IRON KINETICS IN INDIVIDUAL BONE MARROW REGIONS ( BONES )

Vacha, J. V. Znojil, Jirina Hola, J. Dungel: Iron Kinetics in Individual Bone Marrow Regions (Bones) in Mice. Acta vet. Brno, 53, 1984: 119-136. The course of concentration of labelled iron (.oFe) after its intravenous administration was observed experimentally in plasma, erythrocytes, spleen and individual bones (here in haem and non-haem fraction separately). The optimum number of iron compartments in individual bones and the constants characterizing the flow through these compartments was then sought by means of mathematical modelling. It was possible to type the compartments by comparing flows into the bones of various types and to try to find their physiological interpretation. The following compartments were determined (apart from the circulating plasma and erythrocytes): 1. An iron pool with a half-time of efflux of several tens of minutes to several hours, probably corresponding to the iron contained in the interstitial marrow and splenic spaces, in a rapidly assumed equilibrium with plasma; 2. An iron pool with a half-time of efflux about nine hours, corresponding to the iron incorporating into haem or deposited in the pre-haem pool in erythroblasts; 3. An iron pool with a half-time of efflux of about 1-3 days, having the character of stores, probably deposited both in erythroblasts (half-time 3.15 days) and in cellular elements of the haematopoietic microenvironment (half-time 1.65 days); 4. An iron pool bound in the long-term in the teeth. The bones could be divided according to the representation of compartments into three groups: 1. Long bones and spine, in which intensive erythropoiesis takes place, exhausting the interstitial pool and consuming most of the storage iron pool; 2. Acral bones and the skull, with minimal erythropoiesis, in which the dynamics of the interstitial and storage iron pool localized beyond erythroblasts predominantly occurs; 3. Thoracic bones, representing a group intermediate between the two former ones in several aspects. The model is also in accordance with the independently (chemically) ascertained iron content in the bones. The results represent a considerable refinement of our general model of ferrokinetics in mice (Vacha et al. 1982) and provide a firmer basis for the study of ferrokinetics in bone marrow under pathological states of erythropoiesis. Studies of this type are an inevitable prerequisite of correct interpretation of the labelled iron incorporation method as an indicator of the intensity of erythropoiesis. Ferrokinetics, iron compartments in bones of various types, haem and non-haem iron, .oFe-incorporation method. Knowledge of the internal exchange of iron in an organism is a prerequisite for the correct interpretation of the commonly used method of labelled iron incorporation into the erythropoietic organs. The internal iron kinetics in mice are, however, known only incompletely as yet. In our previous paper (Vacha et al. 1982) we proposed a model of the ferrokinetics of mice encompassing the sizes of basic iron compartments in the erythropoietic organs, the blood and the liver and the extent of flows between them. The employment of a model of ferrokinetics for the study of erythropoiesis presupposes, however, an analysis of iron compartments in the bone marrow more sophisticated than that which can be made in such a general model. The basic idea of the present paper

is to analyse experimental data separately in individual types of bones; a more refined division of iron compartments and a higher degree of conformity of the model with the experimental data could be achieved by comparing or correlating the ferrokinetic characteristics of fifteen different bones.
The method employed was again compartmental analysis.The plot of changes in concentration of tracer (••Fe) in individual bones after its administration into the circulation of an animal can be decomposed into components by means of mathematical modelling, the components corresponding to the dynamically differing pools (compartments) of iron in bones.Indices (constants) characterizing the intensity of the compartment influx or efflux can be found by an optimization method, the fluxes germane to individual bones can be mutually compared and possibly physiologically interpreted.A knowledge of marrow iron compartments in the undisturbed state is a prerequisite of the proposed study of their changes under conditions where erythropoiesis is displaced from the steady state by a pathogenic stimulus.It is also a prerequisite for the employment of labelled iron incorporation into the erythropoietic organs as an indicator of the intensity of erythropoiesis.

Materials and Methods
The experimental techniques used are covered in the previous work and will only be briefly summarized here (for details see Vacha et al. 1982).Most data used in the present article were obtained in the identical animals as in the previous work; mathematical methods are, however, totally different.Where data from our previous works were used for correction and interpretation of the results, they always concern animals of the same strain, sex, age and breeding conditions as in the present article.
1.The Animals.All experiments were performed on males of the strain C57BL/I0ScSnPh bred in our institute, 11.5-14 weeks old, mean body mass 28.8 g.The mice were kept at a temperature of 22°C, diet Larsen (Velaz, CSSR) with an iron content of 6.3-7.5 mmol/kg, water ad libitum.
2. ••Fe Administration.The form of administration used was ferrous citrate labelled with 6•Fe, mfr.Rotop (GDR), supplied in an isotonic buffer containing ascorbic and citric acids, supplemented with 0.9 % benzylalcohol, pH 4-6, specific activity 10.5-34.3GBq/mmol Fe.The preparation was diluted immediately prior to administration with saline to an equivalent of 1.8 or 5.4 nmol elementary iron per mouse, corresponding to a radioactivity of 5.6 -18.5 104 Bq per mouse (in 0.1 ml).However, it follows from the discussion contained in Vacha et al. (1982) that any proportion of iron not bound to transferrin is entirely negligible in our experimental regimen, since it should above all be apparent in a very rapid initial decline in the plasma and the fast depositing of this iron in the liver, which was not the case.The proportion of unbound iron can be estimated at less than 1 % of administered activity.
3. Ii•Fe Clearance from Plasma.At selected intervals following Ii•Fe administration groups of 10-35 mice with Ii•Fe administered were investigated.The animals were sacrificed at 0900 h in all cases by bleeding from the axillar blood vessels under ether narcosis.The blood was transferred using a heparinized pipette to a test-tube containing powdered heparin and after 20 min centrifugation at 1400 g 0.25 ml plasma was taken from each sample, 0.75mldistilled water added, and this mixture was thoroughly shaken and the extinction of oxyhaemoglobin from any haemolysed erythrocytes present was spectrophotometrically measured at a wavelength of 540 nm.Using a calibration curve the proportion of haemolysed erythrocytes in each plasma sample was then calculated.Following the measurement of extinction 2 rnl trichloroacetic acid (c = 1.22 mol/I) was added to each sample, the sample was stirred, centrifuged, and the radioactivity of the supernatant measured.On precipitation of the haemoglobin released by haemolysis using trichloroacetic acid a certain unprecipitable fraction remains; according to our previous results (Hola et al. 1975) the amount of this depends on the time elapsed from Ii•Fe administration.This dependence was utilized in the correction of the influence of haemolysis on plasma radioactivity.The corrections amounted to up to 35 % of the original value after the longer intervals following Ii•Fe application, some other corrections made (for the natural radioactivity of plasma and the body mass of animals) being almost negligible.
The radioactivity of the plasma samples was finally calculated in respect of the total plasma volume determined in turn from the degree of dilution of ••Fe at the time to (at the moment of Ii•Fe application).The conversion factor for 0.25 ml plasma was 7.73.
body mass of animals.The radioactivity of the volume of erythrocytes withdrawn was calculated in respect of the total volume of circulating erythrocytes, determined using erythrocytes labelled with a•Fe in a parallel experiment (Vacha 1975).The conversion factor for samples of 0.1 ml was 7.78 or 7.68 according to the body mass of animals.
5. Organ Measurements.Following the bleeding of the animal the spleen was removed and weighed and its radioactivity measured.The complete skeleton, free of all soft tissue, was obtained by placing the carcasses of the animals in a colony of the beetles Dermestes vulpinus Fbr.(kept according to Grulich 1977); prior to measuring it was divided into individual bones or groups of bones.The teeth were loosened from the mandible and maxilla by trypsin.All organ samples were corrected for natural tissue radioactivity.The mass of bones was determined by weighing in dry state (values were taken from Vacha et al. 1981).
6. •'Fe Incorporation into the Haem and non-Haem Fractions of Iron in Organs.In another group of animals, in addition to the overall radioactivity of the spleen and skeleton, the radioactivity of the haem fraction in these organs was also measured at selected intervals after a'Fe administration.The organs were mechanically homogenized after freezing with liquid nitrogen, the cells haemolyzed with distilled water, and the haem fraction extracted using acid ethylacetate (for details see Vacha et al. 1978).In another group of animals, the absolute quantum of non-haem iron in bone marrow and haem iron in marrow erythroblasts were determined by extraction and mineralization technique (values were taken on from Vacha et al. 1983).
7. Measurement of a•Fe Activity.This was measured using an Automatic Gamma Well Counting System (Nuclear Chicago) in the range 0.5-1.4MeV, efficiency 19 %.All samples were formed into approximately similar geometrical shapes and measured together with a standard formed by a known fraction of administered radioactivity.The length of measuring was chosen for the given background so as to give a dispersion of less than 5 % of the measured value even in the least radioactive samples.8. Mathematical Analysis.The pool of iron present in a bone can be divided roughly into two parts: iron contained in the blood circulating in blood vessels of the marrow (as iron-transferrin complex in plasma and as erythrocyte iron) and iron contained in all other marrow components (erythroblasts, cells ofthe hematopoietic microenvironment, interstitial fluid etc.).The model count can be based primarily on the iron contained in circulating blood.The values of the radioactivity of plasmatic and erythrocyte iron were measured as a function of time after "Fe administration and these values could be very well approximated by means of the least squares method with a curve.In the case of plasmatic soFe the approximation was performed by the sum of three exponentials, in the case of erythrocyte 5'Fe it was performed according to the following equation: 2 E(t) = (1 -L aj exp (kjt)) as exp (-kat), j = 1 with the constants aj, kj (j = 1,2,3).

\,7
Ii Compartment 1 r-  Full circles -total "Fe quantity in the bone in % of the quantity administered, empty circles -5"Fe bound into the haem fraction in % of the quantity administered.Bigger circles -Experiment No.6 (see Methods), smaller circles -Experiment No.5.Stars -5"Fe content in plasma (in time T = 0) and in erythrocytes (24 days after the tracer administration) on the basis of the direct measurement of the plasma and erythrocyte volume in a bone (Vacha et aJ. 1981) and in the case of erythrocytes also on the basis of their unitary volume.Each point usually represents 10 to 25 experimental animals, bars indicated S.E.(if greater than 5 % of the mean).

External compartment
All curves derived from a model calculation; light ones represent individual iron compartments, the thick one being a summation of all of them.x-axis -time in hours and days, y-axis -% of radioactivity administered.
The scheme of the model is in Pig. 1.The tracer moves from plasma into N parallel iron compartments in the bone, directly proportionally to the tracer concentration in plasma.The influx rate of labelled iron (at time t) into a compartment is given by the formula A .c (t)/I00, where A is a proportionality constant ("compartment affinity of plasmatic iron" as a percentage of the iron present in plasma,per day) and c(t) is the tracer concentration in plasma as a percentage of the .oPeadministered (it is the same for all compartments and bones).The efflux rate of labelled iron at time T from a compartment equals the product Kq(T), where K is the rate constant of efflux and q(T) is the quantum of labelled iron in a compartment as a percentage of the $OPe administered.Then The total amount of iron Q in a compartment (determinable by chemical analysis) is given by the equation The rate constant of efflux K is equivalent to the half-time of efflux according to the formula: T 1/2 = In 21K (hereafter we shall for clarity's sake mostly use instead of K vaues the T 1/2 values).
The model calculation set out from the entered curves of the .oPecontent in circulating plasma and erythrocytes and from the required number of compartments N, which was selected as minimal.The model enabled us to choose the rate constants A and K of individual compartments by the least squares method, and the quantity of plasma and erythrocytes contained in a bone.As we had plasma and erythrocyte volumes at our disposal, measured independently by means o{13IJ-albumin and 5ler-labelled erythrocytes (Vacha et al. 1981), we could evaluate the adequacy of the results thus obtained.Because the model calculated plasmatic volume by mere extrapolation to the time T = 0, the results ensuing from the model turned out to be less reliable than directly measured values and we used the latter for further calculations.As regards erythrocytes, more reliable values were obtained from the model calculation in most cases than from direct measurement (burdened with a considerable experimental error).An exception was the skull and the mandible, where the iron contained in erythrocytes interfered with the iron of a very slowly disappearing compartment (dental tissue) during the calculation.Therefore the experimentally ascertained quantity of erythrocytes was entered for the model calculation in the case of the skull and the mandible.
The curves of the tracer content in all individual compartments as a function of time after 50Pe administration were calculated, on the basis of the determined values A and K and the .oPecontent in the circulating blood.The calculation was done separately for fifteen bones (or bone groups), for the skeleton as a whole, and for the spleen.Examples of these results for some typical bones are given in Fig. 2-5.
The possibility to compare the iron quantity as between individual compartments and therefore between individual bones follows from equation (3).These results could be compared with the results of chemical analysis of the iron content and the model can be tested in a manner independent of others.The sum of the AIK values for all compartments 2 to 4 (representing the iron content in a bone in relative units) was calculated and converted to the absolute content (by means of an coefficient derived from a comparison of iron flows in plasma and circulating erythrocytes compartments with the elementary iron content in these compartments).The iron content in bones and spleen deduced from the model was then compared with the chemically determined iron quantity (Tab. 2 in Vacha et al. 1983).

Results
A synopsis of calculated constants A and half-times (Tl/2) for individual compartments and bones is given in Tab. 1.It follows from the table that it is possible to distinguish three main components of the total radioactivity of a bone according to the half-time of efflux: (i) A very heterogenous compartment 2 with a half-time of about 0.2 to 7. -time of about 1 to 3.4 days.Besides these compartments occurring in most bones, it was possible to distinguish the compartment 5 in the skull and the mandible, with an extremely long half-time of 108 to 221 days.(Numbering of compartments begins here with No.2 to accord with our general model of ferrokinetics.)Compartment 2. The compartment cannot be demonstrated in most long bones of extremities.It is clearly differentiated from the experimentally determined course of radioactivity in the haem compartment by a high efflux rate (Figs.3-5, compare the curve designated as "interstitial" with the empty rings).
Compartment 3.This compartment is the biggest of all compartments in most bones (it has the highest constant A).Its half-time is the same in all bones within the framework of accuracy of the experiment, in spite of considerable differences in A3 values.This compartment is most prominent in long bones, its value is lower in thoracic bones on average, and it is near zero in acral bones, Table 1 Characteristics of iron compartments in the erythropoietic organs in mice    33 Column adesignation of the compartment in our general model of ferrokinetics (Vacha et a!. 1982), column b -number and designation of the compartment, column ccharacteristics of compartments ascertained by a model calculation.
The constants A2 to A5 express the proportionality between the plasmatic iron concentration and the iron flow into compartments ("affinity of the compartment"), A2/M to AS/M -the same constants divided by bone mass in mg (data regarding the bone maSS taken from Vacha et a!.1981).Tl/2 -half-time of the effl\lx from compatments (Tl/2 = after the conversion of the A3 values to unit mass of bones (taken from Vacha et al. 1981).It was not possible to distinguish this compartment from compartment 4 in the skull as a result of similarity of T1/2 values; it could be assessed only approximately that the A3-4 value is evenly divided between A3 and A4 values in the skull (without the mandible) and in a ratio of 1: 3 in the mandible (T1/2 values are the same for compartment 3 and 4 in both bones).The calculated course of radioactivity of compartment 3 coincides very satisfactorily with the experimentally determined course of WFe incorporated into the haem of the erythropoietic tissue, especially in the period of maximum radioactivity, when the disturbing effect of labelled erythrocytes is not yet too strong (Fig. 2-4, compare the curve designated "erythropoiesis" with the empty circles).In order to obtain more homogenous sub compartments we presupposed accordingly that comparment 4 is a heterogenous one, that its more slowly changing component is closely tied with erythropoiesis, while the more rapidly changing component is not.Under the assumption that the A constant of the slowly changing compartment represents 7.8 % of the A3 value in all bones, it was possible to divide the flow 4 into two components in all bones, designated as A4a and A4b in the two bottom rows of Table 1.The relative magnitude of the two components is given in these rows calculated according to the relations: the relative magnitude A4a = (A4 -0.078 x A3) x 100jA4 %; the relative magnitude A4b = (100 --A4a) %.The half-time of the components 4a and 4b is about 1.65 and 3.15 days, respectively.
The justification of this dividing of the flow 4 follows a. o. (see Discussion) from Fig. 6, from which is apparent a high degree of dependence of the half-time of outflow 4 on the relative magnitude of the A4a component.
Compartment 5.During the calculation of the variations in time of the tracer concentration in the circulating erythrocyte compartment, a certain excess of radioactivity occurred in the skull and the mandible in comparison with experimentally determined values.It was possible on this basis to separate an extremely slow component in these two bones, almost without an efflux.The absolute magnitude of this compartment is not negligible even from the viewpoint of the whole skeleton, and the large incorporation into it suggests that after some time (evidently longer than the duration of the experiment) a sudden efflux must occur.
The behaviour of all compartments mentioned as a function of the time after tracer administration in some typical bones is depicted in Figs.2-5.Both "external" compartments (circulating plasma and erythrocytes in the marrow vessel system) change mutually inversely, as the tracer passes gradually from plasma to erythrocytes.The maximum of the "internal" (fixed) compartments occurs in the order of compartments 2, 3 and 4. It is possible to read from these figures which components the sum 59Fe incorporation is composed of at any moment after the tracer administration, which is of great interest in using the 59Fe incorporation as an indicator of the intensity of erythropoiesis, i. e. of compartment 3.
The values given in Tab. 1 referring to the skeleton as a whole must be regarded as less reliable than the values pertaining to the individual bones.Wherever there .
are numerous sub compartments these become superimposed, which slightly impairs the reliability of the identification of compartments and of the estimation of their dynamic characteristics, together with the effect of measurement errors.
The differences appear predominantly in small compartments (4 and 5).
The results of the model calculation of the absolute iron quantities in bone marrow are, along with the results of a direct measurements (Va c h a et a1.1983), given in Tab. 2.

Discussion
It is possible to deduce some hypothetical conclusions about the physiological meaning of the compartments from a comparison of the behaviour of iron compartments in the bones of various types.
Compartment 2. It follows from the discrepancy between the calculated course of tracer concentration in this compartment and experimentally derived values of radioactivity in the haem fraction (Figs. 3 to 5) that iron incorporated into haem is not in question here.Neither, apparently, is a fraction destined for short-term incorporation into the haem (pre-haem pool), because this compartment is absent in most strongly erythropoietic bones (in pelvis, femurs, tibiae and fibulae).We isolated a large pool of non-haem iron in rapidly assumed equilibrium with the plasma compartment in our general model of ferrokinetics (Vacha et a1. 1982), which we interpreted as a pool consisting of iron-transferrin complex in the interstitial ("extravascular") space of various tissues and organs.The dynamics of the present compartment 2 best correspond to this pool in the bone marrow and spleen; the half-time of the efflux from the interstitial pool took 2.29 hours in our previous model, which is a value roughly comparable to the Tl/2 values of our present compartment 2 (Tab.1).Accordingly, compartment 2 could most probably be identified for the most part with the iron in the interstitial space of the bone marrow and the spleen, exchanging rapidly with plasma with a half-time of about one hour.The impossibility demonstrating this compartment in strongly erythropoietic bones by means of a tracer apparently derives from its fast consumption for erythropoiesis, so that its presence is not manifested in the 59Fe incorporation curve.
Compartment 3. The close resemblance of the curves calculated for the A3 flow to experimentally determined 59Fe values in the haem fraction of bone marrow implies that this flow is directly connected with haem production, i. e. with erythropoiesis.A slight shift of the calculated curve in front of that of the experimental data then shows that compartment 3 encompasses not only the iron incorporating immediately into the haem, but also the labile pool of pre-haem iron destined for short-term incorporation, whose existence in maturing red cells is amply documented by biochemical methods (for summary see Romslo 1980).The scheme of our model does not allow differentiation of pre-haem and haem compartments by calculation; it would be possible, however, to assess the ma~ tude of the haem fraction at various points of time from the mentioned shift of the curves in front of the experimentally determined values.But, apart from this differentiation, it appears beyond doubt that the compartment 3 represents an iron pool connected immediately with erythropoiesis localized in maturing red cells.An approximate idea can be formed from the A3/M line in Tab. 1 of the erythropoiesis intensity per unit volume of the marrow of various bones (it must, of course, be assumed that the marrow represents a fixed fraction of the total bone mass).The situation is graphically illustrated in Fig. 7; the most intensive erythropoiesis takes place in long bones of the extremities and in the spine (without tail vertebrae), while the tail, fore and hind feet and skull participate only very slightly in erythropoiesis (4.3 % of the erythropoietic activity of the whole skeleton, in spite of their mass representing 45 % of the whole skeletal mass).Compartment 4.There are several reasons why we must suppose this compartment to consist of at least two components.It is represented both in bones with intensive erythropoiesis (with a high A3/M value) and in bones with a weak one, though the constant A4/M has lower values in these bones; also the Tl/2 values differ in the two types of bones.From the correlation between the A3/M and A4/M constants it can be deduced that a substantial part of the A4 flow is directed into the same structures (cells) as the flow destined for the production of haem immediately, i. e. into the erythroblasts.As the A4 value is dependent on the bone mass in bones with low erythropoiesis while it is more dependent on the intensity of erythropoiesis in the strongly erythropoietic bones, we drew the tentative inference that the behaviour of the compartment 4 as a whole could best be explained by its division into two parts, A4a (which is not connected with erythropoiesis immediately and prevails in acra) and A4b (which displays a close connection with erythropoiesis and prevails in erythropoietic bones).
A prerequisite for the quantitative division of compartment 4 was that the influx into the sub compartment 4b equals 7.8 % of the flow employed for erythropoiesis immediately (i.e. of the flow A3).It is possible to verify whether a flow amounting to 0.078 x A3 corresponds quantitatively to the need of the storage iron in erythroblasts, or whether it perhaps surpasses their capacity for consuming iron.The mass of iron consumed by a tissue is proportional to the A/K ratio (see equation 3).As the K4 value of the strongly erythropoietic bones roughly equals 0.22, the fraction of iron incorporated into maturing red cells as storage iron equals 7.8/0.22= 35.4; the fraction of iron consumed for haem production immediately then equals 100.0/1.745= 57.1 % (the K3 value of strongly erythropoietic bones is about 1.754).So the ratio between the quantity of the storage iron deposited in the marrow and supplied as the flow A4b to the iron brought as A3 flow and incorporated in hemoglobin equals 35 : 57.This ratio approaches the ratio between the quantity of iron deposited in pronormoblasts and early erythroblasts on the one hand and the late erythroblasts on the other (37 : 63) reported on the basis of autoradiographic studies by Harriss (1958).As the haem synthesis in the early phases of the erythroblast development is small, the flow of iron into them could be considered a storage flow and in this case the two data agree quantitatively very well.It could therefore reasonably be supposed that a great part of the A4 flow is directed to the early erythroblasts.The appropriateness of dividing the A4 flow into these two components also follows from the dependence shown in Fig. 6.It is apparent that the fraction of storage iron directed outside erythropoiesis (A4a) is in a close relation to the half--time of iron vanishing from compartment 4.This means that the greater the part of flow 4 consumed for erythropoiesis immediately (the smaller A4a is) the slower the tracer disappears from compartment 4 in the bone; physiologically this apparently means that the bulk of the storage iron is destined for incorporation into the .009005 haem of (early) erythroblasts, with which it leaves the bone after their transit time has elapsed.The Tl/2 4b value equal to 3.15 days is in a reasonable agreement with the transit time of red blood cell from the proerythroblast stage, reported to be equal to 2.7 days in mice (Mary et al. 1980).
It follows also from the above considerations that the slow non-haem flow A4 is composed of two different fluxes, the faster one (Tl/2 = 1.65 days) detectable in bones with a low intensity of erythropoiesis per unit mass, and the slower one (Tl/2 = 3.15 days) localised in strongly erythropoietic bones.While the slower flow of storage iron into erythroblasts prevails in the long bones and in the spine, the storage iron in the skull and acra is directed into the further marrow constituents (i.e. into reticular cells, endothelial cells and RES); the thoracic bones represent an intermediate group in this respect (Fig. 6).The distribution of the storage pool 4 (as a whole) in the skeleton is evident from the row denoted as A4/ M in Tab. 1 and from Fig. 8.This picture does not differ from Fig. 7 too much, as there is a strong correlation between fast (A3/M) and slow (A4jM) reflux components as mentioned above.Compartment 5.The calculated course of the tracer concentration in this compartment coincides very well with the experimentally determined radioactivity in the teeth (the curve designated "teeth" and rectangles in Fig. 5).
The extremely long half-time of this compartment could be explained by deposition of iron into a certain anatomical zone of a tooth; the iron would persist here till the time determined by abrasion of the tooth, when the sudden loss probably occurs (at a time beyond the scope of our experiment).
The adequacy of our model of ferrokinetics in bones could also be assessed independently by comparing an experimentally determined quantity of iron in the erythropoietic tissue with values deduced from the model, Tab. 2. The quantity of iron in stores could not be calculated quite reliably, as the distribution of iron into various types of stores is no doubt determined well with direct incorporation from plasma, but it is not guaranteed that the transfer of iron from the phagocyted elderly erythrocytes in RES is distributed among bones in an identical manner.The iron content in the spleen was experimentally determined only approximately, as the iron in circulating erythrocytes could not be distinguished from the iron contained in erythroblasts in this organ.Subject to these limitations we can, however, consider the agreement of results obtained from the model (i.e. on the basis of dynamic measurement of 59Fe incorporation) with independent chemical determination of absolute quantities of iron to be very satisfactory; the justification of our scheme of compartments and their dynamic characteristics was thus verified independently.
The present model of ferrokinetics enabled us to refine our previous analysis (Vacha et al. 1982).The interstitial pool of iron in erythropoietic organs was differentiated from the pre-haem pool in the erythroblasts.At the same time the parameters of iron kinetics in the interstitial space were more precisely determined; considerable differences were found between them in various bones.The flow of iron destined immediately for erythropoiesis showed roughly identical dynamic characteristics in all bones.The flow into the teeth was differentiated from the flow into stores and the latter was divided into a part destined immediately for erythropoiesis and a part destined for storage outside the erythroblasts.It is important from the practical point of view that the components participating in the radioactivity of the erythropoietic tissue at various time intervals after administration of labelled iron are now known with great certainty, at least in the rest state of erythropoiesis.It is a basic presupposition of employing of the common method of labelled iron incorporation as an indicator of the intensity of erythropoiesis.

Fig. 2 .
Fig. 2. Course of 5"Fe radioactivity in femurs as dependent on time after tracer administration.

Fig. 3 .
Fig. 3. Course of G9Fe radioactivity in scapulae as dependent on time after the tracer administration.Notes as in Fig. 2. Fig. 4. Course of G9Fe radioactivity in tail vertebrae as dependent on time after the tracer administration.Notes as in Fig. 2.

Compartment 4 .=Fig. 5 .Fig. 6 .
Fig. 5. Course of '"Fe radioactivity in the skull (inc!. the mandible) as dependent on time after the tracer administration.Rectangles -.oFe content in the teeth, other notes as in Fig. 2.

Fig. 7 .
Fig. 7. Distribution of the erythropoiesis in the skeleton of mice (A3jM values).

Fig. 8 .
Fig. 8. Distribution of the storage iron in the skeleton of mice (A4/M values).

Table 2
Comparison of iron quantity in bones calculated from the parameters ascertained by the model with the result of the direct chemical determination Arithmetical means ± S. E., values in nmol Fe. 1 -apprOltimativ~ value.Chemically determined iron quantities ("real values") taken fromVacha et aJ.1983.