A Stealthy Dilemma

Carbon monoxide (CO) is a gas produced due to the incomplete combustion of organic material (Luchini et al. 2009). It is odorless, colorless, and tasteless that is inspired into the respiratory system and easily enters the bloodstream. Different sources of carbon monoxide can be enumerated, namely: fires, stoves, automobile exhaust, improperly ventilated furnaces, gasoline engines, among others. Cigarette smoke is also a significant source of carbon monoxide.


What makes Carbon monoxide a significant toxic substance?

The gas being implicated is known to be rapidly absorbed in the blood. CO, though binds hemoglobin (Hb – a red blood cell molecule that carries oxygen) slower than oxygen, has an affinity of 200-255 times greater than that of oxygen and its release is 10,000 times slower. The resulting compound formed from CO and Hb binding is known as carboxyhemoglobin (COHb). The formation of such a compound indicates competition between CO and oxygen for binding sites. An increase, therefore, of carbon monoxide concentration in blood could cause a decrease in the oxygen carrying capacity of the blood. This would lead to a decrease of oxygen delivery to various tissues and organs leading to hypoxia. The effects of such a situation is usually significantly seen in organs that rely heavily on oxygen, e.g. the brain and the heart.


Pathophysiology:

The impaired delivery of oxygen and its utilization is causes by the toxicity of carbon monoxide. This gas affects many sites in the body but it has a profound effect on organs which has high oxygen requirement. This results from cellular hypoxia which is mainly caused by the hindrance in oxygen delivery. Relative functional anemia happens when carbon monoxide binds with hemoglobin. An encompassing atmospheric carbon monoxide level of 100 ppm would produce an HbCO of 16% during equilibration.

Since carbon monoxide have a greater affinity to hemoglobin, an increase in carbon monoxide concentration would cause the increased binding of the molecules of oxygen to other oxygen binding sites and would cause a leftward shift in the oxyhemoglobin dissociation curve which means that there is a decreased availability of oxygen to tissues which are hypoxic. Carbon monoxide has a greater affinity to cardiac myoglobin which may result to myocardial depression and hypotension.

Carbon monoxide has a half-life of 3-4 hours at room temperature and this gas is eliminated through the lungs.

What makes it more lethal is that the presence of CO in the environment cannot be readily felt due to its odorless, colorless and tasteless properties, hence, is termed as the silent killer. Manifestations range from shortness of breath and to death due to continued exposure. No pathognomonic sign is known except for a cherry red color of the face which is a strong clue to acute CO poisoning. (McPherson and Pincus, Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22nd edition, 2012, p. 360) It is while the known treatment prescribed is hyperbaric oxygen therapy.


Due to the aforementioned clinical significance of CO, different methods of measuring the substance were employed overtime. The most common ones are: Gas chromatography, spectrophotometry, and the use of specialized spectrophotometric machines known and CO-oximeters. Such tests will be discussed in the succeeding posts.


Killing Me Softly: Detecting the Culprit (Part 1-Biological Specimens)

          Due to the nature of carbon monoxide, and the implications it could cause to the human body, different tests were developed to detect and, further, to quantitate its levels. Such tests could help the physicians in the diagnosis of a patient in suspicion of carbon monoxide poisoning. Tests for biological specimens are as follows:

1. CO detection by gas chromatography

          A. (Collison et al. 1967)

          One method that could be helpful in determining the carbon monoxide content in our body is by gas chromatography. It is accurate and precise so as that it is treated as the reference method for COHb determination.

          It uses a blood specimen, since carbon monoxide is bound to hemoglobin, this gas is then released through hemolysis and would further react in a closed system with Potassium Ferricyanide (K3Fe(CN)6). If the reaction between hemoglobin to methemoglobin is done, the gases liberated would be transferred to a separating column. The CO that has been separated would be reduced to methane then that would be detected with a flame ionization detector. The evaluation is done by comparing a known concentration of HbCO to the results of the response of the concentration of blood CO.

A schematic diagram is shown below to give illustration to the aforementioned process:


2. Spectrophotometric

          Spectrophotometers use the Beer-Lambert’s Law as their principle in determining concentrations of different substances. The Law states that the absorbance, A, of an analyte at a particular wavelength is proportional to its concentration, C, and the length of the light path, L, in the cuvette in which the analyte is dissolved in a solvent. (McPherson and Pincus, Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22nd edition, 2012, p. 430). Simply speaking, light absorbed is equivalent to the concentration of the substance being determined.

          Radiant energy from a light source passes through a monochromator which determines the wavelength to be transmitted. The light then passes through the sample contained in a cuvette where some of the light is absorbed by the substance being measured, while some are transmitted pointing to the detector where transmitted light is measured. The transmittance is then analyzed by the machine and depending on the level of automation the machine is at, the result would either be shown as the absorbance of the substance (where the medical technician would have to compute for the concentration) or the concentration already.

         CO determination methods using spectrophotometry are based on the principle that different hemoglobin derivatives can be measured using different wavelengths. Absorbance of the different hemoglobin species are measured by at least four to six wavelengths, and their concentrations are determined through calculations.

  • Commins & Lawther method (1965)In this method, the blood sample (can be finger prick) that was taken will be dissolved in ammonia (10 ml, 0-0.4%). This solution would be divided into half, the oxygen would be bubbled, and this is done to convert any COHb to O2Hb. Hemoglobin’s concentration would be the difference in the optical density of the sample which is read at 575 & 559 nm. Then, the concentration of COHb is the difference in the second sample and the amount of O2Hb in the sample. The COHb is read at 414 and 426 nm. In the past, spectrophotometric measurements are said to produce errors but with the Commins and Lawther method, the error in the concentrations of methemoglobin is eliminated since it is present in sample and reference solutions.
  • Beutler and West (1984)This spectrophotometric method is based on the measurement of the absorbance of a two-pigment mixture which is a product of the reduction of oxyhemoglobin and methemoglobin with sodium hydrosulfite. Sosium hydrosulfite reduces both the aforementioned hemoglobin species, but not carboxyhemoglobin. Absorbances were measured at 420nm and 432nm.

The procedure requires collection of venous blood containing either EDTA (with a ratio of 1.5 mg/mL of vlood) or heparin (14.3 USP units/mL of blood). 25uL of blood is added with a hemolyzing solution and is mixed by inversion. Aster 5 minutes, 0.1mL of the previous mixture is pipetted into a cuvette which already contains 1.15mL of a CO-Hb diluting solution. In another way to this, 0.2mL of the hemolysate is added to 2.3 mL of the CO-Hb diluting solution in a larger cuvet. The mixture must be covered, and is mixed by inversion. After allowing to stand for 10 minutes, the mixture is read with a CO-oximeter at 420nm and 432nm.


3. CO – oximeters (Mahoney et al. 1993)

          Clinical laboratories use specialized spectrophotometers to determine COHb in patients suspected to be acutely or chronically exposed to CO. Such machines are called CO – oximeters. CO – oximeters are models of spectrophotometers limited by wavelength selections to the measurement of clinically relevant hemoglobin derivatives, namely: oxyhemoglobin, methemoglobin, carboxyhemoglobin, and HHb among others. For all CO – oximeters, the determination of the said hemoglobin species is based on the difference or ratio of the light absorption of each specie from that of the total light absorption for all Hb derivatives present in the blood sample.


Comparison of the test methods:

Untitled


Additional information about different tests using different biological specimens are tabulated, as follows:


Killing Me Softly: Detecting the Culprit (Part 2 – Environmental detection)

Detection of Carbon Monoxide in environmental samples is very important to detect probability of CO leaks which may lead to CO poisoning. The use of detection of CO were notoriously essential in coal miners during the early days.

Animal sentinels were used for the said purpose. One animal used was the Canary. This method was endorsed by John Scott Haldane. Following an explosion in coal mines, miners get into the area bringing along them canaries caged in wooden frames. If any sign of distress is seen with the bird, it is presumed that the area is unsafe.

(Photo courtesy from Mine Safety and Health Administration - MSHA , United Stated Department of Labor, http://www.msha.gov/CENTURY/CANARY/PAGE2.asp#.U3FCLdJHKIo)

(Photo courtesy from Mine Safety and Health Administration – MSHA , United States Department of Labor, http://www.msha.gov/CENTURY/CANARY/PAGE2.asp#.U3FCLdJHKIo)

Canaries were used because they show more clear signs of distress. An example would be that a canary would sway noticeably from its perch before falling when it is affected by carbon monoxide poisoning.

But today they are not using animal sentinels as detecting medium for obnoxious gases. Electrical detectors are developed which are more reliable.


 

The different detectors are as follows:


Detector strips:

When carbon monoxide molecules move toward the blob detector, Carbon monoxide steals oxygen from the chemical salts on the blob. When it oxidizes itself, it will make the blob chemicals turn black. When the carbon monoxide is not plenty and there are many oxygen molecules around, there is a transition metal salt found on the blob which is responsible for stealing oxygen back and it will turn the blob to its original form.


Electronic alarms:

If carbon monoxide is present, the detector would be changing its color which means the beam was interrupted and the photocell is not able to pick up any light. This would trigger the circuit to alarm.