THE pressure reversal of anesthesia was discovered in the light intensity of bacterial luciferase. 1This in vitro study implies that the partial molar volume of the luciferase is greater during anesthesia than during the awake state. 1This observation was confirmed in vivo in tadpoles, 2newts, 3and rats. 4We found that the light intensity of firefly luciferase (FFL) also responds to high pressure. 5We also measured the effects of anesthetics and myristate (a 14-carbon fatty acid, neutralized by NaOH) on the volume of FFL. 6Halothane 0.5 mm increased the FFL volume by 3.93 cm3mol−1whereas myristate 2.5 μm decreased it by 7.66 cm3mol−1.
We hypothesized that if the molar volume of FFL determines depth of anesthesia, myristate may antagonize anesthesia. As expected, our preliminary study showed that myristate antagonized anesthesia in goldfish. 7Contrary to the general belief that fatty “acids” are toxic to mammals, the intravenous hyperalimentation fluids, Intralipid (Baxter, Deerfield, IL), and others are composed of a variety of long-chain fatty acids: linoleic, oleic, palmitic, linolenic, stearic acids, and so on. These fatty acids are neutralized by triglycerides. They are called essential fatty acids (EFA), and are well tolerated by debilitated patients.
The generally held idea among anesthesiologists that fatty acids do not cross the blood-brain barrier is contradicted by oleamide (18-carbon fatty acid, oleic acid, neutralized by NH4OH) which crosses the blood-brain barrier and induces sleep. 8,9
Materials and Methods
Our protocol was approved by the Animal Experimentation Committee of the Salt Lake City VA Medical Center, Salt Lake City, Utah. Goldfish, obtained at local pet stores, were placed in a glass tub (125 mm in diameter and 65 mm in depth) containing 400 ml dechlorinated tap water. A pair of circular stainless steel screens (120 mm in diameter) was placed on the water surface and bottom of the container.
The effects of anesthetics were quantitated by recording the avoidance response (here, swimming). 10The response of goldfish to electrical stimuli was determined by a programmable constant-current generator (Keithley 220, Cleveland, OH). Three consecutive 0.2-s stimuli, separated by 10-s apart were applied. The occurrence of swimming constituted a positive response. The use of electrical stimuli enabled us to quantify the stimulus by noting the strength of the currents, and in that regard was superior to clamping or surgical incision.
Myristate was dissolved in methanol and added to water at 10, 20, and 40 μm. The amount of methanol in the tub did not exceed 5 mm. The ED50of methanol is about 1,500 mm when estimated by extrapolation of the ED50values of long-chain n -alcohols.
Goldfish were placed in two tubs, one with myristate and the other without, for the control. After 2 h, both groups were stimulated as described. Starting at 0.5 mA, the current was increased at 10 min intervals in increments of 0.5 mA. In agreement with our previous report, 7the myristate group responded identically to the control. Because all goldfish responded to 3.0 mA, the supramaximal 4.0 mA was used for stimulation.
After confirming that both groups would respond to 4.0 mA stimulation, we transferred the goldfish to the tubs with or without myristate, which were preequilibrated with anesthetics. Anesthetics were vaporized by an anesthesia machine and bubbled into the containers for 30 min at an oxygen flow of 2 l/min. For controls, only oxygen was bubbled into the container. Thirty minutes after transfer to the tub, preequilibrated with anesthetics, the goldfish in each group were stimulated with the constant-current of 4.0 mA. Fish that did not start swimming were considered anesthetized. To ensure a constant anesthetic concentration during the experiment, the tubs were encased in a plastic box, and anesthetics were continuously administered in the gas phase of the closed flow-through system. Each group consisted of 30 goldfish.
Figure 1is the logistic plot of the effect of myristate on the dose-response curves of halothane according to the following equation
, 11where P is the partial pressure of anesthetics with two unknown constants (ED50and n ).
This equation shows that the dose-response curves become sigmoidal because when P = 0 then y = 1, and when P =∞ then y = 0. Best-fit values for the two unknowns were obtained by Origin software (Northampton, MA), and shown in table 1.
Waud 11did not define the meaning of n , which represents the steepness of the plot. Logistic equation is formed for the data that represent population distribution (quantal), and the value does not represent the binding number. The n values were halothane 6.01 ± 0.61, isoflurane 5.61 ± 0.61, and enflurane 4.00 ± 0.12 without myristate. The values for n and ED50are summarized in table 1. Myristate 40 μm increased the ED50values of halothane, isoflurane, and enflurane 242% on average.
Yost et al. 12reported that intraperitoneal injection of oleamide 100 mg/kg in rats showed no effect on the minimum alveolar concentration (MAC) of desflurane. They suggested that the 18-carbon fatty acids lack the effect on the central action of anesthetics, possibly because of the failure to achieve effective concentration in the brain. Yang et al. 13however, reported that intraperitoneal injection of oleamide 700 mg/kg in rats potentiated the effects of diazepam but antagonized ethanol, methamphetamine, and caffeine. It is likely that the 100 mg/kg dose by Yost et al. 12was below the ED50.
Weigt et al. 14reported that intravenous nutritional fatty acids activated N -methyl-d-aspartate receptor, implying that some of these intravenous fatty acids may support anesthesia. However, current data show that the 14-carbon fatty acid antagonized the action of volatile anesthetics.
Receptor binders usually have specific competitors. Volatile anesthetics and alcohols do not. This indicates that the myristate action may not involve receptor binding. The current study shows that the anesthetic action is physical and can be nonspecifically antagonized.