Dry Chemistry Testing: Part 2 of 3
In the first blog in this three-part series, we saw how a dry chemistry card could help determine when a woman was most likely to get pregnant. For reference, a few drops of saliva are added to the card, where a color change indicates the period of ovulation in the next five days.
This blog discusses how a dry card chemistry assay can determine if a person or a pet has ingested a toxic alcohol.
The Dangers of Toxic Alcohol Poisoning
Data from the American Poison Control Center’s National Poison and Exposure Database reveals that more than 93,000 cases of toxic alcohol poisoning occurred in 2010. Close to 2,000 of these patients were classified as experiencing major morbidity. Sixty-one of these people died. The same database reports that many of these accidental poisonings occurred in a residential setting, and nearly half of the victims were under the age of five.
Methanol and ethylene glycol are the two most common causes of toxic alcohol poisoning. Methanol (aka, “wood alcohol”) is found in automobile windshield-washer fluid. Similarly, ethylene glycol is the major component of automobile antifreeze fluid. Both products are often packaged as brightly colored liquids and are reported to have a sweet taste. It is easy to see that young children and pets might be tempted to drink them.
The danger that these chemicals pose is due to toxic metabolites that form in the body after ingestion. If treatment is not available or is delayed, the mortality rate of methanol poisoning can be as high as 50–80%. Ethylene glycol is readily absorbed in the gastrointestinal tract, and neurological damage can occur within as little as 30 minutes post-ingestion.
Current Methods for Toxic Alcohol Poisoning
The gold standard for the diagnosis of toxic alcohol poisoning is gas chromatography in blood. Gas chromatography (GC) is well-established technology that is most commonly found in industrial and academic research labs, but not in most hospital labs. The equipment is relatively expensive and is best used by well trained professionals. The columns in GCs are often run at elevated temperatures and frequently employ expensive helium as the carrier gas. GCs are not instruments that can be left unused for long periods of time and suddenly pressed into service. Accurate calibration for various compounds require a GC instrument to be thermally equilibrated, and that takes time.
Immunoassays for these small molecules are impractical, as they are far too small to be immunogenic on their own. Even if they were covalently attached to a carrier protein (e.g., bovine serum albumin), it is not clear that useable antibodies could be generated that would discriminate between these closely related compounds.
Physicians are therefore left to manage their intoxicated patients empirically by monitoring symptoms, osmolar gap, acidosis, and anion gap measurements. These measurements are less precise and more difficult to interpret than direct toxic alcohol measurements in blood.
There are two therapies that can be employed to mitigate the effects of toxic alcohol poisoning. The first is hemodialysis using the same devices that are used to treat patients in renal failure. The other therapy is less obvious: intravenous ethyl alcohol infusions. Ethyl or grain alcohol is the physiological component of alcoholic beverages. It competitively blocks the metabolism of the toxic alcohols so that they can be excreted in urine in their original, unmetabolized form.
Detecting Toxic Alcohol Poisoning with Dry Chemistry Tests
PortaScience, Inc., of Moorestown, New Jersey, was recently acquired by DCN Dx for their expertise in dry chemistry reagents. One PortaScience-developed project is a point-of-care assay for ethanol, ethylene glycol, and methanol. It is based on the use of enzymatic pathways that are specific for each of these compounds. Each enzymatic pathway utilizes an electron transfer system that reacts with an immobilized substrate that produces a color change that is proportional to concentrations of ethanol, ethylene glycol, and methanol.
- For ethylene glycol, the enzyme is glycerol dehydrogenase. This enzyme reacts with ethylene glycol in the sample in the presence of the cofactor NAD+ to generate NADH. The NADH is subsequently reacted with another enzyme—diaphorase—and it converts a chromogenic substrate into a colored product. This substrate is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). It is yellow in its original form and turns a dark purple on reduction with NADH. The purple color is directly proportional to the concentration of ethylene glycol in the sample.
- For methanol, the enzyme used is alcohol dehydrogenase. In the presence of NAD+, this enzyme also generates NADH and formaldehyde. Formaldehyde in the presence of atmospheric oxygen reacts enzymatically with diaphorase with another chromogenic substrate called Purpald. This generates a purple color that is proportional to the concentration of methanol in the sample.
- The ethanol assay uses the same enzymes as the methanol assay, but the chromogenic substrate is MTT, the same substate used in the ethylene glycol assay. Determination of the ethanol concentration is useful in monitoring therapy in the presence of the toxic alcohols.
These systems were first evaluated in solution-phase chemistry and subsequently transferred to a nylon membrane with a pore size of 0.45 microns to make a dry chemistry assay. The nylon membrane was first coated with the appropriate chromogenic substrate in the appropriate buffers to maintain pH. After drying, a second coat consisting of the necessary enzymes, cofactor and surfactants are then applied over the chromogenic substrate.
The assay is run by applying 15 microliters of sample (serum or whole blood) to the coated card. Color develops within a few minutes and is read with a handheld reflectance meter equipped with a 565 nm LED light source. The reduced chromogenic substrates undergo a shift in their absorption maximum from 405 nm (yellow) to 565 nm (purple). The range of these assays is from 10 mg/dL to 200 mg/dL. Ethylene glycol has a toxicity threshold of 25 mg/dL, and methanol’s toxic threshold is 20 mg/dL.
As was the case with the dry card assay for calcium and magnesium in saliva for the monitoring of ovulation, a lot of work goes into making these tests stable and reproducible. Enzymes are proteins, and as such are subject to denaturation on coating on a synthetic polymer such as nylon. Various stabilization strategies have been employed such as the use of hydroxy propyl cellulose, carboxy-methyl cellulose, polyvinyl pyrrolidone (PVP), and xanthan gum. Hungry bacteria and fungi that are omnipresent in the environment must also be considered and prevented from destroying the reagents before they can be used. A commercially viable product should have a shelf life of at least 12 months at refrigerator temperatures. Long-term storage at room temperature is preferred for ease of distribution and storage. Shipping stability is another consideration as brief exposure to temperatures over 100°F are not uncommon during summer in some locations.
Conclusion
If you need an assay for a small organic compound in a point-of-use setting, contact the experts at PortaScience. Their knowledge and experience could turn your needs into a reliable analytical tool. The examples in this blog demonstrate how different alcohols can be coupled to enzyme-cofactor-substrate combinations to produce a color change proportional to the concentration of the analyte. However, it is not hard to imagine how an enzyme inhibitor such as an insecticide or pesticide could be detected by a decrease in enzymatic activity. Even if you have a talented team of chemists and manufacturing personnel in-house, confidential discussions between teams of professionals can produce synergistic results not obtainable by one group working in isolation.
Be sure to check out Part 1 and Part 3 of this series on dry chemistry.
DCN Dx is a globally recognized contract developer of point-of-care devices based in Carlsbad, California. Since its founding, DCN Dx has committed itself to furthering the rapid diagnostic and point-of-care test market through the continued evolution of technologies and applications. DCN Dx’s cross-functional team of scientists and engineers develop and integrate all aspects of the point-of-care device system, including complex binding reactions, cassettes, sample handling devices, and reader systems. The company assists clients in developing entire rapid diagnostic tests from concept to commercialization.
To learn more about DCN Dx’s services, click here.
