No hard and fast line can be drawn between the metabolism of protein and that of nuclein. For though, morphologically speaking, the nuclei of cells are sharply differentiated from the circumambient cytoplasm, and exhibit equally distinct staining reactions, yet, chemically, the differences between them are quantitative rather than qualitative.
But while, as far as chemical changes are concerned, nuclein metabolism is comparable with that of protein, nevertheless the former in respect of its “energy” and its bearing upon growth and production, is infinitely more vital, incomparably more active; for it is in nuclear changes that we may best discern evidence of the initiation of oxidation processes and other varieties of enzymatic activity. Moreover, as Walker Hall points out, “the presence of masked iron phosphorus and certain forms of fat in the cell nucleus strengthens this view, and thus we are led to recognise the important part played by the nucleus in the life of the cell, and to appreciate the influence of nuclein heredity in cellular exchanges.”
So much by way of prelude, but the story of the growth of our knowledge of nuclein as opposed to protein is so fascinating as to be worthy of a slight digression.
The Isolation of Nucleic Acid
Functionally regarded, the nucleus is the essential element of the cell. Embedded within the cytoplasm, its isolation therefrom, and this in quantities sufficient for analysis, may well have dismayed the earlier workers. But the resources of Friedrich Miescher were equal thereto. Treating surgical bandages soaked with pus with a dilute solution of sodium sulphate, he extracted the heavy pus cells. These, then, by careful decantation, were easily disengaged. The pus cells, still intact, were then subjected to the digestive action of artificial gastric juice. The protoplasm was thus dissolved, but not the more resistant nuclei, which remained as an insoluble grey powder. In this manner cell nuclei, free from protoplasm, became available for chemical analysis. Treating the insoluble nuclei thus obtained with dilute sodium carbonate, a solution was formed. Acetic acid added thereto produced a flocculent precipitate which was found to contain phosphorus, and responded to protein colour tests. This substance Miescher christened by the name of nuclein. Subsequent observers prepared nuclein from the nuclei of yeast cells and the red blood corpuscles of birds. All nucleins are insoluble acids which form soluble salts with sodium. But while they respond to protein colour reactions they differ from protein in that they contain phosphorus and resist the solvent action of artificial gastric juice.
Migrating some ten years afterwards (1897) from Tubingen to Basle, Miescher entered upon his celebrated researches into the habits of the Rhine salmon. He found the belief had long been current that the fish, during their passage from the sea up the Rhine to their spawning haunts, never partook of food. That this belief was well founded he was able to prove; for, saving isolated and easily explicable exceptions, he noted that their alimentary canal was devoid of food débris, while their digestive juices were as a rule inert. One startling change he noted, that while, on the one hand, their muscular tissue profoundly wasted during their migration, their organs of reproduction enlarged enormously, the inevitable conclusion being that eggs and spermatozoa had been created from muscle protein.
Researches on Spermatozoa
Struck by the opportunities for scientific investigation during the spawning season, Miescher determined to resume his work upon nuclein. Spermatic fluid or lachsmilch, being readily obtainable in great quantities, he had to hand a mass of material admirably adapted for chemical examination of the cell nucleus. The conclusion that the heads of the spermatozoa might be regarded as a metamorphosed nucleus seemed obvious, and the opportunity too good to be lost.
On examination he found the “sperm heads” protein-free, made up almost entirely of a single chemical entity, a salt of an organic base rich in nitrogen and an organic acid containing phosphorus. The former was protamine, the latter nucleic acid.
The presence of this salt protamine nucleate led to the conclusion that nuclein was merely a salt of protein and nucleic acid.
The Discovery of Purins
Miescher, who had already isolated nuclein and nucleic acid, came nigh to one other equally important discovery. Heating a specimen of protamine with nitric acid, he noted that a yellow spot formed which turned to bright red when moistened with alkali.
Alive to the import of the reaction, Miescher requested Piccard to examine salmon sperm for purin bases. Extracting the same with hydrochloric acid, Piccard found guanine, and what he thought was hypoxanthine, but which was in truth adenine.
Another distinguished worker in this sphere, Kossel, noted that, subjected to the action of hydrolytic agents, nucleins always yield purin derivatives; also that the same were derived, not from the protein of the nuclein, but from the nucleic acid. Thus it was to Kossel that we are indebted for the discovery of the purin bases, hypoxanthine, xanthine, guanine, and lastly adenine. It was, indeed, through his brilliant achievements that nucleic acid became recognisable as a definite entity, distinguishable from proteins and other body elements, this latter differentiation by token of the purin bases which are contained in nucleic acid.
Moreover, it led to the dissipation of the old belief that uric acid was an intermediate product of protein metabolism, for the revelation of purin bases as decomposition products of nucleic acid carried with it the inference that uric acid also had chemical affinities therewith. The chemical structure of the purin bases and that of uric acid betrayed a common likeness, and, therefore, a presumptive physiological connection; in other words, that a chemical nexus obtained between the cell nucleus or nucleic acid and uric acid.
The physiological derivation of uric acid from nucleic acid did not long lack experimental proof. In 1886 Minkowski found that, given extirpation of their livers, the urine of birds contained ammonium lactate, evidently a substitute for the uric acid normally present, notwithstanding the uric acid never entirely disappeared from the urine. This indicated the derivation of uric acid from two sources:—
- (1) Conversion in the liver of ammonium lactate into uric acid.
- (2) Some other, though unknown, process of formation.
To clear up the obscurity regarding the latter, V. Mach, after extirpating the livers of geese, injected them subcutaneously with hypoxanthine, finding that the same was converted into uric acid, which was excreted in the urine. In this way the capacity of the organism to elaborate uric acid from a purin precursor was demonstrated.
Uric Acid a Derivative of Nucleic Acid
Despite V. Mach’s revelation, the origin of uric acid from nucleic acid was still to seek. In the year following (1889) Horbaczewski traced it to this source, and in the following manner. Mixed with water, the pulp of the calf’s spleen was put to digest at 50° until putrefaction began. The fluid was then sterilised with a solution of lead acetate, and arterial blood being added it was kept at 50°, a current of air meanwhile being passed slowly through the mixture. Subsequently the fluid was found to contain uric acid; but the experiment being repeated, without the passage of air, xanthine and hypoxanthine and not uric acid resulted.
While Horbaczewski’s experimental findings were amply confirmed, some of his deductions therefrom were subsequently proved faulty. (Thus, he thought putrefaction an essential factor; also he believed that the formation of uric acid ensued before the purin groups were disengaged from the nucleic acid, and definitely affirmed that the uric acid was not produced by the oxidation of free xanthine or hypoxanthine.)
But, nevertheless, this pioneer established that in both man and rabbits uric acid was derived from nucleic acid. Also, having observed that when after starvation the food intake was resumed, a leucocytosis occurred, he announced his belief in the following theory. Thus, he noted that leukæmics, whose blood showed a high leucocyte count, excreted an unusually large amount of uric acid; consequently, he came to the conclusion that uric acid was formed from defunct leucocytes. Also that nuclein-rich food, when ingested, contributed to the formation of uric acid only in so far as it induced leucocytosis. Hence the origin of the increased uric acid excretion which occurs when feeding is resumed after starvation.
This increased excretion of uric acid after the ingestion of food rich in nucleic acid has been amply confirmed; but all the earlier attempts to achieve an increased excretion by the ingestion of free purin bases, as opposed to the combined purin bases, existing as such in nucleic acid, failed, although tried repeatedly.
So much for the various stages by which our knowledge of the purin derivatives of nucleic acid has been gradually acquired, for though purin bases had, from early times, been known to exist in animal tissues, their presence there could not be rationally accounted for prior to the discovery of nucleic acid.
It still remains for us to deal in detail with the further developments of our knowledge which concern the disruption of nucleic acid in the body and the process by which uric acid is derived therefrom.
But before proceeding to consider in detail the complex series or enzymatic transformation that this entails, it will, I think, be wiser to deal first with the chemistry of uric acid, its solubilities, and its sources, whether exogenous, endogenous or synthetic.
The Chemistry of Uric Acid and the Purin Bodies
Much of the vague philosophy of disease in past times may fairly be attributed to the complexity and mystery of action inherent in living matter. The subjects of physics, chemistry and biology, in their wider acceptation, were unevolved, and scientific pathology, the offspring of this ancestry, was yet unborn. How much we owe to physics, chemistry, and biology, those handmaids of medicine, is inestimable! But something at least of our debt thereto will be revealed in the following pages.
Of the purins in human urine, the most important is uric acid, and far behind comes xanthine, while traces of hypoxanthine, guanine, and adenine are also detectable. Some years ago the current view was that the metabolism of any protein gave rise to uric acid. This assumption has now proved to be erroneous, for it is known that only certain foodstuffs lead to an increase in the uric acid excretion; in other words, on a diet rich in purin the output thereof is considerably higher than on a purin-free diet, this being due to the large amount of nuclein and purin bases in flesh foods, especially those containing glandular substances. Under ordinary conditions the excretion of uric acid ranges from 0·3-1·2 gm. per diem, or 0·02-0·10 per cent. The oscillations in output vary with the state of health, diet, and personal idiosyncrasy.
Chemical Constitution
The empirical formula of the uric acid molecule, C₅H₄N₄O₃, has for long been known, but it was reserved for Emil Fischer to reveal the chemical structure thereof. Through his labours we now know that uric acid is one of a group of substances which owe their kinship to their possession in common of the heterocyclic ring termed by Fischer the “purin nucleus” (1898).
The intimate relations of the purins of bio-chemical interest to the purin nucleus, and alike to each other, will be rendered more intelligible by examination of their structural formulæ as hereafter given. All, as will be seen, are derivatives of a synthetically formed body purin which, though unimportant in itself, is yet interesting in that it is the basic substance from which the following take origin:—
| Purin | C₅H₄N₄ | |||
| Hypoxanthine | C₅H₄N₄O | Monoxy-purin | } | |
| Adenine | C₅H₃N₄NH₂ | Amino-purin | } | |
| Xanthine | C₅H₄N₄O₂ | Dioxy-purin | } | Purin Bases. |
| Guanine | C₅H₃N₄ONH₂ | Aminooxy-purin | } | |
| Uric acid | C₅H₄N₄O₃ | Trioxy-purin | } |
It now devolves upon us to note the arrangement of the atoms in the purin nucleus. To each atom is affixed a number indicating the exact location of the various atoms and groups attached to the said nucleus. The manner in which the various purin bodies are built up around the purin nucleus C₅N₄ will become apparent from a study of the following structural formulæ culled from Wells’ “Chemical Pathology”:—
Structural Formulæ
To describe the individual derivatives of purin we have to indicate to which particular atom of the purin nucleus the combining groups are attached. Thus, for example, adenine in structure is classed as a 6-amino-purin, and accordingly has the following formula:
Other important bodies built up round the purin nucleus C₅N₄, variously designated as xanthine, alloxuric and nuclein bodies:—
It will be seen that the purin bases stand in very close chemical relationship to uric acid in that the latter also is marked by the possession of a group called the purin nucleus; indeed, the relationship of uric acid to the purin bases is more intimate than to urea (CON₂H₄), close though this latter be as may be seen from the study of its constitutional formula. (For uric acid may be regarded as composed of two urea radicles, linked by a tricarbon chain. By oxidation and hydrolysis, two molecules of urea may be obtained from one of uric acid, and conversely uric acid is produced by the condensation of urea with hydroxy acids).
The first product of the oxidation of purin is hypoxanthine, long recognised as a constituent of meat extracts. Adenine, the amino derivative of hypoxanthine, is met with in combination with other substances in nuclear material. The second oxidation product of purin is xanthine, and its amino derivative guanine, both of which are found in the same substances as hypoxanthine and adenine. Further oxidation of purin gives rise to uric acid. We have to recognise, also, that in addition to the purins of animal origin there are some also derived from vegetables, viz., the methyl purins, caffeine, theobromine, and theine.
Now, as will be seen later, certain compounds, containing nitrogen and phosphorus, constitute the chief, if not the exclusive, source of uric acid. These substances, long known as nucleins or nucleo-proteins, exist in the animal tissues, and in special abundance in those largely made up of cell nuclei, viz., thymus, lymph-glands, etc. The important and, indeed, the distinguishing component of the nucleins or nucleo-proteins is nucleic acid. This, in that through the action of ferments, it is from the nucleic acids that uric acid and the purin bases are derived.
But, apart from this, we have to recollect that nucleic acids yield constituents other than purin bases, viz., the pyrimidine bases, phosphoric acid, and a carbohydrate group. From a study of the structural formulæ of the pyrimidine bases it will be seen that they are closely related to the purin bases, lacking, however, one of the urea radicles. Moreover, it is believed that, though included in the makeup of nucleic acid, they are not derived from purin but are primary products.
To sum up, the characteristic constituents of nucleic acid are the purin bases (adenine, guanine, hypoxanthine, and xanthine), pyrimidine bases (uracil, cytosine, thymine), phosphoric acid and a carbohydrate group.
We have now discussed the chemical structure of uric acid and its relationship to the purin bases; but before proceeding to consider the various sources from which uric acid is derived, it will I think be convenient to consider (1) the physical properties of uric acid and (2) the condition in which it circulates in the blood.
Properties of Uric Acid
When pure, uric acid is white in colour and crystallises in rhombic form. In contrast to urea it is very insoluble, but much less so in blood serum than in distilled water, viz., ⅟₄₀₀₀₀ of water as opposed to ⅟₁₀₀₀ parts of plasma. It yields with alkalies two series of salts, viz., the biurate or mono-basic, and the so-called neutral or bi-basic urate, the latter of which is much more soluble. In water the mono-basic urate forms a colloidal solution from which the crystalline salt gradually precipitates.
The greater solubility of uric acid in blood plasma was, by Garrod and Haig, attributed to the alkalinity of the plasma. But it must be recalled that the earlier workers in this sphere judged of the alkalinity of the plasma by its reaction to litmus, a crude procedure as compared with the use of phenol-phthalein, and Frankel’s electro-potential measurements. Working with these as criteria, it has been shown that blood is normally alkaline in only a minority of cases, and indeed, according to Flack and Hill, the plasma is in reality neutral.
In the urine uric acid and the urates are held in solution by the neutral phosphates. This because the decomposition of the urates into uric acid by the acid salts of the urine is inhibited by the di-sodium phosphate present therein. Its maintenance in solution is possibly also reinforced through the influence of other constituents in the urine, notably, the urinary pigments and sodium chloride.
Uric Acid in the Blood
As to the form in which uric acid circulates in the blood, Sir William Roberts believed that when dissolved in blood serum it was transformed into the relatively soluble sodium quadriurate. This authority held that in gout, either through deficient excretion or over-production, the quadriurate accumulates in the blood. Circulating therein, in a medium rich in sodium carbonate, it takes up an additional atom of the base, and is transmuted into the biurate, which is less soluble and less easily excreted by the kidneys; consequently, the biurate is hoarded up in the blood, at first in gelatinous, and later in an almost crystalline form, when its precipitation is imminent or actually ensues. This, moreover, was apt to occur at sites where the circulation was poor, the temperature low, and more particularly in regions in which the plasma contained a relatively high percentage of sodium chloride, e.g., synovial sheaths.
But, unfortunately for the valency of this otherwise plausible theory, it was proved by Tunnicliffe, Rosenheim, and others, that quadriurates do not exist as definite chemical compounds; in short, it is generally conceded that their existence should no longer be accepted.
Gudzent and Schade’s Theories
Gudzent was of opinion that uric acid can only exist in the blood as the mono-sodium-urate, of which there are two isomeric varieties, the easily soluble unstable lactam, and the stable relatively insoluble lactim urate. It is the former, or lactam, variety that accumulates in the blood in gout and, according to Gudzent, it is the transmutation thereof into the lactim modification that determines the precipitation of urates in the tissues. The lactim urate is soluble only to the extent of 8·3 mg. per 100 cc. serum, whereas the lactam form is soluble up to 18 mg.
Others, like Bechhold, maintain that the urates are present in the blood in a colloidal form, impossible of excretion by the kidneys. Thus Schade contends that, in the presence of alkalies (hydrates), uric acid or its salts may pass into a state in which it is far more soluble than usual. Moreover, on its path to crystallisation from this over-saturated solution, it passes through a colloid stage in which it is relatively stable. The maintenance of this colloid stage and consequently the retardation of precipitation is promoted by certain substances, i.e., glycerine, urea, serum, albumen, nucleic acid, etc. But hitherto the therapeutic possibilities suggested have not been invoked.
Organic Combinations
It will be recalled that purin bodies cannot be detected in the blood in health, though their administration by the mouth results in an increase in the excreta. Minkowski, to account for this, suggested that the purins in the blood were circulating in a combination which prevented them from giving the usual reactions, typical of their presence therein. We have an analogy in the masking of arsenic and iron in the cacodyl compounds and the ferrocyanide ion.[8]
The explanation proffered by Minkowski was elaborated by Von Noorden. His view was that lying at the disposal of the normal organism are a certain number of organic substances. These latter can combine with uric acid and render it soluble. It is then in this form passed through the blood in the kidneys, which eliminate from it the uric acid. Now, in gout these organic substances are deficient or wanting, and the result is that the uric acid is passed into the blood in the form of urates, the elimination of which only proceeds with difficulty; in other words, the purins normally circulate in organic combination and abnormally as salts of sodium.
It is worthy of note that, from a solution containing albuminous substances, Burian and Walker Hall found that while it was easy to remove the bulk of the purins, a certain percentage always remained which it was difficult to extract.
The view that uric acid is probably carried in the blood in combination with some other organic body and not, as was formerly supposed, with sodium salts, rapidly gained adherents, but the nature of the organic complex is still not accurately known. Many believe that at least a moiety of the uric acid circulates in combination with nucleic (thyminic) acid, but no such compound has yet been isolated from the blood. Nevertheless, as MacLeod suggests, this theory, were it proved correct, would account for the fact that some purins at least are katabolised in the body when they are given in a combined state, as thyminic acid, but are excreted unchanged when ingested in a free state. Thus, certain purins, e.g., adenine, when given freely, cause inflammation and calculous deposits in the kidneys of dogs which, however, does not ensue when they are fed with thymic acid.
But Walker Hall, discussing the good results obtained by Schmoll and Fenner from the administration of thyminic acid, states that his experiments do not indicate that the improvement is at all associated with any change in the uric acid excretion.
To sum up, it is obvious, from the mere variety of the hypotheses advanced, that we are still much in the dark as to the actual form in which uric acid circulates in the blood. While on the one hand the quadriurate theory appears no longer tenable,[9] on the other the nature of the suggested uric acid organic complex is still unknown.
Nay, more, Walker Hall, writing in 1913-14, states “there are many who consider that the sodium mono-urate is the only possible compound;” while Wells, in his “Chemical Pathology” (1918), claims that the best evidence points to uric acid existing in the blood “in a free state and not combined, as was at one time urged by several students of gout.”
Complexity of the Problem
How complex, indeed, the task of the bio-chemist may be gathered from some reflections of Walker Hall. He reminds us that the oxidation and deaminisation of the nuclein derivatives, nucleins, nucleotides and nucleosides, is never complete. For purin bases and pyrimidin bases run side by side in the blood-stream together with uric acid. Also, that the unstable but soluble biurate is constantly changing into a less soluble type, viz., from one isomer to another. Moreover, since the red blood corpuscles abound in potassium, urates of potassium must also occur, and to these may be added, too, ammonium and calcium compounds in small quantities.
But more striking is his inference that the occurrence of isomeric forms of uric acid suggests that isomers of purins and pyrimidins also may occur. For the purin ring or pyrimidin nucleus, with their numerous receptors for the linking up of other substances, offer wide potentialities in the direction of isomerism.[10] Some of these, he hazards, may be born of one type of cell nucleus, some of another, while it is not inherently improbable that, “In response to abnormal stimuli or excessive demand, other isomers may be formed.”
Now, though uric acid and the urates can be extracted from the blood, it does not, as he remarks, necessarily follow that they circulate as such in vivo; for, despite modern achievements, “the best of the existing methods for the determination of uric acid in the blood are nearly barbarous in their crudity and intensity.” The various procedures available for such estimates fall short of distinction between the several tautomeric forms of uric acid, much less do they furnish any information as to the associations or combinations of purins or pyrimidins with other substances.
For himself, recognising the generally more complex nature of biological processes, he considers that “the circulation of the purins as sodium mono-urate and its simple extraction by kidney cells, seems almost too simple to be true.”
As to the solubilities of uric acid and urates in gouty blood he points out that the suspension capability of the blood-stream for uric acid much transcends the highest amount of uric acid as yet found in the gouty subject. Accordingly, to him, therefore, it seems that “neither chemical nor physico-chemical processes suffice to explain the problem. There must be something more, something vital, biological.”
Having ascertained as far as possible the measure of our knowledge in regard to the foregoing points, we shall, in the succeeding chapter, proceed to discuss the sources of uric acid, whether of intrinsic or extrinsic origin.