Helen Tredway Graham

Measurement and normal range of free histamine in human blood plasma

Abstract A method is described for measuring histamine in plasma by development of fluorescence with o -phthalaldehyde, after separation of the histamine from most other constituents of plasma by: 1) deproteinizing with trichloroacetic acid, 2) adsorption on Decalso, 3) elution with KBr, 4) extraction into butanol at alkaline pH, and 5) extracting back into water after adding acid and heptane. A mean value of 0.6 μg/1. (S.D. 0.3 μg/1). was obtained for 62 samples of fresh plasma from 46 normal young women. Freezing and thawing the plasma did not significantly affect this value. This level is less than a third of the lowest level previously reported by any chemical method. The specificity of the procedure was tested in several ways. A wide variety of amines were tested with pthalaldehyde in regard to capacity to produce fluorescence and the spectrum of the fluorescence when produced. Large plasma samples were carried through the analytical procedure and chromatographed prior to fluorescence development. Other samples were treated with diamine oxidase. The conclusion is reached that 65–100 per cent of the fluorescence is attributable to histamine. Some samples contained material which accompanied histamine through the entire procedure and reacted with phthalaldehyde to give fluorescence at shorter wavelengths than did histamine. Spermidine seems to be a good candidate for this material.

Dependence of tissue histamine content on local histidine decarboxylase activity

Abstract 1. 1. The histidine decarboxylase activity of mammalian tissues has been estimated by measuring the increase in histamine content of tissue homogenates during incubation in a histidine-containing solution. The incubation was carried out at pH 9.2 in the presence of a strong reducing agent to prevent histaminase activity. The histidine decarboxylase activity so measured varied over a hundred-fold range in the tissues investigated. 2. 2. The histidine decarboxylase activity of tissues varied in general in proportion to the histamine content of the tissues. Since there is strong evidence that histamine is localized almost exclusively in mast cells, these are probably also the locus of histidine decarboxylase. 3. 3. The distribution of histaminase activity was found not to be related either directly or inversely to the histamine content of tissues. 4. 4. The amount of combined histamine in the tissues was less than the amount of free histamine and bore little relation to the amount of combined histamine produced during incubation with histidine.

Action of Veratrine on Medullated Nerve

Fromherz 1 has recently investigated the effect of veratrine and other drugs upon the electrical response of medullated nerve, using a moving coil galvanometer as a measuring instrument. He detects little difference in response between normal and veratrinized nerve, if the nerve is suspended in oxygen throughout. If, however, veratrinized nerve be asphyxiated and allowed to recover in oxygen, he finds that the after-potential after a single shock or short tetanus may last as long as 10 minutes. We have reinvestigated the action of veratrine on the sciatic nerve of R. pipiens with the cathode ray oscillograph, and find that if the concentration is suitably adjusted to the temperature, typical veratrine after-potentials as measured out to one-half second can be obtained whether the nerve is in air or in oxygen. The results of Graham and Gasser 2 are therefore not due to partially asphyxial conditions, as suggested by Fromherz 1 and Hill. 5 In order to study the after-potential throughout its course we have modified the usual oscillographic technique by employing a voltage amplifier of the direct-coupled type, especially designed to avoid amplifier drift (the theory of this amplifier has been published by Schmitt 3 and the details of the circuit as modified for electrophysiological use will be published elsewhere). By this method the resting potential is followed continuously so that due allowance for drift in resting potential can be made in estimating the duration of the after-potential. After a shock the potential changes can be followed for the first 3-4 seconds by photographing a slow sweep of the oscillograph beam; thereafter the potential may be measured every few seconds for as long as desired by reading the balancing potentiometer, using the oscillograph at high amplification as a null instrument. Preliminary experiments have revealed after-potentials lasting as long as 6 minutes after a single stimulation of veratrinized nerve in oxygen after previous asphyxiation. Without asphyxiation the potential seldom lasts longer than 10-30 seconds.

Augmentation of the Positive After-Potential of Nerves by Yohimbine

A nerve poisoned by yohimbine exhibits, after a single response, a recovery curve of excitability characterized by a refractory period, a supernormal period, and a subnormal period. 1 Of these the first 2 have been recognized as existing in unpoisoned nerve and have been brought into approximate relationship with the parts of the action-potential known as the spike and negative after-potential. A subnormal period has been recognized in unpoisoned nerve but only after the nerve has been tetanized, in which case the subnormality is associated with the positive after-potential. 2 The latter association suggests that the effect of yohimbine is to augment the process responsible for the positive after-potential. Such being the case, the apparent absence of a subnormal period following a single response in unpoisoned nerve would be interpreted as due to the small size of the positive after-potential which there exists. A few experiments sufficed to demonstrate that the potential is augmented as was anticipated. The nerves (isolated sciatic of Rana pipiens) were treated in all cases as in the excitability experiments, and their potentials recorded on a cathode ray oscillograph after amplification with a direct-current amplifier, the latter being necessary to avoid distortion of potentials of the length in question. The progress of the potential-change can best be followed in connection with Fig. 1. All parts of the figure start from a potential-level reached after a long period (15 minutes or more) of freedom from activity. Activity is induced by a single break induction shock and starts with the spike potential, which throws the spot far off the record. The actual record starts with the negative after-potential and is continued by the positive. In the initial unpoisoned state (Fig. 1 A) the positive after-potential reaches a maximum of about 5μν and is not distinguishable for more than about 0.6 sec.

Active Constituent of Local Anesthetic Solutions

Such local anesthetics as are salts of weak bases and strong acids—and most local anesthetics come under this chemical classification—have repeatedly been shown to be more effective, particularly for surface anesthesia, anesthesia of isolated nerves, etc., when their solutions have been hydrolyzed to some degree by the addition of alkali. This and other similar evidence led to the theory that the base is the only active constituent of such solutions. 1 This theory has been put to direct test by comparing the local anesthetic activity of solutions of an anesthetic base alone with that of solutions containing the same concentrations of base plus varying concentrations of the anesthetic salt. The anesthetic activity was measured by the time required to anesthetize to a strong break induction shock the nerve of a sciatic-gastrocnemius preparation from Rana pipiens; the time for recovery after removal from the anesthetic solution was also sometimes recorded. The presence of the salt (hydrochloride) has been found to make no difference in the time required to anesthetize with γ-4-morpholine-propyl benzoate, one of a series of recently synthesized local anesthetics 2 (Table I). However, in all cases, as in this one, the presence of salt results in a shift of pH towards the acid side, and this might alter the sensitivity of the tissue or otherwise modify the effectiveness of the anesthetic in such a way that, for instance in the example given, a total anesthetic concentration of 0.008 mols per liter had the same activity at pH 6.9 as 0.004 mols at pH 8.1.

A Late Period of Subnormal Irritability Following Nerve Response

The described effects of the passage of an impulse through isolated frog nerve on the irritability of the nerve are (1) the appearance of refractoriness immediately following the response and (2) under certain circumstances the appearance of supernormality, succeeding the refractoriness, and of much longer duration. By means of certain pharmacological agents, a third, still later effect may be made to appear, a depression of irritability differing from refractoriness not only in its time relations but in other respects as well. Yohimbine is the most effective agent as yet found for producing this effect, which will be referred to as “late subnormality”. About an hour after 1/20,000 yohimbine hydrochloride has been applied to the isolated sciatic nerve of Rana pipiens, cathode ray oscillograph observations very clearly reveal the 3 successive phases of irritability modification—-refractory period, supernormal periodr late subnormal period. The relatively refractory period is somewhat (0.5-1.0σ) longer than in the untreated nerve displaying supernormality; the supernormal period thus begins later than in the untreated nerve, while it ends considerably earlier. The maximum degree of supernormality is reduced. In one typical case, the maximum supernormality fell from 104.5% to 103%, and the duration of the supernormal period decreased from 138° to 47σ. The supernormal period is immediately succeeded by the late subnormal period which lasts 5-10 seconds after the conditioning shock in the nerve yohimbinized as described. The minimum irritability during this period occurs 1-2 seconds after the shock, and after a single conditioning shock is usually 97-99% of the resting level in these lightly poisoned nerves. The subnormality may be both prolonged and intensified by stronger yohimbinization. When the drug action is strong enough to reduce the irritability during the subnormal period to about 95% of the resting level, the supernormality has ordinarily been largely or entirely removed, but there remains in the curve of recovery of irritability a crest of irritability in the region of former supernormality, and this crest tends to persist in less marked form even in deeply poisoned nerves. In these nerves the late subnormality has been observed to last 16-32 seconds. A series of shocks, each one timed to fall in the late subnormal period of its predecessor, increases and prolongs the subnormality very markedly. Each successive shock of the series adds something to the depression left by preceding shocks, but each successive shock adds less than its predecessor. The level of minimum irritability eventually reached has been observed to be 50-96% of the resting irritability, varying with the rate of stimulation as well as with the degree of yohimbinization.

Lactic Acid Increase in Muscle Under the Influence of Anesthetics.

The increase of lactic acid in resting muscle under the influence of narcotics is a familiar phenomenon with high concentrations of chloroform, and was observed by Meyerhof 1 for alcohol and urethanes in concentrations about 10 times those sufficient for general anesthesia. It has now been found that the lactic acid content of isolated whole muscle of green frogs is increased on the average 20 to 50% by ether, nitrous oxide and ethylene at tensions sufficient for general mammalian anesthesia (0.028 atmospheres ether vapor, 0.85 atmospheres ethylene, 1 atmosphere nitrous oxide). This increase is comparable to that produced by chloroform at an equivalent tension (0.01 atmospheres). The lactic acid increase is greater with higher tensions of these anesthetics, being 75 to 105% when 5 to 8 times the anesthetic tension is used. These increases occur with 1/2-2 hours exposure to the anesthetic, and do not seem to be greater with the longer exposures. They are measured by comparing the total lactic acid content of several small muscles (one of each of several pairs) exposed to narcotics, with the lactic acid content of the mates to these muscles not so exposed but otherwise similarly treated. The possibility that the increased lactic acid content is caused wholly by narcotic interference with the oxidative removal of lactic acid is eliminated by the fact that the increase under the influence of narcotics, especially in high concentrations, is very much greater than the increase when all oxidations are prevented by cyanide; and by the second fact that treating both sets of muscles with cyanide before application of narcotic to one of them, or keeping them in an atmosphere of nitrogen throughout the experiment, seems not to decrease the difference between the amounts of lactic acid in the two sets.