TOXICOLOGY
AND APPLIED
PHARMACOLOGY
117,26-36 (1992)
Physiologically Based Pharmacokinetic Model for Methanol in Rats, Monkeys, and Humans VICKY L.HoRToN,*,~ *Chemical
Industry
Institute of Toxicology, University of North
MARK A.HIGUCHI,*
ANDDOUGLASE.RICKERT*
Research Triangle Park, North Carolina Carolina-Chapel Hill, Chapel Hill, North
27709: and jCurriculum Carolina 27514
in Toxicology,
Received November 22. 199 1; accepted June 12, 1992
The acute toxic effects induced by methanol have been the focus of extensive study but little information about chronic effects has been reported (Andrews et al., 1987). It may be necessary to test this compound for chronic, lowlevel exposure effects if methanol is utilized as a fuel extender or as a feedstock for fuel synthesis, since the potential for occupational and environmental exposures would increase (Posner, 1975). The selection of an animal model for chronic methanol studies is problematic because there are marked species differences in acute toxicity symptoms and metabolism after large oral or parenteral methanol doses (Tephly et al., 1979; Tephly and McMartin, 1984). The development of acute toxicity in humans and nonhuman primates is apparently a consequence of their slower rate of formate metabolism (McMartin et al., 1977; Noker and Tephly, 1980). It is unknown whether substantial species differences in methanol or formate metabolism exist at the low methanol vapor concentrations relevant to chronic human exposures. To address the problems associated with the appropriate design of chronic methanol studies, methanol pharmacokinetics were characterized in male Fischer-344 rats and rhesus monkeys exposed to atmospheric methanol concentrations ranging from 50 to 2000 ppm for 6 hr. A physiologically based pharmacokinetic model was then developed to simulate the in viva time course data. The results indicate a dose dependency in methanol pharmacokinetics and a similarity between rats and primates in elimination of low methanol doses.
Physiologically Based Pharmacokinetic Model for Methanol in Rats, Monkeys, and Humans. HORTON, V. L., HIGUCHI, M. A., AND RICKERT, D. E. (1992). Toxicol. Appl. Pharmacol. 117,26-36. The pharmacokinetics of methanol and formate were characterized in male Fischer-344 rats and rhesus monkeys exposed to methanol vapor concentrations between 50 and 2000 ppm for 6 hr. End-of-exposure blood methanol concentrations were not directly proportional to the atmospheric concentration. The methanol exposures did not cause an elevation in blood formate concentrations. After an intravenous dose of [14C]methanol in rats, metabolism, exhalation, and renal excretion contributed 96.6, 2.6, and O.S%, respectively, to the elimination of blood methanol concentrations. These values and the calculated renal methanol extraction efficiency (0.007) are nearly identical to those for humans after low doses of methanol. A physiologically based pharmacokinetic model was developed to simulate the in vivo data. In order to simulate the observed blood methanol concentrations in the inhalation studies in rats, a double pathway for methanol metabolism to formaldehyde was used. One path used rodent catalase K,,, and V,,,, values and the other used a smaller K,,, and V,,,,, to simulate an enzyme with a higher affinity and lower capacity. The lack of proportionality observed in endof-exposure blood methanol concentrations may be due to saturation of an enzyme with higher affinity and lower capacity than catalase. The physiologically based pharmacokinetic model was modified to simulate the monkey data and was scaled-up for humans. In order to simulate the monkey blood methanol concentrations, the use of rodent catalase parameters for methanol metabolism was required. This finding suggests that primates and rodents may be similar in the initial step of methanol metabolism after low methanol doses. Previously published human urinary methanol excretion data was successfully simulated by the model. The models were used to predict the atmospheric methanol concentration range over which the laboratory species exhibit quantitative similarities with humans. Below 1200 ppm, all three speciesexhibit similar end-of-exposure blood methanol concentrations and a linear relationship between atmospheric and blood methanol concentrations. At higher atmospheric concentrations, external and internal methanol concentrations increase disparately, suggesting that delivered dose rather than exposure concentration should be used in interpreting data from high-dose studies. 0 1992 Academic Press, Inc. 0041-008X/92 $5.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
METHODS Reagents. The following chemicals were obtained from Fisher Scientific (Pittsburgh, PA): sodium chloride, sodium hydroxide, dichloromethane, isopropanol, methanol (HPLC grade, >99.9% pure), 2-methoxyethanol, ethanolamine, acetonitrile. sulfuric acid, and magnesium perchlorate. Sodium heparin (porcine intestinal mucosa origin), zinc sulfate, potassium phosphate (monobasic and dibasic), sodium formate, /3-nicotinamide adenine dinucleotide, diaphorasc (Type II-L, derived from Clostridium kluyveri; EC 1.6.4.3). formate dehydrogenase (Type I, derived from Pseudomonas oxalaticus: EC 1.2. I .2). [‘%Z]Methanol (>98% pure by gas chromatography and HPLC), and sodium [“Clformate (>98’% pure by HPLC) were purchased from Sigma 26
METHANOL
PHARMACOKINETIC
Chemical Co. (St. Louis, MO). Resazurin and benzalkonium chloride were obtained from the Eastman Kodak Co. (Rochester, NY) and Aldrich Chemical Co. (Milwaukee, WI). respectively. Animals rind /~&a&r. Male Fischer-344 rats (CDF(F-344)/CrlBR), 190-230 g, were obtained from Charles River Laboratories, Inc. (Kingston, NY, and Raleigh, NC) and allowed to acclimate for at least 2 weeks in a mass-air displacement room (Bioclean, Hazleton Systems, Inc., Vienna, VA) maintained at 22 f 1.5”C with a relative humidity of 50 + 10% and a 12-hr 1ight:dark cycle. NIH-07 open formula diet (Ziegler Brothers, Gardner, PA) and water (Nanopure, Sybron-Barnstead, Boston, MA) were provided ud libitum. Sentinel rats were routinely screened and found to be seronegative for mycoplasmal and viral infections (Microbiological Associates, Bethesda, MD). To permit repeated blood sampling, the rats were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Washington Crossing, NJ) and the right jugular vein was cannulated with Silastic tubing (0.025 in. id. X 0.047 in. o.d., 20 cm long; Dow Corning Corporation, Midland, MI); overnight recovery was permitted. Three young adult male rhesus monkeys (Macaca mulatta, 5-7 kg) were obtained from Hazleton Laboratories (Adice, TX). The animals were housed individually in 48 X 32 X 36-in. stainless steel cages with squeezeback assemblies to facilitate handling. Certified Primate Chow (Purina Mills, Inc., St. Louis, MO) and water were provided except during the inhalation exposure. Each monkey was fitted with a plexiglass collar equipped with metal rings to permit poling capture, and each was trained to sit quietly in a primate restraining chair for at least 2 hr before use in the experiment. A vascular-access port (Model GPV; Norfolk Medical Products, Inc., Skokie, IL), equipped with a Hydromer-coated polyurethane catheter (Hydrocath, 34 French v 35 cm: CardioSearch, Tampa, FL), was implanted in the right femoral vein of each monkey. The catheter was inserted proximally into the vein about 14 cm, placing the tip in close proximity to the abdominal vena cava. The procedures were conducted using sterile surgical techniques and a combination of ketamine hydrochloride (Ketaset, Bristol Veterinary Products, Syracuse, NY). thiamylal sodium (Bio-Tal, Bio-Ceutic Laboratories, St. Joseph, MO), and halothane gas (Fluothane; Fort Dodge Laboratories. Inc., Fort Dodge, IA) for anesthesia. A recovery period of 10 days was allowed before exposure to methanol vapors. Patency of the ports was maintained by flushing with I to 5 ml of sterile heparinized saline (1000 units sodium heparin/ml 0.9% saline) 3 days/week. After the study, the animals were anesthetized with a combination of ketamine, atropine, and intravenous thiamylal sodium, and the ports were surgically removed. Blood sampling. Blood samples ( 150 ~1) were collected from rats through the indwelling cannulae and placed in heparinized Microtainers (BectonDickinson, Rutherford. NJ). Samples were collected at selected times during and after each inhalation exposure and after the intravenous [‘4C]methanol dose. A IOO-~1aliquot ofwhole blood was mixed with 140 pl distilled water, 30 ~1 7.5% (w/w) aqueous ZnSO.,. and 30 ~1 0.4 N NaOH to precipitate protein (Somogyi, 1945). After centrifugation (15 min at 10,000 rpm; SureSpin, Helena. TX). the supemate volume was determined before freezing at -20°C. Blood samples from monkeys were collected aseptically through the port with a sterile Huber point needle (22 g, 1 in., deflected tip; Norfolk Medical Products, Inc., Skokie, IL) and syringe. Samples were obtained prior to each exposure and at designated time points after the exposure was terminated. For the initial 2 hr after exposure, the monkey was kept in the primate restraining chair. After this period, the monkey was returned to his cage. For each subsequent sample, the monkey was caught, chaired, the sample obtained, and the animal immediately returned to his cage. The samples for blood methanol and formate concentration measurements (200 ~1) were placed in heparinized microtainers and the samples for blood pH and bicarbonate (250 ~1) were drawn in heparinized syringes. The port was then flushed with 0.3 ml heparinized saline. Determination of inhaled methanol blood kinetics. Four rats per concentration were exposed to nominal methanol atmospheres of 0.200, 1200, or 2000 ppm (0, 260, 1560, or 2600 mg/m3) for 6 hr in a glass, four-station head-only chamber. The atmospheres were generated by blowing nitrogen
27
MODEL
over methanol contained in a 100 ml impinger and then diluting this vapor with air. The chamber methanol concentration was continuously monitored with a Miran 1A infrared gas analyzer (Foxboro-Wilks, Norwalk, CT; wavelength = 9.5 pm) in a closed loop system. Adjustments in chamber air flow rate (-20 liters/min) were used to maintain the desired concentrations. Analytical chamber concentrations were 0 + 0 (means f SEM: n = 6), 197 f 5.5, 1175 f 17.4, and 1994 f 3.8 ppm for nominal concentrations of 0, 200, 1200, and 2000 ppm. respectively. Each monkey was individually exposed to nominal methanol atmospheres of 0, 50,200, 1200. and 2000 ppm (0,65: 260, 1560, and 2600 mg/m’) in random order with a 2-week recovery period between exposures. The monkeys were exposed in a 16-m’ stainless-steel whole-body chamber (Hazleton Systems, Inc., Aberdeen, MD). Methanol atmospheres were generated with a J-tube evaporation system (Miller et al., 1980) with the rate of methanol delivery to the J-tube controlling the chamber concentration. The atmospheric methanol concentration was monitored as above. Analytical chamber concentrations during the 6 hr exposures to 0, 50, 200, 1200, or 2000 ppm nominal methanol atmospheres were 0 + 0 (means i SEM; n = 18), 49 + 0.5, 205 * 2.7, 1206 + 5.3, and 2006 f 3.8 ppm, respectively. Blood methanol concentrations were measured by gas chromatography using a 5850A Hewlett-Packard gas chromatograph (Hewlett-Packard Instrument Company, Avondale, PA) equipped with a flame ionization detector and a glass column (10 ft X 2 mm i.d.) packed with SO/l00 Carbonpack C coated with 0.1% SP-1000 (Supelco. Bellefonte. PA) held at 60°C. A helium flow rate of 20 ml/min and an isopropanol internal standard (final concentration of 20 pg/ml) were used in the assay. One to 2 ~1 of supernate from blood was analyzed in duplicate. Standards (1 to 400 pg methanol/ml) were also analyzed. The pre-exposure methanol concentration in each rat was used to correct for endogenous levels. Blood formate concentrations were measured by the coupled formate dehydrogenase-diaphorase enzymatic method (Makar and Tephly, 1982; Black, 1985). Duplicate 30-~1 aliquots of supemate from blood were analyzed. The fluorescence of each sample was determined with a Perkin-Elmer LS5 fluorescence spectrophotometer (Oak Brook Instrument Division, Oak Brook, IL). Standards (I to 25 pg sodium formate/ml) were also analyzed. A blank, to which formate dehydrogenase was not added. was prepared for each biological sample to correct for background. Blood-gas analyses. An automated blood-gas analyzer (RadiometerCopenhagen ABL2 Acid-Base Laboratory, The London Company. Cleveland, OH) was used to immediately measure blood pH, bicarbonate, and pCOz in whole blood samples (heparinized). Determination kinetics. Two
of intravenous
[“Cjmethanol
blood,
urine,
and exhalation
groups of rats (four rats/group) were given 100 mg [‘4C]methanol/kg (specific activity = 36.6 pCi/mmol; -24 &i/animal) through their jugular cannulae (flushed with a volume of saline equivalent to the dose solution volume) and placed in metabolism cages. Group 1 was used to determine blood concentration-time courses and cumulative urinary excretion of [?]methanol and [‘4C]formate. Blood samples were collected and handled as previously described. The urine traps, placed in dry ice, were changed and the volume excreted was determined before refreezing the samples at -20°C. Blood and urine were analyzed for [“Clmethanol and [?]formate using an HPLC system (Waters Associates, Inc., Milford, MA) equipped with a Rezex ROA-BR-organic acid column (300 X 7.5 mm i.d.; Phenomenex, Ranch0 Pales Verdes, CA) and precolumn (50 X 4.6 mm i.d.). The mobile phase was 0.043 N HzS04 with 1% acetonitrile (flow rate = I ml/min). Radiolabeled methanol and formate were detected and quantitated by an in-line Ramona-5-LS radioactivity detector (Raytest GmbH, Straubenharbt 1. Germany): Flo-Stint 11(Radiomatic Instruments and Chemical Co., Inc., Tampa, FL) was used as the scintillant. Supernate from blood (100 ~1) was injected directly and urine was centrifuged (5 min at 10.000 rpm) to remove large debris before analyzing 100 ~1. Group 2 was used to determine the cumulative exhalation time courses of [‘4C]methanol and “‘C02. Room air (500 ml/min) was drawn through an activated charcoal filter and then through the metabolism cage by a vac-
28
HORTON.
HIGUCHI,
TABLE 1 Parameters Used in the Physiologically Based Methanol Pharmacokinetic Model Rat Body weight (kg) Cardiac output (liters/hr) Alveolar ventilation (liters/hr) Methanol partition coefficients Blood-to-air Liver-to-blood Rich-to-blood Kidney-to-blood Slow-to-blood Blood flows (% cardiac output) Liver Kidney Richly perfused Slowly perfused Compartment volumes (% body weight) Liver Kidney Richly perfused Slowly perfused Metabolism constants Methanol V,,,,I (mg/hr. kg”.74) rC,i (mg/liter) Vmaxz(mg/hr . kg”.74) K,,,z (mg/liter) Formaldehyde V,,,,, (mmol/hr . kg”.74) K,,, (mmol/liter) Formate C,,, (mmol/hr . kg”.14) K,,, (mmol/liter) Renal extraction efficiencies Methanol Formate
AND APPLIED
PHARMACOLOGY
117,26-36 (1992)
Physiologically Based Pharmacokinetic Model for Methanol in Rats, Monkeys, and Humans VICKY L.HoRToN,*,~ *Chemical
Industry
Institute of Toxicology, University of North
MARK A.HIGUCHI,*
ANDDOUGLASE.RICKERT*
Research Triangle Park, North Carolina Carolina-Chapel Hill, Chapel Hill, North
27709: and jCurriculum Carolina 27514
in Toxicology,
Received November 22. 199 1; accepted June 12, 1992
The acute toxic effects induced by methanol have been the focus of extensive study but little information about chronic effects has been reported (Andrews et al., 1987). It may be necessary to test this compound for chronic, lowlevel exposure effects if methanol is utilized as a fuel extender or as a feedstock for fuel synthesis, since the potential for occupational and environmental exposures would increase (Posner, 1975). The selection of an animal model for chronic methanol studies is problematic because there are marked species differences in acute toxicity symptoms and metabolism after large oral or parenteral methanol doses (Tephly et al., 1979; Tephly and McMartin, 1984). The development of acute toxicity in humans and nonhuman primates is apparently a consequence of their slower rate of formate metabolism (McMartin et al., 1977; Noker and Tephly, 1980). It is unknown whether substantial species differences in methanol or formate metabolism exist at the low methanol vapor concentrations relevant to chronic human exposures. To address the problems associated with the appropriate design of chronic methanol studies, methanol pharmacokinetics were characterized in male Fischer-344 rats and rhesus monkeys exposed to atmospheric methanol concentrations ranging from 50 to 2000 ppm for 6 hr. A physiologically based pharmacokinetic model was then developed to simulate the in viva time course data. The results indicate a dose dependency in methanol pharmacokinetics and a similarity between rats and primates in elimination of low methanol doses.
Physiologically Based Pharmacokinetic Model for Methanol in Rats, Monkeys, and Humans. HORTON, V. L., HIGUCHI, M. A., AND RICKERT, D. E. (1992). Toxicol. Appl. Pharmacol. 117,26-36. The pharmacokinetics of methanol and formate were characterized in male Fischer-344 rats and rhesus monkeys exposed to methanol vapor concentrations between 50 and 2000 ppm for 6 hr. End-of-exposure blood methanol concentrations were not directly proportional to the atmospheric concentration. The methanol exposures did not cause an elevation in blood formate concentrations. After an intravenous dose of [14C]methanol in rats, metabolism, exhalation, and renal excretion contributed 96.6, 2.6, and O.S%, respectively, to the elimination of blood methanol concentrations. These values and the calculated renal methanol extraction efficiency (0.007) are nearly identical to those for humans after low doses of methanol. A physiologically based pharmacokinetic model was developed to simulate the in vivo data. In order to simulate the observed blood methanol concentrations in the inhalation studies in rats, a double pathway for methanol metabolism to formaldehyde was used. One path used rodent catalase K,,, and V,,,, values and the other used a smaller K,,, and V,,,,, to simulate an enzyme with a higher affinity and lower capacity. The lack of proportionality observed in endof-exposure blood methanol concentrations may be due to saturation of an enzyme with higher affinity and lower capacity than catalase. The physiologically based pharmacokinetic model was modified to simulate the monkey data and was scaled-up for humans. In order to simulate the monkey blood methanol concentrations, the use of rodent catalase parameters for methanol metabolism was required. This finding suggests that primates and rodents may be similar in the initial step of methanol metabolism after low methanol doses. Previously published human urinary methanol excretion data was successfully simulated by the model. The models were used to predict the atmospheric methanol concentration range over which the laboratory species exhibit quantitative similarities with humans. Below 1200 ppm, all three speciesexhibit similar end-of-exposure blood methanol concentrations and a linear relationship between atmospheric and blood methanol concentrations. At higher atmospheric concentrations, external and internal methanol concentrations increase disparately, suggesting that delivered dose rather than exposure concentration should be used in interpreting data from high-dose studies. 0 1992 Academic Press, Inc. 0041-008X/92 $5.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
METHODS Reagents. The following chemicals were obtained from Fisher Scientific (Pittsburgh, PA): sodium chloride, sodium hydroxide, dichloromethane, isopropanol, methanol (HPLC grade, >99.9% pure), 2-methoxyethanol, ethanolamine, acetonitrile. sulfuric acid, and magnesium perchlorate. Sodium heparin (porcine intestinal mucosa origin), zinc sulfate, potassium phosphate (monobasic and dibasic), sodium formate, /3-nicotinamide adenine dinucleotide, diaphorasc (Type II-L, derived from Clostridium kluyveri; EC 1.6.4.3). formate dehydrogenase (Type I, derived from Pseudomonas oxalaticus: EC 1.2. I .2). [‘%Z]Methanol (>98% pure by gas chromatography and HPLC), and sodium [“Clformate (>98’% pure by HPLC) were purchased from Sigma 26
METHANOL
PHARMACOKINETIC
Chemical Co. (St. Louis, MO). Resazurin and benzalkonium chloride were obtained from the Eastman Kodak Co. (Rochester, NY) and Aldrich Chemical Co. (Milwaukee, WI). respectively. Animals rind /~&a&r. Male Fischer-344 rats (CDF(F-344)/CrlBR), 190-230 g, were obtained from Charles River Laboratories, Inc. (Kingston, NY, and Raleigh, NC) and allowed to acclimate for at least 2 weeks in a mass-air displacement room (Bioclean, Hazleton Systems, Inc., Vienna, VA) maintained at 22 f 1.5”C with a relative humidity of 50 + 10% and a 12-hr 1ight:dark cycle. NIH-07 open formula diet (Ziegler Brothers, Gardner, PA) and water (Nanopure, Sybron-Barnstead, Boston, MA) were provided ud libitum. Sentinel rats were routinely screened and found to be seronegative for mycoplasmal and viral infections (Microbiological Associates, Bethesda, MD). To permit repeated blood sampling, the rats were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Washington Crossing, NJ) and the right jugular vein was cannulated with Silastic tubing (0.025 in. id. X 0.047 in. o.d., 20 cm long; Dow Corning Corporation, Midland, MI); overnight recovery was permitted. Three young adult male rhesus monkeys (Macaca mulatta, 5-7 kg) were obtained from Hazleton Laboratories (Adice, TX). The animals were housed individually in 48 X 32 X 36-in. stainless steel cages with squeezeback assemblies to facilitate handling. Certified Primate Chow (Purina Mills, Inc., St. Louis, MO) and water were provided except during the inhalation exposure. Each monkey was fitted with a plexiglass collar equipped with metal rings to permit poling capture, and each was trained to sit quietly in a primate restraining chair for at least 2 hr before use in the experiment. A vascular-access port (Model GPV; Norfolk Medical Products, Inc., Skokie, IL), equipped with a Hydromer-coated polyurethane catheter (Hydrocath, 34 French v 35 cm: CardioSearch, Tampa, FL), was implanted in the right femoral vein of each monkey. The catheter was inserted proximally into the vein about 14 cm, placing the tip in close proximity to the abdominal vena cava. The procedures were conducted using sterile surgical techniques and a combination of ketamine hydrochloride (Ketaset, Bristol Veterinary Products, Syracuse, NY). thiamylal sodium (Bio-Tal, Bio-Ceutic Laboratories, St. Joseph, MO), and halothane gas (Fluothane; Fort Dodge Laboratories. Inc., Fort Dodge, IA) for anesthesia. A recovery period of 10 days was allowed before exposure to methanol vapors. Patency of the ports was maintained by flushing with I to 5 ml of sterile heparinized saline (1000 units sodium heparin/ml 0.9% saline) 3 days/week. After the study, the animals were anesthetized with a combination of ketamine, atropine, and intravenous thiamylal sodium, and the ports were surgically removed. Blood sampling. Blood samples ( 150 ~1) were collected from rats through the indwelling cannulae and placed in heparinized Microtainers (BectonDickinson, Rutherford. NJ). Samples were collected at selected times during and after each inhalation exposure and after the intravenous [‘4C]methanol dose. A IOO-~1aliquot ofwhole blood was mixed with 140 pl distilled water, 30 ~1 7.5% (w/w) aqueous ZnSO.,. and 30 ~1 0.4 N NaOH to precipitate protein (Somogyi, 1945). After centrifugation (15 min at 10,000 rpm; SureSpin, Helena. TX). the supemate volume was determined before freezing at -20°C. Blood samples from monkeys were collected aseptically through the port with a sterile Huber point needle (22 g, 1 in., deflected tip; Norfolk Medical Products, Inc., Skokie, IL) and syringe. Samples were obtained prior to each exposure and at designated time points after the exposure was terminated. For the initial 2 hr after exposure, the monkey was kept in the primate restraining chair. After this period, the monkey was returned to his cage. For each subsequent sample, the monkey was caught, chaired, the sample obtained, and the animal immediately returned to his cage. The samples for blood methanol and formate concentration measurements (200 ~1) were placed in heparinized microtainers and the samples for blood pH and bicarbonate (250 ~1) were drawn in heparinized syringes. The port was then flushed with 0.3 ml heparinized saline. Determination of inhaled methanol blood kinetics. Four rats per concentration were exposed to nominal methanol atmospheres of 0.200, 1200, or 2000 ppm (0, 260, 1560, or 2600 mg/m3) for 6 hr in a glass, four-station head-only chamber. The atmospheres were generated by blowing nitrogen
27
MODEL
over methanol contained in a 100 ml impinger and then diluting this vapor with air. The chamber methanol concentration was continuously monitored with a Miran 1A infrared gas analyzer (Foxboro-Wilks, Norwalk, CT; wavelength = 9.5 pm) in a closed loop system. Adjustments in chamber air flow rate (-20 liters/min) were used to maintain the desired concentrations. Analytical chamber concentrations were 0 + 0 (means f SEM: n = 6), 197 f 5.5, 1175 f 17.4, and 1994 f 3.8 ppm for nominal concentrations of 0, 200, 1200, and 2000 ppm. respectively. Each monkey was individually exposed to nominal methanol atmospheres of 0, 50,200, 1200. and 2000 ppm (0,65: 260, 1560, and 2600 mg/m’) in random order with a 2-week recovery period between exposures. The monkeys were exposed in a 16-m’ stainless-steel whole-body chamber (Hazleton Systems, Inc., Aberdeen, MD). Methanol atmospheres were generated with a J-tube evaporation system (Miller et al., 1980) with the rate of methanol delivery to the J-tube controlling the chamber concentration. The atmospheric methanol concentration was monitored as above. Analytical chamber concentrations during the 6 hr exposures to 0, 50, 200, 1200, or 2000 ppm nominal methanol atmospheres were 0 + 0 (means i SEM; n = 18), 49 + 0.5, 205 * 2.7, 1206 + 5.3, and 2006 f 3.8 ppm, respectively. Blood methanol concentrations were measured by gas chromatography using a 5850A Hewlett-Packard gas chromatograph (Hewlett-Packard Instrument Company, Avondale, PA) equipped with a flame ionization detector and a glass column (10 ft X 2 mm i.d.) packed with SO/l00 Carbonpack C coated with 0.1% SP-1000 (Supelco. Bellefonte. PA) held at 60°C. A helium flow rate of 20 ml/min and an isopropanol internal standard (final concentration of 20 pg/ml) were used in the assay. One to 2 ~1 of supernate from blood was analyzed in duplicate. Standards (1 to 400 pg methanol/ml) were also analyzed. The pre-exposure methanol concentration in each rat was used to correct for endogenous levels. Blood formate concentrations were measured by the coupled formate dehydrogenase-diaphorase enzymatic method (Makar and Tephly, 1982; Black, 1985). Duplicate 30-~1 aliquots of supemate from blood were analyzed. The fluorescence of each sample was determined with a Perkin-Elmer LS5 fluorescence spectrophotometer (Oak Brook Instrument Division, Oak Brook, IL). Standards (I to 25 pg sodium formate/ml) were also analyzed. A blank, to which formate dehydrogenase was not added. was prepared for each biological sample to correct for background. Blood-gas analyses. An automated blood-gas analyzer (RadiometerCopenhagen ABL2 Acid-Base Laboratory, The London Company. Cleveland, OH) was used to immediately measure blood pH, bicarbonate, and pCOz in whole blood samples (heparinized). Determination kinetics. Two
of intravenous
[“Cjmethanol
blood,
urine,
and exhalation
groups of rats (four rats/group) were given 100 mg [‘4C]methanol/kg (specific activity = 36.6 pCi/mmol; -24 &i/animal) through their jugular cannulae (flushed with a volume of saline equivalent to the dose solution volume) and placed in metabolism cages. Group 1 was used to determine blood concentration-time courses and cumulative urinary excretion of [?]methanol and [‘4C]formate. Blood samples were collected and handled as previously described. The urine traps, placed in dry ice, were changed and the volume excreted was determined before refreezing the samples at -20°C. Blood and urine were analyzed for [“Clmethanol and [?]formate using an HPLC system (Waters Associates, Inc., Milford, MA) equipped with a Rezex ROA-BR-organic acid column (300 X 7.5 mm i.d.; Phenomenex, Ranch0 Pales Verdes, CA) and precolumn (50 X 4.6 mm i.d.). The mobile phase was 0.043 N HzS04 with 1% acetonitrile (flow rate = I ml/min). Radiolabeled methanol and formate were detected and quantitated by an in-line Ramona-5-LS radioactivity detector (Raytest GmbH, Straubenharbt 1. Germany): Flo-Stint 11(Radiomatic Instruments and Chemical Co., Inc., Tampa, FL) was used as the scintillant. Supernate from blood (100 ~1) was injected directly and urine was centrifuged (5 min at 10.000 rpm) to remove large debris before analyzing 100 ~1. Group 2 was used to determine the cumulative exhalation time courses of [‘4C]methanol and “‘C02. Room air (500 ml/min) was drawn through an activated charcoal filter and then through the metabolism cage by a vac-
28
HORTON.
HIGUCHI,
TABLE 1 Parameters Used in the Physiologically Based Methanol Pharmacokinetic Model Rat Body weight (kg) Cardiac output (liters/hr) Alveolar ventilation (liters/hr) Methanol partition coefficients Blood-to-air Liver-to-blood Rich-to-blood Kidney-to-blood Slow-to-blood Blood flows (% cardiac output) Liver Kidney Richly perfused Slowly perfused Compartment volumes (% body weight) Liver Kidney Richly perfused Slowly perfused Metabolism constants Methanol V,,,,I (mg/hr. kg”.74) rC,i (mg/liter) Vmaxz(mg/hr . kg”.74) K,,,z (mg/liter) Formaldehyde V,,,,, (mmol/hr . kg”.74) K,,, (mmol/liter) Formate C,,, (mmol/hr . kg”.14) K,,, (mmol/liter) Renal extraction efficiencies Methanol Formate
