Current Protocols in Toxicology
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This Featured Protocol presents the full text of a cutting-edge Unit from Current Protocols in Toxicology, including expert commentary sections with critical information designed to ensure the success of your experiments.3000099 From UNIT 9.2 Detection of Heme Oxygenase Activity by Measurement of CO

Contributed by Contributed by Hendrik J. Vreman and David K. Stevenson
Stanford University Medical Center
Stanford, California

Heme oxygenase (HO, E.C. 1.14.99.3) is the first and rate-limiting enzyme in the heme degradation pathway. In the presence of NADPH-cytochrome P-450 reductase, HO catalyzes the following reaction, producing equimolar amounts of carbon monoxide (CO) and biliverdin:

The biliverdin is immediately reduced to bilirubin, the pigment associated with jaundice. HO exists as two active isozymes, the inducible HO-1 and the constitutive HO-2. A third isozyme (HO-3) is ~90% identical to HO-2 in its amino-acid sequence but shows little HO activity (McCoubrey et al., 1997).

HO activity can be assayed by several methods, most of which rely on measurements of the rate of product formation. Bilirubin formation can be monitored in purified microsomal preparations by a coupled enzyme reaction using biliverdin reductase (see UNIT 9.3). Such assays require labor-intensive sample preparation and involve spectrophotometric quantification of the bilirubin formed in the aqueous phase or extracted into an organic phase.

A more direct procedure, described in this unit, involves the quantification, by gas chromatography, of HO-generated CO. This method is specific, sensitive, reproducible, and simple. CO determination can be used to study HO activity in all types of tissue at various stages of tissue fractionation and in tissue slices. The assay is also useful for studying, in vitro, the effects of various HO inhibitors, including the highly colored metalloporphyrin derivatives of heme.

HO activity in preparations from animal or plant tissue can be determined by measuring the production of CO. The protocol described in this unit measures the CO produced in a sealed reaction vial following the interaction of HO in a tissue preparation with hemin and NADPH.

Tissue homogenates are centrifuged at 13,000 × g and the supernatants (or other fractions purified to a greater or lesser extent) are incubated for 15 min at 37°C with 50 µM methemalbumin as substrate in the absence (blank) or presence (total) of NADPH in septum-sealed, CO-free vials. The reaction is terminated by quick-freezing samples to -78°C. The CO thus produced diffuses into the headspace of the reactor, where it can be quantified by gas chromatography (GC) on a molecular-sieve column with a reduction gas detector.

HO activity is expressed as nanomoles of CO produced per hour per milligram of protein. The method allows analysis of as little as 2 µl of rat tissue homogenate (20% w/v), prepared from 0.4 mg of liver (~40 µg total protein), for example. The assay measures the CO produced by all HO isozymes present in the sample.

When analyzing a large number of samples in 1 day, having two analysts work in conjunction can increase the sample throughput rate to up to ~300 vials per day. One analyst can isolate the tissues, perform the HO assays, and determine the amount of protein in the samples, while the other processes the tissues, prepares the samples, calibrates the instrument, and analyzes the CO content of the samples.

NOTE: Because CO can be produced through photooxidative reactions between organic molecules in the samples and endogenous (e.g., riboflavin) or exogenous photooxidizers (e.g., metalloporphyrins), it is important that the steps involving CO generation and quantification be performed under conditions of reduced light.

Materials

Tissue or cell samples

0.1 M potassium phosphate, pH 7.4 (APPENDIX 2A)

0.9% (w/v) NaCl (optional, APPENDIX 2A)

HO substrate (see recipe)

4.5 mM NADPH (see recipe)

NADPH-cytochrome P-450 reductase (optional)

Anhydrous magnesium perchlorate (anhydrone; Fisher)

GC calibration gas: 10.8 µl CO/liter air (Scott Specialty Gases)

CO gas, 99.9% pure (e.g., Matheson Gas Products), optional

12 × 32-mm amber vials with polypropylene screw caps fitted with septa (e.g., Alltech Associates)

Vial racks (e.g., Fisher or equivalent)

Hamilton gas-tight syringe with repeating dispenser

2.5-mm-thick blue silicone sheets (Alltech Associates), for use in making septa

Hopcalite (CuO/MnO) catalytic converter (Trace Analytical)

Vial-purging assembly (see Fig. 9.2.1A)

Headspace-sampling assembly (see Fig. 9.2.1B)

0.0625-in. (1.59-mm) o.d. sleeve connector

18-G side-port needles, 5 and 7 cm

68 × 0.53-cm (i.d.) stainless steel column

40- to 60-mesh molecular sieve, 13X (Alltech Associates)

Gas-flow meter (J & W Scientific)

Gas-chromatograph system with reduction gas detector (Trace Analytical)

Sample injection valve with Model 451 Recycling Intervalometer (Gralab Instruments Division)

Recorder: 10-mV recorder (Linear Instrument) or integrating recorder (e.g., CR-3A, Shimadzu Scientific Instruments)

20-G side-port needle.

Additional reagents and equipment for determination of protein concentration (APPENDIX 3)

Prepare sample

1. Collect tissues or cells and rinse with ice-cold 0.1 M potassium phosphate, pH 7.4, or 0.9% NaCl. Keep tissue on ice.

   
   Whenever possible, flush or blanch tissue in situ or immediately after removal to eliminate circulating hemoglobin (a potential source of substrate). The assay can be performed on any cell or tissue type.

2. Homogenize fresh tissue in 4 vol ice-cold 0.1 M potassium phosphate (or 9 vol potassium phosphate for tissue with high levels of HO activity, e.g., spleen). Transfer tissue homogenates into microcentrifuge tubes.

3. Centrifuge homogenate 1 min at 13,000 × g, 4°C.

   
   A minimum of 100 µl of supernatant is needed for the assay, so prepare at least 200 µl of homogenate. The assay may be performed on crude enzyme preparations or on HO enzymes purified from specific subcellular compartments, e.g., from nuclear, mitochrondrial, microsomal, or soluble fractions. Purified isozymes can also be used.

4. Aspirate and discard any lipid from the supernatant surface. Transfer supernatant to a clean microcentrifuge tube without disturbing pellet.

5. Remove an aliquot of supernatant for determination of protein concentration.
    

Perform HO reaction

6. For each sample, assemble a set of five reaction vials into a rack. Label three vials "total" and the other two "blank."

   
   Total CO production (from HO activity and other sources) will be measured from the vials labeled "total," and the blanks will be used to measure any CO generated by non-HO activity.

   For conveniently handling large numbers of reaction vials, use commercially available 102-peg racks with 5 × 16 pegs, modified by shortening the pegs from 45 mm to 22 mm. These racks are also useful for storing clean vials.

7. Using 1.0-ml Hamilton syringes with repeating dispensers, add to all sample vials 20 µl HO substrate and 20 µl tissue preparation (from step 4). To the total samples, add 20 µl NADPH; to the blank samples, add 20 µl 0.1 M potassium phosphate, pH 7.4.

   
   Reactants should be pipetted in a manner that will prevent cross contamination: i.e., pipet substrate onto the bottom of the vial, tissue preparation onto the vial wall close to the bottom, and NADPH and buffer onto the wall just above the tissue preparation.

   The production of CO also requires the presence of NADPH-cytochrome P-450 reductase. This enzyme should be added when using highly purified tissue preparations from organs that have small amounts of reductase activity (e.g., brain or heart), or when reductase inhibitors are included in the reaction medium.

8. Seal vials with septum-fitted screw caps and mix liquids by swirling rack.

   
   Septa (8 mm diameter) are cut from 2.5-mm-thick, high-temperature blue silicone sheets (Alltech Associates).

9. Transfer rack to water bath and incubate 5 min at 37°C.

10. Prepare CO-free air by passing compressed air through a Hopcalite catalytic converter (see Fig. 9.2.1A).

11. Purge each vial for 2 sec with CO-free air at a flow rate of 200 to 300 ml/min using the vial-purging assembly (t= 0 min).

    
    If caps are wetted with distilled water prior to purging, the vial-purging assembly will penetrate the septa more smoothly (see Fig. 9.2.1A).

12. Incubate samples an additional 15 min at 37°C (t= 15 min). Remove vials from the water bath in the same sequence and at the same rate that they were purged. Dry vials with a towel and place them into a rack set in crushed dry ice.

13. Cover vials with a black plastic sheet and analyze headspace gas for CO as soon as possible.

    
    The septum material produces small amounts of CO at ambient temperatures, but the production rate is negligible at -78°C.

Quantify CO

14. Confirm that the GC injection-valve controller, detector, and recorder are functioning properly. Check and record carrier flow rate, column temperature, detector settings, and recorder settings. Verify that the carrier gas flow rate is ~30 ml/min and column temperature is 140°C.

    
    The ten-port pneumatic sample injection valve, fitted with a headspace-sampling assembly (Fig. 9.2.1B), injects the gas sample through a water-vapor trap filled with anhydrous magnesium perchlorate (Fisher) , onto the 68 × 0.53-cm (i.d.) stainless steel column packed with 40- to 60-mesh molecular sieve 13X (Alltech Associates). The carrier gas flows through the column at a rate of ~30 ml/min to separate the vial gases on the basis of molecular size. The carrier gas then transports the reactor gas to the detector.

    Under these operating conditions, the elution time for CO is ~90 sec, and as little as 1 pmol of CO is detectable. A 10-mV recorder can be used to chart the detector response (peak height) at a speed of 20 cm/hr. Integrating recorders are more efficient and convenient for determining CO peak areas accurately and rapidly.

15. Attach a clean, septum-fitted, capped vial to the headspace-sampling assembly (Fig. 9.2.1B) and turn on the valve controller to initiate the cycling of the valve between the injection phase (15 sec) and analysis phase (90 sec). Release carrier gas by puncturing the septum with a 20-G side-port needle.

    
    Injection of the vial headspace gas onto the GC column will purge the vial of CO but will also leave the vial pressurized with carrier gas. This pressure must be released before standard gas is added to the vial.

16. Prepare a standard curve by injecting into the depressurized vial, one at a time, aliquots of 50, 100, 150, 200, and 250 µl standard gas (representing ~24, 48, 72, 96, and 120 pmol CO, respectively).

    
    If CO production is expected to be very high, greater volumes (up to 1000 µl) of CO standard gas may be introduced. However, with most GC systems, the standard curve ceases to be linear for quantities of CO >120 pmol. Higher quantities can be determined quite accurately through interpolation.

    Standard gas may be purchased or can be prepared by mixing 20 µl pure CO gas (99.9%; e.g., Matheson Gas Products) with 1000 µl of CO-free gas in a 1000-µl Hamilton acrylic syringe sealed with a septum fitted into an 18-G needle hub without the needle. The CO concentration in the syringe should remain stable for ~8 hr.

17. Plot the detector response (peak area, mV·sec) against pmol CO injected. Calculate the slope, in pmol CO per mV·sec.

    
    The plot for detector response should be linear from the origin up to 120 pmol.

18. Attach each HO reaction vial to the headspace-sampling assembly during the analysis phase of the previously injected sample. Record the detector response to CO as the area under the CO peak (retention time of ~0.7 min), expressed in mV·sec.

    
    If numerous samples are to be analyzed, check the slope of the standard curve hourly by injecting 250 µl CO standard gas into a CO-free depressurized vial. Compare with the peak area determined during the preparation of the initial standard curve.

19. Record data. Calculate the mean peak heights for total and blank samples during the analysis phase.

20. Measure protein content of processed tissue samples.

    
    HO activity can be expressed or normalized in several ways. The most widely accepted practice involves expressing activity as nanomoles of product formed (in this case CO) per hour per milligram protein. Thus determination of HO activity requires measurement of the protein concentration of the sample.

    Many protein determination methods can be used. Each methodology has its own sets of advantages and disadvantages and not all yield the same values for a given tissue preparation. The authors use the Lowry method (Lowry et al., 1951) because of its widespread use and sensitivity.

Calculate HO activity

21. Calculate HO activity as follows:

    

    The factor 15 min is the reaction time. In some instances, HO activity may be more appropriately expressed in terms of activity per unit (mg) fresh weight of tissue. Normalization of activity on the basis of fresh weight may be preferred, for example, when working with intact tissue slices, cells, and preparations with widely varying concentrations of HO-inactive protein. In such cases, calculate HO activity according to the following equation:

    

Use Milli-Q-purified water or equivalent for all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

HO substrate (150 µM heme/15 µM albumin)

Dilute 1 vol of 1.5 mM heme/0.15 mM albumin methemalbumin (see recipe) with 9 vol of 0.1 M potassium phosphate, pH 7.4 (APPENDIX 2A).

Prepare fresh daily

Methemalbumin, 1.5 mM heme/0.15 mM albumin

Dissolve 9.9 mg hemin (Sigma) in 2.5 ml of 0.4 M Na₃PO₄. Add H₂O to 8 ml and dissolve 100 mg of bovine serum albumin (A7030, Sigma). Gradually adjust to pH 7.4 using 1.0 N HCl (~0.75 ml) in a 1-ml gas-tight Hamilton syringe with a repeating dispenser while stirring vigorously. Add H₂O to 10.0 ml. Store up to 14 days at 4°C.

NADPH, 4.5 mM

4.3 mg b-nicotinamide adenine dinucleotide phosphate, reduced form (Na₄NADPH; Sigma)

1.0 ml 0.1 M potassium phosphate, pH 7.4

Prepare fresh daily

Background Information
Heme oxygenase plays an important role in homeostasis in cells and tissues. Not only does the enzyme play a key role in the degradation of heme (Maines, 1992), but it also produces CO, a gas with potential physiologically activity. Like nitric oxide, CO has been shown to modulate cyclic guanosine 5´-monoposphate concentrations (Maines, 1997). Furthermore, bilirubin, a prominent end product of the heme degradation pathway, has been shown to possess significant antioxidant properties, particularly in neonates just after birth (Dennery et al., 1995). Finally, HO has been shown to be a heat-shock protein and to respond to oxidative stress (Maines, 1997). Although activation of HO during heat shock or oxidative stress is being examined through measurements of HO gene transcription (mRNA) and translation (HO protein), it will be of interest to determine whether gene regulation and protein production also modulate HO activity. Thus, HO activity measurements are becoming increasingly relevant and important.

Several methods for the determination of HO activity in animal tissues have been described. The most commonly used method measures spectrophotometrically the amount of bilirubin produced by the sequential reactions of HO and biliverdin reductase in reconstituted microsomal preparations (Tenhunen et al., 1968; Tenhunen, 1972; Maines, et al., 1977). The use of a linked, multienzyme assay for the determination of HO presents difficulties for both experimental design and interpretation of results (Lodola et al., 1979). First, sample preparation and activity measurements are time consuming. Second, the spectrophotometric technique for the determination of the bilirubin produced requires a low absorptivity, transparent sample matrix, and an excess of biliverdin reductase. Furthermore, the molar extinction coefficient for bilirubin needed for the calculation of HO activity depends on the reaction matrix (aqueous or organic), and must be determined for each tissue preparation if accuracy is to be achieved (Tenhunen et al., 1968).

A different and more direct HO assay was first described by Cavallin-Stahl et al. (1978) using methodology initially developed for the determination of CO in blood. This method, which was subsequently modified by Sunderman et al. (1982), does not require biliverdin reductase and measures the CO produced from the oxidation of heme to biliverdin by HO. CO is trapped with added hemoglobin and is subsequently released into the headspace of a second reaction vessel by the action of potassium ferricyanide [K₃Fe(CN)₆]. The CO in the headspace is then reduced to methane and quantified by GC. HO activity measurements using the GC method are reported to correlate well with those of the spectrophotometric assay (Vreman et al., 1988).

The protocol presented in this unit provides a simpler, more sensitive, and better integrated method by omitting the trapping step. Instead, CO is measured directly from the reaction vial headspace by a reduction gas detector (Vreman et al., 1984; Vreman and Stevenson, 1988). The detector can specifically measure trace levels (parts per billion or pmol) of reducing gases-i.e., those that are capable of combining with oxygen (O₂), including CO and hydrogen (H₂).

When directed through a heated bed of mercuric oxide (HgO), gases such as CO undergo the following reaction:

The amount of mercury vapor produced during this reactions is directly proportional to the inlet CO concentration. It is detected by means of an ultraviolet light photometer located immediately downstream from the reaction bed. The high molar ultraviolet light absorption coefficient gives this detector its unusual sensitivity.

Further, because CO is the only gas generated, in equimolar quantities, from the degradation of heme to biliverdin or bilirubin in the presence of NADPH and O2 (Vreman et al., 1988), use of the reduction gas detector makes this assay specific for the detection of HO activity.

The procedure is rapid, simple, specific, and accurate. It permits the measurement of HO activity in an unlimited variety of tissue extracts and tissue slices (Meffert et al., 1994). Because no direct spectrophotometric measurements of HO reaction products are involved, highly colored or photosensitizing inhibitors, such as metalloporphyrins, can be studied using this method (Vreman et al., 1993). The original method (Vreman and Stevenson, 1988) involved using 20 µl of undiluted methemalbumin (2 mM heme/0.15 mM albumin) to yield a final reaction concentration of 800 µM heme. This concentration is much higher than that used for spectrophotometric methods (<100 µM). Because HO inhibition is competitive in nature, the authors have subsequently decreased the heme concentration to 50 µM; this change enabled the method to be more responsive to HO inhibitor studies.

Critical Parameters
This protocol is used to accurately detect and measure trace amounts of an odorless and invisible gas (Vreman and Stevenson, 1988). The handling and quantification of gases present unique problems in the laboratory. It is important to recognize that gas molecules are compressible, small, and mobile through Brownian motion. All these factors can promote rapid loss through the inappropriate handling of samples. The protocol describes the dispensing of gas with syringes open to the atmosphere. This technique works well, but must be completed as quickly as possible.

It is also important to recognize issues related to the permeability of gases through materials with relatively open molecular structure, including plastics such as polyethylene or polypropylene (in contrast to glass). Thus the composition of vessels used in the procedure may also play a role in CO losses. Furthermore, some organic materials (including some plastics, silicone, and rubber; see Levitt et al., 1995) may spontaneously produce CO at ambient temperatures or in the presence of light. This could contribute to falsely elevated assay results. Most problems of this nature can be kept to a minimum by keeping assay vials cool and dark, running appropriate blank controls, and by completing analyses as rapidly as possible.

HO activity in tissue samples may not remain stable. Stability varies from preparation to preparation. Timely assay of tissue preparations is important for reproducible and accurate results.

The GC system has great sensitivity towards CO, which readily passes through it. However, a large number of other, mostly organic, molecules can be retained on the molecular sieve column and can thereby affect its molecular separating characteristics or reduce the HgO in the reaction bed. Thus, it is important to keep the headspace gas as simple as possible by using, besides air, only inert gases such as nitrogen (N₂) and O₂ if the gaseous phase of the HO reaction needs to be altered. The use of organic solvent solutions for reactants (e.g., DMSO or pyridine) should be avoided or carefully researched in advance. Call Trace Analytical Customer Service (650-364-6895) with any questions about system compatibility. The system is absolutely and totally incompatible with the use of volatile halogenated hydrocarbons (e.g., chloroform and some anesthetics).

Some processes, such as photooxidation (Vreman et al., 1990a) or lipid peroxidation (Vreman et al., 1998), could contribute to CO generation in reaction vials. However, analysis of a blank reaction mixture will correct for these possible sources of error.

H₂, produced by microorganisms, is another reduction gas that may sometimes be present with CO. This potential interferant (with a retention time of 0.3 min) will be separated from CO (retention time of 0.7 min) on the molecular sieve column (Ostrander et al., 1982).

The combination of these discriminating steps contributes to the great specificity of this HO assay.

Troubleshooting
Because CO is a gas, its quantification with the GC system poses unique problems. Unlike working with liquids, handling and transferring gases can result in a loss of material that cannot be detected until it is too late for recovery. However, loss of sample can be prevented through the careful use of well-planned techniques. The GC system itself can also, sometimes spontaneously, cause analysis failure.

When no CO peaks, or peaks with erratic area and retention times, emerge during analysis of CO-containing samples, the causes listed in Table 9.2.1 should be considered.

Table 9.2.1 also lists the possible causes and solutions for a gradual loss of sensitivity to CO in the GC system. Because the detection of CO depends on the chemical conversion of HgO to Hg gas, the reaction bed will become depleted of HgO as the Hg evaporates. Thus, the system will gradually (over ~2 year) lose its sensitivity to CO. This diminishing sensitivity should not affect daily CO analyses. With normal use, the column should be usable for up to 2 years. When the column fails, as detected by unstable and high baseline, it should be disconnected from the detector and reconditioned for 16 hr at 200°C, repacked with new column or replaced with a new column.

Anticipated Results
Table 9.2.2 shows representative results obtained using this methodology.

The results are not given as absolute values to be used for comparisons. They should be considered as representative values or trends for HO activity that may be encountered when different tissues from different species in various developmental stages are assayed. These results also represent HO activities in tissues observed after in vivo and in vitro perturbations that either increase (heme) or inhibit (metalloporphyrins) HO activity. In addition to these perturbations, there are many other parameters that may also play a significant role in the magnitude of the measured HO activities. These include, for instance, the route of administration of HO affectors (IP, IV, etc.), the time between administration and HO activity measurements, and the HO purification and concentration steps. It is our policy to always include an appropriate reference tissue as an assay check.

Linearity.
In preparations from rat liver and other tissues, HO activity is linear for up to 30-min of reaction time and up to 500 µg protein from the supernatant resulting from a 1-min 13,000 × g centrifugation. CO quantitation by this method is linear to 120 pmol.

Reproducibility.
As an example of the within-run reproducibility of the method, assay of CO from 20-µl samples (n= 10) of adult rat liver and spleen yielded the following results [mean CO (pmol) ± SD (C.V.)]: for liver, 137 ± 4 (4%) total CO, 30 ± 4 (13%) blank, and 106 ± 4 (4%) net CO; for spleen, 440 ± 10 (2%) total CO, 49 ± 2 (4%) blank, and 391 ± 10 (2%) net CO.

Sensitivity.
The limit of detection for CO is ~1 pmol. However, 10 pmol CO per vial is generally recommended as a practical working limit. Under current reaction conditions, ~1 µl (10 to 20 µg protein) of a 1-min 13,000 × g rat liver supernatant will produce 10 pmol of CO.

Time Considerations
The typical HO activity assay for ~6 tissue samples or 30 reactions requires ~4 to 5 hr. Sample preparation, including organ dissection and pretreatment, homogenization, and microsome isolation, should take 1 to 2 hr. Setting up and performing the HO reaction requires 1.5 hr. Quantitation of CO, including instrument calibration and sample analysis, requires 1.5 hr. Protein concentrations can be determined while CO is being measured. Finally, calculations can be performed in an hour, with most completed during the CO quantification phase.

An experienced analyst can perform and analyze ~160 HO reactions in a typical 8-hr day. With two analysts working together, the throughput rate can be increased to ~300 reactions per day.
Literature Cited
Cavallin-Stahl, E., Jonsson, F.-I., and Lundh, B. 1978. A new method for determination of microsomal haem oxygenase (E.C. 1.14.99.3) based on quantitation of carbon monoxide formation. Scand. J. Clin. Lab. Invest. 38:69-76.

Dennery, P.A., McDonagh, A.F., Spitz, D.R., Rodgers, P.A., and Stevenson, D.K. 1995. Hyperbilirubinemia results in reduced oxidative injury in neonatal Gunn rats exposed to hyperoxia. Free Radic. Biol. Med. 19:395-404.

Levitt, M.D., Ellis, C., Springfield, J., and Engel, R.R. 1995. Carbon monoxide generation from hydrocarbons at ambient and physiological temperature: A sensitive indicator of oxidant damage? J. Chromatogr. 695:324-328.

Lodola, A., Hendry, A.F., and Jones, O.T.G. 1979. Haem oxygenase: A reappraisal of the stoichiometry. FEBS Lett. 104:45-50.

Lowry, O.H., Rosebrough, H.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

Maines, M.D. 1992. Heme Oxygenase: Clinical Applications and Functions. CRC Press, Boca Raton, Fla.

Maines, M.D. 1997. The heme oxygenase system: A regulator of 2nd-messenger gases. Annu. Rev. Pharmacol. Toxicol. 37:517-554.

Maines, M.D., Ibrahim, N.G., and Kappas, A. 1977. Solubilization and partial purification of heme oxygenase from rat liver. J. Biol. Chem. 252:5900-5903.

McCoubrey, W.K., Jr., Huang, T.J., and Maines, M.D. 1997. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur. J. Biochem. 247:725-732.

Meffert, M.K., Haley, J.E., Schuman, E.M., Schulman, H., and Madison, D.V. 1994. Inhibition of hippocampal heme oxygenase, nitric oxide synthase, and long term potentiation by metalloporphyrins. Neuron 13:1225-1233.

Ostrander, C.R., Stevenson, D.K., Neu, J., Kerner, J.A., and Moses, S.W. 1982. A sensitive analytical apparatus for measuring hydrogen production rates. I. Application to studies in small animals. Evidence of the effects of an a-glucoside-hydrolase inhibitor in the rat. Anal. Biochem. 119:378-386.

Sunderman, F.W., Jr., Downs, J.R., Reid, M.C., and Bibeau, L.M. 1982. Gas chromatographic assay for heme oxygenase activity. Clin. Chem. 28:2026-2032.

Tenhunen, R. 1972. Method for microassay of microsomal heme oxygenase activity. Anal. Biochem. 45:600-607.

Tenhunen, R., Marver, H.S., and Schmid, R. 1968. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc. Nat. Acad. Sci. U.S.A. 61:748-755.

Vallier, H.A., Rodgers, P.A., and Stevenson, D.K. 1993. Inhibition of heme oxygenase after oral vs intraperitoneal adminstration of chromium porphyrins. Life Sci. 52:79-84.

Vreman, H.J. and Stevenson, D.K. 1988. Heme oxygenase activity as measured by CO production. Anal. Biochem. 168:31-38.

Vreman, H.J., Kwong, L.K., and Stevenson, D.K. 1984. Carbon monoxide in blood: An improved micro-blood sample collection system, with rapid analysis by gas chromatography. Clin. Chem. 30:1382-1386.

Vreman, H.J., Stevenson, D.K., Henton, D., and Rosenthal, P. 1988. Correlation of carbon monoxide and bilirubin production by tissue homogenates. J. Chromatog. Biomed. Appl. 427:315-319.

Vreman, H.J., Gillman, M.J., Downum, K.R., and Stevenson, D.K. 1990a. In vitro generation of carbon monoxide from organic molecules and synthetic metalloporphyrins mediated by light. Dev. Pharmacol. Ther. 15:112-124.

Vreman, H.J., Rodgers, P.A., and Stevenson, D.K. 1990b. Zinc protoporphyrin administration for suppression of increased bilirubin production by iatrogenic hemiolysis in rhesus neonates. J. Pediatr. 117:292-297.

Vreman, H.J., Lee, O.K., and Stevenson, D.K. 1992. In vitro and in vivo characteristics of the heme oxygenase inhibitor: ZnBG. Am. J. Med. Sci. 302:335-341.

Vreman, H.J., Ekstrand, B.C., and Stevenson, D.K. 1993. Selection of metalloporphyrin heme oxygenase inhibitors based on potency and photoreactivity. Pediatr. Res. 33:195-200.

Vreman, H.J., Wong, R.J., Sanesi, C.A., Dennery, P.A., and Stevenson, D.K. 1998. Simultaneous production of carbon monoxide and thiobarbituric acid reactive substances in rat tissue preparations by an iron/ascorbate system. Can. J. Physiol. Pharmacol. 76:1057-1065.

Key References
Maines, 1992. See above.
This book provides a comprehensive review of heme oxygenase.

Maines, 1997. See above.
This review presents the most up-to-date information on the heme oxygenase system.

Vreman and Stevenson, 1988. See above.
This paper provides technical details on measuring HO activity by assaying CO production.

Vreman et al., 1984. See above.
This article presents technical details on determining CO concentrations by GC.
 

Figure 9.2.1
Double-needle assemblies for vial purging (A) or headspace sampling (B). The assemblies are identical except for the direction of the gas flow through the needles. (A) Compressed air is passed through a heated (~120°C) catalytic converter containing Hopcalite (CuO/MnO) catalyst. The catalyst oxidizes any CO to CO₂. The resulting CO-free gas is used to purge the headspace of the reaction vial just prior to incubation. When the assembly is removed from the vial after purging, the vial pressure will equilibrate to atmospheric pressure via the long needle after the shorter purging gas-inlet needle has been withdrawn. (B) The long needle introduces carrier gas into the vial and the short needle serves as an outlet. Such an arrangement will prevent vial liquid from being forced into the injection valve by the flow of carrier gas. 
 

Table 9.2.1 GC Troubleshooting Guide
 

Problem	Possible cause	Solution
No CO peaks or peaks with erratic area and retention time	Faulty CO standard or poor sampling or transfer of standard	Check for plugged or leaky syringe
	Poor or no carrier flow through the vial or column	Check the detector outlet flow rate (which should be 30 ml/min) and peak retention time
		Check for proper position needle assembly through septum
	Reaction vial septum leaks when attached to headspace-sampling assembly	Check septum with soap solution with controller in inject mode
	Loose valve or carrier line fittings	Check valves and line fittings with soap solution
	Poor injection valve functioning	Check operation and actuation of valve and valve controller
	Inappropriate column or detector temperature	Column temperature should be 140°C; detector temperature should be 270°C
	Ultraviolet light source burned out	Replace light source (Trace Analytical)
Gradual loss of sensitivity to CO	Introduction of water vapor onto column	Check column moisture trap or replace anhydrone and perform a new calibration curve
	Depletion of reaction bed	Replace reaction bed (Trace Analytical)
Baseline drift	Column contaminated	Recondition or replace column

Table 9.2.2 Representative HO Activity Values Obtained Using the CO Method ^a,b
 

	HO activity (nmol CO/hr/mg protein
Treatment	Liver	Spleen	Brain	Kidney	Skin	Intestine
Newborn rhesus monkey (in vivo intravenous treatment) c
Control	0.19 ± 0.10	0.62 ± 0.20	0.46 ± 0.14	0.12 ± 0.07	0.04 d	0.30 ± 0.12
Heme	0.33 ± 0.23	0.78 ± 0.14	0.68 ± 0.28	0.32 ± 0.15	0.12 ± 0.02	0.41 ± 0.42
Heme + ZnPP b	0.08 ± 0.06	0.28 ± 0.03	0.60 ± 0.26	0.30 ± 0.13	0.10 ± 0.12	0.23 ± 0.12
Newborn Wistar rat (in vivo intraperitoneal treatment) e
Control	0.43 ± 0.21	2.19 ± 0.66	-	-	-	-
CrMP	0.08 ± 0.04	0.98 ± 0.65	-	-	-	-
Adult Wistar rat (in vitro treatment) f
Control	0.47 ± 0.10	2.23 ± 0.96	0.97 ± 0.34	0.17 ± 0.12	-	-
ZnBG	0.06 ± 0.03	0.04 ± 0.06	0.34 ± 0.20	ND	-	-

^aAbbreviations: ZnPP, zinc protoporphyrin; CrMP, chromium mesoporphyrin; ZnBG, zinc deuteroporphyrin bis glycol; ND, not determined.
^bAll HO activity values measured from 1-min 13,000 × g supernatants from homogenized tissue using 50 µM heme as a substrate.
^cData taken from Vreman et al. (1990b). For each group of animals, control or treated, n = 4 except where otherwise noted.
^d n=1.
^eData taken from Vallier et al. (1993). For control animals, n = 10; for treated animals n = 11.
^fData taken from Vreman et al. (1992). For control and treated animals, n= 3.

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