Biological effects of short-term, high-concentration exposure to methyl isocyanate. IV. Influence on the oxygen-binding properties of guinea pig blood.

Whole blood oxygen equilibrium curves (O2 ECs), blood buffer lines, and several hematologic properties were determined for adult guinea pigs exposed to 700 ppm methyl isocyanate (MIC) for 15 min. MIC inhalation effected a significant reduction of blood O2 affinity; the half-saturation pressure (P50) at 38 degrees C increased from the control (untreated) level of 22.8 +/- 0.1 mm Hg to values ranging from 28.5 to 43.7 mm Hg for experimental animals. MIC exposure had no apparent influence on O2 EC shape or CO2 Bohr effect. Erythrocyte volume, [metHb], O2 binding capacity, and combined red cell organic phosphate concentration (DPG + ATP) were not affected by MIC treatment. However, experimental animals experienced a severe metabolic acid-base disturbance; blood lactate concentration ranged from 8.6 to 24.0 mmole/L. Results indicate that lactic acidosis was solely responsible for increased blood P50 of MIC-treated animals. No direct effects of MIC on hemoglobin function were observed. Reduced Hb-O2 affinity, in conjunction with severe hypoxemia, compromised the guinea pigs' capacity for pulmonary O2 loading; at PaO2 of 30 mm Hg, Hb-O2 saturation (S) decreased from 66% S for controls to 42% S for MIC-treated animals.


Introduction
Carbamylation of amino terminal residues of hemoglobin (Hb) by cyanate compounds alters the physical and functional properties of the protein tetramer (1). Cyanate and isocyanate have been investigated extensively as potential anti-aggregation agents for treatment of sickle cell disease (2,3). The effect of cyanate carbamylation on Hb-02 binding has also provided a valuable research tool for testing the adaptive significance of increased blood oxygen affinity for high altitude exposure (4,5). These reported effects of cyanates on Hb-02 transport prompted speculation that methyl isocyanate (MIC) impaired tissue oxygen delivery among victims of the Bhopal tragedy. This hypothesis suggested that MIC carbamylation significantly increased Hb-02 affinity, which inhibited peripheral 02 unloading and resulted in tissue hypoxia. This investigation reports the effects of MIC inhalation at a high and lethal concentration on the blood oxygen transport properties of spontaneously breathing guinea pigs. Results showed a notable reduction of Hb-*Section ofPhysiology and Biophysics, Division

Materials and Methods
Animals, Treatment, and Blood Collection Adult female guinea pigs (Hartley strain) weighing 424 to 568 g were exposed to a mean methyl isocyanate concentration of 698 ppm (range 618-804 ppm) for 15 min. A detailed description of methods for MIC treatment is presented elsewhere (6). Immediately following exposure, animals were lightly anesthetized with Halothane and blood drawn from the retro-orbital sinus into heparinized Vacutainers (Becton-Dickinson, Rutherford, NJ). Control guinea pigs were exposed to air alone and bled in an identical manner. Blood samples were immediately packed in ice and transported to Brown University by air. Experimental measurements commenced approximately 5 hr after blood collection.

Oxygen Equilibrium Curves (02 ECs)
Multiple-point isocapnic 02 ECs were generated for whole blood of control and experimental guinea pigs at 38°C by using microtechniques previously described (7). Briefly, a small aliquot of blood (0.5 to 1.0 ,uL) was gently spread between gas-permeable Teflon membranes and the blood-membrane trilayer secured by 0ring to an opaque carier-disk with 7 mm center hole. The blood film was then mounted horizontally in a single compartment sample chamber (1 mL internal volume) and equilibrated with a humidified CO2/N2 gas mixture.
Following desaturation, the blood sample was equilibrated with 24 to 34 (X = 28) isocapnic gas mixtures of increasing 02 tension. For each static point, blood film Po2 was determined by measuring the 02 tension of the surrounding gas phase by electrode oximetry. Simultaneously, Hb-02 saturation (S) was determined by dual wavelength spectrophotometry (542, 560 nm), light being transmitted to and from the blood film by optical fiber bundles. When 02 tension in the cuvette produced a saturation greater than 95% S, the blood film was exposed to C02/02 (Po2 > 600 mm Hg) to obtain a 100% S signal. Complete 02 ECs were generated in approximately 20 min, and data were transmitted directly to an IBM PC programmed for data acquisition and analysis. A fresh blood film was prepared for each 02 EC to minimize the potential effects of erythrocyte metabolism on blood 02 affinity.
Three isocapnic 02 ECs were measured for each blood sample at 2, 5, and 8% CO2. Blood film pH was estimated for each equilibrium curve from two-point Astrup blood buffer lines (8) determined with a microtonometer (AMT1, Radiometer, Copenhagen), thermostatted glass electrode, and pH meter (pHM 84, Radiometer). Po2 values were read for each 02 EC at 5% saturation increments between 5 and 95% S. CO2 Bohr coefficients (A log Po2/ApH) were then determined by least-square regression (5-95% S), and a standard 02 EC was calculated for each individual at the appropriate blood pH or Pco2.

Results and Discussion
Blood Oxygen-Binding Properties  coefficient was somewhat lower than P50 values previously reported for guinea pigs (11). Differences may be related to animal age, methods of anesthesia and/or experimental techniques for generating equilibrium data. 02 EC values for MIC-treated animals at a common Pco2 of 40 mm Hg were significantly right-shifted and exhibited substantial individual variability (Fig. 1). The P50 for the five experimental animals ranged from 28.5 to 43.7 mm Hg. Figure 2 illustrates the effects of methyl isocyanate exposure on the shape of the 02 equilibrium curve. For this analysis, the individual 02 ECs for experimental animals were scaled to the control P50 (22.8 mm Hg).
The vertical bars, plotted at 5% saturation increments between 5 and 95% S, encompass the P02 ranges for the five MIC-treated data sets. The solid curve is the mean 02 EC for control animals illustrated in Figure 1. Results indicate minimal P02 variability among the five scaled experimental 02 ECs. Furthermore, the normalized data described an 02 EC shape almost identical to the control curve. MIC inhalation, at a high and lethal concentration, had no apparent effect on equilibrium curve shape. The CO2 Bohr effect at half-saturation (A log P5/ ApH) was not different for control (-0.62 ± 0.03) and MIC-treated animals (-0.60 ± 0.05). CO2 Bohr slopes were also saturation-independent between 10 and 90% S for both animal groups.
Results ofthese oxygen-binding studies revealed that MIC inhalation significantly increased P5O but had no influence on 02 EC shape or the effect of carbon dioxide on blood 02 affinity. Several hematologic properties relevant to blood oxygen transport were evaluated to determine the factor(s) responsible for the decreased Hb-02 affinity.
Hematologic Properties MIC inhalation for 15 min at a concentration of 700 ppm produced significant increases in hematocrit ratio and [Hb] ( Table 1). The mean corpuscular hemoglobin concentration (MCHC), however, remained unchanged (Table 1). These findings suggest that MIC treatment had no effect on erythrocyte volume. Reduced Hb-02 affinity of experimental animals, therefore, cannot be attributed to the potential consequences of cell volume change, i.e., effects of volume-induced changes in [Hb] (12) and intracellular pH (13). Furthermore, methyl isocyanate exposure did not promote Hb oxidation; [metHb] was approximately 1% of total [Hb] for both animal groups (Table 1).
MIC treatment had no effect on oxygen binding capacity of guinea pig blood (Table 1); the slightly higher capacity value reported for experimental animals reflects their increased [Hb]. The calculated oxygen to hemoglobin ratio (mL 02/g Hb) for air-equilibrated blood samples was approximately 1.3 for both control and MIC-treated animals.
The organic phosphates DPG and ATP, important allosteric modifiers of Hb function, exhibited small but significant differences between animal groups (Table 1). MIC-treated animals had decreased [DPG] and increased [ATP]. The net effect was a minimal change in combined erythrocyte organic phosphate concentration. These observed changes in RBC organic phosphates are consistent with severe acidosis (1).
MIC-treated guinea pigs experienced a metabolic acid-base disturbance. Blood lactate concentrations among these spontaneously breathing animals ranged from 8.6 to 24.0 mmole/L (Table 1). Blood gas and acidbase measurements also revealed a metabolic acidosis for pump-ventilated guinea pigs following 15 min exposure to 675 ppm MIC (14). [Lactate] for control animals was also elevated (2.6-7.4 mmole/L); these latter findings may reflect a metabolic acid-base disturbance resulting from halothane-induced ventilatory depression.

Effect of Metabolic Acidosis on Hb-02 Affinity
Increased blood [lactate] resulting from methyl isocyanate inhalation was apparently the sole cause for the observed reduction of Hb-02 affinity among the experimental animals. The 02 EC for MIC-treated guinea pigs are reported at a standard mammalian arterial Pco2 of 40 mm Hg (Fig. 1). The corresponding blood pH values ranged from 7.19 to 6.79, reflecting the severe metabolic acidosis. Furthermore, there was a direct relationship between blood [lactate] and P50 for MIC-treated guinea pigs, i.e., animals with the highest [lactate] exhibited the highest 02 affinity coefficient.
The effect of metabolic acidosis on P50 was evaluated by calculating the 02 affinity coefficients for expenmental animals at blood pH 7.40 using the measured CO2 Bohr slopes. At pH 7.40, the half-saturation Po2 for MIC-treated animals (20.7 + 0.7 mm Hg) approximated the control P50 (22.8 + 0.1 mm Hg). In a more definitive study, blood from three experimental guinea pigs was titrated to the control base excess with NaHCO3. The measured P50 for titrated blood from experimental animals (22.9 + 1.3 mm Hg at pH 7.40) was virtually identical to the control value. These findings strongly suggest that the reduced Hb-02 affinity in MIC-treated animals resulted from the lactic acidosis.
Functional Consequences of MIC Treatment on Blood 02 Delivery MIC inhalation (675 ppm) caused rapid and severe lung injury (15), resulting in significant intrapulmonary shunts and ventilation-perfusion mismatch (14). The functional consequences of this pulmonary damage was hypoxemia; arterial Po2 ranged from 35 to 40 mm Hg for pump-ventilated animals following 15 min MIC exposure (14). For spontaneously breathing guinea pigs, a lower Pao2 would be predicted. The present investigation also revealed a metabolic acid-base disturbance; blood [lactate] in the MIC-treated animals was significantly elevated (Table 1). These latter findings are indicative of tissue hypoxia. Systemic 02 delivery for the MIC-treated guinea pig was apparently inadequate to sustain the animal's aerobic energy requirements, necessitating the added contribution of anaerobic glycolysis.
The acid-induced reduction of Hb-02 affinity, in con-junction with severe hypoxemia, further jeopardized the guinea pigs' capacity for blood oxygen transport. To substantiate this conclusion, Hb-02 saturation was calculated for control and experimental animals at an assumed Pao2 of 30 mm Hg. At pHa 7.40, control guinea pig blood would be 66% saturated with oxygen at Pao2 = 30 mm Hg. For MIC-treated animals (Pco2 = 40 mm Hg), the right-shifted equilibrium curve would reduce arterial saturation to 42%, values ranging from 31 to 53% S for the five individuals. This analysis assumes only a metabolic acid-base disturbance for experimental animals. Inclusion of the respiratory acidosis reported for MIC-exposed guinea pigs (14) would further right-shift the 02 EC, reduce arterial saturation to a lower level, and hence further compromise pulmonary oxygen loading. This investigation provided no evidence for a direct effect of methyl isocyanate on hemoglobin function. Although MIC is highly reactive with Hb when blood is exposed in vitro (3,16), the reported effect of carbamylation on Hb-02 affinity was not detected for inhalation-treated guinea pigs. One interpretation of these findings suggests that the rapid and devastating effects of high MIC concentrations on pulmonary structure (15), blood-gas exchange properties (14), and possible reflex inhibition of breathing (17) minimized the effective contact of the gas with functional alveoli.