I2/H+ – Remove nitrite by chromatography or reaction
with Griess reagent
3. Quinone/Hydroquinone
• Prepare RSNO by reacting nitrite and thiol
• Use KCN/K3Fe(CN)6 to remove Fe-bound NO
Measurement of Fe-Bound NO
• Measure NO released with and without treatment with KCN/K3Fe(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 when sampling gas that contains <1 ppb NO (Zero Air Filter)
• Step 2: Measure signal when sampling gas of known NO concentration
KMnO4
Carbon
45 ppm NO
Accuracy of ppb Measurements with ppm Calibration
Persson, M.G. et. al., Lancet 1994, 343, 146-147
NO in Exhaled Breath
Controls Asthmatics Smokers
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 ()
P. Silkoff, et al., Am. J. Respir. Crit. Care Med. 1997, 155, 260-267.
NO PLAT ppb vs. Expiratory Flow
Kimberly, B.; et al.,Am. J. Respir. Crit Care Med. 1996 153, 829-836.
Nasal Contribution to Exhaled NO
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 H2O) to close vellum and eliminate nasal NO.
• Monitor and Display exhalation pressure
• Fixed flow rate 50 mL/s BTPS
(always report flow)
Bacterial
and Viral
Filter
NO and
Pressure
Ports
Restrictors
Restricted Exhaled Breath Kit
30 mL/s
250 mL/s
200 mL/s
150 mL/s
100 mL/s
50 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 Children
Duration (sec)
6 6
Plateau (sec)
3 2
Variation 3-5% 3-10%
2-5%
Changes in ENO
VISIT
0
20
40
60
80
100
120
1 2 3 4 5 6 7
INHALED STEROID
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 H2O)
• 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
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.
Silkoff,P.E., Sylvester, J.T., Zamel, N. Permutt, S. Am.
J. Respir. Crit. Care Med. 2000: 161;1218-1228.
Toukias, N.M., George, S. C. J. Appl. Physiol. 1998: 85; 653-666.
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
• DNO and DNOCW higher in
asthmatics vs. normal
subjects and DNO
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
Smoker (n=9)
Comparison of Models
CAlv
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
0
2
4
6
8
10
12
14
20 30 40 50 60 70 80 90
Pre
ss
ure
(to
rr)
Time (sec)
Bronchoconstrictor (Guinea Pig)
0
2
4
6
8
10
12
14
16
20 30 40 50 60 70 80 90
[NO
] (p
pb
)
Time (sec)
Exhaled NO - Horse
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0 10 20 30 40 50 60
[NO
] (p
pb
)
Time (sec)
Future Applications of Exhaled NO Measurements
• Research Use Only
• Diagnosis (Asthma, Rejection, Infections)
• Steroid Dosage
• Patient Compliance
NOAnalysis
• Liquid – Acquire Data, Name Samples, Integrate Peaks, Prepare Calibration Curves and Calculate Concentrations
• REB – Acquire Data, set NO plateaus, Save and Print Results
• Breath – Acquire Data, Calculate Max. Min and plateau for each breath and overall mean and std error.
• Bag – Acquire Data, set NO levels for bags, calculate average for replicates
• Support for Color Printers
• Improved Error Detection
• On-line Help
• File extensions
• Compatible with 2.07 and 3.00 Firmware
• CD with Windows 95/98/NT and Mac Os
NOAnalysis™ Version 3.21
REB
• Flowmeter
• New Results Printout and File Structure
• ATS Criteria
Liquid
• Manual Integration
• Names Tables
• Change Units after Calculations
Hardware and Electronics
Main Components
• Circuit Boards
»Power Supply Board
»Analog Board
»Digital Board
»PMT Amplifier Board
Main Components:
• PMT
• PMT Socket
• Cooler
• Red Pass filter
• Ozone Generator
• High Voltage Transformer
Routine Maintenance
• Vacuum pump
• Hopcalite/Moleculite Trap and Charcoal
Odor Filter
• Reaction Cell
• PMT Cooler
Vacuum Pump
PumpInlet
Oil Fil l Caps
Pump Outlet
Oil LevelSight Glass
Drain Plug
Oil Return/Gas Ballast
Power Switch
Changing Vacuum Pump Oil
• Open oil fill cap
• Position waste oil container under oil drain plug
• Slowly open drain plug until oil begins to drain
• Tilt pump to help drain all of the oil from the pump
• Oil can be disposed of at commercial motor oil recycling centers
• Reinstall drain plug
• Fill pump with ½ quart Mobil oil-view oil level in window on pump
• Fill pump with ½ quart Mobil oil-view oil level in window on pump
• Replace oil fill cap
Changing Hopcalite Trap and Charcoal Odor filter
• Turn NOA and pump OFF before proceeding
• Loosen hose clamps on both ends of the hopcalite trap
and one end of the charcoal odor filter old traps from
tubing
• Remove plastic caps from both ends of new filters
• Attach new filters
• Retighten hose clamps
• Reset the timer for the trap
Cleaning the Reaction Cell
Moving cooler to reach reaction cell
Reaction Cell as viewed from PMT side
Cooler Maintenance
• Over time cooler will pick up water from the atmosphere
• Water must be removed ~ 1 year by baking out cooler
Upgrade NOA 280 280i
• NOA Firmware installation instructions
• Configuration of firmware Version 3.00 for use in
the 280 NOA
• Installation of Flow meter cable
Troubleshooting
• Errors
• Warnings
• Miscellaneous problems
ERRORS
• E 01 Corrupted Setup Data (Very Rare)
• Cause: When NOA is started, the program checks the
data stored in the battery backed RAM. If there is a
problem with this data, E 01 is reported
• Solution: Check constants to verify they have not reset to
default. May need to change out battery back RAM.
ERRORS
• E 02 Cell Pressure was Above the Limit
• Cause: Cell pressure must be above 100 Torr for the
vacuum pump to start
• Solutions: Check power switch on pump, check power
cord is plugged in, check that all traps are secure. Go to
status screen to check cell pressure. If still low from a
previous run, loosen frit restrictor to bring cell pressure
above 100 Torr.
ERRORS
• E 03 Ozone Supply Pressure was Below the limit
• Cause: Oxygen supply must be above 4 psi
• Solutions: Be sure tank is not empty, check that regulator
outlet flow is between 10psi-100psi, check tubing
between regulator and oxygen inlet and check adjustment
of the gas regulator (black knob) on the back of the
analyzer
ERRORS
• E 04 Did not detect ozone
• Cause: During start-up test NOA looks for an increase in
signal. If there is no increase in signal, E 04 will be
reported.
• Solutions: Check ozone fuse, loose cable from PMY
socket to amp board, loose connection on ribbon cable
from amplifier to analog board. Also could require new
ozone generator, new High Voltage transformer
E 04 – More on Troubleshooting
• Could be caused by a bad PMT
• Check by tricking the analyzer into starting
(disconnecting signal cable from amp board),
starting the analyzer, count to 15 reconnect
cable.
ERRORS
• E 05 Cooler Temperature Above the Limit
• Cause: If cooler temperature is > 0 C, the E 05 is
reported.
• Solutions: Check temperature of cooler on status screen.
If temperature is not decreasing it may be the cooler fuse.