Examination of Phosphorus Metabolism in Liver of Rats by Radiophosphorus

Abstract

In the first (experimental) part of the paper, at the beginning, data are presented on the rate of excretion in 16 rats over a period of 18 days. The data are based on test for and measurements of radioactivity in 260 excreta of urine and faces respectively. The particular object of this measurements was to determine the balance of radio phosphorus exchange. Since each of the excreta of faces and urine was measured three times, the total number of measurements of radioactivity in excrements on rats was 1,560.

In a group <of 87 rats the radioactivity of inorganic phosphor-us in the liver was measured three times in each case, so the total number of measurements of activity ‘in the inorganic fraction of the liver was 261.

The measuring of radioactivity in the blood of 20 rats was done 60 times.

Finally, the radioactivity was measured in twelve different organic fractions of the liver involving a group of 87 rats. Since .in these cases each fraction was also measured three times, the total number of measured fractions was 3,132. If the above totals of the measurement of radioactivity in different components and samples obtained from the rats are all added up, the grand total amounts to 5,013, i. e. the number of measurements of radioactivity taken in experiments on rats.

A Chemical quantitative analysis was also done in livers of the same group of rats, concerning the content of ordinary (not radioactive) phosphorus in organic and inorganic factions. AH together, the number of colorimetric measurements of ordinary phosphorus were 3, 393.

The results of 'these measurements formed the basis of experimental

studies concerned with four different problems: 1) the balance of the turnover of radio phosphorus, 2) the distribution of radio phosphorus in several functional systems of the organism of rats, 3) the movement and distribution of radio phosphorus over the organic fractions — considered as a function of time — in the liver of rats, and 4) the distribution of ordinary phosphorus in liver fractions of rats.

1) The study of the radio phosphorus balance came first. The animals were each administered a dose of 10 microcuries of radio phosphorus in the stomach by means of a rubber tube. One 1,000 the part of the dose was kept, each time, as a standard gauge for the testing of momentary radioactivity by a Geiger counter (phosphorus standard gauge). This reference sample was (kept until the end of the experiment, its activity being re-measured at certain fixed intervals. Every twenty-four hours the total quantities of the excreted radio phosphorus in the daily excreta were measured over a period of thirty-six days. During the first few days the measurement was concerned with a larger number of rats but the number of animals to be measured for radioactivity of the excreta gradually decreased (because one group after another had in turns to be sacrificed at fixed periods so that fractions of their livers could be used for an analysis of the content of radioactive and non-radioactive phosphorus). During the final days of the experiments the excremental on only four of the animals were collected, whereas in the first few days as many as 16 animals had been used for the purpose. The results established the fact that the excretion rate was approximately 10 per cent in the course of the initial twenty-four hours; during the 2nd and 3nd days the rate was over 5 and 3 per cent respectively; then the rate pf excretion gradually decreased to about 1 percent. Around this intensity the radioactivity remained stationary for a longer period, at the same time continuing to decline steadily, though at a much slower rate than over the first few days. While the first 10 percent became excreted during the first twenty-four hours, the following 10 percent did so over the successive 3 days. It took another sixteen days before a third of the originally introduced dose was excreted. The excretion of one half of the amount of introduced radio phosphorus took over 50 days. The results of the experiments go to show that radio phosphorus stays in the organism for a long time, which as a rule allows of an effective tracing of its exchange in the organism.

2) The second set of problems studied in the experimental part was concerned with the distribution of inorganic radio phosphorus in particular functional systems of the organism of rats. The main object of the analysis was to establish what part of the retained phosphorus remains 'in the liver, or what the conditions are that affect the content of (radio phosphorus in the liver during the experimental period, for the main theme of our paper is concerned with the exchange (the turnover) of radio phosphorus in the liver. Therefore, concurrently with estimations of radioactivity in the excremental, we also measured radioactivity in the blood, respectively in the liver itself. The measurement concerning the liver was done in all the series of experiment (all together- four- series of rats were used consisting of 20 to 24 animals each) while the radioactivity in the excremental was measured in the first series only, and that of the blood in the second.

Comparative measurements of radioactivity in blood, liver and excreta have shown that a constant rate of distribution of radioactivity soon establishes itself between blood, liver and excreta, which is maintained independently of continual changes in absolute rates of radioactivity in the systems concerned. Unavoidable technical errors aparat (and possibly also

the corresponding difference in the general speed of the process of metabolism in particular groups of rats), we can say that the experiments have abundantly demonstrated that in all blood (i. e. in a mass of 20 gr.) of one rat the radioactivity is maintained in the approximate proportion of from 1:1 to 1:1,3 in relation to the whole of the liver, while the radioactivity in all blood and the excreta is in the ratio of 1:7 — 1:10. These ratios are also borne out by the fact that the curve of decrease in radioactivity is very nearly identical in all three systems. Taking it all together, the absolute rate of radioactivity tends to decrease rapidly in all three systems, and the coefficient of radioactivity decrease in the blood during the 4 to 24-hour period is approximately 5,8:1, and in the liver during the 2 to 24-hour period it is 6,22:1, or 4,5:1 during 4 — 24 hours.

This group of experiments and the data obtained go to show — from the view point of radio phosphorus distribution over the examined 3 functional areas — that the organism of rats behaves as a uniform (unione) reactive medium which, in a comparatively short time, establishes the balance of radio phosphorus distribution over the areas, retaining the same proportions until the end of experimental period. Accordingly, the process of radio phosphorus exchange goes on in a correlative manner, the changes in one of the systems being made adequately manifest in the others.

3) The third and main set of problems dealt with in the paper was concerned with a study of the metabolism of radio phosphorus in the liver of rats and the rate of its distribution, considered as a function of time, in the organic compounds of the liver. This series of experiments was made upon a group of 87 rats. In aether narcosis, the livers were in each case taken out and placed without delay in liquid air. Extractions were then made in three stages, namely a) the excretion of acidosoluble fractions of phosphorus by J. Sachs’s method, d) the exertion of lipoid fractions by the partly modified G. Hevesy ¦test, and c) finally, the residual phosphorus which had been bound in the nucleoproteid fraction was obtained from the remainder of the liver tissue. Liberation of this phosphorus from the tissue was done by the method of Kjeldahl in 'the first two series, and in the last two electric cradles was used.

Separate acidosoluble fractions were then detached (from the general acidosoluble extract) by the J. Sachs method. The phosphorus was finally liberated from these fractions by the technique used according to the directions of the method. From phospholipoids the phosphorus was liberated by the same procedure as in the case of neucleoproteids (L e. by the Kjeldahl method in the first 2 series, and by the use of the electric cradle in the last two). After being liberated from their organic connectives, all the phosphates (of organic as well as inorganic origin) were then left to settle in order to form a sediment as ammonium phosphomolybdates; they were used for the purpose of measuring the quantities of analytical (the ordinary, not radioactive) phosphorus contained in particular fractions, as well as for the counting of beta radiation of the radio phosphorus, which was always found intermixed and combined with the compounds of ordinary phosphorus. Non-active analytical phosphorus was determined by the quantitative-colorimetric method after Benedict. Beta rays were estimated by the use of the Geiger counter produced at the “Boris Kidric” Institute for Nuclear Studies at Vinci. Colorimetric analyses were gauged by the Pulprich photometer with S-69 filter in the experiments conceded with the first 2 series, while all subsequent measurement was done by the use of Becikmann’s spectrophotometer, model DU, by means of light and the wavelength of 750 millimicrons. The following were the fractions isolated from the liver: the inorganic, all sugar fractions (glycogen, glucose, and all intermediary products of its decomposition), the fraction of labile and stable phosphorus from ATP, adenilic acid and the fraction of coenzims (viz. all acidosoluble fractions). The phospholipoid fraction was not subdivided but analysed only as a whole, was also done in respect of the nucleoproteids.

The results of these analyses showed that motion of radioactivity of the organic fractions runs parallel to that of the inorganic ones, but only partly so, for the respective curves of radioactivity are different. The parallelism to inorganic phosphorus is shown by the sugai- group and the labile as well as the stable fractions from ATP. The adenilic acid and coenzyms differ from all the acidsoluble fractions in that they are slow to reach the maximum of radioactivity of the phosphorus contained in the fractions, and also because of the slower rate of decrease in activity following the maximum. The greatest divergences from the inorganic fractions were found in the phospholipoid and the nucleoproteid fractions, for they are the latest to reach the maximum while exceeding by far the activity ate of the other fractions during the period of the decline of radioactivity. The comparisons of radioactivity between the liver fractions were made in accordance to two criteria. The first to be compared were the absolute rates of radioactivity, and then the rations of radio phosphorus distribution in the organic liver fractions were compared at particular successive intervals. It was shown that already after a two-hour interval (the time when the first rats were sacrificed) the distribution over the fractions having a motion of radioactivity parallel to that of the inorganic function proceed-s in the same proportions as those of later periods. On the other hand, as regards the coenzyms and the adenilic acid the constancy of the rate of phosphorus distribution ds realised only after an interval of two hours, that is to say, in the case of the adenilic and the coenzims after 3 and 4,4 respectively. As regards the phospholipoids, the constant rate of radiophosphorus distribution ds realised after 22,8 hours, while the nucleoproteids, the slowest of all, take as much as 30,7 hours. The constancy of the rates of radio phosphorus distribution goes to show by itself that the same specific phosphorus activity comes to be established in all liver fractions; however, this can only be determined directly on the basis of comparisons with the results of a quantitative analysis of ordinary (not radioactive) phosphorus.

4) The fourth series of analyses was concerned with the determination of quantities of ordinary phosphorus in the above liver fractions. The results of the analyses are shown in tabular form. As regards the conclusion, a special mention deserves the fact that the proportions of the ordinary phosphorus distribution in the liver fractions fully correspond with the proportions of their radioactivity. This fact only confirms 'the conclusion that follows from the constancy of percentual distribution of radioactivity after an interval of 30,7 hours. This shows that after the balance of distribution of radioactivity has been reached the specific phosphorus activity of all fractions actually becomes identical with the specific activity of (inorganic fractions of the liver in a given time. The proportions of radio activity for ali time-periods after 30,7 hours being equal to the proportions of the ordinary (not radioactive) phosphorus, the specific activity of organic fractions cannot but decline corresponding to the decrease of specific activity of the inorganic fraction.

In comparing our results of the experiments relative to the ordinary non-active phosphorus with the findings of other authors (in the field, we find that the averages of our organic phosphorus are lower and those of the inorganic phosphorus higher than those reported by, among others, J. Sachs, the increase in the percentage being approximately 30.0 and the decrease about 65.0 with reference to 'inorganic phosphorus and that of organic fractions respectively, as the result of experiments upon our rats and those of Sachs. The difference might be accounted for by the fact (established by Hevesy) that rats when given food rich in sugar tend to increase the organic and to decrease the inorganic fractions of the liver phosphorus. Hevesy’s experimental rats who were given food containing large amounts of sugar showed a rats of phosphorus distribution in their liver fractions similar to that of Sachs’s rats, whereas the rats with large amounts of protein and without sugar in their food showed a phosphorus-distribution rate similar to the one found in our animals.

Since lit is sugar, as suggested' by Hevesy, that determines the dynamics of the building of organic phosphorus compounds in the liver of rats, we decided to re-examine and compare once more our findings with those of Sachs on the basis of equivalency of saccharine phosphorus. We were much impressed on finding very similar proportions of phosphorus distribution within the organic group fractions by the side of totally different proportions of the whole group of organic fractions of our rats and those of Sachs, as compared with the corresponding quantities of their inorganic phosphorus. It was this fact that suggested the idea of organic, as against the inorganic, liver fractions reacting as a uniform reactive system. The question soon arose whether it -was possible to learn — from our experimental facts and data — the nature of the order of organic substances, that brings about this uniform reaction. Since the experimental facts failed to provide a direct answer, it became necessary to discover a method that should make such an attempt practicable. The method which suggested itself consisted in the application of Guldberg-Waage’s law of the action of active masses upon the metabolism of phosphorus in the liver of rats. Since the experiments had pointed to the possibility of the reactive unity being brought about by the irreversible chain of metabolism, one of the co-authors of this paper first expounded the theory of the possibility in question (16), and in the second and third parts of this paper the same co-author applied the methods derived from his theoretical studies to the results of these experiments.