STUDIES OF METABOLIC TURNOVER WITH TRITIUM AS A TRACER

V. THE PREDOMINANTLY NON-DYNAMIC STATE OF BODY CONSTITUENTS IN THE RAT*

BY ROY C. THOMPSON AXD JOHN E. BALLOU

(From the Biology Section, Radiological Sciences Department, General Electric Company, Richland, Washington)

(Received for publication, February 16, 1956)

The pioneering isotopic tracer studies of Schoenheimer and coworkers, in which deuterium- and N16-labeled compounds were utilized in the meas- urement of metabolic turnover rates, have been widely accepted as con- vincing evidence for the essentially dynamic state of most, if not all, body constituents (1). The “dynamic state of body constituents” has become a byword in most modern textbooks of .biochemistry, and Folin’s earlier distinction between endogenous and exogenous metabolism of proteins (2) has been usually treated as a historic, but outgrown concept. In recent years it has become evident, however, that not all body constituents are so very dynamic. Thesefindings have been recently reviewed by Mitchell (3), who finds them in no sense incompatible with the essential features of Folin’s original concept. Despite this more recent evidence for non-dy- namic components in many tissues, the results of short term experiments of isotope retention are still frequently interpreted as indicating rapid turn- over rates for the total amount of the constituent under investigation. Such interpretations ignore the fact that the presence of a long lived frac- tion could not possibly be detected during the short experimental periods employed.

In experiments similar to the early deuterium work of Schoenheimer et al. (I), but with tritium as a tracer, we have previously demonstrated the existence in the rat of metabolically inert components with apparent biological half lives of the order of 100 days or longer (4, 5). These ex- periments involved the administration of a single large dose, or a short series of doses, of tritium oxide. The amount of tritium incorporated in the long lived components was always small compared to that incorporat,ed in the more dynamic components. The long lived components were dis- cernible only by virtue of the wide range of concentration over which tritium may be detected, which allowed us to trace the tritium content of

* This paper is based on work performed under contract No. W-31-109-Eng-52 for the Atomic Energy Commission.

795

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796 TRITIUM AS TRACER IN METABOLISM. V

the animals until the shorter lived components were eliminated. While these experiments clearly demonstrated the existence of metabolically inert components in nearly all tissues, only rough estimates of pool sizes were possible.

We have now completed chronic exposure studies which are susceptible to a more quantitative interpretation. These studies bear out the earlier indications that the concept of the dynamic state of body constituents is more restricted in its applicability than has generally been supposed.

Methods

Twelve female rats of the Sprague-Dawley strain, average weight 235 gm., were injected intraperitoneally with sufficient tritium oxide to bring the concentration of tritium in their body water to approximately 3.4 PC. per ml. Thereafter, for a period of 124 days, all drinking water avail- able to these animals contained 5.0 PC. per ml. of tritium as the oxide. Purina laboratory chow was available ad Zibitum throughout the experi- ment. After 6 weeks on the tritium oxide regimen, the animals were mated. The offspring were exposed to the uniform maternal tritium oxide environment in utero, were nursed by the mothers treated with tritium, and weaned to the regimen of tritium oxide-labeled drinking water, which was maintained until the animals were 6 months old.

After cessation of tritium oxide administration, groups of rats from both generations were killed at intervals as indicated in Tables I and II, and the organically bound tritium content of various organs and tissues was de- termined. Samples were pooled from the two or three animals in each of these groups. Total samples were taken except in the case of fat (genital and perirenal), pelt (dorsal), bone (femora), and muscle (hind leg). The residual carcass was thoroughly ground and the aliquots were analyzed. The methods employed in the removal of water from the tissues and in the combustion of the dry residue have been previously described (6). Sam- ples were not equilibrated with water (7) ; therefore, the values for organi- cally bound tritium include “freely exchangeable” as well as “firmly bound” tritium.

In addition to the samples of organ and tissue, organically bound tritium was determined in fractions separated from the residual carcass. The fractions studied are listed in Tables I and II, the methods employed in their separation having been previously described (5). The fraction listed as “insoluble residue” consists of material left after removal of water- soluble, fat-soluble, and NaOH-soluble fractions.

The tritium-counting procedure has been previously described (7, 8).

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R. C. THOMPSON AND J. E. BALLOU 797

Results

Experimental results are summarized in Table I (first generation rats) and Table II (second generation rats). All the data are calculated in

TABLE I Concentration of !&ilium in Organically Bound Hydrogen of Rats after

Chronic Exposure to Tritium Oxide for 4 Months during Maturity

GroupNo:..................................... Time killed, days Average rat weght, gm.. 1 *,8 1 *:I 1 ,;I / 2% 1 :i 1 ;:I

Tritium concentration in water from combustion as per cent of concentration in body water during exposuret

Tissues

Compound fractions

Carcass 21 Liver 28 Lung 16 Heart 9.5 Kidney 25 Stomach 18 Small intestine 20 Large “ 15 Brain 22 Pelt 24 Muscle 23 Fat 15 Bone 18 Phospholipides 17 Non-saponifiable lipides 17 Saturated fatty acids 15 Unsaturated fatty acids 7.0 Collagen 1.9 Water-soluble 15 Alcohol-ether-insoluble 17 Insoluble residue 9.7

13 8.9

12 11 11 9.7 8.7 8.4

18 14 14 12 4.6

12 15 12 7.0 3.2

13 13

8.4 2.2 3.1 5.3 3.5 4.7 3.8 5.7

12 12 9.2 9.2 3.2 9.2 7.3 8.6 4.6 3.5 7.0 8.9 4.8

4.4 0.57 0.97 1.03 1.2 3.1 1.5 2.8 6.2 7.6 4.8 4.0 3.2 6.0 5.4 5.7 6.0 3.4 3.4 2.2

2.8 0.29 1.03 1.03 0.65 2.3 1.00 1.9 3.8 4.0 3.0 3.6 3.0 4.0 2.4 7.0 8.8 3.4 3.4 0.73 2.8

- 0.81 0.086 0.32 0.25 0.32 0.84 0.33 0.81 1.4 1.7 0.46 0.30 1.8 0.62 1.4 1.4 2.5 2.8 0.24 0.35 1.2

* Each group consisted of two animals, samples from which were pooled prior to analysis.

t Average concentration of tritium in body water during exposure was 3.7 PC. per ml. The values were normalized as explained in the text.

terms of the tritium concentration in water derived from the combustion of the dry tissue, or compound fraction, and expressed as a per cent of the concentration of tritium which was maintained in the body water through- out the exposure period. By expressing tissue-bound tritium in terms of tritium concentration in the water derived from the combustion of the tissue, all figures become proportional to the specific activity of tritium in

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798 TRITIUM AS TRACER IN METABOLISM. V

hydrogen, thus simplifying comparisons among compounds of various hy- drogen contents and body water. The average concentration of tritium in body water maintained during the period of chronic exposure was 3.7

TABLE II Concentration of Tritium in Organically Bound Hydrogen of Rats after Chronic

Exposure to Tritium Oxide from Conception to 6 Months of Age

Group No . . . . . . . Sex............................................ ii. k. k.

4 6 F. & F. 4. M”. $.

No. of rats _. _. _. Timekilled,doy ._....,.... i

3 z

3 3 3

21: 11; 20: 30;

Average rat weight, gm., 368 247 2:: 3;; 2:: 396 508 399

Tritium concentration in water from combustion as per cent of concentration in body water during exposure*

Tissues

Compound fractions

Carcass 19 17 18 Liver 27 27 20 Lung 24 26 16 Heart 21 22 19 Kidney 26 25 20 Stomach 24 26 20 Small intestine 21 21 19 Large “ 20 22 20 Brain 37 39 32 Pelt 28 22 25 Muscle 23 18 20 Fat 12 11 15 Bone 17 16 18 Phospholipides 21 17 20 Non-saponifiablelipides 26 20 15 Saturated fatty acids 11 10.3 13 Unsaturated fatty acids 7.8 5.9 8.1 Collagen 22 20 20 Water-soluble 16 18 18 Alcohol-ether-insoluble 25 24 20 Insoluble residue 14 14 12

15 10.5 5.4 4.6 8.1 2.3 0.62 0.4: 7.6 5.9 2.9 2.7

12 6.5 2.1 1.1 9.5 3.9 1.5 1.2

12 8.3 3.2 3.8 9.7 3.9 2.6 1.7

12 6.5 5.3 2.9 30 11 13 13 21 17 12 8.0 16 11 5.8 4.2 13 8.1 4.6 3.4 14 11 9.5 8.1 17 10.3 7.6 4.6 17 10.310.6 6.0 13 7.8 4.9 3.2 7.3 4.1 3.4 2.1

17 16 15 12 12 6.8 4.0 2.4 17 13 8.6 6.2 9.5 4.0 5.4

4.0 0.41 3 2.5 3.8

3.9 1.6 3.1

10 6.2 3.2 1.9 7.6 4.1 4.3 1.7 1.1

L6 1 0.6: 2 4.4 3.4

1.8 0.19 1.9 1.5 0.62 2.5 0.97 2.1 5.1 2.7 1.3 0.44 4.9 3.0 3.8 0.46 0.46 2 0.32 3.6 3.1

The values were normalized as explained in the text. * The average concentration of tritium in body water during exposure was 3.7

jdc. per ml.

NC. per ml. The percentage values listed in Tables I and II may therefore be converted to microcuries per ml. by multiplying by 0.037.

The decrease in tritium concentration of tissue after exposure may result not only from metabolic processes, but also as a consequence of simple dilution due to growth of the animal. In an effort to eliminate this effect of dilution, data in Tables I and II, where necessary, have been normal- ized to a constant weight of organ, tissue, or compound fraction. For

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R. C. THOMPSON AND J. E. BALLOU 799

those organs analyzed in toto, the standard taken for normalization was the average organ weight in the first group of the second generation rats which were killed. Where only partial tissue samples were taken, normalization was based on total animal weight, with the first group of second generation rats being taken as the standard. Data for male and female animals were separately normalized. With the exception of Group 6 in Table I and Group 8 in Table II, weight differences at death were relatively slight, and the normalization procedure introduced only small changes in tritium con- centrations.

Data from the separated compound fractions, with one exception, were not normalized. It was felt that the small differences in “fraction yield” among the groups of rats were just as attributable to chemical and manipu- lative losses as to differences due to animal growth. The single exception was the case of saturated fatty acids which, in the first generation animals, increased quite significantly in Groups 5 and 6.

DISCUSSION

Radiation Dose-In previous studies in this series, it was sometimes necessary to employ concentrations of tritium which irradiated the rats at levels as high as 30 rads per day for periods of several days (5). While no gross symptoms of radiation damage were observed, such high levels of radiation are undesirable because of their possible effects on the general metabolism of the animal. In the present study, the continuous level of 3.7 PC. of tritium per ml. of body water gave a maximal dose rate to the animal of only 1 rad per day.

Metabolic Incorporation of Hydrogen from Body Water-Organically bound hydrogen in an animal may arise from two sources. (a) It may be present, already bound, in organic molecules taken in as food, these mole- cules or portions thereof being incorporated into the tissue compounds of the animal. (b) It may be incorporated from the hydrogen of body water during the metabolic synthesis of tissue compounds. In the present study the second generation rats killed at the end of the exposure period (Groups 1 and 2, Table II) were exposed throughout their existence to body water hydrogen containing a constant proportion of tritium. The organically bound hydrogen in their food contained no tritium. The tritium content of the tissue compounds of these animals should therefore indicate the extent to which hydrogen is derived from each of the two available sources, for the moment the possibility of isotopic differentiation being excluded. The results for Groups 1 and 2 (Table II) indicate that, for most of the samples analyzed, from 20 to 30 per cent of the organically bound hydrogen was apparently derived from body water. Several of these samples were well outside this range, however, and require additional comment. Brain

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800 TRITIUM AS TRACER IN METABOLISM. V

tissue was uniquely high in bound tritium, with results which indicate that nearly 40 per cent of its hydrogen was derived from body water. This leads to the conclusion that brain, to a greater extent than other tissue, is composed of materials synthesized within the body. Such a finding may be related to the existence of the so called “blood-brain bar- rier” which might be expected to hinder incorporation of large preformed molecules into the brain substance (9, 10). Thus Friedberg and Green- berg have observed an apparent barrier to the uptake of amino acids by the brain (11). The fact that the composition of brain lipides, as com- pared with other organ lipides, is least influenced by diet (12) is also in qualitative accord with this finding.

The separated fatty acid fractions and the gross fat sample were notably low in bound tritium, indicating that these materials, to a greater extent than other tissue components, were derived from preformed organic molecules taken in as food. This conclusion is consistent with many studies which have demonst’rated the extensive direct incorporation of fatty acids from food into depot f&s (13). The lesser incorporation of tritium into the unsaturated as compared to the saturated fatty acids cor- responds to previous findings with deuterium (14).

The tritium concentration in cert.ain tissues from the first generation animals (Table I, Group 1) is significantly lower than in corresponding tissues from second generation animals (Table II, Groups 1 and 2), indi- cating that the 4 months exposure of the first generation rats was insuffi- cient to permit equilibrium levels of tritium to be attained in some of the more metabolically inert fractions. Most notable is the case of collagen, in which tritium incorporation is lo-fold higher in the animals exposed from conception. It seems quite evident that the bulk of the collagen in the first generation rats had been formed prior to the tritium oxide expo- sure and that little degradation and resynthesis of this collagen occurred during the 4 month exposure period. The metabolic inertia of collagen was indicated in previous studies by ourselves (5) and others (15-17) and is borne out by other features of the present investigation, to be discussed subsequently.

In the foregoing discussion no account has been taken of the possibility that the incorporation into tissue compounds of tritium from body mater may not be a true measure of hydrogen incbrporation. It is evident from recent studies by ourselves (7) and others (18, 19) that differences do exist in the rates of incorporation of deuterium and tritium under circum- stances similar to those of the present experiment. Ratios of tritium to deuterium incorporation as low as 0.77 have been reported for the case of mammary gland fatty acids (18). It is likely that even larger differences may exist between tritium and protium (ordinary hydrogen) incorpora-

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R. C. THOMPSON AND J. E. BALLOU 801

tion. It is therefore reasonable to suppose that the incorporation into tissue compounds of hydrogen from body water in the rats of the present experiment was actually somewhat higher than indicated by the results with tritium. While this uncertainty would preclude any inferences based on absolute values of tritium incorporation, it is not felt that it significantly affects any of the comparisons considered in the preceding discussion.

It should also be pointed out that the fraction of organically bound hydrogen which is derived from body water is undoubtedly a function of diet. Thus Bernhard and Schoenheimer found that, on a fat-free diet, approximately 50 per cent of hydrogen in saturated fatty acids was de- rived from body water, as indicated by deuterium incorporation (14). This is in contrast to our result of 10 to 15 per cent as indicated by

---

EXCRETED WAiER EXCRETED -COMPOUNDS

FIG. 1. Simplified scheme of hydrogen metabolism with ingested water labeled with tritium. H, organically bound hydrogen; h, water hydrogen; *, tritium label.

tritium incorporation, and with a diet containing not less than 5 per cent crude fat. The difference in these two results is probably owing largely to differences in the fat content of the diets rather than to an isotope effect.

Metabolic Inertia of Tissue Components As Measured by Tritium Reten- tion--The principal purpose of the present experiment was to obtain data from which the size of the metabolically inert hydrogen pools could be estimated. A schematic representation of the metabolic systems involved is shown in Fig. 1. Tritium-labeled hydrogen, from drinking water, enters the body water pool by Route 1. Non-labeled hydrogen from water, from food, enters by Route 2. Hydrogen also enters the pool of body water from the catabolism of tissue compounds, Route 3 representing the con- tribution from tritium-labeled compounds and Route 4 the contribution from unlabeled compounds. The pool of body water is not separated into compartments, all portions having the same concentration of tritium, which is accurately reflected by any sample of body water.

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802 TRITIUM AS TRACER IN METABOLISM. V

The organically bound hydrogen of tissue compounds is not uniformly labeled with tritium. Bound hydrogen arising from body water via Route 5 will be labeled to the same extent as hydrogen of body water, while bound hydrogen incorporated directly from food (Route 6) mill be unlabeled. The same compound, of course, may, and probably in most cases will, con- tain both hydrogen derived from body mater and hydrogen derived from food. The organically bound hydrogen pool consists of a great many com- partments, the hydrogens of which will usually not equilibrate with each other.

Because of much justifiable criticism of the non-rigorous methods em- ployed in the interpretation of tracer studies in biological systems (e.g., Reiner (20)), it seems desirable at this point to define clearly the recog- nized limitations of the present study. The biological systems involved encompass the total animal. The complexity and lack of understanding of these systems are such that no attempt at a rigorous mathematical interpretation of the results is considered justified. Our aim is nothing more than a gross demonstration of the relative metabolic inertia of a great variety of tissue components and a semiquantitative indication of the magnitude of these relatively inert components. In performing this demonstration we will consider gross retention curves as being the sum of exponential components, and derive half lives for these components. In so doing, it is recognized that these exponential components and their associated half lives bear no clearly definable relationship to any specific biological component. These exponential components do, however, afford a fairly accurate representation of the net rate at which tritium is being lost from the tissue under investigation, and this loss of tritium can be, at least semiquantitatively, related to the rate of degradation of the compounds which compose the tissue.

In Fig. 2, data are presented on the retention of tritium in the second generation rats after removal of tritium from the drinking water. The organically bound tritium curve is derived from analyses on the “residual carcass,” but may be considered as representative of the total animal. Within 10 days the concentration of tritium in hydrogen of body water has dropped to about the same level as tritium in organically bound hydro- gen, and by 25 days the tritium concentration in body mater hydrogen is less than one-tenth of that in organically bound hydrogen. Because of this rapid decrease in body water tritium, there is no possibility of a con- tinued significant recycling of tritium between body water and tissue com- pounds. Upon removal of tritium from Route 1 (Fig. l), tritium incor- poration via Route 5 rapidly dwindles to insignificance, and the loss of organically bound tritium from the tissue compounds becomes a measure of the breakdown or excretion of these compounds via Routes 3 and 3’.

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R. C. THOMPSON Ah-D J. E. BALLOU 803

Although this measure of tissue degradation applies in a strict sense only to those components whose hydrogen was derived from body water, it can be assumed that it reasonably represents the total tissue. In other words, it can be assumed that Routes 3 and 3’ are equivalent to Routes 4 and 4’. There would seem to be no a priori reason for assuming that the origin of a compound’s hydrogen from food molecules or from body water should play a determining role in the subsequent fate of that compound, par- ticularly since most compounds will contain hydrogen from both sources.

By equating loss of tritium via Routes 3 and 3’ with the breakdown or

COMBUSTION WATER H3 B -I\ 1 (RESIDUAL CARCASS) 8 !G

ad . l BODY WATER H3

t- ,,I ’ \HALF LIFE,3.3 DAYS

%L I”

z$a oww

3P-k

!s;oc, ‘, H;F LIFE, ’

0 100 200 300

DAYS

FIG. 2. Retention of body water tritium and organically bound tritium in rats after chronic exposure to tritium oxide from conception to 6 months of age.

excretion of labeled compounds, one assumes that the tritium is not lost by simple exchange with protium from body water. Tritium in labile positions, e.g. attached to oxygen or nitrogen atoms, is certainly rapidly lost by such an exchange process. The proportion of such labile hydrogen in the body is small, however, and much careful work with deuterium has shown that, with few exceptions, hydrogen linked to carbon is not ex- changed under physiological conditions (1). The possibility of a very slow rate of exchange of carbon-linked tritium with body water protium, however, cannot be ruled out. If such an exchange contributes appreci- ably to the loss of tritium from tissue compounds, the neglect of this factor in our calculations will result in an underestima2ion of the biological half lives of the compound turnover processes.

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SO4 TRITIUM AS TRACER IN METABOLISM. V

In the case of the second generation rats, we shall assume that the amount of tritium present in any gross organic tissue component at the conclusion of the tritium oxide feeding period is proportional to the mass of that component. Thus, if half the tritium in a given tissue is lost with a half life of 100 days, we shall assume that half of the tissue is being de- graded and resynthesized with this half life. This amounts to an assump- tion that all organic tissue constituents contained the same proportion of uniformly labeled hydrogen at the conclusion of the exposure period. It was hoped that the long uniform exposure from conception to 6 months of age would result in reasonably uniform tritium labeling. As previously mentioned (Table II, Groups 1 and 2), complete uniformity among tissues and compound fractions was not achieved. It is felt, however, that the assumption of uniform labeling will introduce no greater error than that inherent in other factors in the interpretation.

It should be remarked that the possibility of isotopic differentiation between tritium and protium is of no significant concern in the interpre- tation of tritium retention. Once incorporated in the organic molecule, the release of tritium will be largely determined by the metabolic fate of the entire molecule, which will not be significantly influenced by a mass difference of 2 units in the total molecular weight. This conclusion was borne out in experiments in which the retention of organically bound tritium and deuterium in rats was compared (7).

In accord with the above considerations, we may now return to the in- terpretation of the organically bound tritium retention curve of Fig. 2. This curve may be resolved, as shown, into two exponential components with half lives of 130 and 22 days. Extrapolation of these components to zero time indicates their relative magnitude, the 130 day component ac- counting for nearly half of the total tissue. We shall emphasize again that such resolution of a retention curve does not imply that two discrete bi- ological components with these half lives exist. Rather, one should con- clude that the many components which must exist in the animal may be grouped in this manner and that the “average half life” of the more inert group of components is 130 days, while that of the more dynamic group of components is 22 days. Such a grouping does not preclude the acknowl- edged existence of components with very short half lives. It does, how- ever, indicate that such short half life components are quantitatively insig- nificant in the over-all picture for the total animal.

The biological half life of tritium in the body water of the rat, as deduced from body water analyses on the early groups of the animals killed, was 3.3 days, the same value as that determined in previous acute exposure studies (4). By knowing the rate at which tritium is lost from body water and the rate at which tritium is being introduced into the body water by

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It. C. THOMPSON AND J. E. BALLOU 805

breakdown of tissue compounds (with an assumption that all released tritium follows Route 3 (Fig. l)), one can calculate the concentration of tritium which should be present in the body water throughout the period following cessation of tritium oxide feeding. Such a calculation results in the curve drawn for body water tritium in Fig. 2. The comparison of this curve with the experimentally determined values serves as an excellent internal check on the general validity of the experimental and interpreta- tive procedures.

In a manner similar to that illustrated for the residual carcass in Fig. 2,

DAYS FIG. 3. Retention of organically bound tritium in rat tissues and compound frac-

tions after chronic exposure to tritium oxide from conception to 6 months of age.

retention curves were plotted for all organs, tissues, and separated com- pound fractions by using the data from Tables I and II. Fig. 3 shows several such curves for representative samples. These retention curves mere resolved into exponential components and the half lives and relative magnitudes of the components determined. Table III summarizes the results from the second generation animals, the data from the first genera- tion rats having led to similar results in most cases. The results from the first generation animals are not shown, since the assumption of uniform labeling involved in the evaluation of the magnitude of the components is more questionable for these rats.

It was possible to resolve components with half lives as short as 3 to 5 days for only four organs. This does not mean that such dynamic com-

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806 TRITIUM AS TRACER IN METABOLISM. V

ponents were not present in the other samples, but simply indicates that, owing to the predominance of longer lived components, it was impossible to resolve components of very short half life accurately.

Relatively inert components with half lives, in most cases longer than 100 days, constitute at least 30 per cent of all the samples analyzed, except liver, kidney, lung, stomach, and small intestine. Heart might also be

TABLE III Magnitude and Biological Half Life of Tissue Components of Rat

Tissue

Compound fractions

- 1

Carcass Liver Lung Heart Kidney Stomach Small intestine Large “ Brain Pelt Muscle Fat Bone Phospholipides Non-saponifiable lipides Saturated fatty acids Unsaturated fatty acids Collagen Water-soluble Alcohol-ether-insoluble Insoluble residue

Long lived component Shorter lived components

Magnitude, :r cent tota

tissue or fraction

Biological half life

47 3

14

&YS

130 140 320

&YS

22 12,4.5 10, 3

8 180 11 20 300 20, 5 17 160 9 30 180 13, 5 54 150 16 67 110 11 40 100 16 69 70 17 72 240 16 33 220 20 50 160 20 60 80 15 74 80 10 72 1000 15 50 60 10 36 200 25 40 300 15

expected to fall in this category, but the data obtained for heart were too erratic for accurate resolution of the retention curve. Even these meta- bolically active organs possess well defined components with half lives exceeding 100 days.

The greatest degree of metabolic inertia is exhibited by collagen, with 72 per cent of the fraction exhibiting an apparent half life of 1000 days. From the scatter of points on the collagen retention curve (Fig. 3), it should be evident that the half life could have been assigned any value from 500

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R. C. THOMPSON AND J. E. BALLOU SO7

days to infinity about as reasonably. The conclusion would seem to be justified that most of the collagen of mature rats is not replaced during the lifetime of the animal. This conclusion is supported by the lo-fold lower incorporation of tritium in the first generation animals which were exposed after attaining essentially full growth.

While the existence of metabolically inert collagen (E&17), and protein fractions generally (21-24), has been demonstrated by other investigators, the extreme metabolic inertia of a substantial fraction of body lipides seems to have been first indicated by earlier studies in this series (4, 5). Other workers have determined biological half lives for various lipide components of the rat, using isotopic tracer methods, with resulting values which range from 1 to 35 days (25-27). In all cases, these values were based on obser- vations extending over short time periods (not exceeding 30 days), and the assumption was made that the half life observed applied to the total lipide fraction under investigation. It should be evident from the present study that these short half lives are not representative of the total lipide frac- tions, but apply only to the more dynamic portions of these fractions. The present data indicate that relatively inert components constitute the majority of body lipides generally, and approximately two-thirds of both saturated and unsaturated fatty acids.

A recent report by Steele (28) on the retention of Cl4 in tissues of the mouse after a single ingestion of uniformly labeled sucrose furnishes sup- port for our hypothesis of the prevalence of metabolically inert components in most tissues. Although extending over a period of only 36 days, his data indicate components with apparent half lives of from about 17 to 33 days in all the tissues examined. His value of 33 days for mouse muscle compares with our finding in a 31 day experiment of an apparent half life of approximately 30 days for the organically bound tritium of the total mouse (6). Thus the results with hydrogen labeling are in excellent agree- ment with the results from a comparable experiment in which the carbon was labeled directly. In comparing these two studies, it becomes evident that the use of tritium as a label offers at least two advantages over the apparently more straightforward carbon label. First, the uniform label- ing of tissue compounds is certainly more closely approached with chronic tritium oxide administration than with the feeding of any carbon-labeled substance; and second, the greater probability of carbon reutilization, as compared to hydrogen reutilization, makes quantitative interpretation of Cl4 retention data much more difficult.

SUMMARY

Rats exposed to a constant level of tritium oxide in body water from con- ception to 6 months of age were subsequently killed at time intervals ex-

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808 TRITIUM AS TRACER IN METABOLISM. V

tending to 300 days. Another series of animals, similarly exposed for a period of 124 days after attaining maturity, was subsequently killed at in- tervals extending to 360 days. Organically bound tritium was determined in various organs, tissues, and compound fractions, and the results were interpreted as follows:

1. Under the dietary conditions employed, without consideration for the possible effects of isotopic differentiation, from 20 to 30 per cent of the hydrogen of most tissue compounds was derived from body water. Brain and fatty acids were exceptional, with nearly 40 per cent of brain hydrogen and about 10 per cent of fatty acid hydrogen derived from body water.

2. Dynamic components (half lives of a few days) constitute a very small proportion of the total animal, and in the present experiment were distin- guishable from the predominant “less dynamic” components only in liver, lung, stomach, and intestine.

3. Approximately half of the organic materials constituting the total rat is being degraded and resynthesized with apparent biological half lives longer than 100 days.

4. Collagen was found to be uniquely inert. Most of the collagen of mature rats is apparently not replaced during the lifetime of the animal.

5. The majority of body lipides, including about two-thirds of both saturated and unsaturated acids, exhibit half lives of the order of 70 days or longer.

The authors wish to acknowledge the technical assistance of Elizabeth Desposato, Margaret Lawson, and Alma Crosby, and the services of Arthur Case and coworkers of the Radiochemical Analysis group of t,he Biology Section.

BIBLIOGRAPHY

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Roy C. Thompson and John E. BallouTHE RAT

STATE OF BODY CONSTITUENTS IN PREDOMINANTLY NON-DYNAMIC

WITH TRITIUM AS A TRACER: V. THE STUDIES OF METABOLIC TURNOVER

1956, 223:795-809.J. Biol. Chem. 

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