The metabolic processes characterizing abnormal foci have been investigated in various ways. One method was devised in order to study the acid base changes which take place within abnormal foci created by surgically produced wounds under the influence of various chemical, physical and biological factors. This research was made in collaboration with C. Huesca Mejia. (212)

Adult Carworth Farm female and male albino rats weighing 150 to 200 grams were employed. The animals were separated according to sex and all received a standard diet of Purina Chow and water ad lib. Tests were run on groups of twenty animals, with from two to four control animals in each group.

The animals were divided into groups that were subjected to various experimental conditions for three days, after which time, under ether anesthesia, a wide area of the back of each animal was carefully depilated by hand. A 1 square cm. wound was then produced in the depilated area down to the dorsal aponeurosis. The wound was kept free of blood with dry gauze until bleeding had entirely ceased, and was then left uncovered. All wounds were made between 8:00 a.m. and 10:00 a.m.

A glass electrode made according to the specifications of Mclnnes and Dole (223) was used. As reference, an electrode containing normal saline was employed. (224) The electrodes were mounted on a stand in a fixed position. A model H Beckman pH meter, whose signal was amplified by a 6H6 and 6SN7 push pull, was used as the first amplifier. The readings were made on a 200 micro ampermeter adjusted so as to have the full scale represent one pH unit. The experimental error using this apparatus was found to be less than ± .01.

pH determinations were carried out by bringing the wound area into contact with the tips of the electrodes. The animals were held gently but firmly in one hand until they were completely immobile, at which time a firm contact was established between the center of the wound area and the tip of the glass electrode, and between the reference electrode and the wound periphery. Readings were carried out on the surface of the freshly exposed aponeurosis within ten to fifteen minutes after the surgical procedure. Subsequent readings were made every twenty four hours upon the wound crust, which was washed and then moistened with a drop of an 0.1% sodium chloride solution freshly adjusted to pH 5 as suggested by Blank for use in skin pH determinations. (225) The electrodes were standardized with buffers of different pH before and during each period of testing.

The effect of various chemical agents upon the wound pH was studied by daily oral administration for three days preceding the operation and thereafter until the conclusion of the series of pH determinations. In general, the substances were administered according to weight in quantities corresponding to the therapeutic doses accepted for humans or in amounts equal to 10% of the daily toxic dose. Water soluble substances were administered in drinking water. Oil soluble substances were administered on pieces of bread. In some cases, substances were introduced directly into the stomach through a catheter. In a few cases, substances were injected subcutaneously.

In the 164 control animals, the pH of the wound surface varied between 7.30 and 7.33 when measured ten to fifteen minutes after the completion of the surgical procedure. The pH remained at this level for at least three hours.

Twenty four hours after the wounds were produced, the pH of the moist crust covering the area was found to be between 7.72 and 7.76 in all untreated animals. (Fig. 234)

Fig. 234. Second day wound crust pH values remain in a range from 7.72 and 7.76 in normal rats as seen in the animals which served as controls for the multiple experiments.

At forty eight hours, the pH of the crust was found to be between 7.42 and 7.60, this being the period of greatest variability. At seventy two and ninety six hours, all the readings were between 7.28 and 7.32. After the fifth day, similar values were found and further readings were not taken.

No consistent correlation was found between the values on different days for individual animals. For example, in some animals with a wound pH of 7.30—the lowest value—immediately after the operation. pH reached 7.76, the highest value for the normal range, in twenty four hours. In males, values appeared to be slightly higher than in females.

In 410 of the 860 animals exposed to various chemicals pH value of the wounds was determined several minutes after the operation and found to be between 7.28 and 7.35. Only 22 showed a deviation from the control range of 7.30 to 7.33. No correlation was observed between the minimal changes in these animals and the type of treatment administered.

While the pH values found several minutes after wound induction showed very little or no variation from the control range and no correlation with the various experimental conditions employed, the findings at twenty four hours showed considerable significance for the agents used. At forty eight hours and thereafter, no important differences were observed between values for controls and those for groups treated in different ways. In continuing these studies therefore, determinations were carried out only several minutes after the production of the experimental wounds and then again 24 hours later. Actually, only values for the twenty four hour reading appeared significant and will be discussed. We made 24 hour measurements for all of the 860 animals treated with different agents. For convenience, we refer to the pH value of the crust at twenty four hours after creation of the experimental wound as the s.d.c. pH (second day crust pH).

From four to twelve animals were employed in the assays of the activity of each agent. By applying the same experimental conditions to animals in groups tested at different times, it was possible to determine whether the changes observed were due to some external factor such as temperature, humidity, etc., or were actually due to the imposed experimental conditions. The s.d.c. pH has proven to be of considerable interest because consistently similar changes have been found to be produced by the same agents when applied to animals in different groups tested weeks or months apart.

Considering all the animals treated with various agents, three possibilities have been found to exist: 1) There may be no effect upon the s.d.c. pH, in which case the values will all fall within the control range of 7.72 and 7.76 found in untreated animals; 2) s.d.c. pH may be elevated to values between 7.77 to 7.85; or 3) the s.d.c. pH may be reduced to values of 7.70 to 7.60.

We will present here only the conclusions of these studies as related to the various agents investigated.

Chemical Factors. Cations And Anions

It was interesting to investigate the influence exerted by some cations and anions by first using the same anion with various cations and then using different anions with the same cation. It was apparent in all the experiments that the immediate pH of the wound does not differ from that of the untreated animals, and that the s.d.c. pH data obtained are concordant.

We studied the influence exerted by anions first by investigating the effects of administration of acids. With even strong inorganic acids, no changes were seen in the normal tissues. Definite and opposite effects were thus obtained with acids corresponding to the elements of the Vllth series and with those of the Vlth series. The first group of acids, especially HC1. induced a frank acidifying effect while the second produced an alkalizing effect. The phosphate and nitric ions showed strong acidification, the iodide and bromide ions were weaker than the chloride ion. The bicarbonate ion clearly alkalized, as did sulfate and selenate. The thiosulfate ion had an obvious alkalizing effect, with pH values as high as 7.85. Among the organic acids, citric acid produced one of the strongest effects, even stronger than that of the inorganic acids. The fact that citric acid is only slightly metabolized could explain its strength. The gluconate ion induced a slight acidification. The cacodylate ion seemed to induce no changes although not enough animals were utilized to judge its effect thoroughly.

In studying the effects of salts, the roles of both cation and anion were considered. Using different anions for the same cation, it was possible to judge the effect of the cation.

Sodium and lithium produced relatively slight acidification. Potassium manifestly acidified as did ammonium, the latter, however, to a less marked extent. Marked acidifying effects were seen for iron, mercury and bismuth. A lesser effect was obtained with molybdenum and aluminum. On the other hand, a manifest alkalization was found for bivalent calcium, strontium, copper, barium and cobalt. Manganese and silver cations seemed to influence the second day wound pH only slightly toward alkalization. It seems that there is an additive effect for the different elements in their acidifying or alkalizing influence. This has permitted us to judge the effects of the different ions. Potassium induces greater acidification than sodium, and still greater acidification than ammonium. Potassium chloride, in which the two ions have an additive effect, is thus more frankly acidifying. The same is true for the acid phosphate. The alkalizing tendency of sulfate ion opposes the acidifying effect of potassium and explains the slight acidifying influence of potassium sulfate. We must arrive at the relatively strong alkalizing tendency of the carbonate ion to find an anion able to counteract the acidifying effect of potassium. The s.d.c. pH effect of potassium carbonate is in the normal range. The data obtained through this study led to the research on the intervention of the elements in biology, which is the subject of Chapter 5. Information concerning the effect upon the s.d.c. pH of these elements as well as the relationship between this effect and the structure of the elements is discussed in this chapter.

Calcium ion has an alkalizing influence strong enough to counteract the acidifying tendencies of such anions as chloride and phosphate. Weaker acidifying anions, such as lactate and gluconate, are not sufficiently strong to counteract the alkalizing tendency of calcium, and calcium salts of these acids have a strong alkalizing effect.

This analysis indicates that the effect of a salt upon the local pH of abnormal tissues can be judged by considering the additive influence exerted by anion and cation, the effect increasing if both have tendencies in the same direction, and decreasing if the tendencies are opposed. We will not emphasize here the other antagonistic biological effects of cations and anions, as they pass from acidifying (citric acid and potassium) to alkalizing (calcium and thiosulfate). We will discuss these effects in connection with the pharmacological studies of these agents. For the moment, we want only to emphasize the value of this investigational method.