NUCLEIN METABOLISM
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
N (1)—C (6) N CH
C (2)—C (5)—N (7) HC C NH
C (8) CH
N (3)—C (4)—N (9) N C—N
Purin nucleus. Purin.
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:
N==C—NH₂
| |
HC C—NH
‖ ‖ \
‖ ‖ CH
‖ ‖ /
N—C—N
Adenine
(6-amino-purin).
Other important bodies built up round the purin nucleus C₅N₄, variously
designated as xanthine, alloxuric and nuclein bodies:—
HN—C==O HN—C==O
| | | |
H₂NC C—NH O==C C—NH
‖ ‖ \ | ‖ \
‖ ‖ CH | ‖ CH
‖ ‖ // | ‖ //
N—C—N HN—C—N
Guanine Xanthine
(2-amino-6-oxypurin). (2-6-dioxypurin).
HN—C==O HN—C==O
| | | |
HC C—NH O==C C—NH
‖ ‖ \ | ‖ \
‖ ‖ CH | ‖ C==O
‖ ‖ // | ‖ /
N—C—N HN—C—NH
Hypoxanthine Uric acid
(6-oxypurin). (2-6-8-trioxypurin).
H₃C—N—C==O HN==C==O
| | | |
| | CH₃ | | CH₃
| | / | | /
O==C C—N O==C C—N
| ‖ \ | ‖ \
| ‖ CH | ‖ CH
| ‖ // | ‖ /
H₃C—N—C—N H₃C—N—C—N
Caffeine Theobromine
(1-3-7-trimethyl, (3-7-dimethyl,
1-2-6-dioxypurin). 2-6-dioxypurin).
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.
{ NH—CO { N==C—NH₂ { NH—CO
Thymine { CO C CH₃ Cytocine { CO CH Uracil { CO CH
{ NH—CH { NH—CH { NH—CH
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.