CO-oximetry measurements and antioxidant effects of ascorbic acid and methylene blue in equine methemoglobinemic blood

Objectives: To determine the effects of time after sampling on CO-oximetry measurements of equine blood samples and the effects of adding ascorbic acid (AscAc) and methylene blue (MetBlue) to samples with methemoglobinemia. Design: Experimental study. Setting: University teaching hospital. Animals: Thirty healthy adult horses assigned to 5 groups. Interventions: Repeated CO-oximetry determinations were performed on venous (n = 6) and arterial blood samples (n = 7) stored at 0°C for 48 hours. Methemoglobinemia was induced in vitro in 17 additional blood samples. Six were used as untreated controls, 6 had AscAc added, and 5 had MetBlue added. Total hemoglobin, oxyhemoglobin, carboxyhemoglobin, methemoglobin (MetHb), and oxygen saturation of hemoglobin (SO₂) were measured. Measurements and Main Results: Oxyhemoglobin and SO₂ increased from 69.8% ± 10.2% and 90% ± 3% to 82.8% ± 7.9% and 99% ± 3%, respectively, after 8 hours in venous blood (mean ± SD, P < 0.001). There was an effect of treatment (P = 0.032) and of time (interaction P = 0.003) on MetHb% in methemoglobinemic samples. The difference in absolute MetHb% from time 0 was as follows: 7.0% (interquartile range [IQR] = 21.2), –0.2% (IQR = 3.5), and –4.4% (IQR = 5.2) at 48 hours in control, AscAc, and MetBlue groups, respectively (P < 0.05). There was no effect of time on MetHb% in the AscAc group (23% [IQR = 52.6] at time 0 to 23.2% [IQR = 56.9] after 48 h). Conclusions: Storage of blood in ice water to determine O₂Hb and SO₂ using a CO-oximeter should not exceed 4 hours. Measurement of MetHb% could be delayed by up to 48 hours if AscAc is added to the sample. MetBlue significantly decreased MetHb% over time. The limitations of this study include the fact that the antioxidant effects of AscAc and MetBlue were evaluated in vitro and not in vivo. Further studies are needed to evaluate different storage temperatures and syringe types.