MiniReview
Pharmacology & Toxicology 1991, 69, 157-163.
Methanol and Formic Acid Toxicity: Biochemical Mechanisms Jyrki Liesivuori' and Heikki Savolainen' "Kuopio Regional Institute of Occupational Health P.O. Box 93, SF-70701 Kuopio, Finland and "Institute of Occupational Health Sciences, University of Lausanne, CH-1005 Lausanne, Switzerland (Received September 7, 1990; Accepted March 21, 1991) Abstract: Metabolism of methanol, methyl ethers, esters and amides give rise to formic acid. This acid is an inhibitor of the mitochondria1 cytochrome oxidase causing histotoxic hypoxia. Formic acid is a weaker inhibitor than cyanide and hydrosulphide anions. The body burden of formate in methanol poisoning is high enough to cause acidosis, and other clinical symptoms. Part of the protons can be attributed to formic acid whereas the most significant acid load results from the hypoxic metabolism. The acidosis causes e.g. dilatation of cerebral vessels, facilitation of the entry of calcium ions into cells, loss of lysosomal latency and deranged production of ATP. The latter effect seems to impede parathormonedependent calcium reabsorption in the kidney tubules. Besides, urinary acidification is affected by formic acid. Its excretion causes continuous recycling of the acid by the tubular cell C1-/formate exchanger. This sequence of events may partially explain an accumulation of formate in urine. Occupational exposure to vapours of methanol and formic acid can be quantitatively monitored by urinary formic acid determinations. Formic acid toxicity may prove a suitable model for agents causing histotoxic hypoxia.
Today methanol and methyl tertiary-butyl ether are being added to gasoline as antiknocking agents. Extensive tests are under way to use methanol alone as a fuel for internal combustion engines. In addition to solvent uses, methanol seems predestined to play an increasingly important role as an intermediate for other chemicals, e.g., formaldehyde, formic acid, methylamines and methyl esters. Formic acid has almost replaced mineral acids in textile dyeing, leather tanning, coagulation of latex rubber and electroplating. It is also an important precursor for synthesis of chemicals and pharmaceuticals and a valuable pH regulator in resin manufacturing. Formic acid is widely used as a preservative of silage in the Nordic countries. The first reports on amblyopia and amaurosis caused by skin absorption and ingestion of methanol were published in the beginning of this century. There has been much controversy about the causes of methanol toxicity although the metabolism of methanol to formaldehyde and further to formic acid in the body was understood quite early (R0e 1943). The accumulation of formic acid in the metabolic acidosis by methanol poisoning linked the toxicity of the two chemicals together (R0e 1943; Clay et al. 1975; McMartin ef al. 1980). The studies by Martin-Amat et al. (1978) revealed that the ocular lesion in methanol poisoning essentially represented a toxic optic neuropathy, not retinal oedema, and that it was produced by formate, and not by formaldehyde. The toxic effects of formic acid are due to an inhibition of cytochrome oxidase complex at the terminal end of the respiratory chain in the mitochondria (Nicholls 1976). This leads to "histotoxic hypoxia" (Erecinska & Wilson 1980). Besides the optic nerve, other organs (brain, heart, kidneys) with a high rate of oxygen consumption are possible targets (Zitting et al. 1982). The toxicology of formic acid has
principally been investigated in connection with methanol studies (Rse 1982). There are no recent reviews on formic acid itself although it is a common substance in modern chemical industry. Several attempts have been made to establish a biological monitoring method for determining the occupational exposure to these agents (Liesivuori & Savolainen 1987a). No satisfactory correlations have been found between the methanol exposure and urinary formic acid when urine samples have been taken immediately after work-shift (Heinrich & Angerer 1982). Some methods are impracticable for mass screening as they require multiple samplings per subject (Ferry et al. 1980; Sedivec et al. 1981). Metabolism of methanol and formic acid. Inhalation and skin absorption are the most important entry routes of toxic compounds in occupational exposure. Methanol and formic acid are easily absorbed through the respiratory system. Retention of inhaled methanol in humans was 58% and independent of pulmonary ventilation (Sedivec et al. 1981). The calculated absorption rate of liquid methanol through human skin is of the same magnitude as that of other solvents (Dutkiewicz et al. 1980). Ingested methanol and formic acid are absorbed almost totally as seen in the cases of accident and selfpoisoning (Naik et al. 1980). Both compounds are distributed in the body water compartment. The volume of distribution is 0.6-0.7 l/kg (methanol) and 0.5 l/kg (formic acid) in methanol poisoning cases (Sejersted et al. 1983). Methanol oxidation. The first step in the metabolic pathway of methanol is oxidation to formaldehyde (fig. 1). In rats, a catalase-peroxidase system is primarily responsible for the initial step while
158
MiniRevie w
JYRKI LIESIVUORI AND HEIKKI SAVOLAINEN
in humans and monkeys, alcohol dehydrogenase, ADH, plays a major role. The third possible pathway in methanol oxidation is the hepatic microsomal mixed-function oxidase system. Humans and monkeys do not oxidize methanol through the catalase-dependent system because of the low activities of the peroxide-generating enzymes, e.g., urate oxidase, glycolate oxidase and xanthine oxidase (Goodman & Tephly 1970). Formaldehyde oxidation. Low concentrations of formaldehyde in water exists almost solely in a hydrated form, methanediol (Bieber & Trumpler 1947; Walker 1975). This diol formed from methanol via formaldehyde (fig. 1) is a substrate for the ADH-catalyzed reaction resulting in formic acid formation. Formaldehyde accumulation has not been detected in body fluids or tissues after methanol administration. Following intravenous infusion of formaldehyde into monkeys, clearance of this compound from the blood occurs rapidly (t,,2 1.5 min; McMartin et al. 1979). A formaldehyde dehydrogenase appears to be quite specific for formaldehyde. In the reactions catalyzed by this enzyme, formaldehyde combines with reduced glutathione, GSH, to form S-formyl glutathione. The product hydrolyzes to form formic acid and GSH. In vitro experiments with human blood showed that formaldehyde is quickly oxidized to formic acid. The oxidation is carried out by a NADdependent formaldehyde dehydrogenase in the erythrocytes (Malorny 1969). This supports the observation of significant amounts of radioactivity retained in erythrocytes after injection of I4C-labelledformaldehyde in rats (Upreti et al. 1987). Formaldehyde oxidation can occur in liver mitochondria through a high-activity aldehyde dehydrogenase (Koivula & Koivusalo 1975). Metabolism through the tetrahydrofolic
acid-dependent one-carbon pool provides still another pathway for oxidation of formaldehyde to formic acid. Because of the numerous routes, all formaldehyde derived from methanol will rapidly be metabolized to formic acid as found in a single human case of formaldehyde poisoning (Eells et al. 1981). Formic acid metabolism. Two pathways have been suggested for the disposition of formic acid: oxidation either through the catalase-peroxidative system or through the one-carbon pool. The catalase system appears to be poor in rat and monkey probably due to the low level of peroxidative capacity of the hepatic systems and the low activity of peroxide-generating oxidases (Goodman & Tephly 1970). An alternative pathway for the metabolism of formic acid is a tetrahydrofolic aciddependent one-carbon pool. Formic acid enters this pool by combining with tetrahydrofolic acid, THF, to 1O-formylTHE The ATP-dependent reaction is catalyzed by 10-formyl-THF synthetase, a ubiquitous enzyme in mammalian tissues (Whiteley 1960). Various enzymatic reactions can direct the 10-formyl-THF to other pathways. The rate of disappearance of methanol from the blood in both rats and monkeys is approximately the same (Clay et al. 1975). However, rats metabolize formic acid at about twice the rate of that seen in monkeys (McMartin et al. 1977). Hepatic THF levels in humans and monkeys are only half those in rats, and formylated-THF derivates are 2-fold higher in monkeys than in rats (Black et al. 1985). The activity of 10-formyl-THFdehydrogenase, the enzyme cata-
I
y .284x t12.1
Urinary formic acid (mmol/mol creatinine)
200)-
I cytoplasm
r 0.80
/
H13
/
NADH NAD+ m ltochondria
Fig. 1. Metabolism of methanol. The initial dehydrogenation of methanol is catalyzed by alcohol dehydrogenase, ADH. The resultant formaldehyde is spontaneously hydrated to methanediol (Bieber & Triimpler 1947; Walker 1975) which is the substrate for ADH. The formic acid thus formed is liberated to circulation or it forms coordination species with the cytochrome haem iron in the mitochondria (Nicholls 1976). Cytochrome oxidase may act as peroxidase oxidizing formic acid to carbon dioxide with NAD+ as cosubstrate. This latter hypothetical reaction may be slow while the low-spin association of the formic acid with the cytochrome oxidase haem impedes mitochondria1 oxygen reduction to water.
I
I
5 10 Methanol in a i r (pmol/l) Fig. 2. Urinary formic acid excretion as a function of methanol exposure. Four to 5% of absorbed methanol is excreted as formic acid in the urine. The peak excretion rate is reached slowly, possibly because of cycling of chloride and formate by the tubular Cl-/ formate exchanger in the kidneys (Karniski & Aronson 1985) and slow initial dehydrogenation of methanol to formaldehyde.
MiniReview
159
METHANOL AND FORMIC ACID TOXICITY
lysing the final step of formic acid oxidation to carbon dioxide, is markedly smaller in monkey and human liver, being 20 to 25% of that in rat liver (Johlin et al. 1987). Thus, two mechanisms may be operative in explaining slow formic acid oxidation causing accumulation of the acid in humans, monkeys and pigs, namely low hepatic T H F levels and reduced hepatic 10-formyl-THF dehydrogenase activity (Makar et al. 1990). Accumulation of formic acid. Following its intravenous infusion at doses lower than 100 mg/kg (2.2 mmol/kg), formic acid is cleared from rats with a half-life of 12 min., and from monkeys with a half-life of 3 1 min. The respective half-lives increase with increasing doses in both species, which indicates that formic acid metabolism is a saturable process (Clay et al. 1975). When formate was infused into monkeys, the accumulation in blood lasted ten hours. After this, the concentrations ranged between 10 and 30 mmol/l (Martin-Amat et al. 1978). Formate elimination from the blood of pigs gave a half-life of 87 min. (Makar et al. 1990). The calculated urinary halflife of formic acid in rabbits is 7.2 hr (Liesivuori & Savolainen 1986). After oral administration of methanol. the amount of
Urinary formic acid
y 1 . 5 -70.5 ~
(mmol/rnol creatinine) 300
r:0.88
//
NzlO
excreted formic acid varies among species, in man being 5% and in dogs 20%. After intravenous administration of sodium formate, dogs eliminated 42% of the injected amount in 48 hr (Lund 1948a & b). Approximately 5% of the formic acid dose was excreted in the urine within 40 hr when given by gastric gavage to rabbits (Liesivuori & Savolainen 1986). When sodium formate was given orally to humans 2.1 % of the dose was detected in urine collected over 24 hr (Malorny 1969). Despite the slow clearance and the accumulation of formic acid in many species (Clay et al. 1979, only few studies have stated the figures of formic acid concentration in different organs. The accumulation of the acid varied from 0.4 mmol/g in the brain and liver to 0.6 mmol/g in the heart and kidney when 100 mg of formic acid/kg body weight (2.2 mmol/kg) when injected into the ear vein of the rabbits each day for a period of five days (Liesivuori et al. 1987). The formic acid concentrations in dogs after peroral administration of methanol were higher than in rabbits but decreased in the same order, i.e., kidney, liver, and brain (Lund 1948a). The formate level in the cerebrospinal fluid of methanol poisoned monkey (0.1 mol/kg) was measured to be about one half of the blood level. The formate concentration was 0.002 mmol/g in the liver and 0.0004 mmol/g in the kideny (McMartin et al. 1979). When disposition of formaldehyde after inhalation exposures was investigated in rats, the terminal half-life for radioactivity was estimated to be 55 hr. It was concluded that the oxidation of formaldehyde to formate and its incorporation via one-carbon metabolism are of major importance in the long-term pharmacokinetics of formaldehyde following inhalation exposures (Heck el al. 1983). Inhibition of cytochrome oxidase activity. Methanol and formic acid have common mechanisms of toxicity since formic acid is a metabolic end product of methanol and mainly responsible for the toxic inhibition of the cytochrome oxidase. Cytochrome oxidase is the terminal member of the eukar-
if
loo 50J'1 $1
1. Inhibited cyt a_,
I
I
300 Formic acid in a i r (nmol/l) 100
200
Fig. 3. Urinary formic acid as a function of formic acid exposure. Morning urinary formic acid excretion after a preceding 8-hr occupational exposure to the acid 17 hr earlier. A major part of the absorbed acid is oxidised to carbon dioxide. Nevertheless, urinary acid is linearly related to exposure. By coincidence, the current hygienic limit for methanol (200 p.pm.) and for formic acid (5 p p m . ) correspond to almost identical urinary formic acid concentration (200 mmol/mol creatinine), a value adopted also a biological exposure indicator by the American Conference of Governmental Industrial Hygienists (1990-1991 Threshold limit values for chemical substances and physical agents and biological exposure indices, ACGIH, Cincinnati, USA 1990).
5. Mitochondria1 damage
+-
\
a,
+ 2. Lactic acidosis
4. Enhanced generation of Of,HOZ and
3. Can+influx in cells
OHaL
5. Membrane damage Fig. 4. Model of formic acid effects: acidosis and histotoxicity. The initial effect of formic acid is its association with the cytochrome oxidase haem. Inhibited oxidative metabolism leads to acidosis and accumulation of lactate. Protons are known to increase the cell membrane calcium ion permeability and they may favour the generation of reactive oxygen species. The latter can cause cell and mitochondrial membrane damage aggravating the mitochondrial dysfunction and augmenting e.g. Ca2+ ion permeability.
160
JYRKI LIESIVUORI AND HEIKKI SAVOLAINEN
yotic mitochondrial electron transport chain and an integral protein complex of the inner mitochondrial membrane. This enzyme participates into the four electron reduction of oxygen molecule to water with concomitant synthesis of ATP. Formic acid inhibits the activity of cytochrome oxidase (fig. 1) by binding at the sixth coordination position of ferric haem iron (Keyhani & Keyhani 1980). Cyanide, carbon monoxide and sulfide in this order are more potent inhibitors than formic acid (Erecinska & Wilson 1980). The Ki for the inhibition of the oxidase by formic acids is 1 mmol (Nicholls 1976). The maximum concentrations of formic acid measured in blood, brain, heart, liver and kidney after five daily intravenous doses to rabbits were roughly similar to that causing impaired oxidative metabolism and damage at cellular level (Liesivuori et al. 1987). The inhibition by formic acid of cytochrome oxidase increases with decreasing pH, suggesting that the active inhibitor is the undissociated acid. The acid is permeable through the inner mitochondrial membrane only in this form (Nicholls 1976). The cytochrome oxidase catalyzed oxidation of formic acid results in the formation of the socalled mixed-valence state, a form of the enzyme in which one site has been reduced and to which the acid is bound, whereas the other site remains oxidized (Brittain et al. 1977). These findings have been summarized in a proposed reaction mechanism of formic acid oxidation catalysed by cytochrome oxidase (fig. 1). Effects of metabolic acidosis. The methanol poisoning is characterized by a severe metabolic acidosis (McMartin et al. 1980; Sejersted et al. 1983; Becker 1983). The Henderson-Hasselbalch equation predicts that with a pH drop of 0.3, which is commonly observed in methanol poisonings (Osterloh et al. 1986), the concentration of undissociated formic acid (pK, 3.7) doubles. Thus, acidosis may potentiate the inhibition of cellular respiration and hasten the onset of cellular injury. Also the progressive acidosis will induce circulatory failure. This leads to tissue hypoxia and lactic acid production, both of which further increase the acid load, in turn increasing undissociated formic acid. This cycle is termed “circulus hypoxicus” (Jacobsen & McMartin 1986). Formic acid levels in methanol poisonings can account only for part of the late increase in the anion gap (McMartin et al. 1980) whereas lactic acid may offer a more significant contribution (Smith et al. 1981; Shahangian & Ash 1986). Metabolic acidosis is characterized by an increase in the excretion of calcium (Schieppati et al. 1985), ammonia and protons (Simon et al. 1985; Silbernagl & Scheller 1986). Calcium has important regulatory roles in many metabolic reactions, e.g., the release of acetylcholine, the protein kinase reaction and the activation of neutral proteinase. The extracellular concentration of Ca2+ is 5,000- to 10,000-fold higher than the cytosolic one.. Renal tubular acidosis leads to altered whole body calcium metabolism (Sly et al. 1985). Formic acid in the kidneys appears to interfere with the tubular reabsorption mechanism for cal-
MiniReview
cium (Liesivuori & Savolainen 1987b). A linear increase in the urinary calcium and formic acid concentrations in the air was seen in workers exposed to either methanol or formic acid (Liesivuori & Savolainen 1987b). It seems that the formic acid concentration in the kidneys is sufficiently high to impede cyclic AMP-mediated parathormone-controlled Ca2+ reabsorption in the distal tubuli (Savolainen 1989). Nevertheless, the results are in agreement with those in sustained renal tubular acidosis (Sly et al. 1985). The chloride/formate exchanger recycles formate by nonionic diffusion in parallel with Na+/H+ exchange across the luminal membrane in the proximal tubule for the reabsorption of chloride (Karniski & Aronson 1985). This may delay excretion of the formic acid in the urine after formate exposure (Liesivuori & Savolainen 1987a). Addition of formate in proximal tubules reversibly increases the net volume reabsorption (Schild et al. 1987). It is a well known fact that renal ammoniagenesis in the proximal tubule cell is highly increased by chronic metabolic acidosis. Glutamine is the major substrate in ammoniagenesis. Deamidation by mitochondrial glutaminase yields ammonium and glutamate ions. Ammonium is secreted as ammonia and hydrogen ion by separate mechanisms reforming ammonium cation in the lumen (Silbernagl & Scheller 1986). The urinary ammonia concentrations from the workers occupationally exposed to methanol or formic acid were of an order of magnitude greater than the formic acid concentrations suggesting that formic acid interferes with ammoniagenesis (Liesivuori & Savolainen 1987a). This is consistent with the results from an animal study on cyanide, which is another effective inhibitor of cytochrome oxidase and inhibits glutamine deamination (Preuss et al. 1974). Cyanide has been shown to produce also a rapid dysfunction of hydrogen ion handling mechanisms (Maduh et al. 1990). Histological and ultrastructural alterations caused by formate. Identical changes in the ocular system, e.g., optic disc oedema, optic nerve lesions, and pupillary reflex changes, have been produced by methanol and formic acid in monkeys (Martin-Amat et al. 1978). Formate action in methanol amblyopia in humans is based on the histotoxic hypoxia in water-shed areas of the cerebral and distal optic nerve circulations. In cases of severe methanol intoxication, pathological changes also in central nerves system have been revealed by computerized axial tomography (Henze et al. 1986). The alterations in the brain specimens from the formic acid-exposed rabbits (Liesivuori et al. 1987) were less grave than the ones seen in monkeys with higher formic acid doses (Martin-Amat et al. 1978). However, the calcium deposits in the cerebral hemispheres and the hippocampus were in good agreement with the intracerebral calcifications associated with renal tubular acidosis (Sly et al. 1985). The increase in brain formic acid concentrations of the rabbits 1 hr after the last dose might be explained by acidification of blood which leads to less dissociated formic acid and in-
MiniReview
METHANOL AND FORMIC ACID TOXICITY
creased concentrations in cerebrospinal fluid (Liesivuori et al. 1987; McMartin et al. 1979). Furthermore, the increased H + concentration is a contributing factor in the increased permeability of cell membrane to Ca2+ (Iijima et al. 1986). Because of its higher oxygen requirements for cellular functions, the kidney functions on the verge of hypoxia even under normal conditions (Preuss et al. 1974). Mitochondria1 respiration and cellular calcium content and kinetics have been found to be profoundly altered in ischaemic renal failure (Schieppati et al. 1985). Thus, kidneys should be especially vulnerable to formic acid (Nicholls 1976; Karniski & Aronson 1985; Schild et al. 1987). This'seems to be the case for rabbits (Liesivuori et al. 1987). This was further supported by changes, e.g. oedematous degenerated epithelial cells, in the kidneys of rats exposed to vapours of formaldehyde and formic acid (Zitting et al. 1982). The formic acid induced acidosis (Liesivuori & Savolainen 1987a) exacerbates spontaneous sarcoplasmic reticulum Ca2+ release in myocardium and may be arrhythmogenic (Orchard et al. 1987). Since mitochondrial respiration produces most of the cellular ATP in the myocardium, it has been suggested that mitochondrial dysfunction may be a crucial factor in the transition from reversible to irreversible cardiac injury (Cheung et al. 1986). Furthermore, vasoconstriction has been demonstrated by formate infusion in dog (DeFelice et al. 1976a, b). The histotoxic changes by formic acid were marked in rabbit heart muscle seen as calcium aggregates and myelin figures. Liver is clearly less sensitive to hypoxic episodes (Zitting et al. 1982), although calcium deposits surrounded by inflammatory cells were observed as well (Liesivuori et al. 1987). Model of formic acid eflects at cellular level. The abovementioned aspects of formic acid toxicity at cellular level are summarized in a simplified model of calcium influx (fig. 4). Exposure to either methanol or formic acid leads to accumulation of acid in the body. Formic acid inhibits cytochrome oxidase, causing decreased synthesis of ATP (Nicholls 1976). This is followed by anaerobic glycolysis and lactic acidosis. At the same time, and also because of acidosis, the generation of superoxide anions and hydroxyl radicals is enhanced leading to membrane damage, lipid peroxidation and mitochondrial damage (Bralet et al. 1991; Chacon & Acosta 1991). This, and the decreased pH in acidosis, allows the influx of calcium into the cells (Iijima et al. 1986; Liesivuori et al. 1987; Liesivuori & Savolainen 1986). Although the mitochondrial dysfunction may be secondary to calcium overload in the mitochondria, the final consequence is cell death (Cheung et al. 1986; Richter & Kass 1991). The proposed model (fig. 4) is supported by the findings on cyanide-induced toxicity (Johnson et al. 1986; Maduh et al. 1990). Biological monitoring of occupational exposure to methanol and formic acid. The accumulation of formic acid has been detected in many human methanol poisoning cases (Lund 1984b; McMartin
161
et al. 1980; Sejersted et al. 1983; Shahangian et al. 1984). The formic acid concentrations in blood and urine correlated well with the severity of toxic effects as seen in experimental and clinical studies. This was not the case for methanol (Martin-Amat et al. 1978; Osterloh et al. 1986). No satisfactory correlations between occupational methanol exposure and urinary methanol concentrations have been found (Heinrich & Angerer 1982; Liesivuori & Savolainen 1987b). This makes formic acid a better indicator of methanol poisoning and of workers' exposure to methanol or formic acid (fig. 2 and 3). The elimination of formic acid from the body seems to be an unexpectedly slow process which might not have been observed in previous studies on monitoring variables in the exposure to methanol (Ferry et al. 1980; Sedivec et al. 1981; Heinrich & Angerer 1982). The half-life of formic acid determined in human methanol poisoning cases has been as long as 20 hr (Shahangian et al. 1984). This favours the delayed collection of a urine sample in the monitoring of the occupational exposure (Liesivuori 1986). The slow renal clearance of formic acid after exposure to methanol or formic acid may be caused by zero order kinetics in methanol and formic acid metabolism (Clay et al. 1975) as well as in urinary clearance of formic acid (fig. 3). Another aspect of slow excretion is the continuous recycling of formic acid and protons with chloride in the kidneys (Karniski & Aronson 1985). For practical reasons, sampling in biological monitoring is carried out either becore the work-shift or at the end of the work-shift. The former reflects accumulation of a toxic substance in repeated exposure while the latter is used for compounds with rather fast elimination. Establishment of a sampling strategy in biological monitoring of occupational exposure to methanol and formic acid (Liesivuori 1986) was needed before a practical relationship could be shown between inhaled methanol or formic acid vapour and the concentration of urinary formic acid (Liesivuori & Savolainen 1987b). In a study where urine samples were collected at the end of the work-shift, the levels of urinary formic acid in the methanol-exposed group were increased only to about 2-fold compared with those in the control group (Heinrich & Angerer 1982). The exposures to methanol and formic acid at the current occupational exposure limit values caused concentrations of four to six times those in the non-exposed group (Liesivuori & Savolainen 1987b). The correction of urinary formic acid for the excretion of creatinine has proved to be important in several works (Ferry et al. 1980; Liesivuori & Savolainen 1986; Ogata et al. 1989). Concluding remarks. Although the highest concentrations of formic acid detected in silage making and workshops (Liesivuori 1986; Liesivuori & Savolainen 1987a) constitute a significant health hazard, especially to individuals with cardiovascular disease or kidney ailments, there are only few reported cases of formic acid poisoning in the current literature. In addition to the local effects, the major complications included metabolic
162
JYRKI LIESIWORI AND HEIKKI SAVOLAINEN
acidosis, renal insuficiency and respiratory failure as expected from methanol poisoning cases (Naik et al. 1980). The symptoms may be delayed up to 24 hr due to methanol oxidation to formic acid, followed by more severe symptoms: vomiting, Kussmaul’s respiration, pain in the back and abdominal pain. Simultaneously, amblyopia sets in which may develop rapidly into amaurosis (R0e 1982). At the late stage of poisoning, acidosis progresses to increased lactate production and decreased pH. This leads to histotoxic hypoxia and circulatory failure (Jacobsen 8z McMartin 1986). The treatment of methanol poisoning includes administration of ethanol to block formate production and of folic acid to enhance formate oxidation to carbon dioxide (Becker 1983). Dialysis should be continued for a longer period of time to eliminate formic acid completely. This may be due to deranged cellular proton excretion mechanism which is a comparable situation to cyanide poisoning (Maduh et ul. 1990). Alkalinization of urine would also hinder the recycling of formic acid. An anion exchange inhibiting drug, e.g., furosemide, may be of potential benefit. By blocking the formate chloride exchanger, the gradient of formic acid non-ionic diffusion may be reversed favouring the excretion of formic acid rather than its reabsorption (Karniski & Aronson 1985).
References Becker, C. E.: Methanol poisoning. J. Emerg. Med. 1983, 1, 51-58. Bieber, R. & G. Triimpler: Angenaherte, spektrographische Bestimmung der Hydrations-gleichgewichtskonstantenwasserige Formaldehydlosungen. Helv. Chim. Acta 1947, 30, 186Cb1865. Black, K. A., J. T. Eells, P.E. Noker, C. A. Hawtrey & T. R. Tephly: Role of hepatic tetrahydrofolate in the species difference in methanol toxicity. Proc. Natl. Acad, Sci. USA 1985, 82, 3854-3858. Bralet, J., C. Bouvier, L. Schreiber & M. Boquillon: Effect of acidosis on brain lipid peroxidation in brain slices. Brain Res. 1991,539, 175-177.
Brittain, T., C. Greenwood & A. Johnson: Mixed-valence cytochrome oxidase-formate complex. Biochem. J . 1977, 167, 531-534.
Chacon, E. & D. Acosta: Mitochondrial regulation of superoxide by Ca2+: An alternate mechanism for the cardiotoxicity of doxorubicin. Toxicol. Appl. Pharmacol. 1991, 107, 117-128. Cheung, J. Y., A. Leaf & J. V. Bonventre: Mitochondria1 function and intracellular calcium in anoxic cardiac myocytes. Amer. J. Physiol. 1986, 250, C18-C25. Clay, K. L., R. C. Murphy & W. D. Watkins: Experimental methanol toxicity in the primate: analysis of metabolic acidosis. Toxicol. Appl. Pharmacol. 1975, 34, 49-61, DeFelice, A., W. Wilson & J. Ambre: Acute cardiovascular effects of intravenous methanol in the anesthetized dog. Toxicol. Appl. Pharmacol. 1976a, 38, 631438. DeFelice, A., W. Wilson & J. Ambre: Vasoactive effects of methanol and sodium formate on isolated canine basilar artery. Toxicol. Appl. Pharmacol. 1976b, 38, 595601. Dutkiewicz, B., J. Konczalik & W. Karwacki: Skin absorption and per 0s administration of methanol in men. Znt. Arch. Occup. Environ. Health 1980, 47, 81-88. Eells, J. T., K. E. McMartin, K. Black, V. Virayotha, R. H. Tisdell & T. R. Tephly: Formaldehyde poisoning. Rapid metabolism to formic acid. J . Amer. Med. Assoc. 1981, 246, 1237-1238.
MiniReview
Erecinska, M. & D. F. Wilson: Inhibitors of cytochrome c oxidase. Pharmacol. Therap. 1980, 8, 1-20. Ferry, D. G., W. A. Temple & E. G. McQueen: Methanol monitoring. Comparison of urinary methanol concentration with formic acid excretion rate as a measure of occupational exposure. In?. Arch. Occup. Environ. Health 1980, 47, 155-163. Goodman, J. I. & T. R. Tephly: Peroxidative oxidation of methanol in human liver: the role of hepatic microbody and soluble oxidases. Res. Commun. Chem. Pathol. Pharmacol. 1970,1,441-450. Heck, H., T. Y. Chin & M. C. Schmitz: Distribution of 14C-formaldehyde in rats inhalation exposure. In: Formaldehyde toxicity. Ed.: J. E. Gibson. Hemisphere Publishing Corporation, Washington D. C., 1983, pp. 26-37. Heinrich, R. & J. Angerer: Occupational chronic exposure to organic solvents. X. Biological parameters for methanol exposure. Int. Arch. Occup. Environ. Health 1982, 50, 341-349. Henze, T., P.Scheidt & H. W. Prange: Die Methanol-Intoxikation. Klinische, neuropathologische und computertomografische Befunde. Nervenarzt 1986,57, 658-661. Iijima, T., S. Ciani & S. Hagiwara: Effects of the external pH on Ca channels: experimental studies and theoretical considerations using a two-site, two-ion model. Proc. Natl. Acad. Sci. USA 1986, 83, 654-658.
Jacobsen, D. & K. E. McMartin: Methanol and ethylene glycol poisonings. Mechanism of toxicity, clinical course, diagnosis and treatment. Med. Toxicol. 1986, 1, 309-334. Johlin, F. C., C. S . Fortman, D. D. Nghiem & T. R. Tephly: Studies of the role of folk acid and folate-dependent enzymes in human methanol poisoning. Mol. Pharmaocl. 1987, 31, 557-561. Johnson, J. D., T. L. Meisenheimer & G. E. Isom: Cyanide-induced neurotoxicity: role of neuronal calcium. Toxicol. Appl. Pharmacol. 1986, 84, 464469. Karniski, L. P. & P. S . Aronson: Chloride/formate exchange with formic acid recycling: a mechanism of active chloride transport across epithelial membranes. Proc. Natl. Acad. Sci. USA 1985, 82, 63626365.
Keyhani, J. & E. Keyhani: EPR study of the effect of formate on cytochrome c oxidase. Biochem. Biophys. Res. Commun. 1980,92, 327-333.
Koivula, T. & M. Koivusalo: Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution. Biochim. Biophys. Acta 1975,397, 9-23. Liesivuori, J.: Slow urinary elimination of formic acid in occupationally exposed farmers. Ann. Occup. Hyg. 1986, 30, 329333.
Liesivuori, J. & H. Savolainen: Urinary excretion of formic acid in rabbit. Acta pharmacol. et toxicol. 1986, 58, 161-162. Liesivuori, J. & H. Savolainen: Urinary formic acid as an indicator of occupational exposure to formic acid and methanol. Amer. Ind. Hyg. Assoc. J . 1987a, 48, 32-34, Liesivuori, J., V.-M. Kosma, A. Naukkarinen & H. Savolainen: Kinetics and toxic effects of repeated intravenous dosage of formic acid in rabbits. Brit. J. Exp. Pathol. 1987, 68, 853-861. Liesivuori, J. & H. Savolainen: Effect of renal formic acid excretion on urinary calcium and ammonia concentrations. Klin. Wochenschr. 1987b, 65, 860-863. Lund, A.: Metabolism of methanol and formic acid in dogs. Acfa pharmacol. et toxicol. 1948a, 4, 108-121. Lund, A.: Excretion of methanol and formic acid in man after methanol consumption. Acta pharmacol. et toxicol. 1948b, 4, 205-212.
Maduh, E. U., J. L. Borowitz & G. E. Isom: Cyanide-induced alteration of cytosolic p H Involvement of cellular hydrogen ion handling processes. Toxicol.Appl. Pharmacol. 1990,106,20 1-208. Makar, A. B., T. R. Tephly, G. Sahin & G. Osweiler: Formate metabolism in young swine. Toxicol. Appl. Pharmacol. 1990,105, 315-320.
Malorny, G.: Stoffwechselversuchemit Natrium-fonniat und Ameisensaure beim Menschen. Z . Eriihrungswiss. 1969,9, 340-348.
MiniReview
METHANOL AND FORMIC ACID TOXICITY
Martin-Amat, G., K. E. McMartin, S. S. Hayreh & T. R. Tephly: Methanol poisoning: ocular toxicity produced by formate. Toxicol. Appl. Pharmacol. 1978, 45, 201-208. McMartin, K. E., J. J. Ambre & T. R. Tephly: Methanol poisoning in human subjects: role of formic acid accumulation in the metabolic acidosis. Amer. J . Med. 1980, 68, 414418. McMartin, K. E., G. Martin-Amat, A. B. Makar & T. R. Tephly: Methanol poisoning. V. Role of formate metabolism in the monkey. J . Pharmacol. Exp. Therap. 1977, 201, 564-572. McMartin, K. E., G. Martin-Amat, P. E. Noker & T. R. Tephly: Lack of role for formaldehyde in methanol poisoning in the monkey. Biochem. Pharmacol. 1979, 28, 645-649. Naik, R. B., W. P. Stephens, D. J. Wilson, A. Walker & H. A. Lee: Ingestion of formic acid-containing agents - report of three fatal cases. Postgrad. Med. J. 1980, 56, 451456. Nicholls, P.: The effects of formate on cytochrome aa, and on electron transport in the intact respiratory chain. Biochim. Biophys. Acta 1916, 430, 13-29. Ogata, M., T. Iwamoto & T. Kawai: Enzymatic assay of urinary formic acid as an index of methanol exposure. Ind. Health 1989, 27, 125-129. Orchard, C. H., S. R. Houser, A. A. Kort, A. Bahinski, M. C. Capogrossi & E. G. Lakatta: Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+release in rat myocardium. J. Gen. Physiol. 1987, 90, 145-165. Osterloh, J. D., S. M. Pond, S. Grady & C. E. Becker: Serum formate concentrations in methanol intoxication as a criterion for hemodialysis. Ann. Intern. Med. 1986, 104, 2W203. Preuss, H. G., K. Baird & H. Goldin: Oxygen consumption and ammoniagenesis in isolated dog renal tubules. J. Lab. Clin. Med. 1914,83,937-946. Richter, C. & G. E. N. Kass: Oxidative stress in mitochondria: Its relationship to cellular Ca2+ homeostasis, cell death, proliferation, and differentiation. Chem.-Biol. Interact. 1991, 77, 1-23. Rse, 0.: Clinical investigations of methyl alcohol poisoning with special reference to the pathogenesis and treatment of amblyopia. Acta med. scand. 1943, 113, 558-608. Rse, 0.:Species differences in methanol poisoning. CRC Crit. Rev. Toxicol. 1982, 10, 215-286. Savolainen, H.: New uses for old urine tests. Brit. J. Ind. Med. 1989, 46,361-363. Schieppati, A,, P. D. Wilson, T. J. Burke & R. W. Schrier: Effect of renal ischemia on cortical microsomal calcium accumulation. Amer. J . Physiol. 1985, 249, C476c483.
163
Schild, L., G. Giebiesch, L. P. Karniski & P. S. Aronson: Effect of formate on volume reabsorption in the rabbit proximal tubule. J. Clin. Invest. 1987, 79, 32-38. Sedivec, V., M. Mraz & J. Flek: Biological monitoring of persons exposed to methanol vapours. Int. Arch. Occup. Environ. Health 1981, 48, 257-271. Sejersted, 0. M., D. Jacobsen, S. Ovrebo & H. Jansen: Formate concentrations in plasma from patients poisoned with methanol. Acta med. scand. 1983, 213, 105-1 10. Shahangian, S. & K. 0. Ash: Formic and lactic acidosis in a fatal case of methanol intoxication. Clin. Chem. 1986, 32, 395-397. Shahangian, S., V. L. Robinson & T. A. Jennison: Formate concentrations in a case of methanol ingestion. Clin. Chem. 1984, 30, 1413-1414. Silbernagl, S. & D. Scheller: Formation and excretion of NH, < - > NH,+. New aspects of an old problem. Klin. Wochenschr. 1986, 64, 862-870. Simon, E., D. Martin & J. Buerkert: Contribution of individual superficial nephron segments to ammonium handling in chronic metabolic acidosis in the rat. J. Clin. Invest. 1985, 76, 855864. Sly, W. S., M. P. Whyte, V. Sundaram, R. E. Tashian, D. HewettEmmett, P. Guibaud, M. Vainsel, H. J. Baluarte, A. Gruskin, M. Al-Mosawi, N. Sakati & A. Ohlsson: Carbonic anhydrase I1 deficiency in 12 families with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. New Engl. J. Med. 1985, 313, 139-145. Smith, S. R., S. J. M. Smith & B. M. Buckley: Combined formate and lactate acidosis in methanol poisoning. Lancet 1981, ii, 1295-1 296. Upreti, N. K., M. Y.H. Farooqui, A. E. Ahmed & G. A. S. Ansari: Toxicokinetics and molecular interaction of ''C-formaldehyde in rats. Arch. Environ. Contam. Toxicol. 1987, 16, 263-273. Walker, J. F.: State of dissolved formaldehyde. In: Formaldehyde. Ed.: J. F. Walker. Robert E. Krieger Publishing Company, New York, 1975, pp. 52-61. Whiteley, H. R.: The distribution of formate-activating enzyme and other enzymes involving tetrahydrofolic acid in animal tissues. Comp. Biochem. Physiol. 1960, 1, 222-247. Zitting, A., H. Savolainen & J. Nickels: Biochemical and toxicological effects of single and repeated exposures to polyacetal thermodegratation products. Environ. Res. 1982, 29, 287-296.
Pharmacology & Toxicology 1991, 69, 157-163.
Methanol and Formic Acid Toxicity: Biochemical Mechanisms Jyrki Liesivuori' and Heikki Savolainen' "Kuopio Regional Institute of Occupational Health P.O. Box 93, SF-70701 Kuopio, Finland and "Institute of Occupational Health Sciences, University of Lausanne, CH-1005 Lausanne, Switzerland (Received September 7, 1990; Accepted March 21, 1991) Abstract: Metabolism of methanol, methyl ethers, esters and amides give rise to formic acid. This acid is an inhibitor of the mitochondria1 cytochrome oxidase causing histotoxic hypoxia. Formic acid is a weaker inhibitor than cyanide and hydrosulphide anions. The body burden of formate in methanol poisoning is high enough to cause acidosis, and other clinical symptoms. Part of the protons can be attributed to formic acid whereas the most significant acid load results from the hypoxic metabolism. The acidosis causes e.g. dilatation of cerebral vessels, facilitation of the entry of calcium ions into cells, loss of lysosomal latency and deranged production of ATP. The latter effect seems to impede parathormonedependent calcium reabsorption in the kidney tubules. Besides, urinary acidification is affected by formic acid. Its excretion causes continuous recycling of the acid by the tubular cell C1-/formate exchanger. This sequence of events may partially explain an accumulation of formate in urine. Occupational exposure to vapours of methanol and formic acid can be quantitatively monitored by urinary formic acid determinations. Formic acid toxicity may prove a suitable model for agents causing histotoxic hypoxia.
Today methanol and methyl tertiary-butyl ether are being added to gasoline as antiknocking agents. Extensive tests are under way to use methanol alone as a fuel for internal combustion engines. In addition to solvent uses, methanol seems predestined to play an increasingly important role as an intermediate for other chemicals, e.g., formaldehyde, formic acid, methylamines and methyl esters. Formic acid has almost replaced mineral acids in textile dyeing, leather tanning, coagulation of latex rubber and electroplating. It is also an important precursor for synthesis of chemicals and pharmaceuticals and a valuable pH regulator in resin manufacturing. Formic acid is widely used as a preservative of silage in the Nordic countries. The first reports on amblyopia and amaurosis caused by skin absorption and ingestion of methanol were published in the beginning of this century. There has been much controversy about the causes of methanol toxicity although the metabolism of methanol to formaldehyde and further to formic acid in the body was understood quite early (R0e 1943). The accumulation of formic acid in the metabolic acidosis by methanol poisoning linked the toxicity of the two chemicals together (R0e 1943; Clay et al. 1975; McMartin ef al. 1980). The studies by Martin-Amat et al. (1978) revealed that the ocular lesion in methanol poisoning essentially represented a toxic optic neuropathy, not retinal oedema, and that it was produced by formate, and not by formaldehyde. The toxic effects of formic acid are due to an inhibition of cytochrome oxidase complex at the terminal end of the respiratory chain in the mitochondria (Nicholls 1976). This leads to "histotoxic hypoxia" (Erecinska & Wilson 1980). Besides the optic nerve, other organs (brain, heart, kidneys) with a high rate of oxygen consumption are possible targets (Zitting et al. 1982). The toxicology of formic acid has
principally been investigated in connection with methanol studies (Rse 1982). There are no recent reviews on formic acid itself although it is a common substance in modern chemical industry. Several attempts have been made to establish a biological monitoring method for determining the occupational exposure to these agents (Liesivuori & Savolainen 1987a). No satisfactory correlations have been found between the methanol exposure and urinary formic acid when urine samples have been taken immediately after work-shift (Heinrich & Angerer 1982). Some methods are impracticable for mass screening as they require multiple samplings per subject (Ferry et al. 1980; Sedivec et al. 1981). Metabolism of methanol and formic acid. Inhalation and skin absorption are the most important entry routes of toxic compounds in occupational exposure. Methanol and formic acid are easily absorbed through the respiratory system. Retention of inhaled methanol in humans was 58% and independent of pulmonary ventilation (Sedivec et al. 1981). The calculated absorption rate of liquid methanol through human skin is of the same magnitude as that of other solvents (Dutkiewicz et al. 1980). Ingested methanol and formic acid are absorbed almost totally as seen in the cases of accident and selfpoisoning (Naik et al. 1980). Both compounds are distributed in the body water compartment. The volume of distribution is 0.6-0.7 l/kg (methanol) and 0.5 l/kg (formic acid) in methanol poisoning cases (Sejersted et al. 1983). Methanol oxidation. The first step in the metabolic pathway of methanol is oxidation to formaldehyde (fig. 1). In rats, a catalase-peroxidase system is primarily responsible for the initial step while
158
MiniRevie w
JYRKI LIESIVUORI AND HEIKKI SAVOLAINEN
in humans and monkeys, alcohol dehydrogenase, ADH, plays a major role. The third possible pathway in methanol oxidation is the hepatic microsomal mixed-function oxidase system. Humans and monkeys do not oxidize methanol through the catalase-dependent system because of the low activities of the peroxide-generating enzymes, e.g., urate oxidase, glycolate oxidase and xanthine oxidase (Goodman & Tephly 1970). Formaldehyde oxidation. Low concentrations of formaldehyde in water exists almost solely in a hydrated form, methanediol (Bieber & Trumpler 1947; Walker 1975). This diol formed from methanol via formaldehyde (fig. 1) is a substrate for the ADH-catalyzed reaction resulting in formic acid formation. Formaldehyde accumulation has not been detected in body fluids or tissues after methanol administration. Following intravenous infusion of formaldehyde into monkeys, clearance of this compound from the blood occurs rapidly (t,,2 1.5 min; McMartin et al. 1979). A formaldehyde dehydrogenase appears to be quite specific for formaldehyde. In the reactions catalyzed by this enzyme, formaldehyde combines with reduced glutathione, GSH, to form S-formyl glutathione. The product hydrolyzes to form formic acid and GSH. In vitro experiments with human blood showed that formaldehyde is quickly oxidized to formic acid. The oxidation is carried out by a NADdependent formaldehyde dehydrogenase in the erythrocytes (Malorny 1969). This supports the observation of significant amounts of radioactivity retained in erythrocytes after injection of I4C-labelledformaldehyde in rats (Upreti et al. 1987). Formaldehyde oxidation can occur in liver mitochondria through a high-activity aldehyde dehydrogenase (Koivula & Koivusalo 1975). Metabolism through the tetrahydrofolic
acid-dependent one-carbon pool provides still another pathway for oxidation of formaldehyde to formic acid. Because of the numerous routes, all formaldehyde derived from methanol will rapidly be metabolized to formic acid as found in a single human case of formaldehyde poisoning (Eells et al. 1981). Formic acid metabolism. Two pathways have been suggested for the disposition of formic acid: oxidation either through the catalase-peroxidative system or through the one-carbon pool. The catalase system appears to be poor in rat and monkey probably due to the low level of peroxidative capacity of the hepatic systems and the low activity of peroxide-generating oxidases (Goodman & Tephly 1970). An alternative pathway for the metabolism of formic acid is a tetrahydrofolic aciddependent one-carbon pool. Formic acid enters this pool by combining with tetrahydrofolic acid, THF, to 1O-formylTHE The ATP-dependent reaction is catalyzed by 10-formyl-THF synthetase, a ubiquitous enzyme in mammalian tissues (Whiteley 1960). Various enzymatic reactions can direct the 10-formyl-THF to other pathways. The rate of disappearance of methanol from the blood in both rats and monkeys is approximately the same (Clay et al. 1975). However, rats metabolize formic acid at about twice the rate of that seen in monkeys (McMartin et al. 1977). Hepatic THF levels in humans and monkeys are only half those in rats, and formylated-THF derivates are 2-fold higher in monkeys than in rats (Black et al. 1985). The activity of 10-formyl-THFdehydrogenase, the enzyme cata-
I
y .284x t12.1
Urinary formic acid (mmol/mol creatinine)
200)-
I cytoplasm
r 0.80
/
H13
/
NADH NAD+ m ltochondria
Fig. 1. Metabolism of methanol. The initial dehydrogenation of methanol is catalyzed by alcohol dehydrogenase, ADH. The resultant formaldehyde is spontaneously hydrated to methanediol (Bieber & Triimpler 1947; Walker 1975) which is the substrate for ADH. The formic acid thus formed is liberated to circulation or it forms coordination species with the cytochrome haem iron in the mitochondria (Nicholls 1976). Cytochrome oxidase may act as peroxidase oxidizing formic acid to carbon dioxide with NAD+ as cosubstrate. This latter hypothetical reaction may be slow while the low-spin association of the formic acid with the cytochrome oxidase haem impedes mitochondria1 oxygen reduction to water.
I
I
5 10 Methanol in a i r (pmol/l) Fig. 2. Urinary formic acid excretion as a function of methanol exposure. Four to 5% of absorbed methanol is excreted as formic acid in the urine. The peak excretion rate is reached slowly, possibly because of cycling of chloride and formate by the tubular Cl-/ formate exchanger in the kidneys (Karniski & Aronson 1985) and slow initial dehydrogenation of methanol to formaldehyde.
MiniReview
159
METHANOL AND FORMIC ACID TOXICITY
lysing the final step of formic acid oxidation to carbon dioxide, is markedly smaller in monkey and human liver, being 20 to 25% of that in rat liver (Johlin et al. 1987). Thus, two mechanisms may be operative in explaining slow formic acid oxidation causing accumulation of the acid in humans, monkeys and pigs, namely low hepatic T H F levels and reduced hepatic 10-formyl-THF dehydrogenase activity (Makar et al. 1990). Accumulation of formic acid. Following its intravenous infusion at doses lower than 100 mg/kg (2.2 mmol/kg), formic acid is cleared from rats with a half-life of 12 min., and from monkeys with a half-life of 3 1 min. The respective half-lives increase with increasing doses in both species, which indicates that formic acid metabolism is a saturable process (Clay et al. 1975). When formate was infused into monkeys, the accumulation in blood lasted ten hours. After this, the concentrations ranged between 10 and 30 mmol/l (Martin-Amat et al. 1978). Formate elimination from the blood of pigs gave a half-life of 87 min. (Makar et al. 1990). The calculated urinary halflife of formic acid in rabbits is 7.2 hr (Liesivuori & Savolainen 1986). After oral administration of methanol. the amount of
Urinary formic acid
y 1 . 5 -70.5 ~
(mmol/rnol creatinine) 300
r:0.88
//
NzlO
excreted formic acid varies among species, in man being 5% and in dogs 20%. After intravenous administration of sodium formate, dogs eliminated 42% of the injected amount in 48 hr (Lund 1948a & b). Approximately 5% of the formic acid dose was excreted in the urine within 40 hr when given by gastric gavage to rabbits (Liesivuori & Savolainen 1986). When sodium formate was given orally to humans 2.1 % of the dose was detected in urine collected over 24 hr (Malorny 1969). Despite the slow clearance and the accumulation of formic acid in many species (Clay et al. 1979, only few studies have stated the figures of formic acid concentration in different organs. The accumulation of the acid varied from 0.4 mmol/g in the brain and liver to 0.6 mmol/g in the heart and kidney when 100 mg of formic acid/kg body weight (2.2 mmol/kg) when injected into the ear vein of the rabbits each day for a period of five days (Liesivuori et al. 1987). The formic acid concentrations in dogs after peroral administration of methanol were higher than in rabbits but decreased in the same order, i.e., kidney, liver, and brain (Lund 1948a). The formate level in the cerebrospinal fluid of methanol poisoned monkey (0.1 mol/kg) was measured to be about one half of the blood level. The formate concentration was 0.002 mmol/g in the liver and 0.0004 mmol/g in the kideny (McMartin et al. 1979). When disposition of formaldehyde after inhalation exposures was investigated in rats, the terminal half-life for radioactivity was estimated to be 55 hr. It was concluded that the oxidation of formaldehyde to formate and its incorporation via one-carbon metabolism are of major importance in the long-term pharmacokinetics of formaldehyde following inhalation exposures (Heck el al. 1983). Inhibition of cytochrome oxidase activity. Methanol and formic acid have common mechanisms of toxicity since formic acid is a metabolic end product of methanol and mainly responsible for the toxic inhibition of the cytochrome oxidase. Cytochrome oxidase is the terminal member of the eukar-
if
loo 50J'1 $1
1. Inhibited cyt a_,
I
I
300 Formic acid in a i r (nmol/l) 100
200
Fig. 3. Urinary formic acid as a function of formic acid exposure. Morning urinary formic acid excretion after a preceding 8-hr occupational exposure to the acid 17 hr earlier. A major part of the absorbed acid is oxidised to carbon dioxide. Nevertheless, urinary acid is linearly related to exposure. By coincidence, the current hygienic limit for methanol (200 p.pm.) and for formic acid (5 p p m . ) correspond to almost identical urinary formic acid concentration (200 mmol/mol creatinine), a value adopted also a biological exposure indicator by the American Conference of Governmental Industrial Hygienists (1990-1991 Threshold limit values for chemical substances and physical agents and biological exposure indices, ACGIH, Cincinnati, USA 1990).
5. Mitochondria1 damage
+-
\
a,
+ 2. Lactic acidosis
4. Enhanced generation of Of,HOZ and
3. Can+influx in cells
OHaL
5. Membrane damage Fig. 4. Model of formic acid effects: acidosis and histotoxicity. The initial effect of formic acid is its association with the cytochrome oxidase haem. Inhibited oxidative metabolism leads to acidosis and accumulation of lactate. Protons are known to increase the cell membrane calcium ion permeability and they may favour the generation of reactive oxygen species. The latter can cause cell and mitochondrial membrane damage aggravating the mitochondrial dysfunction and augmenting e.g. Ca2+ ion permeability.
160
JYRKI LIESIVUORI AND HEIKKI SAVOLAINEN
yotic mitochondrial electron transport chain and an integral protein complex of the inner mitochondrial membrane. This enzyme participates into the four electron reduction of oxygen molecule to water with concomitant synthesis of ATP. Formic acid inhibits the activity of cytochrome oxidase (fig. 1) by binding at the sixth coordination position of ferric haem iron (Keyhani & Keyhani 1980). Cyanide, carbon monoxide and sulfide in this order are more potent inhibitors than formic acid (Erecinska & Wilson 1980). The Ki for the inhibition of the oxidase by formic acids is 1 mmol (Nicholls 1976). The maximum concentrations of formic acid measured in blood, brain, heart, liver and kidney after five daily intravenous doses to rabbits were roughly similar to that causing impaired oxidative metabolism and damage at cellular level (Liesivuori et al. 1987). The inhibition by formic acid of cytochrome oxidase increases with decreasing pH, suggesting that the active inhibitor is the undissociated acid. The acid is permeable through the inner mitochondrial membrane only in this form (Nicholls 1976). The cytochrome oxidase catalyzed oxidation of formic acid results in the formation of the socalled mixed-valence state, a form of the enzyme in which one site has been reduced and to which the acid is bound, whereas the other site remains oxidized (Brittain et al. 1977). These findings have been summarized in a proposed reaction mechanism of formic acid oxidation catalysed by cytochrome oxidase (fig. 1). Effects of metabolic acidosis. The methanol poisoning is characterized by a severe metabolic acidosis (McMartin et al. 1980; Sejersted et al. 1983; Becker 1983). The Henderson-Hasselbalch equation predicts that with a pH drop of 0.3, which is commonly observed in methanol poisonings (Osterloh et al. 1986), the concentration of undissociated formic acid (pK, 3.7) doubles. Thus, acidosis may potentiate the inhibition of cellular respiration and hasten the onset of cellular injury. Also the progressive acidosis will induce circulatory failure. This leads to tissue hypoxia and lactic acid production, both of which further increase the acid load, in turn increasing undissociated formic acid. This cycle is termed “circulus hypoxicus” (Jacobsen & McMartin 1986). Formic acid levels in methanol poisonings can account only for part of the late increase in the anion gap (McMartin et al. 1980) whereas lactic acid may offer a more significant contribution (Smith et al. 1981; Shahangian & Ash 1986). Metabolic acidosis is characterized by an increase in the excretion of calcium (Schieppati et al. 1985), ammonia and protons (Simon et al. 1985; Silbernagl & Scheller 1986). Calcium has important regulatory roles in many metabolic reactions, e.g., the release of acetylcholine, the protein kinase reaction and the activation of neutral proteinase. The extracellular concentration of Ca2+ is 5,000- to 10,000-fold higher than the cytosolic one.. Renal tubular acidosis leads to altered whole body calcium metabolism (Sly et al. 1985). Formic acid in the kidneys appears to interfere with the tubular reabsorption mechanism for cal-
MiniReview
cium (Liesivuori & Savolainen 1987b). A linear increase in the urinary calcium and formic acid concentrations in the air was seen in workers exposed to either methanol or formic acid (Liesivuori & Savolainen 1987b). It seems that the formic acid concentration in the kidneys is sufficiently high to impede cyclic AMP-mediated parathormone-controlled Ca2+ reabsorption in the distal tubuli (Savolainen 1989). Nevertheless, the results are in agreement with those in sustained renal tubular acidosis (Sly et al. 1985). The chloride/formate exchanger recycles formate by nonionic diffusion in parallel with Na+/H+ exchange across the luminal membrane in the proximal tubule for the reabsorption of chloride (Karniski & Aronson 1985). This may delay excretion of the formic acid in the urine after formate exposure (Liesivuori & Savolainen 1987a). Addition of formate in proximal tubules reversibly increases the net volume reabsorption (Schild et al. 1987). It is a well known fact that renal ammoniagenesis in the proximal tubule cell is highly increased by chronic metabolic acidosis. Glutamine is the major substrate in ammoniagenesis. Deamidation by mitochondrial glutaminase yields ammonium and glutamate ions. Ammonium is secreted as ammonia and hydrogen ion by separate mechanisms reforming ammonium cation in the lumen (Silbernagl & Scheller 1986). The urinary ammonia concentrations from the workers occupationally exposed to methanol or formic acid were of an order of magnitude greater than the formic acid concentrations suggesting that formic acid interferes with ammoniagenesis (Liesivuori & Savolainen 1987a). This is consistent with the results from an animal study on cyanide, which is another effective inhibitor of cytochrome oxidase and inhibits glutamine deamination (Preuss et al. 1974). Cyanide has been shown to produce also a rapid dysfunction of hydrogen ion handling mechanisms (Maduh et al. 1990). Histological and ultrastructural alterations caused by formate. Identical changes in the ocular system, e.g., optic disc oedema, optic nerve lesions, and pupillary reflex changes, have been produced by methanol and formic acid in monkeys (Martin-Amat et al. 1978). Formate action in methanol amblyopia in humans is based on the histotoxic hypoxia in water-shed areas of the cerebral and distal optic nerve circulations. In cases of severe methanol intoxication, pathological changes also in central nerves system have been revealed by computerized axial tomography (Henze et al. 1986). The alterations in the brain specimens from the formic acid-exposed rabbits (Liesivuori et al. 1987) were less grave than the ones seen in monkeys with higher formic acid doses (Martin-Amat et al. 1978). However, the calcium deposits in the cerebral hemispheres and the hippocampus were in good agreement with the intracerebral calcifications associated with renal tubular acidosis (Sly et al. 1985). The increase in brain formic acid concentrations of the rabbits 1 hr after the last dose might be explained by acidification of blood which leads to less dissociated formic acid and in-
MiniReview
METHANOL AND FORMIC ACID TOXICITY
creased concentrations in cerebrospinal fluid (Liesivuori et al. 1987; McMartin et al. 1979). Furthermore, the increased H + concentration is a contributing factor in the increased permeability of cell membrane to Ca2+ (Iijima et al. 1986). Because of its higher oxygen requirements for cellular functions, the kidney functions on the verge of hypoxia even under normal conditions (Preuss et al. 1974). Mitochondria1 respiration and cellular calcium content and kinetics have been found to be profoundly altered in ischaemic renal failure (Schieppati et al. 1985). Thus, kidneys should be especially vulnerable to formic acid (Nicholls 1976; Karniski & Aronson 1985; Schild et al. 1987). This'seems to be the case for rabbits (Liesivuori et al. 1987). This was further supported by changes, e.g. oedematous degenerated epithelial cells, in the kidneys of rats exposed to vapours of formaldehyde and formic acid (Zitting et al. 1982). The formic acid induced acidosis (Liesivuori & Savolainen 1987a) exacerbates spontaneous sarcoplasmic reticulum Ca2+ release in myocardium and may be arrhythmogenic (Orchard et al. 1987). Since mitochondrial respiration produces most of the cellular ATP in the myocardium, it has been suggested that mitochondrial dysfunction may be a crucial factor in the transition from reversible to irreversible cardiac injury (Cheung et al. 1986). Furthermore, vasoconstriction has been demonstrated by formate infusion in dog (DeFelice et al. 1976a, b). The histotoxic changes by formic acid were marked in rabbit heart muscle seen as calcium aggregates and myelin figures. Liver is clearly less sensitive to hypoxic episodes (Zitting et al. 1982), although calcium deposits surrounded by inflammatory cells were observed as well (Liesivuori et al. 1987). Model of formic acid eflects at cellular level. The abovementioned aspects of formic acid toxicity at cellular level are summarized in a simplified model of calcium influx (fig. 4). Exposure to either methanol or formic acid leads to accumulation of acid in the body. Formic acid inhibits cytochrome oxidase, causing decreased synthesis of ATP (Nicholls 1976). This is followed by anaerobic glycolysis and lactic acidosis. At the same time, and also because of acidosis, the generation of superoxide anions and hydroxyl radicals is enhanced leading to membrane damage, lipid peroxidation and mitochondrial damage (Bralet et al. 1991; Chacon & Acosta 1991). This, and the decreased pH in acidosis, allows the influx of calcium into the cells (Iijima et al. 1986; Liesivuori et al. 1987; Liesivuori & Savolainen 1986). Although the mitochondrial dysfunction may be secondary to calcium overload in the mitochondria, the final consequence is cell death (Cheung et al. 1986; Richter & Kass 1991). The proposed model (fig. 4) is supported by the findings on cyanide-induced toxicity (Johnson et al. 1986; Maduh et al. 1990). Biological monitoring of occupational exposure to methanol and formic acid. The accumulation of formic acid has been detected in many human methanol poisoning cases (Lund 1984b; McMartin
161
et al. 1980; Sejersted et al. 1983; Shahangian et al. 1984). The formic acid concentrations in blood and urine correlated well with the severity of toxic effects as seen in experimental and clinical studies. This was not the case for methanol (Martin-Amat et al. 1978; Osterloh et al. 1986). No satisfactory correlations between occupational methanol exposure and urinary methanol concentrations have been found (Heinrich & Angerer 1982; Liesivuori & Savolainen 1987b). This makes formic acid a better indicator of methanol poisoning and of workers' exposure to methanol or formic acid (fig. 2 and 3). The elimination of formic acid from the body seems to be an unexpectedly slow process which might not have been observed in previous studies on monitoring variables in the exposure to methanol (Ferry et al. 1980; Sedivec et al. 1981; Heinrich & Angerer 1982). The half-life of formic acid determined in human methanol poisoning cases has been as long as 20 hr (Shahangian et al. 1984). This favours the delayed collection of a urine sample in the monitoring of the occupational exposure (Liesivuori 1986). The slow renal clearance of formic acid after exposure to methanol or formic acid may be caused by zero order kinetics in methanol and formic acid metabolism (Clay et al. 1975) as well as in urinary clearance of formic acid (fig. 3). Another aspect of slow excretion is the continuous recycling of formic acid and protons with chloride in the kidneys (Karniski & Aronson 1985). For practical reasons, sampling in biological monitoring is carried out either becore the work-shift or at the end of the work-shift. The former reflects accumulation of a toxic substance in repeated exposure while the latter is used for compounds with rather fast elimination. Establishment of a sampling strategy in biological monitoring of occupational exposure to methanol and formic acid (Liesivuori 1986) was needed before a practical relationship could be shown between inhaled methanol or formic acid vapour and the concentration of urinary formic acid (Liesivuori & Savolainen 1987b). In a study where urine samples were collected at the end of the work-shift, the levels of urinary formic acid in the methanol-exposed group were increased only to about 2-fold compared with those in the control group (Heinrich & Angerer 1982). The exposures to methanol and formic acid at the current occupational exposure limit values caused concentrations of four to six times those in the non-exposed group (Liesivuori & Savolainen 1987b). The correction of urinary formic acid for the excretion of creatinine has proved to be important in several works (Ferry et al. 1980; Liesivuori & Savolainen 1986; Ogata et al. 1989). Concluding remarks. Although the highest concentrations of formic acid detected in silage making and workshops (Liesivuori 1986; Liesivuori & Savolainen 1987a) constitute a significant health hazard, especially to individuals with cardiovascular disease or kidney ailments, there are only few reported cases of formic acid poisoning in the current literature. In addition to the local effects, the major complications included metabolic
162
JYRKI LIESIWORI AND HEIKKI SAVOLAINEN
acidosis, renal insuficiency and respiratory failure as expected from methanol poisoning cases (Naik et al. 1980). The symptoms may be delayed up to 24 hr due to methanol oxidation to formic acid, followed by more severe symptoms: vomiting, Kussmaul’s respiration, pain in the back and abdominal pain. Simultaneously, amblyopia sets in which may develop rapidly into amaurosis (R0e 1982). At the late stage of poisoning, acidosis progresses to increased lactate production and decreased pH. This leads to histotoxic hypoxia and circulatory failure (Jacobsen 8z McMartin 1986). The treatment of methanol poisoning includes administration of ethanol to block formate production and of folic acid to enhance formate oxidation to carbon dioxide (Becker 1983). Dialysis should be continued for a longer period of time to eliminate formic acid completely. This may be due to deranged cellular proton excretion mechanism which is a comparable situation to cyanide poisoning (Maduh et ul. 1990). Alkalinization of urine would also hinder the recycling of formic acid. An anion exchange inhibiting drug, e.g., furosemide, may be of potential benefit. By blocking the formate chloride exchanger, the gradient of formic acid non-ionic diffusion may be reversed favouring the excretion of formic acid rather than its reabsorption (Karniski & Aronson 1985).
References Becker, C. E.: Methanol poisoning. J. Emerg. Med. 1983, 1, 51-58. Bieber, R. & G. Triimpler: Angenaherte, spektrographische Bestimmung der Hydrations-gleichgewichtskonstantenwasserige Formaldehydlosungen. Helv. Chim. Acta 1947, 30, 186Cb1865. Black, K. A., J. T. Eells, P.E. Noker, C. A. Hawtrey & T. R. Tephly: Role of hepatic tetrahydrofolate in the species difference in methanol toxicity. Proc. Natl. Acad, Sci. USA 1985, 82, 3854-3858. Bralet, J., C. Bouvier, L. Schreiber & M. Boquillon: Effect of acidosis on brain lipid peroxidation in brain slices. Brain Res. 1991,539, 175-177.
Brittain, T., C. Greenwood & A. Johnson: Mixed-valence cytochrome oxidase-formate complex. Biochem. J . 1977, 167, 531-534.
Chacon, E. & D. Acosta: Mitochondrial regulation of superoxide by Ca2+: An alternate mechanism for the cardiotoxicity of doxorubicin. Toxicol. Appl. Pharmacol. 1991, 107, 117-128. Cheung, J. Y., A. Leaf & J. V. Bonventre: Mitochondria1 function and intracellular calcium in anoxic cardiac myocytes. Amer. J. Physiol. 1986, 250, C18-C25. Clay, K. L., R. C. Murphy & W. D. Watkins: Experimental methanol toxicity in the primate: analysis of metabolic acidosis. Toxicol. Appl. Pharmacol. 1975, 34, 49-61, DeFelice, A., W. Wilson & J. Ambre: Acute cardiovascular effects of intravenous methanol in the anesthetized dog. Toxicol. Appl. Pharmacol. 1976a, 38, 631438. DeFelice, A., W. Wilson & J. Ambre: Vasoactive effects of methanol and sodium formate on isolated canine basilar artery. Toxicol. Appl. Pharmacol. 1976b, 38, 595601. Dutkiewicz, B., J. Konczalik & W. Karwacki: Skin absorption and per 0s administration of methanol in men. Znt. Arch. Occup. Environ. Health 1980, 47, 81-88. Eells, J. T., K. E. McMartin, K. Black, V. Virayotha, R. H. Tisdell & T. R. Tephly: Formaldehyde poisoning. Rapid metabolism to formic acid. J . Amer. Med. Assoc. 1981, 246, 1237-1238.
MiniReview
Erecinska, M. & D. F. Wilson: Inhibitors of cytochrome c oxidase. Pharmacol. Therap. 1980, 8, 1-20. Ferry, D. G., W. A. Temple & E. G. McQueen: Methanol monitoring. Comparison of urinary methanol concentration with formic acid excretion rate as a measure of occupational exposure. In?. Arch. Occup. Environ. Health 1980, 47, 155-163. Goodman, J. I. & T. R. Tephly: Peroxidative oxidation of methanol in human liver: the role of hepatic microbody and soluble oxidases. Res. Commun. Chem. Pathol. Pharmacol. 1970,1,441-450. Heck, H., T. Y. Chin & M. C. Schmitz: Distribution of 14C-formaldehyde in rats inhalation exposure. In: Formaldehyde toxicity. Ed.: J. E. Gibson. Hemisphere Publishing Corporation, Washington D. C., 1983, pp. 26-37. Heinrich, R. & J. Angerer: Occupational chronic exposure to organic solvents. X. Biological parameters for methanol exposure. Int. Arch. Occup. Environ. Health 1982, 50, 341-349. Henze, T., P.Scheidt & H. W. Prange: Die Methanol-Intoxikation. Klinische, neuropathologische und computertomografische Befunde. Nervenarzt 1986,57, 658-661. Iijima, T., S. Ciani & S. Hagiwara: Effects of the external pH on Ca channels: experimental studies and theoretical considerations using a two-site, two-ion model. Proc. Natl. Acad. Sci. USA 1986, 83, 654-658.
Jacobsen, D. & K. E. McMartin: Methanol and ethylene glycol poisonings. Mechanism of toxicity, clinical course, diagnosis and treatment. Med. Toxicol. 1986, 1, 309-334. Johlin, F. C., C. S . Fortman, D. D. Nghiem & T. R. Tephly: Studies of the role of folk acid and folate-dependent enzymes in human methanol poisoning. Mol. Pharmaocl. 1987, 31, 557-561. Johnson, J. D., T. L. Meisenheimer & G. E. Isom: Cyanide-induced neurotoxicity: role of neuronal calcium. Toxicol. Appl. Pharmacol. 1986, 84, 464469. Karniski, L. P. & P. S . Aronson: Chloride/formate exchange with formic acid recycling: a mechanism of active chloride transport across epithelial membranes. Proc. Natl. Acad. Sci. USA 1985, 82, 63626365.
Keyhani, J. & E. Keyhani: EPR study of the effect of formate on cytochrome c oxidase. Biochem. Biophys. Res. Commun. 1980,92, 327-333.
Koivula, T. & M. Koivusalo: Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution. Biochim. Biophys. Acta 1975,397, 9-23. Liesivuori, J.: Slow urinary elimination of formic acid in occupationally exposed farmers. Ann. Occup. Hyg. 1986, 30, 329333.
Liesivuori, J. & H. Savolainen: Urinary excretion of formic acid in rabbit. Acta pharmacol. et toxicol. 1986, 58, 161-162. Liesivuori, J. & H. Savolainen: Urinary formic acid as an indicator of occupational exposure to formic acid and methanol. Amer. Ind. Hyg. Assoc. J . 1987a, 48, 32-34, Liesivuori, J., V.-M. Kosma, A. Naukkarinen & H. Savolainen: Kinetics and toxic effects of repeated intravenous dosage of formic acid in rabbits. Brit. J. Exp. Pathol. 1987, 68, 853-861. Liesivuori, J. & H. Savolainen: Effect of renal formic acid excretion on urinary calcium and ammonia concentrations. Klin. Wochenschr. 1987b, 65, 860-863. Lund, A.: Metabolism of methanol and formic acid in dogs. Acfa pharmacol. et toxicol. 1948a, 4, 108-121. Lund, A.: Excretion of methanol and formic acid in man after methanol consumption. Acta pharmacol. et toxicol. 1948b, 4, 205-212.
Maduh, E. U., J. L. Borowitz & G. E. Isom: Cyanide-induced alteration of cytosolic p H Involvement of cellular hydrogen ion handling processes. Toxicol.Appl. Pharmacol. 1990,106,20 1-208. Makar, A. B., T. R. Tephly, G. Sahin & G. Osweiler: Formate metabolism in young swine. Toxicol. Appl. Pharmacol. 1990,105, 315-320.
Malorny, G.: Stoffwechselversuchemit Natrium-fonniat und Ameisensaure beim Menschen. Z . Eriihrungswiss. 1969,9, 340-348.
MiniReview
METHANOL AND FORMIC ACID TOXICITY
Martin-Amat, G., K. E. McMartin, S. S. Hayreh & T. R. Tephly: Methanol poisoning: ocular toxicity produced by formate. Toxicol. Appl. Pharmacol. 1978, 45, 201-208. McMartin, K. E., J. J. Ambre & T. R. Tephly: Methanol poisoning in human subjects: role of formic acid accumulation in the metabolic acidosis. Amer. J . Med. 1980, 68, 414418. McMartin, K. E., G. Martin-Amat, A. B. Makar & T. R. Tephly: Methanol poisoning. V. Role of formate metabolism in the monkey. J . Pharmacol. Exp. Therap. 1977, 201, 564-572. McMartin, K. E., G. Martin-Amat, P. E. Noker & T. R. Tephly: Lack of role for formaldehyde in methanol poisoning in the monkey. Biochem. Pharmacol. 1979, 28, 645-649. Naik, R. B., W. P. Stephens, D. J. Wilson, A. Walker & H. A. Lee: Ingestion of formic acid-containing agents - report of three fatal cases. Postgrad. Med. J. 1980, 56, 451456. Nicholls, P.: The effects of formate on cytochrome aa, and on electron transport in the intact respiratory chain. Biochim. Biophys. Acta 1916, 430, 13-29. Ogata, M., T. Iwamoto & T. Kawai: Enzymatic assay of urinary formic acid as an index of methanol exposure. Ind. Health 1989, 27, 125-129. Orchard, C. H., S. R. Houser, A. A. Kort, A. Bahinski, M. C. Capogrossi & E. G. Lakatta: Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+release in rat myocardium. J. Gen. Physiol. 1987, 90, 145-165. Osterloh, J. D., S. M. Pond, S. Grady & C. E. Becker: Serum formate concentrations in methanol intoxication as a criterion for hemodialysis. Ann. Intern. Med. 1986, 104, 2W203. Preuss, H. G., K. Baird & H. Goldin: Oxygen consumption and ammoniagenesis in isolated dog renal tubules. J. Lab. Clin. Med. 1914,83,937-946. Richter, C. & G. E. N. Kass: Oxidative stress in mitochondria: Its relationship to cellular Ca2+ homeostasis, cell death, proliferation, and differentiation. Chem.-Biol. Interact. 1991, 77, 1-23. Rse, 0.: Clinical investigations of methyl alcohol poisoning with special reference to the pathogenesis and treatment of amblyopia. Acta med. scand. 1943, 113, 558-608. Rse, 0.:Species differences in methanol poisoning. CRC Crit. Rev. Toxicol. 1982, 10, 215-286. Savolainen, H.: New uses for old urine tests. Brit. J. Ind. Med. 1989, 46,361-363. Schieppati, A,, P. D. Wilson, T. J. Burke & R. W. Schrier: Effect of renal ischemia on cortical microsomal calcium accumulation. Amer. J . Physiol. 1985, 249, C476c483.
163
Schild, L., G. Giebiesch, L. P. Karniski & P. S. Aronson: Effect of formate on volume reabsorption in the rabbit proximal tubule. J. Clin. Invest. 1987, 79, 32-38. Sedivec, V., M. Mraz & J. Flek: Biological monitoring of persons exposed to methanol vapours. Int. Arch. Occup. Environ. Health 1981, 48, 257-271. Sejersted, 0. M., D. Jacobsen, S. Ovrebo & H. Jansen: Formate concentrations in plasma from patients poisoned with methanol. Acta med. scand. 1983, 213, 105-1 10. Shahangian, S. & K. 0. Ash: Formic and lactic acidosis in a fatal case of methanol intoxication. Clin. Chem. 1986, 32, 395-397. Shahangian, S., V. L. Robinson & T. A. Jennison: Formate concentrations in a case of methanol ingestion. Clin. Chem. 1984, 30, 1413-1414. Silbernagl, S. & D. Scheller: Formation and excretion of NH, < - > NH,+. New aspects of an old problem. Klin. Wochenschr. 1986, 64, 862-870. Simon, E., D. Martin & J. Buerkert: Contribution of individual superficial nephron segments to ammonium handling in chronic metabolic acidosis in the rat. J. Clin. Invest. 1985, 76, 855864. Sly, W. S., M. P. Whyte, V. Sundaram, R. E. Tashian, D. HewettEmmett, P. Guibaud, M. Vainsel, H. J. Baluarte, A. Gruskin, M. Al-Mosawi, N. Sakati & A. Ohlsson: Carbonic anhydrase I1 deficiency in 12 families with the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. New Engl. J. Med. 1985, 313, 139-145. Smith, S. R., S. J. M. Smith & B. M. Buckley: Combined formate and lactate acidosis in methanol poisoning. Lancet 1981, ii, 1295-1 296. Upreti, N. K., M. Y.H. Farooqui, A. E. Ahmed & G. A. S. Ansari: Toxicokinetics and molecular interaction of ''C-formaldehyde in rats. Arch. Environ. Contam. Toxicol. 1987, 16, 263-273. Walker, J. F.: State of dissolved formaldehyde. In: Formaldehyde. Ed.: J. F. Walker. Robert E. Krieger Publishing Company, New York, 1975, pp. 52-61. Whiteley, H. R.: The distribution of formate-activating enzyme and other enzymes involving tetrahydrofolic acid in animal tissues. Comp. Biochem. Physiol. 1960, 1, 222-247. Zitting, A., H. Savolainen & J. Nickels: Biochemical and toxicological effects of single and repeated exposures to polyacetal thermodegratation products. Environ. Res. 1982, 29, 287-296.
