British Journal of Haematology, 1999, 104, 296–302
`
`Pharmacokinetics and red cell utilization of iron(III)
`hydroxide–sucrose complex in anaemic patients:
`a study using positron emission tomography
`
`SOHEIR BESHARA,1 HANS LUNDQVIST,2,3 JOHANNA SUNDIN, 2,3 MARK LUBBERINK, 2 VLADIMIR TOLMACHEV,2
`1 1Department of Medical Sciences,
`3
`SVEN VALIND,3,4 GUNNAR ANTONI,3 BENGT LA˚ NGSTRO¨ M
`AND BO G. DANIELSON
`Renal Section, 2Department of Biomedical Radiation Sciences, 3PET Centre, and 4Department of Clinical Physiology,
`University Hospital, Uppsala, Sweden
`
`Received 10 August 1998; accepted for publication 9 November 1998
`
`Summary. The pharmacokinetics of a single intravenous
`injection of 100 mg iron hydroxide–sucrose complex labelled
`with a tracer in the form of 52Fe/59Fe was followed in six
`anaemic patients for a period ranging from 6 to 8·3 h using
`positron emission tomography (PET). Red cell utilization of
`the labelled iron was followed for 4 weeks. PET data showed
`radioactive uptake by the liver, spleen and bone marrow. The
`uptake by the macrophage-rich spleen demonstrated the
`reticuloendothelial uptake of this iron preparation, with
`subsequent effective release of
`that
`iron for marrow
`utilization. Red cell utilization,
`followed for 4 weeks,
`ranged from 59% to 97%. The bone marrow influx rate
`
`constant was independent of blood iron concentration,
`indicating non-saturation of the transport system in bone
`marrow. This implied that higher doses of the iron complex
`can probably be used in the same setting. A higher influx
`rate into the marrow compared with the liver seemed to be
`consistent with higher red cell utilization. This would
`indicate that early distribution of the injected iron complex
`may predict the long-term utilization.
`
`positron
`pharmacokinetics,
`iron sucrose,
`Keywords:
`emission tomography (PET), 52Fe,59Fe, red cell utilization.
`
`Erythropoietic activity is known to be closely associated with
`marrow iron uptake (Cazzola et al, 1987) and parenteral
`pharmaceutical iron administration has been applied for the
`treatment of iron deficiency in different clinical settings. In a
`previous study (Beshara et al, 1999), the kinetic analysis of
`the early distribution characteristics of the 52Fe-labelled
`iron(III) hydroxide–sucrose complex, an increasingly used
`new form of parenteral iron therapy, was assessed using
`positron emission tomography (PET) in pigs. The quantita-
`tive ability of the PET technique was verified and the
`methodological approach using 52Fe was assessed. Various
`calibration factors involved in the quantification of 52Fe
`using PET were described in detail. In addition, the longer
`half-life of 59Fe (T1/2 ¼ 45 d) (Browne & Firestone, 1986)
`enabled a thorough follow-up of red cell utilization of iron
`from the injected labelled iron complex. Whether similar
`uptake and distribution characteristics of the complex could
`be found in anaemic patients subjected to iron therapy and
`
`Correspondence: Dr Soheir Beshara, Department of Medical
`Sciences, University Hospital, S-751 85 Uppsala, Sweden.
`
`296
`
`how readily the iron is utilized once it has entered tissues are
`questions of particular importance.
`The aim of this study was to assess the early pharmaco-
`kinetics of the labelled iron(III) hydroxide–sucrose complex
`in patients with anaemia using the PET technique, as well as
`the long-term red cell utilization of this iron complex.
`
`METHODS
`
`Patients. Six patients (two males and four females) aged
`24–63 years were included in the study. Baseline haemo-
`globin ranged from 7·7 to 12·1 g/dl. Four patients were
`treated with recombinant human erythropoietin (rHuEpo)
`with a dose which ranged from 30 to 150 U/kg/week. None
`of the patients had received blood transfusions or iron
`supplementation during the 4 weeks prior to inclusion in the
`study. Causes of anaemia other than iron deficiency (IDA),
`renal anaemia (RA) or functional iron deficiency (FID) were
`excluded and iron supplementation therapy outside the
`study protocol was not allowed. According to the definition
`of FID and RA, patient 6 could be classified as either FID or
`
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`Table I. Baseline characteristics of the patients.
`
`Pharmacokinetics and Red Cell Utilization of 52Fe/59Fe-sucrose
`
`297
`
`Age
`(yr)
`
`No.
`
`Sex Diagnosis
`
`Hb
`(g/dl)
`
`S-creatinine S-Fe
`((cid:109)mol/l)
`((cid:109)mol/l)
`
`S-ferritin TIBC
`((cid:109)g/l)
`((cid:109)mol/l)
`
`TS
`(%)
`
`Dose
`(U/kg/week)
`
`Duration
`(months)
`
`rHuEpo therapy
`
`1
`2
`3
`4
`
`5
`6
`
`63
`38
`52
`24
`
`55
`38
`
`Polycystic kidney þ RA
`f
`Medullary spone kidney þ IDA
`f
`m Diabetic nephropathy þ FID
`m Status post renal transplantation
`þ RA
`7·7 253
`Ulcerative colitis, renal stones þ IDA 10·0
`86
`Congenital cystic disease þ FID
`11·6 157
`
`11·2 653
`10·9
`72
`12·1 261
`
`f
`f
`
`9
`12
`11
`
`30
`3
`7
`
`81
`3
`95
`
`501
`8
`37
`
`42
`101
`58
`
`46
`85
`51
`
`50
`22
`12 —
`18
`30
`
`90
`65
`4 —
`14
`150
`
`4
`—
`44
`
`12
`—
`2
`
`S-Fe ¼ serum iron; TIBC ¼ total iron binding capacity; TS ¼ transferrin saturation; rHuEpo ¼ recombinant human erythropoietin; IDA ¼ iron
`deficiency anaemia; RA ¼ renal anaemia; FID ¼ functional iron deficiency.
`
`RA (Ho¨rl et al, 1996). Baseline and clinical characteristics of
`the patients are described in Table I. The study was approved
`by the Ethics Committee and the Isotopes Committee of the
`Medical Faculty of Uppsala University and informed consent
`was obtained from all patients. The PET centre is licensed
`by the Medical Products Agency, Uppsala, Sweden,
`to
`manufacture radiolabelled pharmaceuticals
`for use in
`humans.
`Radiopharmaceutical preparation. Radiolabelled iron(III)
`hydroxide–sucrose complex was prepared as described
`previously (Beshara et al, 1999). The cyclotron at the
`Svedberg Laboratory, Uppsala, Sweden, was used to produce
`52Fe. 59Fe was added as a second tracer for the evaluation of
`red cell utilization. 59Fe as iron chloride was purchased from
`Amersham, U.K., as a solution in 0·1 M HCl. Before the
`synthesis of the iron complex, 59Fe and 52Fe were mixed with
`activities in the ratio of 1 to 40 respectively and the total
`volume was adjusted to 1 ml. 100 mg of sucrose were
`dissolved in the solution of radioiron chloride. The solution
`was sterile filtered and NaOH (10 N) was added to adjust the
`pH to 11·8. Finally, 5 ml of iron hydroxide–sucrose complex
`(Venofer(cid:210)
`, 20 mg Fe/ml), containing 100 mg iron, were
`added to obtain the injection bolus, according to the
`manufacturer’s
`instructions
`(VIFOR International AG,
`Switzerland).
`injected radioactivity given to the
`The amount of
`individual patients ranged from 8·4 to 21 MBq. The effective
`dose equivalent was calculated using a simple model
`according to the International Committee for Radiation
`Units and Measurements (1979). The effective dose equiva-
`lent was found to be <0·5 mSv/MBq and was in accordance
`with those presented by Ferrant et al (1993). The dose level
`in our patient studies was considerably below 10 mSv and
`was found to be at an acceptable level
`for diagnostic
`purposes.
`PET imaging. PET measurements were performed with the
`General Electric Medical Systems (GEMS) PC 4096-15 WB
`scanner, as described elsewhere (Beshara et al, 1999). A
`standardized supine position was selected so as the heart,
`liver, spleen and thoracic vertebrae were situated in the field
`
`of view. Emission measurement times were 1 min for the first
`15 frames, then 15 min for the following frames. Intercali-
`bration of the tomograph versus ionization chamber and
`well counters was performed regularly. Emission scans were
`performed for the first 90 min, followed by transmission and
`emission scans at 3, 4, 5, 6, 7 and 8 h following tracer
`injection.
`Correction52mMn in the blood and calibration procedure. A
`complication when using 52Fe in physiological studies is the
`radioactive daughter 52mMn, which also emits positrons. The
`technique used to compensate for this effect in order to allow
`quantitative kinetic information and the standard technique
`applied for general calibration of the ionization chamber,
`tomograph and well counters have been described (Beshara
`et al, 1999).
`Data analysis. Analysis of PET data in order to calculate
`standardized uptake values was applied as described before
`(Beshara et al, 1999). The net influx from blood to tissue and
`the sizes of the reversible pools were analysed using a three-
`compartment model, namely blood, reversible and irrever-
`sible tissue pools (Rutland, 1979; Patlak et al, 1983; Patlak &
`Blasberg, 1985).
`two separate
`Sampling. Before the PET examination,
`venous Venflon(cid:210)
`cannulae were inserted to enable tracer
`injection and blood sampling, respectively. Nine blood
`samples of 2 ml each were drawn during the scanning
`procedure and the radioactivity in each sample was then
`measured in a well counter that was cross-calibrated with
`the scanner. Additional blood samples were withdrawn at
`1 and 2 h to correct for the influence of varying 52mMn
`radioactivity concentration in blood. Samples for red cell
`utilization of the labelled iron and for assessment of iron
`status were drawn on an outpatient clinic basis.
`Red cell utilization (%) of the labelled iron. Samples were
`withdrawn three times in the first week, then twice weekly
`for the following 3 weeks. Samples were divided into two
`portions, one of which was kept as whole blood and the other
`portion was centrifuged and packed red cells, washed twice,
`were collected. The radioactivity was measured in whole
`blood, plasma, and packed red cells separately to define the
`
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`298
`Soheir Beshara et al
`rate of disappearance of radioactivity from the plasma and to
`confirm that the whole blood radioactivity would represent
`the radioiron red cell incorporation. The red cell utilization
`(%) was then calculated as follows:
`
`total blood volume (ml) · 100
`
`Red cell utilization (%) on day n ¼
`WBA/ml on day n
`injected 59Fe radioactivity
`where WBA is whole blood radioactivity. No correction was
`applied for the whole body/venous haematocrit (Hct), since
`the total blood volume calculations were made directly
`from measurements of blood radioactivity concentrations
`determined in the left ventricle.
`Determination of total blood volume. This was based on the
`initial dilution of the administered isotope. Linear regression
`was applied, using PET data from the heart, typically points
`between 10 and 30 min and extrapolated to the time of
`injection. PET data were corrected for 52mMn equilibration
`
`factor. In one patient (no. 3), the regression line was
`extrapolated back to 5 min as the infusion was slow.
`
`Total blood volume (ml) ¼
`injected activity (Bq)
`whole blood radioactivity concentration
`extrapolated to 0 min (Bq/ml)
`
`The blood volumes as determined from the PET data were
`compared with the predicted blood volumes (Nadler et al,
`1962).
`iron
`Measurement of Hb and iron status. Assessment of
`iron
`status included measurement of serum iron, total
`binding capacity, transferrin saturation and serum ferritin.
`They were measured with the standard methods at baseline
`and followed for 4 weeks.
`Statistical analysis. Data were analysed with the Excel SE þ
`Graphics package (Microsoft Excel version 7.0, Microsoft
`Corporation, U.S.A.). The graphical method used to analyse
`
`Fig 1. Changes in the concentration of 52Fe in the tissues over time are shown in two PET tomographs in one of the patients. L ¼ liver; BM ¼ bone
`marrow; S ¼ spleen.
`
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`

`RESULTS
`
`Pharmacokinetics and Red Cell Utilization of 52Fe/59Fe-sucrose
`299
`isotope in relation to the amount of injected isotope/g body
`PET data (Rutland, 1979; Patlak et al, 1983; Patlak &
`Blasberg, 1985) was applied to study the kinetics of 52Fe in
`weight (bw). Mean uptake values of 10 and 5 were reached,
`followed by a slower steady increase. A slight decrease in
`the tissues. The linear part of the graphical analysis was
`radioactivity was observed in the liver in all the patients by
`analysed by linear regression as described earlier (Beshara
`the end of the PET investigation, whereby the radioactivity
`et al, 1999). The slope was interpreted as an influx rate
`reached an average of 75% of the peak value. In the bone
`constant while the intercept gave an understanding of the
`marrow uptake curves, rapid radioactivity uptake was seen
`apparent distribution volume of the injected iron for the
`during the first 100 min,
`followed by an influx of the
`different tissues. Linear regression was applied for blood
`radioactivity into the bone marrow at a lower but steady
`volume calculation.
`rate. The maximum uptake by the marrow was higher than
`that by the liver in all the patients but two (nos. 3 and 4).
`Representative uptake curves in the liver, spleen and the
`bone marrow in one of the patients are shown in Fig 2.
`Correction of blood data for 52mMn activity. The activity ratio
`52mMn/52Fe, which at physical equilibrium should be close
`to 1, was found in the blood to be 0·22 ⫾ 0·07 (mean ⫾ SD).
`Taking into account
`the positron abundance for each
`radionuclide, this gave a PET signal that was 0·51 times
`lower than during physical equilibrium (Beshara et al,
`1999). In tissues, where the daughter 52mMn is trapped, 52Fe
`and 52mMn can be assumed to be in physical equilibration.
`Therefore, when comparing tissue radioactivity concentra-
`tions with blood values obtained from the heart, the latter
`values were divided by 0·51.
`Graphical analysis. In the bone marrow, equilibration
`between blood and reversible tissue pool was very rapid
`and a linear relationship was found from the very beginning
`of the experiment. For the liver, with a longer equilibration
`phase, the linear relationship was not obtained until 90 min.
`Therefore the last data point in the first scanning session
`obtained at 90 min and the following data points were used
`in the linear regression, whereas for the bone marrow all
`data points were used. The intercepts of the linear part of the
`liver in the individual patients ranged from 1·18 to 2·24,
`whereas the linear part of the bone marrow gave a very small
`intercept which ranged from ¹0·41 to 0·63. The slopes of
`the linear part of the graphical analysis of the bone marrow
`were 1·3–4·5 times higher than that of the liver. Results
`from the linear regression are seen in Table II.
`
`Changes in the radioactivity concentration in the different
`tissues over time are shown in Fig 1.
`
`Pet measurements
`The time span of the kinetic studies in the patients ranged
`from 6 to 8·3 h. Only one tomograph position was used in
`each patient for viewing the liver, thoracic vertebrae and
`heart. Data for analysis of blood kinetics were obtained from
`an area of interest which was selected in the centre of the
`cardiac left ventricle. However, since the patients were
`allowed to leave the camera after every scanning session and
`exact repositioning could not be guaranteed, blood data from
`the well counter were obtained simultaneously with the PET
`data during the first session and a correction factor was
`determined so that the well counter data taken during the
`following sessions could be applied to later points when
`calculating blood kinetics. The factor calculated to adjust
`both measurements ranged from 0·3 to 0·4. Such a factor
`was calculated and applied in each patient.
`Blood kinetics. The radioactivity in the blood, measured by
`PET in the left ventricle of the heart, was found to have a
`rapid clearance phase followed by a slow one, reaching a
`value which ranged from 2% to 13% of the peak activity by
`the end of the study.
`Organ uptake. Fast radioactivity uptake was seen in the
`liver and spleen during the first 60 and 20 min, respectively.
`Uptake values were measured as tissue concentration of the
`
`䉷 1999 Blackwell Science Ltd, British Journal of Haematology 104: 296–302
`
`Fig 2. Representative 52Fe uptake curves in the
`liver, spleen and bone marrow in one of the
`patients.
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`300
`
`Soheir Beshara et al
`Table II. Results of the PET investigation.
`
`Graphical analysis
`
`Liver
`
`Kt (min¹1)
`
`0·0052
`0·0023
`0·0071
`0·0071
`0·0040
`0·0039
`
`Pt
`
`1
`2
`3
`4
`5
`6
`
`Vd
`
`2·24
`1·28
`1·37
`1·77
`1·18
`1·61
`
`Bone marrow
`
`Spleen
`
`Kt (min¹1)
`
`Vd
`
`Kt (min¹1)
`
`0·0152
`0·0058
`0·0096
`0·0092
`0·0123
`0·0176
`
`¹0.41
`0.34
`¹0.38
`0.25
`0.63
`0.50
`
`0·0041
`0·0020
`—
`0·0061
`0·0030
`—
`
`Vd
`
`1·05
`0·72
`—
`0·72
`0·56
`—
`
`Change in Hb and red cell utilization (%) of the labelled iron
`Determined and predicted blood volumes are presented in
`Table III. In all the patients, predicted blood volumes were
`within ⫾10% of the calculated blood volumes.
`The red cell utilization of the administered iron ranged
`from 59% to 97% by the end of the study. In patient 4, six
`erythrocyte concentrates were given for severe anaemia on
`days 10–13. Red cell utilization was followed again from day
`23 to 28. The red cell utilization in this patient reached 57%
`by day 6, dropped to 36% on day 23 and rose again to 59%
`on day 28. In all the patients but two (nos. 3 and 4),
`
`elevation in Hb levels ranged from 0·2 to 2·0 g/dl above the
`baseline by the end of the study. Follow-up of 59Fe red cell
`utilization is shown in Fig 3.
`
`Iron status
`In all the patients but two (nos. 3 and 6), elevations in the
`serum ferritin and transferrin saturation levels above the
`baseline were seen within 24 h and 1 week, but declined to
`baseline within 3–4 weeks. In patient 2 the elevation seen in
`the serum ferritin level at 1 week was accompanied by a
`decrease in transferrin saturation and serum iron values.
`
`Fig 3. Follow-up of 59Fe red cell utilization.
`
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`

`Pharmacokinetics and Red Cell Utilization of 52Fe/59Fe-sucrose
`Table III. Determined and predicted blood volumes.
`
`301
`
`Pt
`
`1
`2
`3
`4
`5
`6
`
`Weight
`(kg)
`
`Height
`(m)
`
`Determined blood
`volume (ml)*
`
`Predicted blood volume
`(ml)†
`
`79
`58
`97
`89
`64
`68
`
`1·72
`1·58
`1·76
`1·95
`1·62
`1·70
`
`4772
`3433
`5178‡
`5927
`3794
`4370
`
`4609
`3507
`5727
`6190
`3814
`4182
`
`* Determined blood volume was calculated using PET data points from the heart between
`10 and 30 min. The regression line was extrapolated to time 0.
`† Predicted blood volume was calculated from the weight and height according to the
`equations described by Nadler et al (1962).
`‡ In this patient, the infusion time was longer than 5 min, therefore the regression line was
`extrapolated to 5 min.
`
`Adverse events
`No adverse events were experienced by any of the patients
`during or following the injection of the drug. One patient (no.
`4) received 6 units of erythrocyte concentrate 9 d after the
`PET investigation over 4 consecutive days due to severe
`anaemia. He was hospitalized for this reason and was
`temporarily withdrawn from the study on day 9 and
`reincluded on day 23.
`
`DISCUSSION
`
`There was a pronounced uptake of the radioactive iron by
`the liver, spleen and the bone marrow which was consistent
`with our earlier results found in pigs (Beshara et al, 1999)
`and in accordance with the major pathways of internal iron
`exchange as previously described (Finch et al, 1970). Of
`particular importance here is whether the injected iron
`entered the macrophages or parenchymal cells. This could be
`relevant to both its availability and potential toxicity. The
`radioactive uptake by the macrophage-rich spleen in this
`study was thought to be representative of the reticulo-
`endothelial iron uptake of this intravenous iron preparation.
`The graphical analysis showed a fairly high distribution
`volume but a low influx rate, which was constant in the liver
`and spleen as compared with the bone marrow, and varied in
`the patients according to baseline iron status. This may reveal
`that, although the immediate distribution is mainly influenced
`by the iron stores, the reticuloendothelial handling of the
`complex acts as a short-term storage to be followed by a
`subsequent effective release of this iron for marrow utilization.
`In our previous study the blood to tissue transfer rate
`constants were found to be steady over the range observed
`(Beshara et al, 1999). A similar finding was found in patients
`who underwent PET scanning for longer periods than those
`for pigs. Of particular interest is that the marrow uptake did
`not reach a plateau. Moreover, the blood to marrow transfer
`rate constants were found to be stable from the very
`beginning and were independent of the variation in blood
`iron concentration. These results indicate that, despite the
`
`high doses of iron administered, the transport system to bone
`marrow was not overloaded. This may imply that higher
`doses than the one given in this study can probably be given
`in the same setting.
`Using the PET data, the blood volume seems to be well
`estimated, since the determined blood volumes were within
`⫾10% of the predicted ones according to weight and height
`(Nadler et al, 1962). This has the advantages that the very
`early changes in blood kinetics can be visualized and the
`frequent observations of 52Fe in the blood can be made
`without withdrawing any blood from the subject, which is of
`special importance in anaemic patients. PET data points
`between 10 and 30 min were used for blood volume
`calculation. These data points were chosen as representative
`of the early steady phase of the blood kinetics. Data points
`earlier than 10 min were excluded in order to allow a
`thorough distribution of the injected isotope and to minimize
`the influence of the varying 52mMn (Buck et al, 1996).
`Red cell iron utilization showed values which ranged from
`59% to 97%. A higher influx rate into the marrow compared
`with the liver seems to be consistent with better utilization,
`as seen in patient 6. In contrast, a higher uptake by the liver
`than that by the marrow was associated with lower
`utilization as in patient 3. The low utilization in this patient
`was also consistent with elevated iron variables by the end of
`the study. This would indicate that the early pharmaco-
`kinetics of the injected iron complex using PET may predict
`the long-term utilization.
`Patient 4 had a red cell utilization of 57% by the sixth day,
`probably due to severe anaemia. The decreased red cell
`utilization seen after erythrocyte concentrate transfusion
`can be explained by the haemolytic component described in
`the anaemia of renal failure (Gutmann & Schwartz, 1990) in
`addition to the depression of erythropoiesis in response to
`transfusion. Extended follow-up of this patient revealed a
`regain in red cell utilization of the injected complex, which
`reached 59% by the end of the study.
`The return of iron variables to baseline levels by the end of
`the study indicated processing and utilization of the injected
`
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`302
`Soheir Beshara et al
`iron. In patient 2, replenishment of the depleted stores was
`shown by the elevated S-ferritin associated with decrease in
`S-iron and transferrin saturation at 1 week. However, high
`red cell utilization was seen during follow-up and was
`associated with the return of S-ferritin to baseline levels.
`
`ACKNOWLEDGMENTS
`
`The authors thank Kathrin Lindstro¨m and Yvonne Lund-
`holm for their assistance with patient data collection and Iva
`Kulhanek for secretarial assistance.
`
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