In vivo distribution and speciation of [^114mIn]InCl₃ in the Wistar rat

Abstract

Five Wistar rats were given an intraperitoneal injection of [^114mIn]InCl₃ during four consecutive days. One hour after the last injection the rats were sacrificed. The in vivo distribution of ^114mIn was studied in the blood and in different organs. Differential centrifugation was used to study the distribution in liver, kidney and spleen homogenate. Rat serum, packed cell lysate, urine and the cytosol of liver, kidney and spleen homogenate were examined by size exclusion fast protein liquid chromatography. The results showed that serum accounts for 90% of the indium activity in whole blood. Indium is preferentially accumulated within the liver, spleen and kidney, the highest amount of ^114mIn being localised in the cytosolic fraction followed by the mitochondria. Size exclusion experiments showed that, in rat serum, indium is exclusively bound to transferrin. These results differed from earlier in vitro incubation experiments of human serum with ^114mIn. It was not possible, from the experiments described herein, to conclude unequivocally whether indium is bound to haemoglobin of packed cell lysate or to another high molecular mass compound. Indium is associated with the high molecular mass fraction in liver, kidney and spleen cytosol; only in kidney are small amounts of ^114mIn found in the low molecular mass fraction. The in vivo inhibitory effect of indium on the δ-aminolaevulinic acid dehydratase (ALAD) enzymatic activity in red blood cells and kidney tissue, well documented by other researchers, could not be attributed to direct binding of indium with this enzyme.

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Introduction

Indium is widely used in the micro-electronics industry as a component in III–V semiconductor materials. InAs, InP and CuInSe₂ are used in photovoltaic cells, light emitting diodes, laser diodes, high frequency microwave devices, millimetre wave telecommunications and ultrafast supercomputers. Indium is also incorporated in several alloys, solders, thermometers, infrared detectors, etc. Radioactive isotopes of indium have applications as diagnostic agents in cancer treatment and are used for organ scanning in nuclear medicine.^1–3 Although the world annual production of indium amounted only to 200 tonnes in 1996,⁴ the superior performance characteristics of III–V indium-containing semiconductors will increase their use and therefore production. Indium is considered to be highly toxic to mammals, with the kidneys and liver being the major target organs, depending on the physicochemical form in which indium is administered.⁵In vivo experiments on test animals have proven that indium causes teratogenic and embryopathic effects.^6,7 The testicular toxicity of several indium-containing compounds has been recognised.⁸ Indium is capable of altering the haem biosynthetic pathway in liver, kidney and erythrocytes.^9–11 Indium induces apoptosis in rat thymocytes at low doses and causes necrosis at higher doses.¹² Indium causes severe damage to the lungs of animals.^13,14 For these reasons, indium has become an element of interest in clinical, toxicological and analytical studies. However, only minor attention has been given to fractionation and speciation techniques. As the toxicity, bioavailability and metabolism are highly dependent on the chemical form in which the metal is present, speciation experiments are important.^15–18 It is the aim of this work to study the in vivo distribution of indium in different organs, the partitioning of indium within the major target organs and the chemical speciation of indium in serum, packed cell lysate, urine and the cytosoplasmic fraction of the main target organs. The in vivo partitioning within liver, kidney and spleen tissue was studied with the aid of differential centrifugation analysis, and size exclusion fast protein liquid chromatography was used for fractionation and speciation experiments of tissue homogenate. In vivo experiments were carried out on five male Wistar rats subjected to intraperitoneal injection of carrier-free ^114mIn tracer.

Experimental

Reagents

Human albumin (96–99%), apotransferrin (>98%) and N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid) (HEPES, 99.5%) were purchased from Sigma (St. Louis, MO, USA). NaCl (p.a.), NaOH (p.a.), NaHCO₃ (p.a.), HNO₃ (65%, p.a.), HCl (32%, p.a.) and HOAc (99–100%, p.a.) were purchased from Vel (Leuven, Belgium). HBr (47%, p.a.), 8-hydroxyquinoline (p.a.) and NH₃ (25%, p.a.) were obtained from Merck (Darmstadt, Germany). Fluka (Milwaukee, WI, USA) supplied Dowex 50Wx4 ion exchange resin. The nitric acid and hydrochloric acid were further purified by sub-boiling. Deionised water was further purified using a Milli-Q (Millipore, Bedford, USA) system. The antisera for albumin, transferrin and immunoglobulin G, used for the nephelometric determinations of these proteins, were purchased from Dade Behring (Liederbach, Germany).

Production of the ^114mIn radiotracer

The production of ^114mIn is based on the nuclear reaction ¹¹⁴Cd (p,n) ^114mIn. A cadmium foil (99.999%, Alfa, Karlsruhe, Germany) was irradiated with protons (energy, 18 MeV) in the cyclotron at the institute. After a cooling period of 30 days, necessary for the decay of ¹¹¹In (t_1/2 = 2.8 days), the cadmium foil containing the ^114mIn tracer (t_1/2 = 49.5 days, E_γ = 190.3 keV) was separated from the cadmium target by means of liquid extraction and ion chromatography.¹⁹ The procedures were adapted from Katheryn²⁰ and Stary,²¹ and Chattopadhyay,²² respectively. The activity in the samples was measured with an NaI(Tl) (3 in × 3 in well type) scintillation detector coupled to a single channel analyser, or with a Ge(Li) semiconductor detector (Canberra series 40 MCA, CT, USA).

Treatment of animals

Five male Wistar rats weighing between 200 and 250 g were maintained in individual polycarbonate cages (Iffa Credo, Milan, Italy) in an environmentally controlled facility on a 12 h light/dark cycle. The animals were allowed to acclimatise for 5 days. Food and water was supplied ad libitum. Each rat was given a daily intraperitoneal injection of 500 µl containing carrier-free 17.5 µCi [^114mIn]InCl₃ on four consecutive days. A minimal amount of 70 µCi was necessary for later measurements. Consecutive injections were preferred to mimic steady state conditions. The indium tracer was dissolved in a physiological, HEPES buffered saline solution. 1 h after the last injection the animals were sacrificed. Blood was taken from the arteria carotis. Urine was collected only on the second day. Kidney, liver, spleen, heart, lung, testes, bone, skin, thyroid glands, bladder, stomach and small intestine were removed and washed with 0.9% NaCl solution. Blood, urine and organs were stored at −20 °C prior to analysis.

Sample preparation, centrifugation and chromatographic separations

Centrifugation of blood and the differential centrifugation of tissue homogenate up to 10 000g were performed with a Centra MP4R centrifuge (IEC, Needham Heights, USA). Differential centrifugation of tissue homogenate at higher speed (between 10 000g and 100 000g) was performed with the Optima^™ TLX System with a TLA-120.2 fixed angle rotor (Beckman, Palo Alto, CA, USA). In the latter case, 1 ml polycarbonate centrifuge tubes (Beckman, Palo Alto, CA, USA) were used.

Depending on the amount of ^114mIn and the mass of the organ, the total organ or a portion of it was transferred into polyethylene counting vials and its radioactivity was measured with a NaI(Tl) scintillation detector for 5 min.

Blood was centrifuged twice at 2000g and 4 °C for 20 min. The packed cells were washed twice with 0.9% NaCl solution. Both the serum fraction and the packed cell fraction were diluted to the same volume and their ^114mIn activity was measured with a Ge(Li) detector. The measurement time was set to keep the relative standard deviation below 1%.

Organs were minced and a 1 + 4 suspension in 0.25 M sucrose solution containing 15 mM HEPES, pH 7.4 was lysed in a borosilicate Potter–Elvehjem type homogeniser fitted with a Teflon pestle. For the intracellular fractionation of ^114mIn in liver, kidney and spleen, the following five step centrifugation programme was used: 300g, 10 min, 4 °C for the removal of intact cells and plasma membrane; 1000g, 10 min, 4 °C for the precipitation of nuclei, cytoskeleton (A); 10 000g, 20 min, 4 °C for the precipitation of mitochondria (B); 30 000g, 30 min, 4 °C for the precipitation of lysosomes, peroxisomes (C); and 100 000g, 75 min, 4 °C for the precipitation of ribosomes and small vesicles (D); the filtrate consists of the cytosolic fraction (E). After centrifugation, all pellets and homogenates were diluted to the same volume and the ^114mIn activity was measured with a Ge(Li) detector. The counting time was set variable in order to keep the relative standard deviation below 2%.

The chromatographic separations were performed with a fast protein liquid chromatography system (Äkta Purifier 10, Amersham Pharmacia Biotech, Uppsala, Sweden). Before injection into the system, all samples were filtered through 0.22 µm syringe filters (Acrodisc LC PVDF, Gelman Sciences, Ann Arbor, MI, USA). All buffers were filtered through a 0.22 µm surfactant-free cellulose acetate (SFCA) filter (Nalgene, New York, USA) and degassed. The detection of proteins was obtained by UV/visible absorption at 254, 280 and 410 nm. The detection and quantification of albumin, transferrin and immunoglobulins were performed by means of nephelometry (Behring Nephelometer 100 Analyser, Behringwerke, Marburg, Germany).

Rat serum was diluted 1 + 2 in elution buffer and 200 µl was injected on a Superose 12 HR (Amersham Pharmacia Biotech, Uppsala, Sweden) size exclusion (range, 5 000–300 000 Da) column and on an Asahipak 520-GS 7G (Showa Denko, Tokyo, Japan) multimode (exclusion limit, 30 000 Da) column. The latter column separates molecules on the basis of their molecular mass, but also shows an affinity for albumin. As a result, albumin is separated from the high molecular mass fraction. The Asahipak column has been successfully applied for metal speciation in serum.²³ A 10 mM HEPES + 5 mM NaHCO₃ + 0.15 M NaCl, pH 7.4 elution buffer was used. Fractions of 0.5 or 1 ml were collected and the ^114mIn activity was measured by NaI(Tl) scintillation counting.

Packed cells were lysed prior to elution. After successive thawing and freezing, the partially lysed cells were diluted 1 + 5 in elution buffer and further disrupted in a Potter–Elvehjem homogeniser. Afterwards, the homogenate was centrifuged at 13 000g and 4 °C for 40 min to remove the remaining cell membranes. No separation was made between red (>99%) and white (<1%) blood cells. A volume of 200 µl was injected on a Superose 12 HR; 1 ml was brought onto a self-packed Sephacryl S-200 HR size exclusion (range, 5 000–250 000 Da) column. Fractions of 1 and 2 ml, respectively, were measured for their ^114mIn activity with an NaI(Tl) scintillation detector. Elution was performed with 15 mM HEPES + 0.15 M NaCl, pH 7.2 buffer.

Undiluted urine (200 µl) was injected on a Superdex Peptide HR (Amersham Pharmacia Biotech, Uppsala, Sweden) size exclusion column. The working range of this column covers the low molecular mass fraction (100–7 000 Da). Fractions of 1 ml were taken and the activity was measured with an NaI(Tl) scintillation detector. Elution was performed with 15 mM HEPES + 0.15 M NaCl, pH 6.5 buffer.

Liver, kidney and spleen were minced and homogenised as described above and the soluble fraction was obtained after centrifugation at 100 000g and 4 °C for 100 min. A volume of 200 µl of 1 + 1 diluted or undiluted solution was injected on a Superose 12 HR column. Fractions of 0.5 or 1 ml were taken and the activity was measured with an NaI(Tl) scintillation detector. Elution was performed with 15 mM HEPES + 0.15 M NaCl, pH 7.2 buffer.

Results and discussion

The results for the in vivo organ distribution of ^114mIn, shown in Table 1, are listed as percentage per gram of organ (wet weight) and percentage per organ (wet weight). In both cases, the values are expressed as the percentage of the total amount of ^114mIn activity found in all measured organs. The large amount of indium in the kidney, liver and spleen is consistent with the findings of other studies by Castronovo and Wagner.⁵ In that study, it is stated that the distribution of indium in body tissues is largely determined by the chemical form of the metal. The kidney extensively accumulates ionic indium, while colloidal indium oxide is accumulated by the liver, spleen and the reticuloendothelial system. The experiments were performed on mice. In the present study, ^114mIn, originally dissolved in concentrated hydrochloric acid, was adjusted to physiological pH with NaOH and then dissolved in a HEPES buffered saline solution. Base-induced hydrolysis of In³⁺ solutions causes precipitation at physiological pH. The outcome depends on the nature of the counter-anion and on the particular base, and is influenced by temperature and by ageing of the precipitate. If halide ions are present, they are likely to be incorporated into the precipitate [In₂(OH)₃Cl₃], but in their absence In(OH)₃ (hydrated indium oxide) is formed.²⁴ Considering that both kidney on the one hand and liver and spleen on the other account for a large amount of ^114mIn activity, we might suppose that the injected compound has mixed ionic and colloidal properties. Goodwin et al.²⁵ developed a method for liver scanning using a ^113mIn colloid. They found that 80% of the injected dose was localised in the liver. On a percentage per organ basis, we found a value of 69.9 ± 7.3% for liver.

Table 1 In vivo organ distribution of ^114mIn in rats expressed as a percentage per gram organ of the total activity in all measured organs and as a percentage per organ of the total activity in all measured organs

Organ	% g^−1 organ	% per organ
a Total bone mass not measured.
Liver	16.5 ± 4.5	69.2 ± 7.3
Kidney	17.1 ± 2.8	10.8 ± 2.8
Heart	2.7 ± 0.5	0.7 ± 0.3
Testes	2.8 ± 0.6	2.3 ± 0.6
Thyroid glands	3.7 ± 0.5	0.5 ± 0.1
Lung	4.4 ± 1.1	2.0 ± 0.9
Spleen	21.8 ± 2.1	4.2 ± 0.6
Stomach	6.6 ± 2.1	3.0 ± 1.1
Bladder	13.4 ± 5.6	0.9 ± 0.3
Small intestine	6.1 ± 2.9	6.4 ± 3.1
Bone	4.9 ± 1.0	^a

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Table 2 shows the results of the in vivo intracellular distribution of ^114mIn in kidney, liver and spleen. In all cases, the cytosolic fraction accounts for the highest activity of ^114mIn, followed by the mitochondrial fraction. Fowler et al.²⁶ found that after rats were given a single intraperitoneal injection of InCl₃ in doses up to 40 mg kg⁻¹, an increased number of In-containing autophagic lysosomes could be observed. This finding could not be confirmed. However, the difference might be due to the fact that, in our study, non-toxic tracer doses of indium were used.

Table 2 In vivo intracellular distribution of ^114mIn in kidney, liver and spleen. A: pellet of nucleus; B: pellet of mitochondria; C: pellet of lysosomes and peroxisomes; D: pellet of ribosomes and small vesicles; E: homogenate containing cytoplasm. Results are expressed as the percentage of total activity within each tissue homogenate

	Kidney	Liver	Spleen
% in A	9.7 ± 4.1	6.3 ± 0.3	3.4 ± 1.4
% in B	22.5 ± 1.0	14.1 ± 2.2	18.7 ± 2.9
% in C	5.7 ± 2.6	1.7 ± 0.5	7.7 ± 1.6
% in D	15.3 ± 1.0	4.6 ± 0.4	11.8 ± 1.5
% in E	46.8 ± 3.6	73.3 ± 3.3	58.4 ± 5.3

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Serum carries the major share of ^114mIn activity in the blood. About 90.2 ± 1.8% (n = 4) of total blood ^114mIn could be found in serum. One rat did not absorb the last injected dose. In that case the amount of ^114mIn in serum was only 75.0%. This is possibly due to the fact that the clearance of indium from blood cells is slower than the clearance of indium from serum.

The results of the chromatographic separation of rat serum are shown in Fig. 1. In contrast to earlier in vitro incubation experiments of indium in human serum,¹⁹ only one peak at the retention time of transferrin can be seen. It seems that, in vivo, indium is bound exclusively to transferrin. In vitro, there is a varying amount of indium bound to transferrin and to a low molecular mass compound; a negligible amount of indium might be bound to albumin.

Fig. 1 Elution profile of [^114mIn]InCl₃ in rat serum separated by size exclusion chromatography on Asahipak 520-GS 7G; buffer: 10 mM HEPES + 5 mM NaHCO₃ + 0.15 M NaCl, pH 7.4; UV absorption at 280 nm.

The chromatogram of the in vivo speciation of ^114mIn in blood cell lysate is shown in Fig. 2. The indium elutes slightly ahead of haemoglobin. Therefore, further investigations are needed to determine whether haemoglobin is the main carrier of indium in red blood cells, the dislocation of In from haemoglobin occurs during elution, or indium is bound to another high molecular mass compound. It is unlikely that traces of serum transferrin account for the high activity at that retention volume. Serum was removed from the packed cells by centrifugation in two consecutive steps, followed by washing of the packed cell fraction twice with 0.9% NaCl solution. Indium is said to influence haem biosynthesis in red blood cells by inhibiting the enzymatic activity of δ-aminolaevulinic acid dehydratase (ALAD).¹⁰ ALAD catalyses the second step of the pathway, i.e. the condensation of aminolaevulinic acid to form the monopyrrole, porphobilinogen.²⁷ From the chromatogram it can be seen that there is no major association between indium and the enzyme (M = 290 000 Da), which must be eluted at the retention volume of 7–9 ml. It could be postulated that the influence of indium on the haem biosynthetic pathway is caused by an indirect interaction, rather than by direct binding to the enzyme, as has been stated for lead.²⁸ In the latter case, lead displaces zinc from its SH-mediated binding site, thereby producing an inhibitory effect. In order to confirm the finding regarding no association between ALAD and indium, the experiments were repeated on a Sephacryl S-200 HR column. The results were the same.

Fig. 2 Elution profile of [^114mIn]InCl₃ in rat blood cell lysate separated by size exclusion chromatography on Superose 12 HR; buffer: 15 mM HEPES + 0.15 M NaCl, pH 7.2; UV absorption at 280 nm.

The separation of rat urine by size exclusion chromatography is shown in Fig. 3. In urine, indium is mostly bound to the low molecular mass fraction, with a maximum at the retention volume of 16.5–17.5 ml, corresponding to a molecular mass of 300–1000 Da; only a small amount of ^114mIn can be found in the void volume (>7000 Da). In previous studies of the binding of indium to the low molecular mass fraction in serum (unpublished work), we have postulated that indium occurs as In(OH)₃, although possibly complexed by the buffer (HEPES and TRIS). Taillefert et al.²⁹ have reported a similar behaviour for soluble Fe³⁺ solutions complexed to TRIS buffer. In urine, similar complexation between free indium and elution buffer might also occur.

Fig. 3 Elution profile of [^114mIn]InCl₃ in rat urine separated by size exclusion chromatography on Superdex Peptide; buffer: 15 mM HEPES + 0.15 M NaCl, pH 6.5; UV absorption at 280 nm.

The results of the separation of liver, kidney and spleen cytosol by size exclusion chromatography are shown in Figs. 4, 5 and 6, respectively. In all cases, it appears that indium preferentially (kidney) or exclusively (liver, spleen) binds to the high molecular mass fraction. This large amount of ^114mIn might be partly due to the presence of serum transferrin in the organs after homogenisation. However, the activity peak in both cases is much broader than in the case of rat serum and the concentration of the residual transferrin in the organs should be much lower than in serum. Thus, it is expected that another high molecular mass compound is capable of binding indium, leading to an unresolved ^114mIn activity peak. Only in the case of kidney cytosol does a considerable amount of indium appear in the low molecular mass fraction. Indium influences hepatic and renal ALAD activity in vitro; however, indium inhibits only renal ALAD in vivo.³⁰ Woods and Fowler postulated that this tissue-specific effect is due to the preferential accumulation of indium by the kidney proximal tubule cell with selective localisation in the cytosol. Zinc reverses the indium-induced inhibition of renal ALAD in vitro and in vivo, suggesting that indium acts by binding groups on the enzyme. Although we also found that indium preferentially accumulates in the cytosolic fraction, the chromatogram shows no direct binding of indium with ALAD and thus replacement of zinc. Therefore the inhibition may rather be due to some other indirect interaction. Again, the fact that background levels far below toxic doses have been used throughout these experiments might be the cause of the differences between the two sets of findings.

Fig. 4 Elution profile of [^114mIn]InCl₃ in rat liver cytosol separated by size exclusion chromatography on Superose 12 HR; buffer: 15 mM HEPES + 0.15 M NaCl, pH 7.2; UV absorption at 280 nm.

Fig. 5 Elution profile of [^114mIn]InCl₃ in rat kidney cytosol separated by size exclusion chromatography on Superose 12 HR; buffer: 15 mM HEPES + 0.15 M NaCl, pH 7.2; UV absorption at 280 nm.

Fig. 6 Elution profile of [^114mIn]InCl₃ in rat spleen cytosol separated by size exclusion chromatography on Superose 12 HR; buffer: 15 mM HEPES + 0.15 M NaCl, pH 7.2; UV absorption at 280 nm.

Acknowledgements

The authors wish to thank Dr. Karel Strijckmans for irradiation of the target, Louis Mees and Inge Van Vynckt for technical assistance, Tommy D'Heuvaert for assistance during the animal experiments and the Flemish Institute for the Promotion of Scientific-Technological Research in Industry (IWT) for financial support.

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Footnote

† Presented at the Whistler 2000 Speciation Symposium, Whistler Resort, BC, Canada, June 25–July 1, 2000.

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