Methanol is a colorless and highly toxic organic solvent and is commonly used in industry. Methanol is rapidly absorbed by oral mucosa and gastrointestinal tract or skin. Subsequently, it is metabolized to formaldehyde by alcohol dehydrogenase, which is in turn converted into formic acid by aldehyde dehydrogenase. It has been reported that formaldehyde stays too transitory to be detected by analytical instruments, while formic acid is easily detected after methanol ingestion [
This research adhered to the tenets of the Declaration of Helsinki or the NIH statement for the Use of Animals in Research.
Thirty adult (200–250 g, National Rodent Laboratory Animal Resources, Shanghai Branch, China) male Sprague-Dawley rats with no ophthalmopathy were supplied with food and water
ERG recordings were performed by the Electrophysiological Test Unit (Roland, Germany) as described by ISCEV. Before test, absolutely dark adaptation for 4 h was necessary. Then animals were anesthetized with 10% chloralhydrate (3.5 mL/kg, intraperitoneal injection) and each pupil was dilated by tropicamide compound. After sufficient dilation, the corneal surface was anesthetized with tetracaine hydrochloride. The recording electrode was positioned on the corneal surface, the reference electrode was penetrated into the middle forehead, and the ground electrode was inserted into the skin of the ipsilateral-mastoid process. In the process of scotopic ERG, the flash light intensity of −25 dB was set for recording Rod-response and 0 dB was set for recording Max-response and OPs. Before the photopic ERG recording, the rats’ eyes needed 5 min for light adaptation. In photopic ERG process, the flash light intensity of 0 dB was set for recording Cone-response. To diminish the external interference, the amplifier was set to be with voltage of ±1 mv and narrow bandpass between 1 and 300 Hz.
Five consecutive wavelets of OPs were carefully measured (Figure
The measurement of amplitudes of OPs response and ET.
SPSS 20.0 software was used to analyze data with
Thirty blood samples (ten samples from group A, group B, and group C resp.) were used to determine methanol concentrations by headspace gas chromatography (GC/HS), as previously described [
The determinations of formic acid concentration were performed on Thermo U3000 HPLC (Thermo Fisher Scientific Inc., Waltham, MA, USA). System parameters included a Calesil ODS-100 C18 chromatographic column (5
A standard curve formula (
Methanol and its metabolite formic acid concentrations in blood
Group | Methanol concentrations in blood (mg/mL) | Formic acid concentrations in blood (mg/mL) |
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Control (A) | <0.05 | <0.05 |
3-day group (B) |
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7-day group (C) | <0.05 |
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Similarly, a standard curve formula (
Compared with methanol, its metabolite, formic acid, persistently accumulated in blood in methanol-treated groups (Figure
Methanol and formic acid concentrations in blood. Compared with methanol, the formic acid metabolite was significantly accumulated in blood.
For Rod-response of ERG, the a and b amplitudes of group B decreased approximately to 53% and 37%, respectively, and b amplitude of group C decreased approximately to 48% compared with the control group (
The latent period and amplitude of Rod-response
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Latent period (ms) | Amplitude ( | |
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For Max-response of ERG, the a and b amplitudes of group B decreased approximately to 53% and 40% and b amplitude of group C decreased approximately to 44% compared with the control group (
The latent period and amplitude of Max-response
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Latent period (ms) | Amplitude ( |
Latent period (ms) | Amplitude ( | |
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The summed amplitudes of oscillation potentials of groups B and C decreased to approximately 53% and 57% compared with the control (
The SAOP and ET of OPs responses
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SAOP ( |
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ET (ms) |
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Comparison of individual amplitude and SAOP between three groups (
Comparison of IPI and ET between the three groups shown above (Figure
For Cone-response, the a and b amplitudes of group B decreased approximately to 53% and 40% and b amplitude of group C decreased approximately to 69% compared with the control group (
The latent period and amplitude of Cone-response
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Latent period (ms) | Amplitude ( |
Latent period (ms) | Amplitude ( | |
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In recent years, methanol poisoning events occurred frequently because of adulterated wine and occupational exposure. Methanol poisoning severely damages the retina and optic nerve resulting in the impairment of vision and visual field [
The successful detection of toxicant is extremely important for diagnosis and treatment of intoxication. Chromatographic methods, including GC/HS and HPLC, are powerful tools in analytical chemistry. GC/HS is widely used to detect volatile compounds, such as ethanol and methanol [
In our present study, although methanol in blood was nearly completely metabolized after 7 days of intoxication, quantified and closed formic acid concentrations were detected in blood in 3- and 7-day intoxication groups. That is to say, formic acid as the continuation of methanol toxicity has a persistently cumulative phenomenon in blood comparing with methanol. It is likely that methanol and its metabolite, formic acid, commonly contributed to the toxic effects of methanol intoxication on retina structure and function and the effect of formic acid might be more persistent.
ERG technique is widely used to assess retinal function. International Society for Clinical Electrophysiology of Vision (ISCEV) Standard for full-field clinical electroretinography (2008 update) specified five responses including Dark-adapted 0.01 ERG (Rod-response), Dark-adapted 3.0 ERG (Max-response), Dark-adapted 3.0 oscillatory potentials (OPs), Light-adapted 3.0 ERG (Cone-response), and Light-adapted 3.0 flicker (30 Hz flicker) [
As a scotopic response of ERG recordings, OPs waves separated from b-wave through fourier spectrum filtering technology can be classified into early, intermediate, and late subgroups [
In this present study, we found that OPs responses were influenced after methanol intoxication. The reductions of SAOP and delays of ET were confirmed in both two methanol-treated groups indicating the damage of neurosensory retinal cell function. The ET of OPs in 7-day intoxication group was more delayed than in the 3-day group, which was not found in other ERG responses. Most of the comparisons of amplitude and latent periods of both acotopic and photopic ERG recordings between 3-day and 7-day groups methanol-treated groups have no remarkable significance, the possible cause of which may be that damage of methanol poisoning was persistent on both acotopic and photopic ERG recordings. Although the methanol was completely metabolized, its metabolite, formic acid, was continuously accumulated in the 7-day group, which might impede the recovery of retinal dysfunction. The other possible reason is that acotopic and photopic ERG recordings reflect the whole retinal function, are less sensitive than OPs, and cannot respond to the subtle changes of retinal function. The severe delay of ET in later methanol-treated rats indicated that OPs might be more sensitive to methanol poisoning. It is found that the amplitudes of later OPs wavelets in methanol-treated groups, such as OS3, OS4, and OS5, were clearly decreased compared with controls. This indicated that later OPs wavelets were more vulnerable to damage after methanol intoxication, which was consistent with reports by Plaziac et al. [
Combined with the methanol and formic acid concentrations in blood, deterioration of retinal function in the 7-day group was more possibly due to formic acid accumulation rather than methanol itself. This present study proposed that methanol and formic acid commonly contributed to retinal dysfunction and formic acid might play a major role in the process.
The present results indicated that both of scotopic and photopic retinal functions were impaired by methanol poisoning and impairment was more serious in the 7-day than in the 3-day group, the reason for which might have been formic acid accumulation in blood after methanol ingestion. Compared with other electroretinogram subcomponents, OPs, especially later OPs and IPI2 were more sensitive to methanol intoxication.
The authors declare that they have no competing interests.
This study was supported by project grants from the Shanghai Science and Technology Commission (Grant no. 14ZR1442400) and the Institute of Forensic Science, Ministry of Justice, China (Grant no. GY2013Z-1).