Research Article: Detecting Methemoglobinemia in Animals with a Drop of Blood

Date Published: December 8, 2016

Publisher: Public Library of Science

Author(s): Toni G. Patton, Stephen L. Blamer, Katherine E. Horak, Christopher James Johnson.


A major concern during pesticide development and use is the impact on non-target species, such as raptors or domestic cats and dogs. Sodium nitrite and para-aminopropiophenone (PAPP) are two toxicants currently being studied for the control of invasive species, such as starlings and feral swine. When given to an animal these compounds oxidize hemoglobin, which renders it unable to carry oxygen resulting in methemoglobinemia. This study developed a method to estimate methemoglobin levels in mammals and birds by examining the efficacy of sodium nitrite to induce the conversion of hemoglobin to methemoglobin. Varying concentrations of sodium nitrite were added to aliquots of coyote, vole, feral swine, starling, and duck blood, collected from captive animals. The blood samples were analyzed spectrophotometrically to determine percent methemoglobin and digitally to determine red color values (RCV) associated with different methemoglobin levels. The avian and mammalian blood reached 100% methemoglobin levels at 200 mM and 15 mM sodium nitrite, respectively. All animals had similar RCV for a given percent methemoglobin. In conclusion, this study developed a procedure to quickly determine methemoglobin levels in mammals and birds. Furthermore, percent methemoglobin can be estimated with one standard curve from any animal species and an image of a blood spot. The technique will be useful during field studies, in agricultural areas, or in a veterinarian’s office for the rapid diagnosis of methemoglobinemia in non-target animals that have eaten toxicants/baits or baited animals.

Partial Text

Invasive species, like starlings and feral swine, inflict extensive damage to ecosystems worldwide and are a growing concern to agricultural industries [1–4]. Many currently used pesticides are costly and are becoming less effective for controlling pests. Furthermore, these compounds are coming under scrutiny related to their mechanisms of action and potential risks to non-target species, like raptors or domestic cats and dogs [1, 5–10]. Several studies have examined methemoglobin-inducing agents to control invasive species populations because some of these compounds, such as para-aminopropiophenone (PAPP) and sodium nitrite, have antidotes [1, 8–17] [18].

As seen in Fig 1. the blood samples had the two characteristic hemoglobin peaks at 540 and 580 nm prior to the addition of sodium nitrite (Fig 1). These two hemoglobin peaks were reduced with increasing concentrations of sodium nitrite (Fig 1). Furthermore, a small peak at 635 nm, representing methemoglobin, began to form at higher sodium nitrite concentrations (Fig 1). These results were consistent across species with all blood samples exhibiting peaks at the same wavelengths. The peak at 635 nm, characteristic of methemoglobin, formed at lower sodium nitrite concentrations in the mammalian samples than in the bird samples. This difference was further observed with the spectrophotometric analysis, revealing that the avian samples reached 100% methemoglobin near 200 mM sodium nitrite and the mammalian blood reached 100% methemoglobin at approximately 15 mM sodium nitrite (Fig 2).

The impact on non-target species, like domestic cats and dogs or raptors, which may eat either the toxicant/bait or baited animals is a concern with current pesticide use and development. Therefore, it is important to have a method to determine potential intoxications of non-target species so antidotes can be administered as soon as possible. In this study, we examined the efficacy of a strong hemoglobin oxidizing compound, sodium nitrite, to estimate methemoglobin levels in mammalian and avian blood samples [21, 23]. We then developed a technique that can be used in veterinarian’s offices, in agricultural areas, or during field trials to estimate methemoglobin levels in mammals and birds.