Neurotransmitter receptors are widely considered the mediators of anesthetic action (1). But why do animals and their neurotransmitter receptors respond to inhaled anesthetics? Few organisms are exposed to such compounds, and anesthetized organisms are virtually never observed in nature. One proposed physical mechanism of anesthesia hypothesizes that receptors are adapted to alterations in bilayer properties induced by the high, fluctuating concentrations of neurotransmitters at synapses (2), and that this produces a selective pressure for susceptibility to anesthetics.
This hypothesis raises the possibility that sustained high concentrations of other endogenous compounds might produce anesthetic effects, albeit without providing any selective pressure to maintain the response to anesthetics. Ketone bodies (acetone, β-hydroxybutyric acid, and acetoacetic acid) provide attractive anesthetic-like possibilities because they span the largest concentration range of all circulating metabolites, the upper extremes of which are associated with central nervous system (CNS) depression. In health, ketone body concentrations are <100 μM (3). In diabetic ketoacidosis, the combined concentration of acetoacetic acid and β-hydroxybutyric acid can exceed 25 mM (4). Ninety percent of these ketoacids are β-hydroxybutyric acid (5). Acetone concentrations approach 9 mM (6).
CNS depression in diabetic ketoacidosis ranges from sedation to unconsciousness, to unresponsiveness to noxious stimuli, a range remarkably similar to that produced by subanesthetic to surgical concentrations of general anesthetics (7). Insulin administration readily reverses the depression. Hyperosmolar nonketotic diabetic coma in non–insulin-dependent diabetes produces similar neurologic findings. The impairments in consciousness associated with this condition and diabetic ketoacidosis have been attributed, in part, to the electrolyte abnormalities and dehydration associated with hyperglycemia. However, when compared with hyperosmolar nonketotic diabetic coma, diabetic ketoacidosis produces lesser glucose levels and dehydration and a greater ketoacidosis (8), suggesting that metabolites generated in diabetic ketoacidosis may provide the basis for impaired consciousness.
In the present study, we investigated whether β-hydroxybutyric acid and acetone have anesthetic-like effects in animals and on ion channels. We used methods widely used in studies of anesthetic mechanisms to investigate the depressant effects of these compounds. In particular, we assessed the capacity of these compounds to reversibly prevent movement in tadpoles (9). We also tested whether, like inhaled anesthetics, these compounds enhanced the function of glycine, GABAA (γ-amino butyric acid type A) receptors (10), or the two-pore domain K channel TRESK (11), and whether they inhibited the function of NMDA (N-methyl-d-aspartate) receptors (12).
METHODS
The University of California San Francisco institutional animal care and use committee approved all animal studies.
Measurement of Anesthetic Potency in Tadpoles
Tadpoles were produced by in vitro fertilization (13). A sexually mature, 9 cm Xenopus laevis female (Nasco, Modesto, CA) was primed with 50 IU human chorionic gonadotropin (hCG) (1 IU/μL) (Sigma, St. Louis, MO) injected into the dorsal lymph sac. Seven days later, ovulation was induced with 800 IU hCG and the frog placed in high salt modified Barth solution (MBS; 108 mM NaCl, 1 mM KCl, 1 mM MgSO4, 5 mM HEPES, 2.5 mM NaHCO3, 0.7 mM CaCl2, adjusted to pH 7.0) in which it laid eggs 6–12 h later.
A male frog was killed and the testes removed and kept in 1× MBS (88 mM NaCl, 1 mM KCl, 1 mM MgSO4, 5 mM HEPES, 2.5 mM NaHCO3, adjusted to pH 7.0) on ice. A sperm slurry was made by crushing the testes in a small amount of 0.1× MMR + gentamicin (10 mM NaCl, 0.18 mM KCl, 0.2 mM CaCl2, 0.1 mM MgCl2, 0.5 mM HEPES, 50 μg/mL gentamicin, adjusted to pH 7.0) and kept on ice until the fertilization step.
Eggs were collected within 10 min of laying, the high salt MBS was removed, and the eggs were covered with the sperm slurry. After upward rotation of the animal pole indicated successful fertilization, eggs were placed in 0.1× MMR. Batches of 30–40 eggs were kept in 100× 15 mM Petri dishes in 0.1× MMR + gentamicin for the first 2 days; the developing embryos then were kept in 0.1× MMR without gentamicin. Media was changed every day.
To test anesthetic potency, 7 to 10-day-old tadpoles were allowed to equilibrate for 30 min in Petri dishes in 0.1× MMR containing the compound under study. Tadpoles were prodded on their side for 30 s with a glass rod. Tadpoles that did not respond to prodding were classified as nonmoving. Tadpoles that responded to any amount of prodding were classified as moving. Nonmoving tadpoles were removed and placed in 0.1× MMR to assess recovery.
Oocyte Preparation for Nucleic Acid Injection
Oocytes were prepared, injected with RNA, and TRESK and NMDA receptors studied by two electrode voltage clamping, with drugs added to frog Ringer perfusate, as previously described (14). Of note, for NMDA receptors, a saturating glutamate concentration was used. Because β-hydroxybutyric acid is largely ionized at pH 7.0, and hence does not readily enter the oocyte, an isoosmotic concentration of sucrose was added to frog Ringer control solutions. Equal osmolarity was confirmed using a vapor pressure osmometer (Vapro vapor pressure osmometer, Logan, UT).
GABAA and Glycine Receptor Studies
Clones of human GABAA α1 and γ2s and rat GABAA β2 subunits in pCIS II vectors, and human α1 glycine receptors in a PBK-CMV vector, were gifts from Professor RA Harris (Univeristy of Texas, Austin, TX). Approximately 1 ng total plasmid DNA, consisting of equal parts GABAA α1 and β2 receptor subunits, and a 10-fold excess of γ2s subunit, or 1 ng total plasmid DNA of the α1 subunit of the glycine receptor, were injected into Xenopus oocyte nuclei and studied 2–3 days later. We tested expression of the γ2s subunit by applying 10 μM zinc chloride for 1 min and then co-applied zinc chloride with GABA; inhibition of currents by 10% or less indicated expression of GABAA receptors containing the γ2s subunit.
Transmembrane potentials were kept constant at −80 mV. Perfusion of oocytes with frog Ringer solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, filtered and adjusted to pH 7.4) for 5 min was changed to perfusion with an identical solution containing agonist (0.03 mM GABA or 0.075 mM glycine, concentrations in the EC10–EC20 range) for 20 s followed by a 5 min washout of frog Ringer solution. These studies then were repeated in the presence of various concentrations of acetone or β-hydroxybutyric acid. A solution containing study compound in frog Ringer was then perfused for 100 s followed by the same concentration in frog Ringer plus agonist. Return to baseline was confirmed after washout with frog Ringer solution. For the studies of β-hydroxybutyric acid, sucrose was added to the frog Ringer solution in the control portion of the study to compensate for the added osmolarity of the β-hydroxybutyric acid.
Acetone Blood-Gas Partition Coefficient
A known quantity of acetone was introduced into a gas-tight syringe containing either blood or frog Ringer solution, and equilibrated at 37°C for 2 h with continuous mixing. At the end of equilibration, acetone concentrations in the gas phase were measured by gas chromatography. The blood-gas and frog Ringer-gas partition coefficient for acetone were calculated as the ratio of the concentration of acetone in blood or frog Ringer solution to that in gas after equilibration (15).
Statistics
For the studies in tadpoles, the fraction of immobile tadpoles as a function of concentration was analyzed by nonlinear regression (SPSS v 11.0, Chicago, IL) to the Hill equation
where F(x) is the fraction of immobile tadpoles at concentration x, n is the Hill coefficient, and EC50 is the concentration at which half of the tadpoles do not move in response to prodding. EC50's, Hill coefficients, and currents through ion channels were compared using a Student's t-test. P < 0.05 was taken as significant.
RESULTS
Both acetone and β-hydroxybutyric acid reversibly anesthetized tadpoles (Fig. 1). The EC50 for acetone was 264 ± 2 mM (mean ± se), with a Hill number of 41 ± 12. The EC50 for β-hydroxybutyric acid was 151 ± 11 mM, with a Hill number of 5 ± 1. The effect of both compounds was reversible: all tadpoles recovered from exposure to acetone and β-hydroxybutyric acid.
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Figure 1.:
Acetone and β-hydroxybutyric acid reversibly prevent movement In response to prodding in Xenopus tadpoles, with EC50's of 264 ± 2 mM for acetone and 151 ± 11 mM for β-hydroxybutyric acid.
Acetone and β-hydroxybutyric acid enhanced the function of α1 glycine receptors (Fig. 2), doing so in the concentration range found in diabetic ketoacidosis. For example, 1, 2.5, and 10 mM acetone produced more than a 200% enhancement in glycinergic currents, and 9 and 12 mM β-hydroxybutyric acid produced more than a 100% enhancement. Beta hydroxybutyric acid enhanced GABAA receptor function at 10 and 20 mM (Fig. 3).
Figure 2.:
Homomeric α1 glycine receptor function is potentiated by acetone and β-hydroxybutyric acid concentrations in the low millimolar concentration range observed in ketoacidosis. Agonist concentration was 75 μM glycine. Five oocytes were studied at each concentration. Potentiations at all concentrations are significantly different from zero.
Figure 3.:
α1β2γ2s GABAA receptor function is enhanced by acetone in concentrations that are below those that produce anesthesia, but above those encountered clinically in ketosis. Beta hydroxybutyric acid enhances the function of this receptor in the clinical concentration range see in diabetic ketoacidosis. Agonist concentration was 30 μM GABA. Five oocytes were studied at 25 and 100 mM acetone, and 6 oocytes at 50 and 400 mM. Seven oocytes were studied at 5 and 20 mM β-hydroxybutyric acid, and 10 oocytes at 10 mM β-hydroxybutyric acid. The current changes observed at the lowest concentration of acetone and β-hydroxybutyric acid were not different from zero; changes in current at other concentrations were all significantly different than zero.
β-Hydroxybutyric acid did not modulate the function of NR1/NR2A NMDA receptors, or TRESK channels (data not shown). Concentrations of acetone exceeding those observed in diabetic ketoacidosis did, however, modulate the function of these channels (Fig. 4) and the GABAA receptor studied (Fig. 3). Fifty millimolar and larger acetone concentrations enhanced α1β2γ2s GABAA receptors. At or above 100 mM, acetone inhibited rather than enhanced TRESK channels. Concentrations of acetone of 200 mM or more inhibited currents through NMDA receptors. We could not study these higher concentrations with β-hydroxybutyric acid owing to the marked attenuation of channel currents from the increased osmolarity of these perfusates.
Figure 4.:
In concentrations that anesthetize tadpoles, but above that observed in ketosis, acetone inhibits NR1/NR2A NMDA receptors, and TRESK channels. For NMDA receptors, n = 5 oocytes at 50 and 400 mM acetone, and n = 7 at other concentrations. Current changes at 200 and 400 mM were different from zero. For TRESK channels, 5 oocytes were studied at each concentration. Current changes at 100, 200, and 400 mM were significantly different from zero.
Current tracings for electrophysiologic studies are in Fig. 5 for β-hydroxybutyric acid and in Fig. 6 for acetone.
Figure 5.:
Current tracings showing the potentiation of GABAA α1β2γ2s receptors (left panel) and α1 glycine receptors (right panel) by 10 mM and 12 mM β-hydroxybutyric acid, respectively. About 30 μM GABA and 75 μM glycine were used to evoke currents in their respective receptors.
Figure 6.:
Upper left panel: 0.05 mM acetone enhances the response of α1 glycine receptors to 75 μM glycine. Upper right panel: 100 mM acetone enhances the response of GABAA α1β2γ2s receptors to 30 μM GABA. Lower left panel: 400 mM acetone inhibits currents through NR1/NR2A NMDA receptors in response to glutamate application. Lower right panel: 400 mM acetone inhibits the response of TRESK channels at +60mV (transmembrane potential).
At room temperature (20°C) the blood-gas partition coefficient for acetone was 611 ± 15, and the frog Ringer or gas partition coefficient was 744 ± 14. At 37°C, the blood-gas partition coefficient for acetone was 281 ± 8, and the frog Ringer-gas partition coefficient was 323 ± 9.
DISCUSSION
We studied the anesthetic effect of two ketone bodies, acetone and β-hydroxybutyric acid. Both study ketone bodies were anesthetics. Both produced a reversible state of unresponsiveness (no movement) to prodding in tadpoles.
The partial pressure of acetone bathing the tadpoles probably equals the partial pressure inside the tadpole because acetone is a small uncharged molecule, which, accordingly, should readily pass into the tadpole. The concentrations inside the tadpole and in the bath should also be similar because the blood-Ringer partition coefficient is not far from 1.0. The EC50 for acetone therefore likely reflects the acetone concentration and partial pressure in the CNS of the tadpole at anesthesia. By contrast, the concentration of β-hydroxybutyric acid bathing tadpoles probably exceeds the concentration in the tadpole, because only the uncharged form of this molecule can penetrate the tissues of the tadpole. Given its pKa of approximately 4.7, only a small fraction of β-hydroxybutyric acid will be uncharged at neutral pH. Thus the EC50 calculated from bath application of β-hydroxybutyric acid probably over-estimates the concentration in the tadpole's nervous system that produces anesthesia. Nonetheless, our results unambiguously show that acetone and β-hydroxybutyric acid are anesthetics.
Both acetone and β-hydroxybutyric acid markedly enhanced glycine receptor function in the concentration range encountered in ketoacidosis. This enhancement approximates that produced by inhaled anesthetics at MAC (10). In addition, β-hydroxybutyric acid enhanced GABAA receptor function, though at the higher concentrations seen in ketoacidosis that produces symptoms of CNS depression. We did not find an effect of β-hydroxybutyric acid or acetone on NR1/NR2A NMDA receptors, or TRESK channels, or of acetone on α1β2γ2s GABAA receptors at the low millimolar concentrations. This stands in contrast to isoflurane which enhances GABAA receptor function (10), inhibits NMDA receptor function (12), and enhances currents through TRESK channels (11).
Our data indicate that enhancement of glycinergic currents is insufficient to produce surgical anesthesia on its own: ketone bodies increase in healthy humans to a few millimolar when intake of carbohydrates and gluconeogenic amino acids is limited, such as in diets high in fat (16), in starvation (17), during fasting, and in prolonged exercise. However, these concentrations do not produce sedation, even though they markedly enhance glycine receptor function. It is not until higher ketone body concentrations are achieved, such as those that occur pathologically in diabetic ketoacidosis, several genetic metabolic diseases, ketotic hypoglycemia of childhood, alcoholic ketoacidosis, corticosteroid or growth hormone deficiency, and ingestion of salicylates that lethargy is seen (3). The development of symptoms at these higher concentrations may be a result of the additional inhibitory effect on GABAA receptors by β-hydroxybutyric acid at these concentrations. That is, the metabolic brain disease seen in ketoacidosis may be a subanesthetic or, in some cases an anesthetic, state resulting, in part, from accumulated metabolites. Enhancement of glycinergic responses may contribute to this depression, as well as GABAA receptors. It may also plausibly involve other anesthetic-sensitive channels (or isoforms which we have not studied), or actions on the channels we have studied by metabolites that we have not investigated, such as acetoacetic acid or the gluconeogenic amino acids that are elevated as the result of protein catabolism in diabetic ketoacidosis.
For acetone, we could study the high concentrations that produced anesthesia both in tadpoles and in expressed receptors. To compare blood concentrations in humans with the concentrations applied to ion channels in perfusates containing frog Ringer solution, we measured the blood-gas and frog Ringer-gas partition coefficients for acetone. Acetone was slightly more soluble in frog Ringer solution (by 15%–20%) compared with blood. Thus, the concentrations we applied to expressed channels were, indeed, in the range produced by ketosis. At concentrations approaching those that produced anesthesia in tadpoles, anesthetic-like effects were produced on some of the channels studied. Concentrations exceeding 50 mM enhanced GABAA receptor currents, while those exceeding 200 mM inhibited NMDA receptor function. These effects may contribute to the anesthesia produced in tadpoles by acetone. Inhibition, rather than enhancement, of TRESK currents was produced by acetone above 100 mM. If anything, such effects might pose an antianesthetic effect.
Ketogenic states associated with fasting or ketogenic diets have anticonvulsant effects (16,18): 40% of patients on a hyperketogenic diet have a 90% reduction in seizures (19). Animal studies corroborate this effect (20,21). The mechanism for the anticonvulsant effect is unknown. One hypothesis suggests that chronic ketosis improves cerebral energy reserves (22,23). Our results suggest that ketone bodies may also enhance chloride conductances at glycinergic and GABAergic synapses. This would be expected to produce neuronal hyperpolarization, thereby increasing the seizure threshold much as benzodiazepines increase seizures thresholds by enhancing chloride currents through GABAA receptors.
In summary, both acetone and β-hydroxybutyric acid are anesthetics. Acetone and β-hydroxybutyric acid produce anesthetic-like enhancement of glycine and GABAA receptor currents at concentrations found in ketosis. At concentrations exceeding those observed in ketosis, but below the concentration producing anesthesia, acetone enhanced GABAA receptor function and inhibited NMDA and TRESK channel function.
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