CARBON DIOXIDE PROCEDURE

For the Quantitative Determination of Carbon Dioxide in Serum

SUMMARY AND EXPLANATION

The measurement of Carbon Dioxide is useful in the assessment of acid-base balance disturbances. Elevated level is observed in metabolic alkalosis and compensated respiratory acidosis (1). A low is observed in compensated respiratory alkalosis and metabolic acidosis (1). Differentiation between the metabolic and respiratory conditions is only possible through additional laboratory testing.

Early methods for the determination of carbon dioxide were based on either volumetric or manometric determination of the released from a sample by acid treatment. These methods used the instruments of Van Slyke (2) until they were replaced by the Natelson microgasometer (3), which still uses manometric determination of the C02.  In the 1970s, Wilson (6), Menson (7) and Norris (8) each presented methods for an enzymatic method of determining C02using phosphoenolpyruvated carboxylase.  The EAGLE CARBON DIOXIDE PROCEDURE is based on their work.

PRINCIPLE

Carbon dioxide (in the form of bicarbonate ions) reacts with phosphoenolpyruvate carboxylase (PEPC),  to form oxaloacetate and phosphate.  The oxaloacetate is then converted to malate by the action of malate dehydrogenase (MDH) and reduced nicotinamide adenine dinucleotide (NADH).  The decrease in absorbency at 340 nm resulting from the oxidation of NADH is proportional to the amount of C02in the sample. Interference from endogenous pyruvate and LDH is eliminated by the inclusion of sodium oxamate.

PEP  +  HC03        PEPC          Oxaloacetate  +  H2PO4

Oxaloacetate  +  NADH  +  H+     MDH         Malate  +  NAD+

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2420 Provides:

CARBON DIOXIDE REAGENT - (Cat. No. 2421)          

REACTIVE INGREDIENTS:

After reconstitution, PEP 1.8 mM. Magnesium Sulfate - 10 mM;

NADH - 0.4 mM; MDH (porcine) - 1250 U/L; PEPC (microbial) 200 U/L; Sodium Oxamate - 2.5 mM; Buffer; non-reactive fillers; Stabilizers and Sodium Azide 0.1% added.

RECONSTITUTION:

Reconstitute the reagent with the amount of diluent stated on the vial label.  Swirl "gently" to dissolve.  DO NOT SHAKE.

PRECAUTIONS:

1. Avoid contact with skin, eyes or clothing. Toxicity has not been established. In case of contact, wash with water.

2. Reagent contains sodium azide. Dispose of with large amount of water to prevent azide reacting with copper plumbing forming an explosive metal azide.

STORAGE AND STABILITY:

Store at 2º- 8º C. Stable until expiration date if vial remains sealed and unopened.  After reconstitution, reagent is stable for 24 hours at room temperature (18º - 25º C) or for 14 days stored at 2º - 8º C. Must be kept tightly capped at all times and avoid excessive shaking of the reagent.

DETERIORATION:

In a sealed vial, the reagent should be a dry powder.  Moisture entering the vial can be observed by observing for caking of the reagent powder. After reconstitution, if the reagent has an absorbency <<1.0 at 340 nm when measured against distilled water, it should not be used.

CARBON DIOXIDE DILUENT - (Cat. No. 2422)

REACTIVE INGREDIENT:

Distilled water with a resin to prevent uptake of atmospheric .

PRECAUTIONS:

Do not ingest. Toxicity  has not been established.

STORAGE AND STABILITY:

Store at 2º - 8º C.  Stable until expiration date on bottle is kept tightly sealed and protected from contamination. NOTE:  Do not pipette by mouth to avoid contamination from the expired air.

DETERIORATION

The diluent should be clear, colorless solution. Turbidity would indicate deterioration.

CARBON DIOXIDE CALIBRATOR - (Cat. No. 2423)

REACTIVE INGREDIENT:

Equivalent to 30 mmol/L in described method.

STORAGE AND STABILITY:

Store at 2º - 8º C.  Dispense with dropper amount sufficient for testing. Do not remove dropper tip in order to assure proper stability.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 340 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Fresh, unhemolyzed serum collected under anaerobic conditions is the recommended specimen.

2. Heparinized plasma collected under anaerobic conditions is acceptable. Oxalate, Citarate and EDTA may not be used.

3. Lipemic, icteric and hemolyzed sample require a Serum Blank.

SAMPLE STORAGE:

The sample may be stored in ice water under anaerobic conditions for up to one hour (9).

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

from air or the breath of the analyst is a major interference in this assay. Reagent preparation, specimen collection and storage instructions must be strictly followed to minimize this interference.

PROCEDURE

MATERIALS PROVIDED:

CARBON DIOXIDE REAGENT (Cat. No. 2421), CARBON DIOXIDE DILUENT (Cat. No.2422) and CARBON DIOXIDE CALIBRATOR (Cat. No. 2423).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.005 mL micropipettor

2. 1.0 mL pipettor or dispenser

3. Incubator capable of maintaining 37ºC

4. Timer, test tubes, rack and timer

REACTION CONDITIONS:

Wavelength 340 nm

Reaction Type Endpoint

Reaction Time 5 minutes

Reaction Temperature 37º C

Sample Volume 0.005 mL

Reagent Volume 1.0 mL

Total Volume 1.005 mL

Low Normal 23 mmol / L

High Normal 34 mmol / L

Calibrator Value 30 mmol / L

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE: For instruments requiring a total volume >>1.0 mL, increase Carbon Dioxide Reagent volume to 2.0 mL and the Sample volume to 0.01 mL. Proceed as outlined.

1. Dispense 1.0 mL CARBON DIOXIDE REAGENT into tubes labeled: Reagent Blank, Calibrator, Control, Sample 1. etc..

2. Place 0.005 mL specimen into appropriately labeled tube. Mix well.

3. Incubate all tubes at 37º C for 5 minutes.

4. Zero instrument at 340 nm with distilled water.

5. Read and record absorbances for Reagent Blank, Calibrator, Control and Unknowns.

6. To obtain values, see "Calculation of Results".

PREPARATION  OF  A  SERUM  BLANK:

Highly lipemic, icteric or hemolyzed samples require a Serum Blank.

1. Add 0.005 mL sample to 1.00 mL saline.

2. Zero instrument at 340 nm with saline.

4. Subtract blank absorbance from sample test absorbance measured in Step 5 of PERFORMANCE OF TEST. Use  this value in calculating the Carbon Dioxide value.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within 10 minutes after completion of incubation.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in the range of 0-40 mmol / L. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed.  Use the CARBON  DIOXIDE CALIBRATOR (Cat. No. 2423) which is provided with the reagent set or other suitable calibration material for this purpose. Specimens exceeding 40 mmol / L should be diluted 1:1 with saline and reassayed.  Multiply the test result by 2 to obtain final answer.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control material  (normal and abnormal) analyzed in the same manner used for the unknown. There are several commercial products available which provide a Buffered solution used for reconstitution and are suitable for this purpose.  Failure to achieve assayed values of freshly prepared control sera must be thoroughly investigated before patient results are reported.

CALCULATION RESULTS

The following equation is used to determine Unknown concentrations:

Unknown (mmol / L =                     

Abs. Rgt Blank - Abs. Sample                

------------------------------------------   X Cal Conc.

Abs. Rgt Blank - Abs. Calibrator

EXAMPLE:

A Reagent Blank has Abs. = 1.800 while the Unknown Abs.= 0.900 and the Calibrator Abs. = 0.800.  The Carbon Dioxide concentration of the Unknown is:

1.800 - 0.900       

-------------------      X 30 mmol / L = 27 mmol / L

1.800 - 0.800

LIMITATION

1. Lipemic, icteric and hemolyzed samples require a Serum Blank.

2. Carbon Dioxide contamination must be avoided.

EXPECTED VALUES ( 9 )                                  

23 - 34 mmol / L

This range represents the 95% confidence interval obtained from a clinically normal population. Each laboratory should establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear in the range of 0 - 40 mmol / L.

PRECISION:

MEAN / STD. DEV. / % CV

Normal 20.7 / 0.48 / 2.3

Abnormal 40.7 / 0.56 / 1.3

Run to Run - Normal and abnormal control sera were assayed for 10 working days to establish run to run precision.

Normal 20.1 / 0.54 / 2.7

Abnormal 39.7 / 1.56 / 3.9

SPECIFICITY:

A comparison of this CARBON DIOXIDE PROCEDURE  with a commercial method using a similar method showed a 98.7 % correlation with samples in the normal and abnormal range.

SENSITIVITY:

This CARBON DIOXIDE PROCEDURE has a sensitivity of 1 mmol / L for each change of 0.033 in absorbance at 340 nm.

REFERENCES

1. Van Slyke, D.D. and Stadie, W.C., J. Biol. Chem. 49:1 (1921).

2. Van Slyke, D.D. and Neil, J.M., J. Biol. Chem. 61:523 (1924).

3. Natelson, S., Microtechniques of Clinical Chemistry, C. Thomas, Springfield, IL., p.147 (1961).

4.   Skeggs, L.T. Jr., Am. J. Clin. Path. 33:181 (1960).

5. Tietz, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders, Philadelphia, PA., pp. 884-887 (1982).

6. Wilson, W., et al, Clin. Chem. 19:640 (1973).

7. Menson, R.C., et al, Clin. Chem. 20:872 (1974).

8. Norris, K.A., et al, Clin. Chem. 20:1093 (1975).

9. Henry, R.J., Clinical Chemistry: Principles and Technics, Harper & Row, New York, NY., p.784 (1974).

10. Young, D.S., et al, Clin. Chem. 21:1D (1975). 11. Martin. E.W., In     Hazard of Medication (Alexander, S.F., Farage, D.J., and Hassan, W.E., Jr. eds.), J.B. Lippincott Co., Philadelphia, PA., and Toronto, Canada, p. 169 (1971).

11. Constantino, N.V., and Kabat, H.F., Am. J. Hosp. Pharm. 30:24 (1973).