The analysis of urine specimens containing peroxides, also has revealed significant amounts of glucuronic acid compounds. We utilized a slightly changed Tollens technique for the dosification of glucuronic acid in the urine, based on the reaction of this acid with naphthoresorcine in an acid medium. To 5 cc. of urine, 0.5 cc. of a 1 % solution in alcohol of naphthoresorcine (1.3 dioxynaphthalene) and 5 cc. of concentrated hydrochloric acid were added. The mixture was boiled for one minute, allowed to stand for another five minutes, then cooled, preferably in an ice water bath. When cold, a mixture of 90% ether and 10% alcohol was added, agitated, and the blue violet color of the ether alcohol measured, using a spectrophotometer. The values obtained in different subjects have shown a definite increase in the amount of glucuronic acid in the urines containing peroxides, indicating a probable relation between them. Based on this correlation, we investigated one of the roles of glucuronic acid in the organism, that of detoxifying agent.

Glucuronic acid could be considered to result, at least in part, from oxidation of glucose. Usually oxygen intervenes in glucose metabolism only after the desmolyse processes * (232), corresponding to the fermentative phase has progressed to the appearance of pyruvic acid. Glucuronic acid could be considered to appear from a more direct fixation of oxygen to the glucose molecule. This fixation has to take place upon C« in order to lead to the appearance of glucuronic acid. Theoretically, the oxygen fixation might be expected to occur at C1 in view of the aldehyde group present at that carbon. This takes place in vitro. It would lead to the appearance of gluconic acid. However, if a phosphoric or other radical is bound to C1 in vivo, fixation of the oxygen at this carbon is prevented. Oxygen attaches itself then at C6 which is the next most reactive carbon in the molecule. This reactivity at C6 is seen when oxidation in vitro is continued beyond gluconic acid, leading to saccharic acid, a bicarboxylic acid with one carboxyl at C1 and another at C6-Glucuronic acid intervenes in the physiological defense processes by combining with certain noxious products and helping to eliminate them in non toxic forms. The resulting compound between glucuronic acid radicals and various substances—and for sulfuric acid radicals as well—has been called "conjugation," the substances being sulfo- or glucurono conjugated compounds. Because of the special attention given in this publication to the conjugation of the double bonds, we will use the term "coupled" for this bond to sulfuric or glucuronic acid.

A certain parallelism exists and has always been emphasized between the detoxifying and eliminating function exerted by the sulfuric and glucuronic radicals. Not only the two derivatives appear in the urine, but it is often noted that glucuronic acid intervenes when large amounts of certain substances, such as menthol or phenol, are present and the sulfuric acid radicals are not in sufficient quantity to insure detoxification and elimination. By administering mineral sulfates to the subject, the proportion of sulfo derivatives is seen to increase. This parallelism appears especially interesting when we recognize that sulfuric acid represents the final stage of the oxidation of sulfur introduced into the organism in combinations in which it enters as a bivalent negative element. Both sulfuric and glucuronic acid result from oxidative processes, one involving the thiol group and the other, glucose.

While glucuronic acid often substitutes for the sulfuric radical in the excreted substances, qualitative differences exist. For example, phenol as well as indoxyl is bound to the sulfuronic radical, while the higher alcohols and especially the cyclic oxyacids do not combine with this acid. For many substances the parallelism that exists in the bond to sulfuric and glucuronic radicals extends only up to a certain point, after which the amount of the sulfuric ester no longer increases. This fact has raised the problem of the action of these two radicals and the differences between them, and can be understood only by considering the substances that are bound by these acids and eliminated as sulfo- or glucurono coupled derivatives.

* The term "desmolyse," widely used by some authors abroad, corresponds to the changes occurring in metabolites under the action of hydrolases, which, when completed, result in a splitting apart of the molecules and the liberation of the energy they possess.

We have seen in Note 19 that, with very few exceptions, such as benzoic acid, almost all the substances excreted or bound to glucuronic acid have an OH as polar group. However, many have more than one polar group.

The bonding with the sulfuric radical, as it occurs in the organism, follows a relatively simple pattern. (Fig. 258a) In a first step, the bonding of one acid function of the sulfuric radical to the substance produces an acid sulfuric ester. This is further changed by combination with a metal, usually potassium, which produces a highly hydrosoluble salt and represents an excremental derivative.

The bond of sulfuric acid in the organism can result in an ester and a saltThe bond of sulfuric acid in the organism can result in an ester and a salt

Fig. 258a. The bond of sulfuric acid in the organism can result in an ester and a salt. Fig. 258b. The bond of glucuronic acid can result in a glucoside, an ester or a salt combination.

The binding is more complicated for glucuronic acid. Glucuronic acid does not realize the bonds, since it is eliminated as such if administered. The bond occurs at the aldehyde group of glucose, forming a glucoside, which is passed further in glucuronic acid. As is seen in Figure 258b, glucose can realize different bonds. It can bind a radical either to the adlehyde group at C1 to form a glucoside, or to its alcoholic hydroxyls to form an ester. The glucuronic acid can bind a metal to its carboxyl to form a salt. The possibility of realizing a glucosidic coupling at C1 or an esteric at C2 has been revealed by Quick. (233) The ability to realize concomitantly a multiple coupling, for which glucuronic acid seems to be highly suitable through its multiple and various functions, appears more interesting. The study of elimination of different oxybenzoic acids shows that it is bound to glycocoll, (Fig 259a) while the para isomer is eliminated coupled by the glucuronic radical. (Fig. 259b)

The difference between sulfuric and glucuronic acid thus appears to be related to double coupling especially if the second function is acid. The presence of two opposite polar groups will thus prevent the bond to sulfuric acid and favor the bond with the glucuronic radical. Organic oxyacids are thus not bound by sulfuric radical as long as their carboxyls are free. The amides, such as salicyl amides—and, the esters such as methyl salicylates— are coupled by the sulfuric radical, while the acids are not. It is through these multiple coupling possibilities—and, in addition the possibility of forming a salt especially with potassium—that glucuronic acid corresponds to a broader activity as a detoxifying and eliminating agent than sulfuric acid.

The ortho oxybenzoic acid (salicylic acid) is bound in the organism to glycocoll

Fig. 259. The ortho oxybenzoic acid (salicylic acid) is bound in the organism to glycocoll, (a) while the para isomer is coupled by the glucuronic radical, (b).

The capacity to utilize glucuronic acid for excretion of noxious substances varies widely between species. Benzoic acid has been shown to appear as a glucuronic derivative in animals by Csouka, Brakefield, and Schmidt (234), but never in humans, a fact that is not confirmed by Quick. (235) Rabbits eliminate the tertiary alcohols as glucuronic derivatives more completely than dogs. While thyroidectomy in rabbits decreases the synthesis of camphoroglucuronic acid (236), the administration of thyroid extract increases it.