Methyl Alcohol Poisoning

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					Methyl Alcohol Poisoning
II. Development of a Model for Ocular Toxicity
in Methyl Alcohol Poisoning Using the Rhesus Monkey
Gladys Martin-Amat, MD; Thomas R. Tephly, MD, PhD; Kenneth                  E. McMartin; Adeeb B. Makar, PhD;
Manmohinder S. Hayreh, MD; Sohan S. Hayreh, MD, PhD, FRCS; Gary Baumbach, MD; Pasquale Cancilla, MD

   \s=b\ Rhesus monkeys were intoxicated               poisoning have been described recentr          al' reported that formic acid accumu-
with methyl alcohol, using an initial dose             ly along with the development of a             lates in the blood of pigtail monkeys
of 2 gm/kg and subsequent doses were                   model for the study of methyl alcohol          concurrent with the development of
administered in order to maintain an at-
tenuated and prolonged state of intoxica-
                                                       toxicity.- '' The delayed onset to toxic-      metabolic acidosis. However, they
tion. Arterial blood samples were drawn                ity, followed by the development of             were unable to reproduce these results
for methyl alcohol, formate, Po2, Pco2, and            metabolic acidosis and the striking            in the rhesus monkey (Macaca mulat-
                                                       ocular toxicity after methyl alcohol           to). Our laboratory has shown the
pH, which were monitored periodically
throughout the course of the experiment.               ingestion, are well known features of          development of metabolic acidosis,
With the use of these procedures mon-                  the toxic syndrome in man.'1" Rhesus           accumulation of formic acid in the
keys developed metabolic acidosis with                 and pigtail monkeys have been recent-          blood followed by coma and death in
the accumulation of formic acid in the                 ly studied and shown to display the            the pigtail monkey (M nemestrina)
blood and a corresponding decrease in                  characteristic metabolic acidosis seen         and the rhesus monkey. Furthermore,
blood bicarbonate. These animals served                in man that occurs coincident with             it has been possible to prevent methyl
as models, which allowed for ocular eval-
                                                       accumulation of formic acid in the             alcohol toxicity through the use of 4-
uation for early signs related to methyl               blood."3 With the suggestion that
alcohol poisoning. A mechanism to                                                                     .methylpyrazole, a potent inhibitor of
explain toxicity is proposed and dis-                  methyl alcohol use may increase, it is         hepatic alcohol dehydrogenase.'"
cussed.                                                .important that we understand the               Whereas metabolic aspects of the
  (Arch Ophthalmol 95:1847-1850, 1977)                 mechanism of its toxicity and derive            methyl alcohol poisoning syndrome
                                                       means to halt its toxic manifestations         were described, no obvious ocular
                                                       when they occur.                                toxicity was seen in monkeys treated
                                                          A marked species difference in the           with 3 gm/kg of methyl alcohol. For
The subject increasing
      toxicology methyl
                   of       alcohol is
      it has received endorsement as
                                                       susceptibility to methyl alcohol ex-
                                                       ists."''' Potts'" has previously de-
                                                                                                       this reason a different protocol for
                                                                                                       intoxication was devised and biochem-
an important energy source for the                     scribed the rhesus monkey to be a               ical measurements were supple-
future.' Important advances in our                      possible model, for methyl alcohol             mented with clinical ophthalmological
understanding of the development of                     poisoning in man.'" However, Cooper            evaluation    and     histopathological
events associated with methyl alcohol
                                                        and Felig17 were unable to confirm             study. This intense and comprehen-
                                                        their results. Comparable doses of             sive experimentation was performed
                                                        methyl alcohol were used but they              in animals that received levels of
                                                        found none of the characteristic com-          methyl alcohol that yielded a pro-
  Accepted for publication Sept 13, 1976.
  From the Toxicology Center, Departments of            ponents of methyl alcohol toxicity,            longed and attenuated state of meta-
Pharmacology (Drs Martin-Amat, Tephly, and              except for a possible CNS depression           bolic acidosis and formic acidemia.
Makar,   and Mr   McMartin), Ophthalmology (Drs         that is usually seen in nonprimate             The dose schedule and biochemical
M. Hayreh and S. Hayreh), and Pathology (Drs
Baumbach and Cancilla), College of Medicine, the        species. Recently, Clay et al1 and             pattern observed throughout the
University of Iowa, Iowa City.                          McMartin et alL' have been able to             course of methyl alcohol poisoning
  Reprint requests    to the   Toxicology Center,       reproduce certain of the results
Department of Pharmacology, the University of                                                          leading to and accompanying ocular
Iowa, Iowa City, IA 52242 (Dr Tephly).                  reported by Gilger and Potts.'1 Clay et        toxicity in rhesus monkeys are re-

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         Fig 1.—Formate accumulation and metabolic acidosis in four rhesus monkeys. Methyl alcohol (2 gm/kg) was
         administered orally at zero time. Arrows indicate times at which methyl alcohol supplements were given (0.5 gm/kg
         except for R6 at 44 and 72 hours when 1 gm/kg was given and at 144 hours when 2 gm/kg was given). Stars indicate
         times at which ophthalmoscope evaluations were performed.

ported here. Clinical ophthalmological         mate levels were determined according to         ble ocular abnormalities. For this
studies and histopathological findings         the method of Makar et al'" and methyl           reason, a different experimental ap-
                                               alcohol was analyzed by modification of the
are reported in subsequent papers (see                                                          proach was employed in order to
p 1851 and 1859).                              gas   Chromatographie procedure described        produce ocular toxicity. In this ap-
                                               by Baker et al.'-'" The animals were allowed
    MATERIALS AND METHODS                      to have fruit and water during the course        proach a lower dose of methyl alcohol
                                                                                                was administered (2 gm/kg) to rhesus
                                               of the experiment. Preparations for oph-
   Male rhesus monkeys (M mulatta),                                                             monkeys in order to produce an atten-
weighing 2.6 to 4.4 kg, were intoxicated by    thalmologic and histopathologic studies are
                                               described elsewhere (see p 1851 and 1859).       uated toxicity syndrome and was
repeated oral administration of methyl                                                          followed by supplemental doses of
alcohol. An initial dose of 2 gm/kg was                       RESULTS
administered orally, followed by subse-                                                         methyl alcohol to maintain a state of
                                                  Since previous studies in our labora-         metabolic acidosis for prolonged peri-
quent doses that were given to maintain
the arterial blood pH between 7.1 and 7.3      tory indicated that we, had been able            ods of time. Using this procedure,
with arterial blood bicarbonate levels at 10   to reproduce certain features of the             monkeys developed the signs of
mEq/liter or above. Animals were pre-          human methyl alcohol intoxication                methyl alcohol toxicity such as meta-
pared as described by McMartin et al.2 The     syndrome in the rhesus monkey, an                bolic acidosis with the accumulation of
femoral artery was cannulated and an           attempt was made to explore whether              formic acid in the blood and decreased
indwelling catheter was fixed in order to      the ocular toxicity observed in man               blood bicarbonate. Furthermore, these
obtain arterial blood samples for methyl       was a feature of the methyl alcohol               animals routinely developed optic disc
alcohol, formate, PO;,, Pco., and pH, which    toxicity syndrome in the monkey. In               edema (see p 1851). The rate of devel-
were monitored throughout the course of
the study. The arterial blood gases and pH     order to establish metabolic acidosis in          opment of metabolic acidosis in these
were measured in a blood gas analyzer and      the monkey a dose of 3 gm/kg has                  monkeys is shown in Fig 1. In each of
bicarbonate concentrations were calculated     been used. These animals usually died             four animals indicated in this figure
 from pH and Pco... values, using the          at about 20 to 30 hours after adminis-            metabolic acidosis develops concur-
 Henderson-Hasselbach relationship. For-        tration- and exhibited no demonstra-             rently with increased formic acid and

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           Biochemical Factors in     Methyl Alcohol-Poisoned        Rhesus    Monkeys               not   yield ocular signs, after a pro-
                                                                                                     longed  chronic acidotic state produced
                                                                CSF Formales             Methyl 11   by the accumulation of formic acid
  Monkey       Formate"            HCO.t        pHt             (Before Death)           Alcohol
     R 1         13.8                4.8        7.13                 13.9                  231       generated from methyl alcohol a
     R 2          8.6                6.3        7.28                                       273
                                                                                                     distinct optic disc edema was observed
     R 3         14.4               10.2        7.25                                       154       (see p 1851). While these findings are
     R 4          7.2                4.0        7.18                                       284       different than those previously re-
     R 5         11.0                9.0        7.24                   7.9                 220       ported by Gilger and Potts,1' they
     R 6         13.1                4.5        7.26                                       220       resemble those reported in the litera-
 "Maximum formate levels   (mEq/llter).                                                               ture relating to human methyl alcohol
 tMinimum arterial blood bicarbonate (mEq/liter).                                                    poisoning.'" '"•':'
 {Minimum arterial blood pH.                                                                              Our recent studies have shown that
 SCerebrospinal fluid formate (mEq/llter) as determined prior   to death.
  [Maximum blood methyl alcohol levels (mg/100 ml).                                                  formic acid accumulation in the blood
                                                                                                     and the onset of metabolic acidosis are
                           METHYL ALCOHOL CONVERSION TO                                              related. Other workers have sug-
                           TOXIC METABOLITE                                                          gested that formaldehyde mediates
                                                                                                     the toxicity of methyl alcohol. These
                                 FORMATE ACCUMULATION
                                                                                                     workers did not have the technology
                                                                                                     available to measure formic acid in a
                                                                                                     specific and sensitive manner, a tech-
                           INHIBITION OF CYTOCHROME                                                  nique that has only recently been
                                    OXIDASE                                                          developed,'" and the inability of other
                                                                                                     workers to find this correlation was
                           INHIBITION OF MITOCHONDRIAL
                                                                                                     probably due to the technological
                                                                                                      problems involved in formate mea-
                             ELECTRON TRANSPORT                                                       surements. In the current studies

                           INHIBITION OF ATP FORMATION
                                                                                                      formaldehyde was not found in body
                                                                                                      fluids of rhesus monkeys poisoned
                                                                                                      with methyl alcohol, but it is not
STASIS OF AXOPLASMIC    /                        1
                                                 *                    \ INHIBITION OF Na.K            possible to eliminate formaldehyde as
                                                                                                      a toxic intermediate in methyl alcohol
           FLOW                                  ;                           ATP.ase ACTIVITY
                                                                                                      poisoning because, as has been
              F                            INTRA.AXONAL                              I
                                                                             INHIBITION OF
                                                                                                      pointed out previously," the lack of
                                                                                                      accumulation of formaldehyde may be
SWELLING                                   SWELLING                          ELECTRICAL               due to its extreme reactivity and to
                                                 I                           CONDUCTION               the abundance of enzyme systems
                                                                                                      available for its transformation to
                                          OPTIC-DISC EDEMA
                                                                                 BLINDNESS             formate. While it is possible that
Fig 2.—Sequence     of events in   methyl   alcohol    poisoning   in relation to ocular
                                                                                       toxicity.       formaldehyde mediates certain as-
                                                                                                       pects of the toxic syndrome of methyl
decreased bicarbonate in the arterial                  levels in the blood range from 9 to 14          alcohol poisoning, its role must be
blood. Formic acid levels were gener-                  mEq/liter. Also, formate levels in CSF          questioned and studied further in
ally maintained at about 7 mEq/liter                   are shown for two animals. They are             order to include it as a possible toxic
or more    at 20 hours or later after the              similar to values obtained in the blood         intermediate in methyl alcohol poison-
initial dose of methyl alcohol. The                    taken at the same time.                         ing. On the other hand, the presence
increases in formate anion corre-                                                                      of formic acid, the production of meta-
                                                                       COMMENT                         bolic acidosis, and ocular toxicity in
sponded to decreases in bicarbonate
anion in arterial blood. The data are                     In previous studies performed in             the monkey are related.
depicted in this fashion because it has                this and other laboratories-:' certain              Recent studies have shown that
been our experience that decreases in                  findings of Potts'" were confirmed               formate inhibits cytochrome oxi-
blood bicarbonate are a more reliable                  that relate to the use of the monkey as          dase'-'--" with Ki values determined to
measure   of the degree of metabolic                   a model for methyl alcohol toxicity in           be between 5 and 30 mM. It is impor-
acidosis after methyl alcohol ingestion                man.                                             tant to note that the formate
than is the arterial blood pH.                           The current study has focused on               concentrations in the blood of the
    The Table shows maximal changes                    the use of the monkey as an experi-              animals in our study reach 7 mEq/
in certain biochemical factors ob-                     mental model to show the develop-                liter or greater, 20 hours after the
tained from the animals shown in Fig                   ment of ocular toxicity due to methyl            first dose of methyl alcohol, and that
 1. Values for arterial blood pH range                 alcohol poisoning. While acute methyl             in the CSF the concentration of
between 7.13 and 7.28 and formate                      alcohol toxicity in the monkey does              formate is similar to that of blood.

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Therefore, diffuse areas of the body,                    activity and     mitochondrial electron              disruption of other adenosine triphos-
including nervous tissue, are exposed                    transport,   severe  disruption of optic             phate (ATP)-dependent systems such
to formate in concentrations that                        nerve function occurs. One conse-                    as membrane ATPase activities that
might inhibit cytochrome oxidase ac-                     quence of this process might be the                  are vital   to neural conduction pro-
tivity. It has been suggested that                       inhibition of neural axoplasmic flow                 cesses. Ultimately, disruption of these
cytochrome oxidase activity in white                     in the optic nerve. Furthermore, it has              functions might be the end result of
matter is low-' and may be critical to                   been reported-"'-" that there is a                   the optic nerve toxicity related to
that tissue. Therefore, there may be a                   dependence of axoplasmic transport                   metabolic products of methyl alcohol.
special sensitivity to agents that can                   in nerves on oxidative metabolism.                   Thus, Fig 2 may describe the sequence
alter cytochrome oxidase activity,                       Cyanide and azide, which are known                   of events in methyl alcohol poisoning
such as formate, and that can achieve                    to disrupt mitochondrial electron                    in relation to ocular toxicity.
concentrations adequate to disrupt                       transport, have been shown to block                     Further work will be needed to
enzymatic activity. The blood flow in                    axoplasmic flow in nerves by virtue of               confirm and revise this hypothesis,
the choroid is extremely high and it                     their block of oxidative metabo-                     but certain features of this scheme
has been shown that substances like                      lism.""'-" It is possible that formate is            must be considered as a possible mech-
peroxidase diffuse from choroidal ves-                   capable of producing similar effects                 anism for ocular toxicity of methyl
sels to the optic disc.-"' It is conceivable             on oxidative metabolism and axoplas-                 alcohol. With future studies, it may be
that formate diffuses through the                        mic flow. Consistent with this hypo-                 possible to elucidate some of the
same area and this may account, in                       thesis is the finding of Baumbach et al              mechanisms involved in the produc-
part, for concentrations of formate                       (see p 1859), who have observed an                  tion of the optic disc edema and cyto-
beyond those of other tissues and that                   increase in the number of mitochon-                  toxic edema in the CNS.
might explain why the optic disc area                    dria in the optic nerve of the animals
is seemingly more sensitive than the                     studied in this report and that may
rest of the white matter.                                indicate stasis of axoplasmic flow.'" "
                                                                                                               This investigation was supported by grants
   It is not inconceivable that through                   Another consequence of the inhibition               GM 19420, GM 12675, and EY 01576 from the
the inhibition of cytochrome oxidase                      of energy production would be the                   National Institutes of Health.


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