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Iron metabolism and placental transfer of iron in the term rhesus monkey (Macaca mulatta): a compartmental analysis
EUROP. J. OBSTET. GYNEC. REPROD. BIOL., 1977,7/3,127-139 0 Elsevier/North-Holland Biomedical Press
Iron metabolism and placental transfer of iron in the term rhesus monkey (Macaca mulatto): a compartmental analysis
J.P. van Dijk Department
of Chemical Pathology, Faculty of Medicine, Erasmus University, Rotterdam,
The Netherlands
VAN DIJK, J.P. (1977): Iron metabolism and placental transfer of iron in the term rhesus monkey (Macaca mulatta): a compartmental analysis. Europ. J. Obstet. Gynec. reprod. Biol., 713, 127--139. The kinetics of iron and the transplacental iron transport in the near-term rhesus monkey were investigated by means of injection of 59Fe(III) bound to rhesus monkey transferrin into the maternal or into the fetal circulation. The experimentally obtained 59Fe disappearance and appearance curves were analyzed. The analyses were based on a 5-compartmental system. Alternative models were considered but found to be inadequate. Very high maternal as well as fetal iron turnover were calculated. The daily reflux of iron in the mother and in the fetus amounted to 75% of the amount of iron cleared from the plasma compartment daily. The calculated iron transport from mother to fetus was about 1.0 pmol/day/lOO g fetus. The reverse transplacental iron transport was negligible. A small placental iron pool of exchangeable iron had to be assumed. There were no clear differences in the distribution of 59Fe over the fetal organs when the tracer dose was injected into the maternal or into the fetal circulation. Only one of the experiments showed a preferential labeling of the fetal liver when the label was given to the mother, a preference which would be expected on the basis of the hypothesis of Fletcher and Suter (1969). ferrokinetics; pregnancy; iron transport; placenta
Introduction
transported from lower maternal plasma iron and transferrin saturation levels into higher levels in the fetal blood (Fletcher and Suter, 1969). In the nearterm rat, rabbit and guinea pig the transplacental iron transport is also directed against a concentration gradient (Nylander, 1953; Bothwell, Pribilla, Mebust and Finch, 1958; Wong and Morgan, 1973). Uphill transports, however, are indirect evidence for active transport mechanisms. Direct evidence has been obtained with metabolic inhibition (rat: Wyllie and Kaufman, 1974; guinea pig: Wong and Morgan, 1973). The results, however, were somewhat contradictory. The uptake of 59Fe by allantoic placenta tissue of the rat is energy dependent (Wyllie and Kaufman, 1974). Transfer of 59Fe through the chorio-allantoic placenta of the guinea pig could be inhibited with
The kinetics or iron during pregnancy and especially the relation between maternal kinetics and fetal need are poorly understood. The iron content of the fetus is proportional to gestational age, weight and blood volume. How does the mother meet with the increasing fetal need? There is evidence that the maternal iron status has practically no influence on the iron status of the fetus. Neither iron deficiency nor iron supplementation of the mother during pregnancy influence serum iron and hemoglobin levels of the newborn (Lanzkowsky, 1961; Harris and Keller1970). Obviously, the placenta and/or the meyer, fetus extract iron from the mother by an active process. In humans after about 20 wk the iron must be 127
128
metabolic inhibitors. The uptake of s9Fe by the yolk sac in the rat is energy dependent (Wyllie and Kaufman, 1974). The maternal transferrin-bound iron is the main source for the fetal iron. The iron should be detached from the transferrin molecule before it is transported across the placenta (Baker and Morgan, 1969). Possibly transferrin receptors on the trophoblast cell surface play an important role (Laurel1 and Morgan, 1964; Mansour, Schubert and Glasser, 1972; King, 1976). In the process of iron transfer across the placenta cells possibly small-molecular-weight carriers are involved (Larking, Weintraub and Crosby, 1970; Mansour et al., 1972). No difference has been found between fetal and maternal transferrin in man. Carbohydrate and amino acid composition are very similar. Moreover, no differences could be detected in their capacity to deliver iron to immature erythrocytes (Fletcher and Suter, 1969; Verhoef and van Eijk, 1975). Therefore, the uphill transfer of iron cannot be explained by differences between maternal and fetal transferrin, but would require a higher fetal binding affinity for iron, which is very unlikely (Verhoef and van Eijk, 1975). There is increasing evidence that the 2 binding sites on the transferrin molecule are functionally heterogeneous (Fletcher and Huehns, 1968; Awai, Chipman and Brown, 197.5) though so far there is no general agreement. One site would preferentially pick up iron from the storage pools and surrender this iron to erythroblast and trophoblast cells. The other site would be mainly involved in absorption and storage of iron (Fletcher and Huehns, 1968; Fletcher and Suter, 1969; Awai et al., 1975). The aim of the present study was to investigate maternal and fetal iron metabolism and transplacental iron transport in the rhesus monkey. The maternal disappearance curve and the fetal appearance curve were analyzed. By means of a 5compartmental model, rate constants for the compartmental iron turnover as well as transplacental iron transport were calculated.
Materials and methods We performed experiments on 9 pregnant rhesus monkeys (Macaca mulutta) obtained from the Primate Center TN0 at Rijswijk, The Netherlands. The
J.P. vanD#k: Iron metabolism in the pregnant rhesus monkey
duration of pregnancy ranged between 120 and 140 days as estimated on the basis of timed mating and early rectal palpation. The animals were starved 12 h prior to operation. Surgical anesthesia was induced with 0.2-O% halothane in oxygen, following premedication with phencyclidine and atropine sulfate and tracheal intubation. A polyvinyl 19 gauge catheter was introduced into the maternal inferior vena cava through the saphenous vein. The placentas and the interplacental vessels were located by transillumination. An interplacental vein was exposed and catheterized with a silicone rubber T-tube (inner diameter 0.5 mm, outer diameter 1 mm). The uterus was returned to the abdominal cavity. After at least 20 min stabilization period the transferrin-bound 59Fe was injected into the maternal or into the fetal circulation. Maternal heparinized blood samples (1 ml) were obtained 2,4, 6, 8, 10, 1.5, 20, 30, 60 and 120 min after introduction of the label. Fetal l-ml samples were obtained 2, 4,8,15,20,30, 60 and 120 min after injection of the label. Moreover, 7 amniotic fluid samples and 8 maternal urine samples were obtained, the urine samples by punctuation of the bladder. After delivery of the fetus and placentas by means of a cesarian section fetal urine was obtained. All mothers and fetuses survived. In 2 experiments we injected the label into the cepahlic vein, about 2 h before cannulation of the interplacental vessels. A first maternal blood sample was taken 10 min after injection of the label. Isolation and putification
of transfenin
The transferrin was isolated from pooled rhesus monkey plasma by means of gel filtration on Sephadex G-150 and was purified twice by ion-exchange chromatography as has been described (Van Eijk and Leijnse, 1968). Subsequently, this purified transferrin preparation was fractionated by electrofocusing within a pH range of 5.5-6.1. The largest and most alkaline fraction was isolated and used in all transport experiments performed. Apotransferrin was prepared by dialysis against an acetate buffer containing EDTA as described previously (Verhoef, Kremers and Leijnse, 1973). EDTA was removed by dialysis against bidistilled water. The freeze-dried transferrin was solubilized in 0.05 M phosphate buffer, pH 7.4, to a final concentration of 2.8 mg/ml.
J.P. vanDijk: Iron metabolismin the pregnantrhesusmonkey The concentration in this solution of purified apotransferrin was determined by stepwise spectrophotometric titration at 470 nm with Fe(H) (Tavenier, 1971). The mean transferrin concentration was 34.5 gmol/l, the standard error 3.2 @mol/l (n = 5). Transferrin labeling procedure To 5 ml of rhesus monkey apotransferrin (35 pmol apotransferrin per liter 0.05 M phosphate buffer, pH 7.4, containing 35 mmol/l NaHCOs) about 40 PCi 59Fe(III)citrate (spec. act. about 5.5 X lo* yCi//_tmol Fe) was added. After 20 h of incubation at 24°C the solution passed a micropore filter. 5 ml were injected into the maternal circulation. The rest, 0.5 ml, was used for determination of the iron concentration, the radiochemical concentration and of the amount of 59Fe bound to transferrin. Electrophoresis on cellulose acetate showed that more than 96% of the radioactivity was bound to transferrin. The final saturation of the transferrin was about 20%. The transferrin to be injected into the fetal circulation (0.8 ml; 0.035 pmol transferrin/ml) was labeled with about 20 &i “Fe(III)citrate. The final transferrin saturation was approx. 60%. The iron concentration in the plasma was determined according to Trinder (1956), the method being adapted to sample volumes of 0.1 ml. In the concentration range of 1O-40 pmol/l we calculated for duplicate determinations a coefficient of variation (c.v.) of 6%. The transferrin concentration in the plasma samples of the rhesus monkey was estimated by immunodiffusion on partigen plates (Behringwerke) containing rabbit antiserum against human transferrin. There is a linear relationship between d2 of the precipitation ring and the concentration of pure monkey transferrin up to 100 mg/lOO ml assuming a molecular weight of 80,000. Immunoelectrophoresis of monkey plasma against antihuman transferrin showed one crossreacting protein only, and the place of precipitation was identical with the place of precipitation of the monkey transferrin/antitransferrin complex. In order to fit the optimal concentration range we had to dilute 1 volume of monkey plasma with 2 volumes of saline. The total iron binding capacity (TYBC) as well as the transferrin saturation were calculated from the measured transferrin concentration. In the range 30-
129
60 pmol Fe/l the TYBC determination (duplicates) showed a C.V. of 6%, in the range 60-80 pmol/l the C.V.was 4%. D~s~iburion of radioactivity over the fetal organs Immediately following spontaneous fetal death the fetuses were transfused with physiological saline by means of a catheter placed into the aorta via an incision in the left ventricle. Blood and saline could escape through an incision in the right ventricle. Transfusion was stopped when the liver was light brown to pale. The net weight of the collected organs was determined, then they were kept in ice-cold saline for 24 h. The saline was changed 3 times. Then the radioactivity was measured in a Packard y-scintillation spectrophotometer. Erythropoietic active bone structures - bones, skull, ribs, etc. - were collected as complete as possible and counted immediately without washing in saline. Maternal and fetal plasma volumes were estimated by means of extrapolation of the 59Fe disappearance curves. The maternal plasma volume (ml) was about 6% of the maternal weight (g). The fetal plasma volume was about 7% of the fetal weight. Experimental set-up We were not entirely free in the set-up of the experiments, because of a simultaneous study of the purine metabolism in the monkeys used. 4 monkeys were used for the study of the iron transport from mother to fetus, denoted in the following as ‘M + F’ experiments. In 3 monkeys the reverse transport was studied (‘F -+ M’ experiments). In these 7 experiments mentioned, sampling of mother and fetus started 2 min after injection of the transferrin-bound 59Fe. Once more 2 ‘M -+ F’ experiments were performed in which the tracer dose was given to the mother some hours before the start of fetal sampling.
Results
In Table I the mean plasma iron concentration, the mean total iron binding capacity and the mean trans-
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
130 TABLE I
Number of experiment
1 2 3 4 5 6 7 8 9
Weight and plasma volume of mother and fetus as well as iron concentration, saturation in the maternal and fetal plasma for experiments 1 to 9
M
F
Mean plasma iron concentration (crmolll) F M
5200 6500 5800 6200 6500 5500 6850 5800 6200
300 180 320 360 390 350 350 310 340
14.2 10.4 11.4 15.5 11.6 12.3 10.4 12.4 11.9
Weight (g)
31.4 29.6 27.0 36.0 32.0 21.4 28.0 30.5 24.8
iron binding capacity and transferrin
Mean TYBC (~molll)
Saturation (%)
Plasma volume (ml)
M
F
M
F
M
F
76 82 62 60 81 72 65 70 74
52 68 35 52 61 46 54 56 45
19 13 18 26 14 17 16 18 16
60 44 77 69 52 47 52 54 55
310 390 345 370 390 330 410 350 370
21 13 22 25 27 25 25 22 24
The fist 4 experiments investigate transport from mother to fetus (‘M + F’ experiments). Experiments 5, 6 and 7 are ‘F 4 M’ experiments. Experiments 8 and 9 are ‘M -+ F’ experiments; however, the label is given to the mother several hours because cannulation of the fetal vessels. TYBC = total iron binding capacity. For details see Results.
ferrin saturation are shown. The mean maternal and fetal values are calculated from the individual values obtained in the 10 maternal samples and in the 8 fetal samples, respectively. There was no trend to lower values during sampling either in the maternal or in the fetal samples. As has been mentioned in Materials and Methods, amniotic fluid, maternal urine as well as fetal urine after delivery of the fetus were sampled. In none of the experiments could we detect any radioactivity in these samples, so these compartments will not play any role in our further considerations. Figure 1 shows the results of one characteristic ‘M + F’ experiment. The maternal disappearance curve is biphasic and can be fitted by linear combination of two exponents. The fetal appearance curve is a complex one with an early well-pronounced S-shape. We shall try to analyze the fetal appearance curves by means of a compartmental model represented in Figure 4. The results of 2 experiments in which the label was injected into the maternal circulation several hours before fetal cannulation and sampling are shown in Figure 2. The maternal disappearance curves are biphasic, the fetal appearance curves are very flat and tend to decline after about 150 min.
dpm /
pmol
lo8
Fe
1
‘“f”--------
0
20
40
60
80
100 time
120
in minutes
Fig. 1. Specific activity of the iron in the maternal (upper curve) and fetal plasma after injection of “Fe into the maternal circulation (experiment 1). When plotted on cartesian scales (open symbols) the fetal specific activity curve shows an early S-shape.
131
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey dpm / pmol
Fe
, LQJ
80
I
I
120
160
1
200
I
time
(
\
240
280 320 in minutes
Fig. 2. Specific activity of the iron in the maternal and fetal plasma (experiment 8,= 0; experiment 9, A A). The 59Fe was injected into the maternal circulation 150 and 200 min prior to fetal sampling.
dpn / pmol Fe
104:
0
lo2 0
I
I
I
20
40
60
I 80 time
Figure 3 shows a characteristic ‘F + M’ experiment. Although there is transport of 59Fe from fetus to mother, the specific activity of the maternal and fetal plasma iron differs by a factor of about 3 X lo3 between the 40th and 120th min. The fetal disappearance curve is biexponential; the maternal appearance curve shows an early S-shape when plotted on a linear scale. The fetuses of the ‘M -+ F’ experiments I,2 and 3 and of the ‘F + M’ experiments 5, 6 and 7 died between 30 min and 1 h after delivery. The distribution of 59Fe over the organs and tissues was measured as described in Materials and Methods. The results are shown in Table II. In the ‘M + F’ experiments 1 and 2 there was no clear difference in the 59Fe distribution pattern as compared with the distribution of 59Fe in the ‘F + M’ experiments. In experiment 3, however, the fetal liver has taken up the 59Fe preferentially.
I
f
100 in minutes
120
Fig. 3. Specific activity of the iron in the fetal (upper curve) and maternal plasma after injection of 59Fe into the fetal circulation (experiment 7).
Theoretical
considerations
The proposed model has to explain the biphasic shape of the disappearance curve at the donor side as well as the flat and early S-shaped appearance curve at the acceptor side. The biexponential disappearance curve of 59Fe can be explained basically in two different ways: 1. By means of early reflux of 59Fe from a second compartment into the plasma iron pool; 2. By means of heterogeneity of the plasma iron compartment in combination with different clearances for the iron specimens. The S-shape of the s9Fe appearance curve can be explained by assuming a placental compartment of exchangeable iron. We constructed 3 models: I. A S-compartmental model (see Fig. 4) in which the maternal compartment 2 and the fetal compartment 5 account for the early maternal and fetal reflux of 59Fe, respectively. A 3rd placental compartment (compartment 3) has been postulated in order to explain the S-shape of the 59Fe appearance curve. It has been assumed that transplacental iron transport occurs in one direction only (M -+ F). A mathematical description of this model is given in the Appendix.
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
132 TABLE II
Distribution of 59Fe over the fetal organs and tissues, the total amount of s9Fe in the fetus as well as the total amount of 59Fe in the fetus relative to the dose of 59Fe injected ‘F-M’
‘M -+ F’
Experiment
Plasma ‘Bone marrow’ Liver Bile Intestine Kidneys Spleen Lung Heart Brain Muscle Total radioactivity in the fetus (dpm) % dose injected
1
2
3
5
6
I
61 14 10 0.1 1.6 0.7 0.3 1.0 0.9 1.5 0.3
62 13 14 0.1 0.5 6.5 0.1 0.4 0.8 1.4 0.2
50 2.0 44 0.2 0.5 0.5 0.2 0.2 0.3 0.8 0.2
48 11 23 0.3 2.0 0.9 0.2 1.2 0.6 1.2 0.3
51 21 12 0.3 1.5 0.7 0.3 1.0 0.4 2.1 0.3
69 14 10 0.1 0.8 0.6 0.3 0.4 0.3 1.0 0.2
2.3 x lo7 98
2.2 x 107 98
6.3 x lo7 97
1.85 X lo6 3.5
1.6 x lo5 0.5
3.2 X lo6 4
The 59Fe distribution is expressed as a percentage of the total amount of 59 Fe present in the fetus at the end of the experiment. The relative amount of s9Fe in the fetus is given as a percentage of the amount of 59 Fe injected into the mother or into the fetus att=O.
II. A 4compartmental bolized by
z(Comp.
1 k
IS
1
k4s
Fig. 4. Five-compartmental model used in our calculations. Compartments 1 and 4 represent the maternal and fetal plasma iron pool. Compartment 3 illustrates a placental iron pool. Compartments 2 and 5 have been introduced in order to explain the early maternal and fetal 59Fe reflux. The rate constant from compartment i to j is given by kij. The subscripts e and s indicate erythropoiesis and storage. Because of the short duration of the experiments it has been assumed that reflux from the e- and s-compartments may be neglected. In addition, one-directional transport from M + F has been assumed; k3r = k43 = 0.
I)$
model which can be sym-
[(Comp. 2)kZ(Comp. ,)l$(Comp.
4)?
Compartments 1 and 2 represent the maternal and the fetal plasma iron compartments, respectively. The ‘maternal’ and ‘fetal’ compartments accounting for the early reflux of 59Fe are postulated in the placenta (compartments 2 and 3). These same compartments also explain the S-shape of the 59Fe appearance curve at the acceptor site. Moreover, one-directional iron transport from compartment 2 to compartment 3 has been assumed. This model did not explain the experimentally obtained fetal appearance curves. A much greater time-lag in the appearance of 59Fe in the fetal circulation, as experimentally observed, is predicted. III. The third model was based on the hypothesis
J.P. vanDijk: Iron metabolism in the pregnant rhesus monkey TABLE III
133
Characterization of the disappearance curves and the calculated values of the rate constants for the experiments l-7 listed in Table I
Number of experiment
nr (min-l)
~Zdn-‘)
m
kr2 (min-l)
kro (min- 1)
kzl (min-‘)
Plasma iron turnover (pmol/day)
Iron reflux (mnol/day)
0.0484 0.0720 0.0831 0.0745
0.0048 0.0040 0.0082 0.0061
0.4923 0.2813 0.3793 0.2223
0.0176 0.0177 0.0245 0.0202
0.0086 0.0054 0.0125 0.0077
0.0269 0.0529 0.0547 0.0527
166 135 208 229
111 103 138 165
41 (min-l)
;In:in-‘)
’
k4s (min -‘I
bo (min-‘)
k.4 (min-‘)
0.0610 0.0485 0.0823
0.0036 0.0030 0.0028
0.3780 0.2381 0.1951
0.0198 0.0105 0.0150
0.0055 0.0038 0.0034
0.0393 0.0376 0.0663
31 11 19
24 8 15
For the meaning of the parameters nr, “2, m and qr, q2, f, refer to eqns. (6) to (13) of the Appendix. The rate constants (k) are defined in the Appendix and are calculated from the same equations. The calculation of the turnover rate and the reflux is shown in the Discussion.
of heterogeneity
sites on the by Fletcher and Huehns (1968). In the sense of this hypothesis the biexponential shape of the 59Fe disappearance curve can be explained by assuming different clearances of the A- and B-site of 59Fe. Mathematically this disappearance curve can be represented by the formula: transferrin
xl(t)
= XlA@)
of
molecule
exp(-6
the
iron
put
forward
It)
’ XIB@)
binding
ex&62t)
in which x(t) represents the amount of 59Fe in the plasma iron compartment at time t. At t = 0 this amount equals xrA t xrn, which means the sum of the total amount of 59Fe bound to the A-site and to the B-site of the transferrin. As has been described in Materials and Methods, we used apotransferrin for the labeling with 59Fe. Since one molecule of transferrin has one A-site and one B-site, statistically we would expect as many Asites as B-sites to be labeled. Extrapolation to t = 0 of the exponents obtained by fitting the disappearance curves shows that xrA and xru are unequal, which can be seen in Table III, where the value of m in 6 out of 7 experiments clearly deviates from 0.5, a value which would be expected in this case. All our further considerations about the kinetics of iron will be based on the assumptions made in
model I. The rate constants ksr and k4s are considered to be negligible, which means that the iron transport is in essence one-directional. This assumption will be discussed below (see Discussion)
Calculation
Here we shall describe the way in which the formulas derived in the Appendix are used to calculate the rate constants. By means of fitting (Riggs, 1970) the biexponential disappearance curves we obtain values for the parameters m, nl, n2 and f, ql and q2. From these parameters we can calculate the rate constants kr2, k2r, kzo and k45r k54 and k40 (see legend to Fig. 4 and eqns. (7) (8) (9) and (1 l), (12) (13) in the Appendix). Table III gives the values of the parameters m, nl, n2 and f, q1 and q2 for the 7 experiments mentioned in Table I. The maternal rate constants kr2, k2r and kr, as well as the fetal rate constants k4s, ks4 and k4, are also shown. The rate constants kra and ka4 can be calculated only when we use the information present in the fetal appearance curves. From the appendix (eqn. (18)) it can be seen that, in order to describe the fetal appearance curve in mathematical terms, knowledge of the
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
134
fetal rate parameters is indispensable. However, in a transport experiment from mother to fetus only the maternal rate constants can be obtained. The fetal rate constants can only be calculated from the reverse experiment. Consequently, krs and ks4 can be calculated only by combining the results from a ‘M --, F’ experiment with the results obtained in a ‘F + M’ experiment. In order to compute the theoretical x4(t) appearance curve (Appendix, eqn. (18)) we need criteria to combine sets of calculated parameters from different experiments. The following quite arbitrary criteria were used: (1) equality within 15% of the maternal and fetal weight ; (2) equality within 15% of the maternal and fetal iron concentration. The computations of the rate constants krs and ks4 are best illustrated by a specific example (combination of experiments 3 and 6). We may write eqn. (18) of the Appendix, describing the fetal appearance curve, as x4(t) = krs *x1(O) *y(ks4, t), which gives: x4(t)
y&34,t) = k13. x1(o) (y represents a function symbol) The only unknown parameter in the function y(k34, t) is the rate constant k34. Values for ql, q2 and f can be found in Table III. b, p and o are calculated from the eqns. (19), (20) and (21) in the Appendix: b= f-q2 +(l - f)qr=ks4=0.038min-’ P = (qr * qz) = k4o * ks4 = 0.0038 X 0.0376 = 1.4 X 10-4min-2 o = (41+ q2) = k 45 + k,, + k40 = 0.0105 t 0.0376 + 0.0038 = 0.052 min-’ We now compute the function y(k34, t) for several values of k34. That value of k34 that gives the best cover of the theoretical function y(ks4, t) with the experimentally obtained fetal appearance curve (experiment 3), when both functions are plotted on the same absolute scale (Fig. 5), is considered the best estimate for ks4. In Figure 5 it can be seen that in this special case k34 = 0.1 results in a reasonably good approximation of the appearance curve.
x4(f) = k13 *x1(0) *Y(~w t) x1(O) = amount of radioactivity (dpm) present in the
16.
8 6 i
*.
2 2
4.
000 0
20
40
60
80
100
120
time in minutes
Fig. 5. The fetal 59Fe appearance curve (experiment 3) and the theoretical y(k34, t) curve calculated for k34 = 0.1 min-l are shown. The y(k34, t) function has been calculated with the rate constants obtained in experiments 3 and 6. For details refer to Calculations.
maternal plasma pool at time 0, which is identical to the injected dose of 59Fe; x1(O) = 11.4 X lo7 dpm (experiment 3) x4(t) = amount of radioactivity present in the fetal plasma pool at time t (experiment 6); the experimentally obtained fetal appearance curve From this equation we can calculate kr3 for several time-points. In the case of an ideal fit ks4 should have the same value at every time-point. In our case, however, kr3 varies between 5.7 X 10M4 and 6.5 X 10B4 with a mean value of about 6.0 X 10T4 min-‘. The assumed steady-state conditions imply the equality kr3 *X1 = k34 *X3, from which we can calculate X3, the total amount of iron present in the placental iron pool. X1 represents the total amount of iron present in the maternal plasma pool (j.dnol). We calculate a placental iron pool of about 2.5 X 10e2 pmol. The daily transplacental iron transport can be computed from the equation: Fe transport = kr3 *X1 . 1440 (pmol/day) We calculate an iron transport of about 3.4 pmol/day (1.06 pmol/day/lOO g fetus). The total amount of radioactivity transported
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
from mother to fetus between time 0 and time t, (A& conforms to the equation: t A:=
s
k,,.xi(t)dt
0
120
AA2’(expt. 3) = 6.0 X 10e4 s
(11.4 X lo7 - 0.3793
0
X e-o.0831 tt 1 1.4 X lo7 - 0.6207 e-“.0082t)dt = 3.3 X lo6 dpm Comparison with the figures given in Table II, experiment 3, reveals AA2’ to be a reasonable estimate. (320 min Moreover, the graphical representation time-scale) of the fitted maternal disappearance curve (experiment 3) and the theoretically obtained x4(t) function (combination of experiments 3 and 6) bears a very close resemblance to the curves shown in Figure 2.
Discussion
Several authors have already stated that the transport of iron from mother to fetus is directed against a concentration gradient (Nylander, 1953; Bothwell et al., 1958). Our results are in agreement with this generally ac, (ted rule (see Table I). The fetal iron binding capacity is a little lower than the maternal one, indicating a lower fetal transferrin concentration. We calculated a mean fetal transferrin saturation of about 57% with a standard deviation of about 11%. The corresponding maternal values are 17% with a standard deviation of 4%. Another commonly accepted rule is that the transplacental iron transport is one-directional, directed from mother to fetus (Bothwell et al., 1958). Comparison of Figures 1 and 3 shows that the transport from mother to fetus is probably much more efficient compared with the transport in the reverse direction. It seems unlikely that this difference can be explained by the relatively large dilution volumes of the maternal serum compartment in combination with somewhat larger rate constants. We therefore assumed a one-directional transport from compartment 1 to compartment 4 (see Fig. 4).
135
As stated in the Introduction, it was our aim to estimate parameters that govern the kinetics of iron in the maternal and fetal serum compartment as well as parameters which determined transplacental iron transport. The model described does not discriminate between active and passive transport mechanism. It only states that the transport is proportional to the amount of iron present in the compartment under study. In the case of fixed concentrations and fixed volumes, such as we assume to exist, the transport is proportional to the concentration. The fact that the 59Fe disappearance curves can be described mathematically by exponential functions means that the statement of proportionality is appropriate. Whenever carrier transports or active transports are involved they must operate far from saturation in this particular case. Consequently the parameters calculated for the transport of 59Fe may be considered as representative for the transport of 56Fe too. From the 3 alternative models mentioned in Theoretical Considerations we chose the Scompartment one because of its better predictive qualities. From Table IV it can be seen that k, 3 is about l/25 klo, which means that the amount of iron transported to the placental compartment and consequently also to the fetus only amounts to 4% of the amount carried to the postulated erythron and storage compartment per unit time. Consequently we may state that where the label is given to the fetus, only a fraction of 59Fe transported to the maternal circulation will be returned to the fetus. This consideration makes it possible to estimate k43 and ksr from eqn. (18) in the Appendix because of the obvious symmetry in the.model. We only have to reinterpret the symbols used. For the combination of the ‘F -+ M’ experiment 6 and the ‘M -+ F’ experiment 3 we calculated a k43 of about 5.5 X lop6 min-’ and a k3r of about 1.2 X lop4 min-‘. In comparison with the product kr3 . X1 the product k43 . X4 is negligible, which indicates that the assumption of transport in one direction is justified. From the rate constants kr2 and kio we can calculate the maternal plasma iron turnover (PIT). PIT = (k,, + kr,) . plasma iron * 1440 @mol/day). The daily reflux (pmol/day) equals k 12 . plasma iron . 1440 because of the equality k,, . X1 = k,, . X2. The calculated maternal and fetal PIT values as well as the reflux values are shown in Table III. The
136
J.P. vanDijk: Iron metabolismin the pregnantrhesusmonkey
TABLE IV
Exp. no.
klZ
3 4
0.0245 0.0189
Characterization of the fetal-maternal S-compartmental system in terms of rate constants, the magnitude of the placental iron compartment X4, and calculated transplacental iron transports kzt
klo
k13
k13
0.0547 0.0755
0.0125 0.0161
6.0 x 1O-4 2.4x 1O-4
(5.7-6.5) X 1O-4 (2.2-2.6)x1O-4
Fe
transport (wnol/day/ 100 g)
k34
k4s
ks4
k40
x4
0.1 0.08
0.0105 0.0105
0.0376 0.0393
0.0038 0.0044
0.025 1.1 0.017 0.8
range
The fetal rate constants obtained from the ‘F + M’ experiments 6 and 5 are taken as representative of the actual values in the ‘M + F’ experiments 3 and 4 respectively. Rate constants in min-‘. Fe transport in pmol/day/lOO g. X4 in pmol Fe.
maternal and fetal iron turnover rates are very high. About 70% of the iron daily leaving the maternal plasma compartment is refluxed. The fetal reflux amounts to 75%. At the moment these results are difficult to interpret. It is known, for instance in the rabbit, that the turnover of the plasma iron as well as the transplacental transport of iron increases with gestation (Douglas, Penton and Watt, 1971). The authors (Douglas et al., 1971) did not calculate which part of the increased turnover was responsible for the transplacental increase of the iron transport. In our case the high maternal PIT values can certainly not be explained by a large transplacental iron transport. Moreover, the same high turnovers are also found in the fetus, suggesting a common principie. In man (Cook, Marsaglia, Eschbach, Funk and Finch, 1970) the mean PIT value was 0.70 mg/lOO ml whole blood per day in the case of iron deficiency. The total reflux - 55% of the PIT value - could be differentiated into an early and a late component (Cook et al., 1970). The early component - 15% of the PIT - had a reentry Tr,a of about 8.5 h and can probably be explained by a lymphatic shunt (Morgan, Marsaglia, Giblett and Finch, 1967; Cook et al., 1970). The late component - 40% of the PIT - with a reentry Tr,a of 7 days is probably wastage iron of erythropoiesis (Cook et al., 1970). Is our reflux identical with the early reflux observed in man, however, with a much higher lymphatic shunt? This question cannot be answered at the moment. The calculated transplacental iron transport (Table IV) amounts to 1 .l and 0.8 pmol/day/lOO g fetus for near-term monkeys. In man this near-term transport is about 4.0 pmol/day/lOO g fetus (Widdowson and
Spray, 1951; Fletcher and Suter, 1969). It can be seen from the appearance curve (Fig. 1) that the transport of 59Fe from M + F is very rapid. In terms of our model this fact indicates a small pool of placental iron. We calculated placental iron compartments of about 0.02 ymol Fe (Table IV). 6 out of 7 fetuses died between 30 min and 1 h after delivery. The distribution of 59Fe over the organs and tissues is shown in Table II. Between the ‘M + F’ experiments 1 and 2 and the ‘F + M’ experiments no clear difference in the 59Fe distribution can be seen. In experiments 3 (‘M + F’), however, the incorporation of 59Fe into the liver is clearly increased, while the 59Fe incorporation into the ‘bone marrow’ is reduced. This observation would be in agreement with the hypothesis of Fletcher and Suter (1969) that the fetal side of the placenta, the ‘storage-directed’ transferrin binding site, preferentially picks up iron from the placenta. However, only 1 of the 3 ‘M + F’ experiments showed a preferential labeling of the liver, so alternative explanations are more likely.
Appendix
Mathematical description of the model illustrated in Figure 4 Let Xi be the amount of labeled iron (dpm) in compartment i. Xi represents the total amount of iron (pmol) in compartment i. Let krj represent the rate constant (min-‘) from compartment i to j. The erythropoietic and storage pools are indicated by e and s, respectively, so k,, represents the rate constant from compartment 1 to the erythropoietic
137
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
compartment(s) (see Fig. 4). For kr, t k,, t k,, we write k,,; the sum k,, + kas will be given by k4c. Between compartments 1, 3 and 4 we assume onedirectional transport. The rate constants kG3 and kar are considered to be negligible (see Theoretical Considerations and Discussion). The following set of equations describes the kinetics of 59Fe in the indicated compartments (see Fig. 4) after injection of the label (bound to transferrin) into the maternal plasma iron compartment: x;(t) = -(krz
+ kzr * x2(t)
+ k,o)xr(t)
(1)
x;(t) = k12 * xl(t)
- kzl * x2(t)
(2)
x;(t)
- k34 * x3(t)
(3)
= k13 * xl(t)
x;(t) = -(k4s +k&q(t)+ks4
*xa(t)+k~.xs(t)
(4) (5)
x;(t) = k4s s x4(t) - kw * xs(t)
Because of the one-directional transport assumed, we may consider eqns. (1) and (2) and eqns. (4) and (5) as two independent sets of equations. When the label is injected into the maternal circulation, the first set of equations can be solved, assuming boundary conditions x2(O) = 0 and x1(O) = A,, A, being the total amount of 59Fe (dpm) injected into the maternal circulation. The general resolution for xl(t) is
amount of 59Fe injected into the fetal circulation. X4(t)
=
x4(0) * f - exp(-qr
+ x4(0) * (1 - f) - exp(-q2t)
k4s + k4o + ks4 = qr + q2
(11)
ks4 * ho = 91.
(12)
b4=f.q2+(1
q2
(13)
-0%
Derivation of the theoretical x4(t) appearance function in the case of injection of s9Fe into the maternal plasma iron compartment 1: substitution of eqn. (6) in eqn. (3) and resolution for x3(t) by Laplace transformation gives: x3(t) = kr3 - xr(O)m * L-’
tkrs*xr(O)(l Application
-m)L-’
of the convolution
(6)
-p
x3(t)= kr3.xr(0)
m + -----exp(-k34t) nr-ka4
kr2+kr0+k2r=nr
+ ----exp(-kk4t) n2 - ks4
+n2
(7)
k2r - kr, = nl - n2
(8)
k2r=m*n:!t(l
(9)
-m)nr
When the tracer dose has been injected into the fetal circulation, x4(t) can be solved from eqns. (4) and (5) assuming boundary conditions x5(O) = 0 and x4(O) = Ar. The symbol Af represents the total
x4(t)
= I_- l
-___-__
t)
- seexp(-n,t)
(1 -m)
(15)
Rearranging eqn. (5) and taking the Laplace transformation, we obtain:
k45* Lb,(t))
(16)
x5(t) = ~-__
s+ kw After rearranging eqn. (4) and substituting and (16) in eqn. (4) we resolve for x4(t):
k13 . elm kl3 ~.xdW k34)(s + nr) ’ (nr - k,,)(s + k34) - (n2 - k,,)(s -__
exp(-nr nr-k34
Values of m, nl and n2 can be obtained by fitting the biphasic maternal disappearance curves. The rate donstants kr2, kro and k2r can be calculated from the following set of equations:
-
theorem results in:
m
-m)*exp(-nzt)
34
(10)
Values of f, q1 and q2 can be obtained by fitting the biphasic fetal disappearance curves. The rate constants kd5, kaO and ks4 can be calculated from the following equations:
x1(t) = x1(O) * m - exp(-ntt) +x,(0)(1
t)
__-_____
. k45 s + k4s + krlo - kss: k 54
-4 + n,)tc
k13 . xdW1 n2 -
b4)(s
eqns. (14)
-m> + k,,)
(17) I
138
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
The inverse Laplace transformation can be taken by application of the convolution theorem, which yields:
We may now write eqn. (18) in a condensed
x4(t)
=
x4(t)=k13.~1(0).Y(k34,t) where y represents a function
form:
(22)
symbol only.
Acknowledgement
-(nr
- b)qr + bnr t p - ob
(n: _onl+pKr
’ exp(-q2t)
-
x exP(-qtt)
- G-_
---
(1 - m)ks4 n2 -
-(n2
k34
We wish to thank Dr. H.C.S. Wallenburg, Department of Gynecology and Obstetrics, Erasmus University, Rotterdam, for the opportunity given to us to perform this study simultaneously with his own investigation on the purine metabolism in the rhesus monkey.
_ q2)
nl- b onl + p) exp(-nr
t)
- b)qs + bn2 + p - ob
G-2
+ p)
ev-q20
(sl -
q2)
References
- b)qr t bn2 + p - ob
-(n2
~---
-IS
exp(-q
on2
P) (41 -
+
1 t>
Awai, M., Chipman, B. and Brown, E.B. (1975): In vivo evidence for the functional heterogeneity of transferrin bound iron. II. Studies in pregnant rats. J. Lab. clin. Med.,
q2)
n, - b
-(n22-
x
on2
-(k,,
85, 785-796. +
Baker, E. and Morgan, E.H. (1969): The role of transferrin in placental iron transfer in the rabbit. Quart. J. exp. Phy siol., 54, 173-186. Bothwell, T.H., Pribilla, W.F., Mebust, W. and Finch, C.A. (1958): Iron metabolism in the pregnant rabbit: iron transfer across the placenta. Amer. J. Physiol., 193, 615-
P)
622.
- b)q2 + b * ks4 + p - ob wGq2
(kg4
-
-
ok34
+
t)
P) (qr - 92)
-(k34
- b)q, t bk34 + p - ob -__ exp(-qr (k& - ok34 + p)(qr - 92)
k,, - b
- ~-_._exp(-ks4t) (k$ - ok34 + P)
t)
(18)
As can be seen, x4(O) = 0. The dimension of x4(t) is dpm. The formula within square brackets is a function of t and k34; all other parameters can be calculated from the individual ‘M + F’ and ‘F + M’ experiments by fitting the obtained disappearance curves (eqns. (6) and (10)). The parameters b, p and o are calculated from the following equations: b=f*q,+(l-f)q,=ks4
(19)
P = qt . qz = ks4 * k4o
(20)
o = qr + qz = k4s + k,o + ks4
(21)
Cook, J.D., Marsaglia, G., Eschbach, J.W., Funk, D.D. and Finch, C.A. (1970): Ferrokinetics: a biological model for plasma iron exchange in man. J. clin. Invest., 49, 197205. Douglas, T.A., Penton, J.P. and Watt, C. (1971): Placental transfer of iron in the rabbit. &it. J. Haemat., 20, 185194. Fletcher, J. and Huehns, E.R. (1968): Function of the transferrin. Nature (Lond.), 218, 12 1 l- 1214. Fletcher, J. and Suter, P.E.N. (1969): The transport of iron by the human placenta. Clin. Sci., 36, 209-220. Harris, J.W. and Kellermeyer, R.W. (1970): The Red Cell, p. 95. Harvard University Press, Cambridge, Mass. King, B.F. (1976): Localisation of transferrin on the surface of the human placenta by electron microscopic immunocytochemistry. Anat. Rec., 186, 15 l-160. Lanzkowsky, P. (1961): The influence of maternal iron deticiency anemia on the hemoglobin of the infant. Arch. Dis. Childh., 36, 205-209.
Larking, C.E., Weintraub, L.R. and Crosby, W.H. (1970): Iron transport across rabbit allantoic placenta. Amer. J. Physiol., 218, 7-l 1. Laurell, C. and Morgan, E. (1964): Iron exchange between transferrin and the placenta in the rat. Actu physiol. stand., 62, 271-279.
139
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey Mansour, M.M., Schubert, A.R. and Glasser, S.R. (1972): Mechanism of placental iron transfer in the rat. Amer. J. Physiol., 22, 1628-1633. Morgan, E.H., Marsaglia, G., Giblett, E.R. and Finch, C.A. (1967): A method of investigating internal iron exchange utilizing twu types of transferrin. J. Lab. clin. Med., 69, 370-381. Nplander, G. (1953): On the placental transfer of iron: an experimental study in the rat. Acta physiol. stand., 29, Suppl. 107. Riggs, D.S. (1970): The Mathematical Approach to Physiological Problems, pp. 146-161. MIT Press, Cambridge, Mass. and London. Tavenier, P. (1971): Onderzoek naar het niet transferrinegebonden ijzer in het plasma. Thesis, Medical Faculty, Rotterdam. Trinder, P. (1956): The improved determination of iron in serum. J. clin. Pathol., 9, 170-172. Van Eijk, H.G. and Leijnse, B. (1968): The iron transport-
ing system
in the body.
I. Isolation
and partial
charac-
terization of rabbit transferrin. Biochim. biophys. Acra, 160, 126-128. Verhoef, NJ., Kremers, J.H.W. and Leijnse, B. (1973): The effect of heterologous transferrin on the uptake of iron and haem synthesis by bone marrow cells. Biochim. biophys. Acta, 304, 114-122. Verhoef, N.J. and Van Eijk, H.G. (1975): Isolation, characterization and function of cord blood transferrin. Clin. Sci. molec. Med., 48, 335-440. Widdowson, E.M. and Spray, C.M. (1951). Chemical development in vitro. Arch. Dis. Childh., 26, 204-214. Wong, C.T. and Morgan, E.H. (1973). Placental transfer of iron in the guinea pig. Quart. J. exp. Physiol., 58, 47-58. Wyllie, J.C. and Kaufman, N. (1974): Effect of metabolic inhibitors and chelating agents on 59Fe uptake by the placental tissues of the rat, Rattus norvegicus. Comp. Biothem. Physiol., 48A, 361-367.
Iron metabolism and placental transfer of iron in the term rhesus monkey (Macaca mulatto): a compartmental analysis
J.P. van Dijk Department
of Chemical Pathology, Faculty of Medicine, Erasmus University, Rotterdam,
The Netherlands
VAN DIJK, J.P. (1977): Iron metabolism and placental transfer of iron in the term rhesus monkey (Macaca mulatta): a compartmental analysis. Europ. J. Obstet. Gynec. reprod. Biol., 713, 127--139. The kinetics of iron and the transplacental iron transport in the near-term rhesus monkey were investigated by means of injection of 59Fe(III) bound to rhesus monkey transferrin into the maternal or into the fetal circulation. The experimentally obtained 59Fe disappearance and appearance curves were analyzed. The analyses were based on a 5-compartmental system. Alternative models were considered but found to be inadequate. Very high maternal as well as fetal iron turnover were calculated. The daily reflux of iron in the mother and in the fetus amounted to 75% of the amount of iron cleared from the plasma compartment daily. The calculated iron transport from mother to fetus was about 1.0 pmol/day/lOO g fetus. The reverse transplacental iron transport was negligible. A small placental iron pool of exchangeable iron had to be assumed. There were no clear differences in the distribution of 59Fe over the fetal organs when the tracer dose was injected into the maternal or into the fetal circulation. Only one of the experiments showed a preferential labeling of the fetal liver when the label was given to the mother, a preference which would be expected on the basis of the hypothesis of Fletcher and Suter (1969). ferrokinetics; pregnancy; iron transport; placenta
Introduction
transported from lower maternal plasma iron and transferrin saturation levels into higher levels in the fetal blood (Fletcher and Suter, 1969). In the nearterm rat, rabbit and guinea pig the transplacental iron transport is also directed against a concentration gradient (Nylander, 1953; Bothwell, Pribilla, Mebust and Finch, 1958; Wong and Morgan, 1973). Uphill transports, however, are indirect evidence for active transport mechanisms. Direct evidence has been obtained with metabolic inhibition (rat: Wyllie and Kaufman, 1974; guinea pig: Wong and Morgan, 1973). The results, however, were somewhat contradictory. The uptake of 59Fe by allantoic placenta tissue of the rat is energy dependent (Wyllie and Kaufman, 1974). Transfer of 59Fe through the chorio-allantoic placenta of the guinea pig could be inhibited with
The kinetics or iron during pregnancy and especially the relation between maternal kinetics and fetal need are poorly understood. The iron content of the fetus is proportional to gestational age, weight and blood volume. How does the mother meet with the increasing fetal need? There is evidence that the maternal iron status has practically no influence on the iron status of the fetus. Neither iron deficiency nor iron supplementation of the mother during pregnancy influence serum iron and hemoglobin levels of the newborn (Lanzkowsky, 1961; Harris and Keller1970). Obviously, the placenta and/or the meyer, fetus extract iron from the mother by an active process. In humans after about 20 wk the iron must be 127
128
metabolic inhibitors. The uptake of s9Fe by the yolk sac in the rat is energy dependent (Wyllie and Kaufman, 1974). The maternal transferrin-bound iron is the main source for the fetal iron. The iron should be detached from the transferrin molecule before it is transported across the placenta (Baker and Morgan, 1969). Possibly transferrin receptors on the trophoblast cell surface play an important role (Laurel1 and Morgan, 1964; Mansour, Schubert and Glasser, 1972; King, 1976). In the process of iron transfer across the placenta cells possibly small-molecular-weight carriers are involved (Larking, Weintraub and Crosby, 1970; Mansour et al., 1972). No difference has been found between fetal and maternal transferrin in man. Carbohydrate and amino acid composition are very similar. Moreover, no differences could be detected in their capacity to deliver iron to immature erythrocytes (Fletcher and Suter, 1969; Verhoef and van Eijk, 1975). Therefore, the uphill transfer of iron cannot be explained by differences between maternal and fetal transferrin, but would require a higher fetal binding affinity for iron, which is very unlikely (Verhoef and van Eijk, 1975). There is increasing evidence that the 2 binding sites on the transferrin molecule are functionally heterogeneous (Fletcher and Huehns, 1968; Awai, Chipman and Brown, 197.5) though so far there is no general agreement. One site would preferentially pick up iron from the storage pools and surrender this iron to erythroblast and trophoblast cells. The other site would be mainly involved in absorption and storage of iron (Fletcher and Huehns, 1968; Fletcher and Suter, 1969; Awai et al., 1975). The aim of the present study was to investigate maternal and fetal iron metabolism and transplacental iron transport in the rhesus monkey. The maternal disappearance curve and the fetal appearance curve were analyzed. By means of a 5compartmental model, rate constants for the compartmental iron turnover as well as transplacental iron transport were calculated.
Materials and methods We performed experiments on 9 pregnant rhesus monkeys (Macaca mulutta) obtained from the Primate Center TN0 at Rijswijk, The Netherlands. The
J.P. vanD#k: Iron metabolism in the pregnant rhesus monkey
duration of pregnancy ranged between 120 and 140 days as estimated on the basis of timed mating and early rectal palpation. The animals were starved 12 h prior to operation. Surgical anesthesia was induced with 0.2-O% halothane in oxygen, following premedication with phencyclidine and atropine sulfate and tracheal intubation. A polyvinyl 19 gauge catheter was introduced into the maternal inferior vena cava through the saphenous vein. The placentas and the interplacental vessels were located by transillumination. An interplacental vein was exposed and catheterized with a silicone rubber T-tube (inner diameter 0.5 mm, outer diameter 1 mm). The uterus was returned to the abdominal cavity. After at least 20 min stabilization period the transferrin-bound 59Fe was injected into the maternal or into the fetal circulation. Maternal heparinized blood samples (1 ml) were obtained 2,4, 6, 8, 10, 1.5, 20, 30, 60 and 120 min after introduction of the label. Fetal l-ml samples were obtained 2, 4,8,15,20,30, 60 and 120 min after injection of the label. Moreover, 7 amniotic fluid samples and 8 maternal urine samples were obtained, the urine samples by punctuation of the bladder. After delivery of the fetus and placentas by means of a cesarian section fetal urine was obtained. All mothers and fetuses survived. In 2 experiments we injected the label into the cepahlic vein, about 2 h before cannulation of the interplacental vessels. A first maternal blood sample was taken 10 min after injection of the label. Isolation and putification
of transfenin
The transferrin was isolated from pooled rhesus monkey plasma by means of gel filtration on Sephadex G-150 and was purified twice by ion-exchange chromatography as has been described (Van Eijk and Leijnse, 1968). Subsequently, this purified transferrin preparation was fractionated by electrofocusing within a pH range of 5.5-6.1. The largest and most alkaline fraction was isolated and used in all transport experiments performed. Apotransferrin was prepared by dialysis against an acetate buffer containing EDTA as described previously (Verhoef, Kremers and Leijnse, 1973). EDTA was removed by dialysis against bidistilled water. The freeze-dried transferrin was solubilized in 0.05 M phosphate buffer, pH 7.4, to a final concentration of 2.8 mg/ml.
J.P. vanDijk: Iron metabolismin the pregnantrhesusmonkey The concentration in this solution of purified apotransferrin was determined by stepwise spectrophotometric titration at 470 nm with Fe(H) (Tavenier, 1971). The mean transferrin concentration was 34.5 gmol/l, the standard error 3.2 @mol/l (n = 5). Transferrin labeling procedure To 5 ml of rhesus monkey apotransferrin (35 pmol apotransferrin per liter 0.05 M phosphate buffer, pH 7.4, containing 35 mmol/l NaHCOs) about 40 PCi 59Fe(III)citrate (spec. act. about 5.5 X lo* yCi//_tmol Fe) was added. After 20 h of incubation at 24°C the solution passed a micropore filter. 5 ml were injected into the maternal circulation. The rest, 0.5 ml, was used for determination of the iron concentration, the radiochemical concentration and of the amount of 59Fe bound to transferrin. Electrophoresis on cellulose acetate showed that more than 96% of the radioactivity was bound to transferrin. The final saturation of the transferrin was about 20%. The transferrin to be injected into the fetal circulation (0.8 ml; 0.035 pmol transferrin/ml) was labeled with about 20 &i “Fe(III)citrate. The final transferrin saturation was approx. 60%. The iron concentration in the plasma was determined according to Trinder (1956), the method being adapted to sample volumes of 0.1 ml. In the concentration range of 1O-40 pmol/l we calculated for duplicate determinations a coefficient of variation (c.v.) of 6%. The transferrin concentration in the plasma samples of the rhesus monkey was estimated by immunodiffusion on partigen plates (Behringwerke) containing rabbit antiserum against human transferrin. There is a linear relationship between d2 of the precipitation ring and the concentration of pure monkey transferrin up to 100 mg/lOO ml assuming a molecular weight of 80,000. Immunoelectrophoresis of monkey plasma against antihuman transferrin showed one crossreacting protein only, and the place of precipitation was identical with the place of precipitation of the monkey transferrin/antitransferrin complex. In order to fit the optimal concentration range we had to dilute 1 volume of monkey plasma with 2 volumes of saline. The total iron binding capacity (TYBC) as well as the transferrin saturation were calculated from the measured transferrin concentration. In the range 30-
129
60 pmol Fe/l the TYBC determination (duplicates) showed a C.V. of 6%, in the range 60-80 pmol/l the C.V.was 4%. D~s~iburion of radioactivity over the fetal organs Immediately following spontaneous fetal death the fetuses were transfused with physiological saline by means of a catheter placed into the aorta via an incision in the left ventricle. Blood and saline could escape through an incision in the right ventricle. Transfusion was stopped when the liver was light brown to pale. The net weight of the collected organs was determined, then they were kept in ice-cold saline for 24 h. The saline was changed 3 times. Then the radioactivity was measured in a Packard y-scintillation spectrophotometer. Erythropoietic active bone structures - bones, skull, ribs, etc. - were collected as complete as possible and counted immediately without washing in saline. Maternal and fetal plasma volumes were estimated by means of extrapolation of the 59Fe disappearance curves. The maternal plasma volume (ml) was about 6% of the maternal weight (g). The fetal plasma volume was about 7% of the fetal weight. Experimental set-up We were not entirely free in the set-up of the experiments, because of a simultaneous study of the purine metabolism in the monkeys used. 4 monkeys were used for the study of the iron transport from mother to fetus, denoted in the following as ‘M + F’ experiments. In 3 monkeys the reverse transport was studied (‘F -+ M’ experiments). In these 7 experiments mentioned, sampling of mother and fetus started 2 min after injection of the transferrin-bound 59Fe. Once more 2 ‘M -+ F’ experiments were performed in which the tracer dose was given to the mother some hours before the start of fetal sampling.
Results
In Table I the mean plasma iron concentration, the mean total iron binding capacity and the mean trans-
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
130 TABLE I
Number of experiment
1 2 3 4 5 6 7 8 9
Weight and plasma volume of mother and fetus as well as iron concentration, saturation in the maternal and fetal plasma for experiments 1 to 9
M
F
Mean plasma iron concentration (crmolll) F M
5200 6500 5800 6200 6500 5500 6850 5800 6200
300 180 320 360 390 350 350 310 340
14.2 10.4 11.4 15.5 11.6 12.3 10.4 12.4 11.9
Weight (g)
31.4 29.6 27.0 36.0 32.0 21.4 28.0 30.5 24.8
iron binding capacity and transferrin
Mean TYBC (~molll)
Saturation (%)
Plasma volume (ml)
M
F
M
F
M
F
76 82 62 60 81 72 65 70 74
52 68 35 52 61 46 54 56 45
19 13 18 26 14 17 16 18 16
60 44 77 69 52 47 52 54 55
310 390 345 370 390 330 410 350 370
21 13 22 25 27 25 25 22 24
The fist 4 experiments investigate transport from mother to fetus (‘M + F’ experiments). Experiments 5, 6 and 7 are ‘F 4 M’ experiments. Experiments 8 and 9 are ‘M -+ F’ experiments; however, the label is given to the mother several hours because cannulation of the fetal vessels. TYBC = total iron binding capacity. For details see Results.
ferrin saturation are shown. The mean maternal and fetal values are calculated from the individual values obtained in the 10 maternal samples and in the 8 fetal samples, respectively. There was no trend to lower values during sampling either in the maternal or in the fetal samples. As has been mentioned in Materials and Methods, amniotic fluid, maternal urine as well as fetal urine after delivery of the fetus were sampled. In none of the experiments could we detect any radioactivity in these samples, so these compartments will not play any role in our further considerations. Figure 1 shows the results of one characteristic ‘M + F’ experiment. The maternal disappearance curve is biphasic and can be fitted by linear combination of two exponents. The fetal appearance curve is a complex one with an early well-pronounced S-shape. We shall try to analyze the fetal appearance curves by means of a compartmental model represented in Figure 4. The results of 2 experiments in which the label was injected into the maternal circulation several hours before fetal cannulation and sampling are shown in Figure 2. The maternal disappearance curves are biphasic, the fetal appearance curves are very flat and tend to decline after about 150 min.
dpm /
pmol
lo8
Fe
1
‘“f”--------
0
20
40
60
80
100 time
120
in minutes
Fig. 1. Specific activity of the iron in the maternal (upper curve) and fetal plasma after injection of “Fe into the maternal circulation (experiment 1). When plotted on cartesian scales (open symbols) the fetal specific activity curve shows an early S-shape.
131
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey dpm / pmol
Fe
, LQJ
80
I
I
120
160
1
200
I
time
(
\
240
280 320 in minutes
Fig. 2. Specific activity of the iron in the maternal and fetal plasma (experiment 8,= 0; experiment 9, A A). The 59Fe was injected into the maternal circulation 150 and 200 min prior to fetal sampling.
dpn / pmol Fe
104:
0
lo2 0
I
I
I
20
40
60
I 80 time
Figure 3 shows a characteristic ‘F + M’ experiment. Although there is transport of 59Fe from fetus to mother, the specific activity of the maternal and fetal plasma iron differs by a factor of about 3 X lo3 between the 40th and 120th min. The fetal disappearance curve is biexponential; the maternal appearance curve shows an early S-shape when plotted on a linear scale. The fetuses of the ‘M -+ F’ experiments I,2 and 3 and of the ‘F + M’ experiments 5, 6 and 7 died between 30 min and 1 h after delivery. The distribution of 59Fe over the organs and tissues was measured as described in Materials and Methods. The results are shown in Table II. In the ‘M + F’ experiments 1 and 2 there was no clear difference in the 59Fe distribution pattern as compared with the distribution of 59Fe in the ‘F + M’ experiments. In experiment 3, however, the fetal liver has taken up the 59Fe preferentially.
I
f
100 in minutes
120
Fig. 3. Specific activity of the iron in the fetal (upper curve) and maternal plasma after injection of 59Fe into the fetal circulation (experiment 7).
Theoretical
considerations
The proposed model has to explain the biphasic shape of the disappearance curve at the donor side as well as the flat and early S-shaped appearance curve at the acceptor side. The biexponential disappearance curve of 59Fe can be explained basically in two different ways: 1. By means of early reflux of 59Fe from a second compartment into the plasma iron pool; 2. By means of heterogeneity of the plasma iron compartment in combination with different clearances for the iron specimens. The S-shape of the s9Fe appearance curve can be explained by assuming a placental compartment of exchangeable iron. We constructed 3 models: I. A S-compartmental model (see Fig. 4) in which the maternal compartment 2 and the fetal compartment 5 account for the early maternal and fetal reflux of 59Fe, respectively. A 3rd placental compartment (compartment 3) has been postulated in order to explain the S-shape of the 59Fe appearance curve. It has been assumed that transplacental iron transport occurs in one direction only (M -+ F). A mathematical description of this model is given in the Appendix.
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
132 TABLE II
Distribution of 59Fe over the fetal organs and tissues, the total amount of s9Fe in the fetus as well as the total amount of 59Fe in the fetus relative to the dose of 59Fe injected ‘F-M’
‘M -+ F’
Experiment
Plasma ‘Bone marrow’ Liver Bile Intestine Kidneys Spleen Lung Heart Brain Muscle Total radioactivity in the fetus (dpm) % dose injected
1
2
3
5
6
I
61 14 10 0.1 1.6 0.7 0.3 1.0 0.9 1.5 0.3
62 13 14 0.1 0.5 6.5 0.1 0.4 0.8 1.4 0.2
50 2.0 44 0.2 0.5 0.5 0.2 0.2 0.3 0.8 0.2
48 11 23 0.3 2.0 0.9 0.2 1.2 0.6 1.2 0.3
51 21 12 0.3 1.5 0.7 0.3 1.0 0.4 2.1 0.3
69 14 10 0.1 0.8 0.6 0.3 0.4 0.3 1.0 0.2
2.3 x lo7 98
2.2 x 107 98
6.3 x lo7 97
1.85 X lo6 3.5
1.6 x lo5 0.5
3.2 X lo6 4
The 59Fe distribution is expressed as a percentage of the total amount of 59 Fe present in the fetus at the end of the experiment. The relative amount of s9Fe in the fetus is given as a percentage of the amount of 59 Fe injected into the mother or into the fetus att=O.
II. A 4compartmental bolized by
z(Comp.
1 k
IS
1
k4s
Fig. 4. Five-compartmental model used in our calculations. Compartments 1 and 4 represent the maternal and fetal plasma iron pool. Compartment 3 illustrates a placental iron pool. Compartments 2 and 5 have been introduced in order to explain the early maternal and fetal 59Fe reflux. The rate constant from compartment i to j is given by kij. The subscripts e and s indicate erythropoiesis and storage. Because of the short duration of the experiments it has been assumed that reflux from the e- and s-compartments may be neglected. In addition, one-directional transport from M + F has been assumed; k3r = k43 = 0.
I)$
model which can be sym-
[(Comp. 2)kZ(Comp. ,)l$(Comp.
4)?
Compartments 1 and 2 represent the maternal and the fetal plasma iron compartments, respectively. The ‘maternal’ and ‘fetal’ compartments accounting for the early reflux of 59Fe are postulated in the placenta (compartments 2 and 3). These same compartments also explain the S-shape of the 59Fe appearance curve at the acceptor site. Moreover, one-directional iron transport from compartment 2 to compartment 3 has been assumed. This model did not explain the experimentally obtained fetal appearance curves. A much greater time-lag in the appearance of 59Fe in the fetal circulation, as experimentally observed, is predicted. III. The third model was based on the hypothesis
J.P. vanDijk: Iron metabolism in the pregnant rhesus monkey TABLE III
133
Characterization of the disappearance curves and the calculated values of the rate constants for the experiments l-7 listed in Table I
Number of experiment
nr (min-l)
~Zdn-‘)
m
kr2 (min-l)
kro (min- 1)
kzl (min-‘)
Plasma iron turnover (pmol/day)
Iron reflux (mnol/day)
0.0484 0.0720 0.0831 0.0745
0.0048 0.0040 0.0082 0.0061
0.4923 0.2813 0.3793 0.2223
0.0176 0.0177 0.0245 0.0202
0.0086 0.0054 0.0125 0.0077
0.0269 0.0529 0.0547 0.0527
166 135 208 229
111 103 138 165
41 (min-l)
;In:in-‘)
’
k4s (min -‘I
bo (min-‘)
k.4 (min-‘)
0.0610 0.0485 0.0823
0.0036 0.0030 0.0028
0.3780 0.2381 0.1951
0.0198 0.0105 0.0150
0.0055 0.0038 0.0034
0.0393 0.0376 0.0663
31 11 19
24 8 15
For the meaning of the parameters nr, “2, m and qr, q2, f, refer to eqns. (6) to (13) of the Appendix. The rate constants (k) are defined in the Appendix and are calculated from the same equations. The calculation of the turnover rate and the reflux is shown in the Discussion.
of heterogeneity
sites on the by Fletcher and Huehns (1968). In the sense of this hypothesis the biexponential shape of the 59Fe disappearance curve can be explained by assuming different clearances of the A- and B-site of 59Fe. Mathematically this disappearance curve can be represented by the formula: transferrin
xl(t)
= XlA@)
of
molecule
exp(-6
the
iron
put
forward
It)
’ XIB@)
binding
ex&62t)
in which x(t) represents the amount of 59Fe in the plasma iron compartment at time t. At t = 0 this amount equals xrA t xrn, which means the sum of the total amount of 59Fe bound to the A-site and to the B-site of the transferrin. As has been described in Materials and Methods, we used apotransferrin for the labeling with 59Fe. Since one molecule of transferrin has one A-site and one B-site, statistically we would expect as many Asites as B-sites to be labeled. Extrapolation to t = 0 of the exponents obtained by fitting the disappearance curves shows that xrA and xru are unequal, which can be seen in Table III, where the value of m in 6 out of 7 experiments clearly deviates from 0.5, a value which would be expected in this case. All our further considerations about the kinetics of iron will be based on the assumptions made in
model I. The rate constants ksr and k4s are considered to be negligible, which means that the iron transport is in essence one-directional. This assumption will be discussed below (see Discussion)
Calculation
Here we shall describe the way in which the formulas derived in the Appendix are used to calculate the rate constants. By means of fitting (Riggs, 1970) the biexponential disappearance curves we obtain values for the parameters m, nl, n2 and f, ql and q2. From these parameters we can calculate the rate constants kr2, k2r, kzo and k45r k54 and k40 (see legend to Fig. 4 and eqns. (7) (8) (9) and (1 l), (12) (13) in the Appendix). Table III gives the values of the parameters m, nl, n2 and f, q1 and q2 for the 7 experiments mentioned in Table I. The maternal rate constants kr2, k2r and kr, as well as the fetal rate constants k4s, ks4 and k4, are also shown. The rate constants kra and ka4 can be calculated only when we use the information present in the fetal appearance curves. From the appendix (eqn. (18)) it can be seen that, in order to describe the fetal appearance curve in mathematical terms, knowledge of the
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
134
fetal rate parameters is indispensable. However, in a transport experiment from mother to fetus only the maternal rate constants can be obtained. The fetal rate constants can only be calculated from the reverse experiment. Consequently, krs and ks4 can be calculated only by combining the results from a ‘M --, F’ experiment with the results obtained in a ‘F + M’ experiment. In order to compute the theoretical x4(t) appearance curve (Appendix, eqn. (18)) we need criteria to combine sets of calculated parameters from different experiments. The following quite arbitrary criteria were used: (1) equality within 15% of the maternal and fetal weight ; (2) equality within 15% of the maternal and fetal iron concentration. The computations of the rate constants krs and ks4 are best illustrated by a specific example (combination of experiments 3 and 6). We may write eqn. (18) of the Appendix, describing the fetal appearance curve, as x4(t) = krs *x1(O) *y(ks4, t), which gives: x4(t)
y&34,t) = k13. x1(o) (y represents a function symbol) The only unknown parameter in the function y(k34, t) is the rate constant k34. Values for ql, q2 and f can be found in Table III. b, p and o are calculated from the eqns. (19), (20) and (21) in the Appendix: b= f-q2 +(l - f)qr=ks4=0.038min-’ P = (qr * qz) = k4o * ks4 = 0.0038 X 0.0376 = 1.4 X 10-4min-2 o = (41+ q2) = k 45 + k,, + k40 = 0.0105 t 0.0376 + 0.0038 = 0.052 min-’ We now compute the function y(k34, t) for several values of k34. That value of k34 that gives the best cover of the theoretical function y(ks4, t) with the experimentally obtained fetal appearance curve (experiment 3), when both functions are plotted on the same absolute scale (Fig. 5), is considered the best estimate for ks4. In Figure 5 it can be seen that in this special case k34 = 0.1 results in a reasonably good approximation of the appearance curve.
x4(f) = k13 *x1(0) *Y(~w t) x1(O) = amount of radioactivity (dpm) present in the
16.
8 6 i
*.
2 2
4.
000 0
20
40
60
80
100
120
time in minutes
Fig. 5. The fetal 59Fe appearance curve (experiment 3) and the theoretical y(k34, t) curve calculated for k34 = 0.1 min-l are shown. The y(k34, t) function has been calculated with the rate constants obtained in experiments 3 and 6. For details refer to Calculations.
maternal plasma pool at time 0, which is identical to the injected dose of 59Fe; x1(O) = 11.4 X lo7 dpm (experiment 3) x4(t) = amount of radioactivity present in the fetal plasma pool at time t (experiment 6); the experimentally obtained fetal appearance curve From this equation we can calculate kr3 for several time-points. In the case of an ideal fit ks4 should have the same value at every time-point. In our case, however, kr3 varies between 5.7 X 10M4 and 6.5 X 10B4 with a mean value of about 6.0 X 10T4 min-‘. The assumed steady-state conditions imply the equality kr3 *X1 = k34 *X3, from which we can calculate X3, the total amount of iron present in the placental iron pool. X1 represents the total amount of iron present in the maternal plasma pool (j.dnol). We calculate a placental iron pool of about 2.5 X 10e2 pmol. The daily transplacental iron transport can be computed from the equation: Fe transport = kr3 *X1 . 1440 (pmol/day) We calculate an iron transport of about 3.4 pmol/day (1.06 pmol/day/lOO g fetus). The total amount of radioactivity transported
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
from mother to fetus between time 0 and time t, (A& conforms to the equation: t A:=
s
k,,.xi(t)dt
0
120
AA2’(expt. 3) = 6.0 X 10e4 s
(11.4 X lo7 - 0.3793
0
X e-o.0831 tt 1 1.4 X lo7 - 0.6207 e-“.0082t)dt = 3.3 X lo6 dpm Comparison with the figures given in Table II, experiment 3, reveals AA2’ to be a reasonable estimate. (320 min Moreover, the graphical representation time-scale) of the fitted maternal disappearance curve (experiment 3) and the theoretically obtained x4(t) function (combination of experiments 3 and 6) bears a very close resemblance to the curves shown in Figure 2.
Discussion
Several authors have already stated that the transport of iron from mother to fetus is directed against a concentration gradient (Nylander, 1953; Bothwell et al., 1958). Our results are in agreement with this generally ac, (ted rule (see Table I). The fetal iron binding capacity is a little lower than the maternal one, indicating a lower fetal transferrin concentration. We calculated a mean fetal transferrin saturation of about 57% with a standard deviation of about 11%. The corresponding maternal values are 17% with a standard deviation of 4%. Another commonly accepted rule is that the transplacental iron transport is one-directional, directed from mother to fetus (Bothwell et al., 1958). Comparison of Figures 1 and 3 shows that the transport from mother to fetus is probably much more efficient compared with the transport in the reverse direction. It seems unlikely that this difference can be explained by the relatively large dilution volumes of the maternal serum compartment in combination with somewhat larger rate constants. We therefore assumed a one-directional transport from compartment 1 to compartment 4 (see Fig. 4).
135
As stated in the Introduction, it was our aim to estimate parameters that govern the kinetics of iron in the maternal and fetal serum compartment as well as parameters which determined transplacental iron transport. The model described does not discriminate between active and passive transport mechanism. It only states that the transport is proportional to the amount of iron present in the compartment under study. In the case of fixed concentrations and fixed volumes, such as we assume to exist, the transport is proportional to the concentration. The fact that the 59Fe disappearance curves can be described mathematically by exponential functions means that the statement of proportionality is appropriate. Whenever carrier transports or active transports are involved they must operate far from saturation in this particular case. Consequently the parameters calculated for the transport of 59Fe may be considered as representative for the transport of 56Fe too. From the 3 alternative models mentioned in Theoretical Considerations we chose the Scompartment one because of its better predictive qualities. From Table IV it can be seen that k, 3 is about l/25 klo, which means that the amount of iron transported to the placental compartment and consequently also to the fetus only amounts to 4% of the amount carried to the postulated erythron and storage compartment per unit time. Consequently we may state that where the label is given to the fetus, only a fraction of 59Fe transported to the maternal circulation will be returned to the fetus. This consideration makes it possible to estimate k43 and ksr from eqn. (18) in the Appendix because of the obvious symmetry in the.model. We only have to reinterpret the symbols used. For the combination of the ‘F -+ M’ experiment 6 and the ‘M -+ F’ experiment 3 we calculated a k43 of about 5.5 X lop6 min-’ and a k3r of about 1.2 X lop4 min-‘. In comparison with the product kr3 . X1 the product k43 . X4 is negligible, which indicates that the assumption of transport in one direction is justified. From the rate constants kr2 and kio we can calculate the maternal plasma iron turnover (PIT). PIT = (k,, + kr,) . plasma iron * 1440 @mol/day). The daily reflux (pmol/day) equals k 12 . plasma iron . 1440 because of the equality k,, . X1 = k,, . X2. The calculated maternal and fetal PIT values as well as the reflux values are shown in Table III. The
136
J.P. vanDijk: Iron metabolismin the pregnantrhesusmonkey
TABLE IV
Exp. no.
klZ
3 4
0.0245 0.0189
Characterization of the fetal-maternal S-compartmental system in terms of rate constants, the magnitude of the placental iron compartment X4, and calculated transplacental iron transports kzt
klo
k13
k13
0.0547 0.0755
0.0125 0.0161
6.0 x 1O-4 2.4x 1O-4
(5.7-6.5) X 1O-4 (2.2-2.6)x1O-4
Fe
transport (wnol/day/ 100 g)
k34
k4s
ks4
k40
x4
0.1 0.08
0.0105 0.0105
0.0376 0.0393
0.0038 0.0044
0.025 1.1 0.017 0.8
range
The fetal rate constants obtained from the ‘F + M’ experiments 6 and 5 are taken as representative of the actual values in the ‘M + F’ experiments 3 and 4 respectively. Rate constants in min-‘. Fe transport in pmol/day/lOO g. X4 in pmol Fe.
maternal and fetal iron turnover rates are very high. About 70% of the iron daily leaving the maternal plasma compartment is refluxed. The fetal reflux amounts to 75%. At the moment these results are difficult to interpret. It is known, for instance in the rabbit, that the turnover of the plasma iron as well as the transplacental transport of iron increases with gestation (Douglas, Penton and Watt, 1971). The authors (Douglas et al., 1971) did not calculate which part of the increased turnover was responsible for the transplacental increase of the iron transport. In our case the high maternal PIT values can certainly not be explained by a large transplacental iron transport. Moreover, the same high turnovers are also found in the fetus, suggesting a common principie. In man (Cook, Marsaglia, Eschbach, Funk and Finch, 1970) the mean PIT value was 0.70 mg/lOO ml whole blood per day in the case of iron deficiency. The total reflux - 55% of the PIT value - could be differentiated into an early and a late component (Cook et al., 1970). The early component - 15% of the PIT - had a reentry Tr,a of about 8.5 h and can probably be explained by a lymphatic shunt (Morgan, Marsaglia, Giblett and Finch, 1967; Cook et al., 1970). The late component - 40% of the PIT - with a reentry Tr,a of 7 days is probably wastage iron of erythropoiesis (Cook et al., 1970). Is our reflux identical with the early reflux observed in man, however, with a much higher lymphatic shunt? This question cannot be answered at the moment. The calculated transplacental iron transport (Table IV) amounts to 1 .l and 0.8 pmol/day/lOO g fetus for near-term monkeys. In man this near-term transport is about 4.0 pmol/day/lOO g fetus (Widdowson and
Spray, 1951; Fletcher and Suter, 1969). It can be seen from the appearance curve (Fig. 1) that the transport of 59Fe from M + F is very rapid. In terms of our model this fact indicates a small pool of placental iron. We calculated placental iron compartments of about 0.02 ymol Fe (Table IV). 6 out of 7 fetuses died between 30 min and 1 h after delivery. The distribution of 59Fe over the organs and tissues is shown in Table II. Between the ‘M + F’ experiments 1 and 2 and the ‘F + M’ experiments no clear difference in the 59Fe distribution can be seen. In experiments 3 (‘M + F’), however, the incorporation of 59Fe into the liver is clearly increased, while the 59Fe incorporation into the ‘bone marrow’ is reduced. This observation would be in agreement with the hypothesis of Fletcher and Suter (1969) that the fetal side of the placenta, the ‘storage-directed’ transferrin binding site, preferentially picks up iron from the placenta. However, only 1 of the 3 ‘M + F’ experiments showed a preferential labeling of the liver, so alternative explanations are more likely.
Appendix
Mathematical description of the model illustrated in Figure 4 Let Xi be the amount of labeled iron (dpm) in compartment i. Xi represents the total amount of iron (pmol) in compartment i. Let krj represent the rate constant (min-‘) from compartment i to j. The erythropoietic and storage pools are indicated by e and s, respectively, so k,, represents the rate constant from compartment 1 to the erythropoietic
137
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
compartment(s) (see Fig. 4). For kr, t k,, t k,, we write k,,; the sum k,, + kas will be given by k4c. Between compartments 1, 3 and 4 we assume onedirectional transport. The rate constants kG3 and kar are considered to be negligible (see Theoretical Considerations and Discussion). The following set of equations describes the kinetics of 59Fe in the indicated compartments (see Fig. 4) after injection of the label (bound to transferrin) into the maternal plasma iron compartment: x;(t) = -(krz
+ kzr * x2(t)
+ k,o)xr(t)
(1)
x;(t) = k12 * xl(t)
- kzl * x2(t)
(2)
x;(t)
- k34 * x3(t)
(3)
= k13 * xl(t)
x;(t) = -(k4s +k&q(t)+ks4
*xa(t)+k~.xs(t)
(4) (5)
x;(t) = k4s s x4(t) - kw * xs(t)
Because of the one-directional transport assumed, we may consider eqns. (1) and (2) and eqns. (4) and (5) as two independent sets of equations. When the label is injected into the maternal circulation, the first set of equations can be solved, assuming boundary conditions x2(O) = 0 and x1(O) = A,, A, being the total amount of 59Fe (dpm) injected into the maternal circulation. The general resolution for xl(t) is
amount of 59Fe injected into the fetal circulation. X4(t)
=
x4(0) * f - exp(-qr
+ x4(0) * (1 - f) - exp(-q2t)
k4s + k4o + ks4 = qr + q2
(11)
ks4 * ho = 91.
(12)
b4=f.q2+(1
q2
(13)
-0%
Derivation of the theoretical x4(t) appearance function in the case of injection of s9Fe into the maternal plasma iron compartment 1: substitution of eqn. (6) in eqn. (3) and resolution for x3(t) by Laplace transformation gives: x3(t) = kr3 - xr(O)m * L-’
tkrs*xr(O)(l Application
-m)L-’
of the convolution
(6)
-p
x3(t)= kr3.xr(0)
m + -----exp(-k34t) nr-ka4
kr2+kr0+k2r=nr
+ ----exp(-kk4t) n2 - ks4
+n2
(7)
k2r - kr, = nl - n2
(8)
k2r=m*n:!t(l
(9)
-m)nr
When the tracer dose has been injected into the fetal circulation, x4(t) can be solved from eqns. (4) and (5) assuming boundary conditions x5(O) = 0 and x4(O) = Ar. The symbol Af represents the total
x4(t)
= I_- l
-___-__
t)
- seexp(-n,t)
(1 -m)
(15)
Rearranging eqn. (5) and taking the Laplace transformation, we obtain:
k45* Lb,(t))
(16)
x5(t) = ~-__
s+ kw After rearranging eqn. (4) and substituting and (16) in eqn. (4) we resolve for x4(t):
k13 . elm kl3 ~.xdW k34)(s + nr) ’ (nr - k,,)(s + k34) - (n2 - k,,)(s -__
exp(-nr nr-k34
Values of m, nl and n2 can be obtained by fitting the biphasic maternal disappearance curves. The rate donstants kr2, kro and k2r can be calculated from the following set of equations:
-
theorem results in:
m
-m)*exp(-nzt)
34
(10)
Values of f, q1 and q2 can be obtained by fitting the biphasic fetal disappearance curves. The rate constants kd5, kaO and ks4 can be calculated from the following equations:
x1(t) = x1(O) * m - exp(-ntt) +x,(0)(1
t)
__-_____
. k45 s + k4s + krlo - kss: k 54
-4 + n,)tc
k13 . xdW1 n2 -
b4)(s
eqns. (14)
-m> + k,,)
(17) I
138
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey
The inverse Laplace transformation can be taken by application of the convolution theorem, which yields:
We may now write eqn. (18) in a condensed
x4(t)
=
x4(t)=k13.~1(0).Y(k34,t) where y represents a function
form:
(22)
symbol only.
Acknowledgement
-(nr
- b)qr + bnr t p - ob
(n: _onl+pKr
’ exp(-q2t)
-
x exP(-qtt)
- G-_
---
(1 - m)ks4 n2 -
-(n2
k34
We wish to thank Dr. H.C.S. Wallenburg, Department of Gynecology and Obstetrics, Erasmus University, Rotterdam, for the opportunity given to us to perform this study simultaneously with his own investigation on the purine metabolism in the rhesus monkey.
_ q2)
nl- b onl + p) exp(-nr
t)
- b)qs + bn2 + p - ob
G-2
+ p)
ev-q20
(sl -
q2)
References
- b)qr t bn2 + p - ob
-(n2
~---
-IS
exp(-q
on2
P) (41 -
+
1 t>
Awai, M., Chipman, B. and Brown, E.B. (1975): In vivo evidence for the functional heterogeneity of transferrin bound iron. II. Studies in pregnant rats. J. Lab. clin. Med.,
q2)
n, - b
-(n22-
x
on2
-(k,,
85, 785-796. +
Baker, E. and Morgan, E.H. (1969): The role of transferrin in placental iron transfer in the rabbit. Quart. J. exp. Phy siol., 54, 173-186. Bothwell, T.H., Pribilla, W.F., Mebust, W. and Finch, C.A. (1958): Iron metabolism in the pregnant rabbit: iron transfer across the placenta. Amer. J. Physiol., 193, 615-
P)
622.
- b)q2 + b * ks4 + p - ob wGq2
(kg4
-
-
ok34
+
t)
P) (qr - 92)
-(k34
- b)q, t bk34 + p - ob -__ exp(-qr (k& - ok34 + p)(qr - 92)
k,, - b
- ~-_._exp(-ks4t) (k$ - ok34 + P)
t)
(18)
As can be seen, x4(O) = 0. The dimension of x4(t) is dpm. The formula within square brackets is a function of t and k34; all other parameters can be calculated from the individual ‘M + F’ and ‘F + M’ experiments by fitting the obtained disappearance curves (eqns. (6) and (10)). The parameters b, p and o are calculated from the following equations: b=f*q,+(l-f)q,=ks4
(19)
P = qt . qz = ks4 * k4o
(20)
o = qr + qz = k4s + k,o + ks4
(21)
Cook, J.D., Marsaglia, G., Eschbach, J.W., Funk, D.D. and Finch, C.A. (1970): Ferrokinetics: a biological model for plasma iron exchange in man. J. clin. Invest., 49, 197205. Douglas, T.A., Penton, J.P. and Watt, C. (1971): Placental transfer of iron in the rabbit. &it. J. Haemat., 20, 185194. Fletcher, J. and Huehns, E.R. (1968): Function of the transferrin. Nature (Lond.), 218, 12 1 l- 1214. Fletcher, J. and Suter, P.E.N. (1969): The transport of iron by the human placenta. Clin. Sci., 36, 209-220. Harris, J.W. and Kellermeyer, R.W. (1970): The Red Cell, p. 95. Harvard University Press, Cambridge, Mass. King, B.F. (1976): Localisation of transferrin on the surface of the human placenta by electron microscopic immunocytochemistry. Anat. Rec., 186, 15 l-160. Lanzkowsky, P. (1961): The influence of maternal iron deticiency anemia on the hemoglobin of the infant. Arch. Dis. Childh., 36, 205-209.
Larking, C.E., Weintraub, L.R. and Crosby, W.H. (1970): Iron transport across rabbit allantoic placenta. Amer. J. Physiol., 218, 7-l 1. Laurell, C. and Morgan, E. (1964): Iron exchange between transferrin and the placenta in the rat. Actu physiol. stand., 62, 271-279.
139
J.P. van Dijk: Iron metabolism in the pregnant rhesus monkey Mansour, M.M., Schubert, A.R. and Glasser, S.R. (1972): Mechanism of placental iron transfer in the rat. Amer. J. Physiol., 22, 1628-1633. Morgan, E.H., Marsaglia, G., Giblett, E.R. and Finch, C.A. (1967): A method of investigating internal iron exchange utilizing twu types of transferrin. J. Lab. clin. Med., 69, 370-381. Nplander, G. (1953): On the placental transfer of iron: an experimental study in the rat. Acta physiol. stand., 29, Suppl. 107. Riggs, D.S. (1970): The Mathematical Approach to Physiological Problems, pp. 146-161. MIT Press, Cambridge, Mass. and London. Tavenier, P. (1971): Onderzoek naar het niet transferrinegebonden ijzer in het plasma. Thesis, Medical Faculty, Rotterdam. Trinder, P. (1956): The improved determination of iron in serum. J. clin. Pathol., 9, 170-172. Van Eijk, H.G. and Leijnse, B. (1968): The iron transport-
ing system
in the body.
I. Isolation
and partial
charac-
terization of rabbit transferrin. Biochim. biophys. Acra, 160, 126-128. Verhoef, NJ., Kremers, J.H.W. and Leijnse, B. (1973): The effect of heterologous transferrin on the uptake of iron and haem synthesis by bone marrow cells. Biochim. biophys. Acta, 304, 114-122. Verhoef, N.J. and Van Eijk, H.G. (1975): Isolation, characterization and function of cord blood transferrin. Clin. Sci. molec. Med., 48, 335-440. Widdowson, E.M. and Spray, C.M. (1951). Chemical development in vitro. Arch. Dis. Childh., 26, 204-214. Wong, C.T. and Morgan, E.H. (1973). Placental transfer of iron in the guinea pig. Quart. J. exp. Physiol., 58, 47-58. Wyllie, J.C. and Kaufman, N. (1974): Effect of metabolic inhibitors and chelating agents on 59Fe uptake by the placental tissues of the rat, Rattus norvegicus. Comp. Biothem. Physiol., 48A, 361-367.