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.