A new Tc-complex with a germanium-hydrazide (GeTH) ligand

A new tetrahydrazido-germanium (GeTH) ligand was synthesized, characterized and complexed with ^99mTc. The negatively charged ^99mTc-chelate was shown to form in high yields at neutral pH in the absence of other reducing agents and exhibits high in vitro and in vivo stability.     

Can a cysteine challenge assay predict the in vivo behavior of Tc-labeled antibodies?

Recent investigations have shown that transchelation to cysteine is a principal mode of in vivo instability of ^99mTc-labeled antibodies. In this investigation, a cysteine challenge assay was used to measure the in vitro instability of ^99mTc directly labeled to two IgG antibodies (B72.3 and C110) via two established direct labeling methods employing mercaptoethanol and stannous ion for antibody reduction and by a novel method using glutathione for this purpose. For both antibodies, the greatest instability to cysteine occurred with stannous ion reduction. The stability of glutathione-reduced B72.3 was indistinguishable from mercaptoethanol-reduced B72.3 whereas glutathione-reduced C110 showed stability roughly intermediate between that of the other reducing agents for this antibody. Results obtained in normal mice were in the direction predicted by the assay: for both antibodies, urinary clearance of ^99mTc was fastest in mice receiving antibodies labeled via stannous ion reduction, presumably because of the increased transchelation of label to cysteine in vivo. Urinary clearance was slower and identical in mice receiving B72.3 labeled via glutathione or mercaptoethanol whereas clearance in the case of glutathione-reduced C110 was intermediate between that of the other two reducing agents. At both time points, higher radioactivity levels were observed in kidneys and lower levels in blood and most other tissues for both antibodies in the case of stannous ion reduction as expected for the label of greatest instability. In the B72.3 case, with only one exception, tissue and blood levels following administration of glutathione-reduced antibody were indistinguishable from that following administration of mercaptoethanol-reduced antibody. In the C110 case, significant differences in activity levels were observed in several tissues between glutathione- and mercaptoethanol-reduced antibodies. In conclusion, the relative in vivo behaviour of ^99mTc when administered to mice while labeled to two IgG antibodies were successfully predicted based on the results of an in vitro cysteine challenge assay.     

Evaluation of possible interference of anti-inflammatory drugs upon scintigraphic imaging with macrophage targeting Tc-J001X: Effects of methylprednisolone, dexamethasone, indomethacin and methotrexate

J001X, an acylated poly-(1,3)-galactoside isolated from Klebsiella pneumoniae proteoglycan, has been developed to target cells from the monocyte-macrophage lineage. Recent experimental work and initial clinical trials have proved the potential of this molecule labeled with ^99mTc for the scintigraphy of inflammatory foci. In a model of radiation-induced inflammation in pigs, the scintigraphic contrast was observed to be very sensitive to a single injection of methylprednisolone given 12 h before scintigraphy. The present study was undertaken to confirm this effect and to estimate the possible interference of various anti-inflammatory agents on the in vivo targeting of macrophages by J001X. Methylprednisolone, dexamethasone, indomethacin and methotrexate used at an immunosuppressive dose were tested to assess the possible risk of false-negative examinations in patients thus treated. Analysis of the results indicated that among the four drugs tested, only methylprednisolone at 0.5–1 mg/kg could interfere with J001X scintigraphy.     

A solid-phase technique for preparation of no-carrier-added technetium-99m radiopharmaceuticals: application to the streptavidin/biotin system

A high effective specific activity (HESA) formulation of a biotin-containing ^99mTc ligand [RP488: dimethyl-Gly-Ser-Cys(Acm)-Lys(Biotin)-Gly] conveniently prepared from solid phase was compared to a typical low effective specific activity (LESA) solution formulation to demonstrate improved targeting to streptavidin in an in vitro assay and in an in vivo rat model. RP488 was coupled to a maleimide-functionalized polyethylene glycol resin via a thiol ether linkage and labeled with ^99mTc-gluconate at room temperature, followed by elution of the HESA ^99mTc-RP488 in saline (minimum specific activity 1000 TBq/mmol by amino acid analysis). Both HESA and LESA ^99mTc-RP488 labeled at > 90% purity. In vitro, HESA ^99mTc-RP488 incubated with streptavidin-agarose was bound quantitatively, but there was competition from addition of increasing amounts of cold RP488. In rats, radiotracer uptake was evident at the site of implantation of streptavidin-agarose beads for the HESA dose, less uptake of low effective specific activity (LESA) material, and no appreciable uptake in the control rats of the LESA or HESA dose. The target-to-background ratio for HESA ^99mTc-RP488 was 5.4 times that of the control. The solid-phase technology offers a convenient way to prepare high specific activity receptor-targeting ^99mTc radiopharmaceuticals.     

In Vivo localization of diglycylcysteine-bearing synthetic peptides by nuclear imaging of oxotechnetate transchelation

A phenomenon of in vivo transchelation of oxotechnetate from a complex with glucoheptonic acid to synthetic peptides bearing oxotechnetate-binding motifs and a technique for in vivo visualization of these peptides are described. Using two model peptides bearing two tandem diglycylcysteine (GGC) motifs (P1) or three GGC motifs (P2), we demonstrated that: (i) these peptides efficiently transchelated oxo-[^99mTc]technetate from a complex with glucoheptonic acid in vitro (a complex with peptides was stable at least 24 h; radiochemical purity exceeded 95% by high performance liquid chromatography); (ii) injection of peptides into the rectus femoris muscle (at 0.5-1 μmol of SH groups) followed by an intravenous injection of ^99mTc-glucoheptonate (0.25-0.5 mCi per animal) yielded visualization of the injected muscle by nuclear imaging within 1 h after injection; (iii) the experimental/control (contralateral) thigh muscle ratio was 1.80 ± 0.05 for peptide P1 and 3.0 ± 0.1 for P2; (iv) the injection of a control peptide P2 with SH groups covalently modified with N-ethylmaleimide resulted in a ratio of 1.4 ± 0.2. These findings argue for specific association of oxo-[^99mTc]technetate with free thiols within the binding motif of injected peptides in vivo. In vivo transchelation of oxo-[^99mTc]technetate may be useful for the purpose of noninvasive imaging of gene expression, i.e., when the expression product bears GGC motifs.     

Molecular recognition and stability of Tc-UBI 29–41 based on experimental and semiempirical results

^99mTc-UBI 29–41 is an antimicrobial peptide fragment that directly radiolabeled with ^99mTc shows high in vitro and in vivo stability, rapid background clearance, minimal accumulation in non-target tissues and rapid detection of infection sites. Molecular mechanics (MM) calculation has been an essential tool in explaining experimental results associated with molecular recognition and stability. This work is an attempt to explain the ^99mTc-UBI 29–41 specificity for bacteria and to understand from a structural point of view, the experimental results indicative of a molecular recognition and stability not well favored for two other cationic peptides (^99mTc-Tat-1-Scr and ^99mTc-Tat-2-Scr ) used as control. Structures of ^99mTc-UBI, ^99mTc-Tat-1-Scr, ^99mTc-Tat-2-Scr and of the corresponding free cationic peptides were built and the optimized structures, in the best stable configurations, were calculated by a MM procedure. In order to correlate the calculated and experimental results, in vitro stability tests with cysteine challenge and stability to dilution in human serum and in saline solution, were performed for the three labeled cationic peptides. The three complexes can be represented by the general formula [Tc(V)(O)(H₂O)₂(Lys_n=1,2-Arg_n=0,1-peptide)]^10+,11+. The potential energies were 104.5, 95.6 and 90.8 kcal/mol for ^99mTc-Tat-1-Scr, ^99mTc-Tat-2-Scr and ^99mTc-UBI 29–41, respectively. Experimental and calculated results were in good agreement. It is thus possible to predict and explain that in similar solution media ^99mTc-Tat-2-Scr would be more stable than ^99mTc-Tat-1-Scr and why ^99mTc-UBI shows the highest stability. In conclusion, the in vitro specific binding to bacteria and the accumulation at infection sites in humans of ^99mTc-labeled UBI could be the result of its high thermodynamic stability, selectivity and stereospecificity.     

Evaluation of Tc-labeled modified serum albumin for tumor detection

Serum albumin (SA) modified and labeled with ¹³¹l-tyramine N-1′-desoxysorbitol (¹³¹I-TDS) has been shown to localize in tumors [Sinn et al., (1990) Nucl. Med. Biol. Part B 17, 819–827]. We prepared similar TDS complexes labeled with ^99mTc and evaluated their potential for tumor imaging. Derivatization of SA with TDS was optimized using cyanuric chloride or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as coupling agents. A high TDS loading yield of 38 mol/mol SA was obtained with the latter reagent. Modified SA (8 and 38 mol TDS/mol SA) were labeled with ^99mTc via the stannous reduction method and injected i.v. into EMT-6 tumor bearing mice. ¹²⁵I-TDS-SA (8 mol ¹²⁵I-TDS/mol SA) revealed a high tumor uptake of 10% ID/g at 3 h post-injection. The ^99mTc-labeled SA and TDS-SA complexes lacked tumor specificity, instead TDS loading of SA resulted in increased liver/spleen uptake, suggesting colloid formation. This study confirms the potential of modified SA for tumor imaging but highlights the importance of choice of radioisotope, as well as site of attachment of the radiolabel to the modified SA for optimal tumor localization.     

Scintigraphic potentials of J001X acylated poly-galactoside for imaging inflammatory lesions in pigs

Although several approaches already exist for the imaging of inflammatory foci, new specific strategies providing functional data on the lesions are required to determine the extent of the disease and also to assess anti-inflammatory treatment. In our study, we investigated the scintigraphic potential of ^99mTc-J001X, an agent developed for the targeting of macrophages. Due to its well documented and progressive evolution of lesions, a model of radiation-induced inflammation in pigs was chosen. Our results demonstrated the ability of J001X to provide images of inflammatory foci with a high contrast. The contribution of some specific and non-specific parameters possibly involved in the scintigraphic behavior of J001X is discussed.     

Evaluation of novel cationic 99mTc(I)-tricarbonyl complexes as potential radiotracers for myocardial perfusion imaging

This report describes the evaluation of three cationic ^99mTc(I)–tricarbonyl complexes — [^99mTc(CO)₃(L)]⁺ (L=N-methoxyethyl-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (ME-PNP), N-[15-crown-5)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (15C5-PNP) and N-[18-crown-6)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (18C6-PNP)) — as potential radiotracers for myocardial perfusion imaging. Biodistribution, imaging and metabolism studies were performed using Sprague–Dawley rats. It was found that bisphosphine ligands have a significant impact on the biodistribution characteristics and clearance kinetics of their cationic ^99mTc(I)–tricarbonyl complexes. Among the three radiotracers evaluated in this study, [^99mTc(CO)₃(15C5-PNP)]⁺ has a very high initial heart uptake and is retained in the rat myocardium for >2 h. It also shows rapid clearance from the liver and lungs. The heart/liver ratio of [^99mTc(CO)₃(15C5-PNP)]⁺ is 2.5 times better than that of ^99mTc-sestamibi at 30 min postinjection. [^99mTc(CO)₃(15C5-PNP)]⁺ is almost identical to ^99mTcN-DBODC5 with respect to heart uptake, heart/lung ratio and heart/liver ratio. Results from metabolism studies show that there is no significant metabolism for [^99mTc(CO)₃(15C5-PNP)]⁺ in the urine, but it does show a small metabolite peak (<10%) in the radio high-performance liquid chromatography chromatogram of the feces sample at 120 min postinjection. Results planar imaging studies demonstrate that [^99mTc(CO)₃(15C5-PNP)]⁺ has a much better liver clearance profile than ^99mTc-sestamibi and might give clinically useful images of the heart as early as 30 min postinjection. [^99mTc(CO)₃(15C5-PNP)]⁺ is a very promising candidate for more preclinical evaluations in various animal models.     

Cellular accumulation and retention of the technetium-99m-labelled hypoxia markers BRU59-21 and butylene amine oxime

BRU59-21 and ^99mTc-butylene amine oxime (BnAO, HL91) are being evaluated for imaging hypoxia in tumors. Both tracers: 1) rapidly reached a plateau in aerobic Chinese hamster ovary cells in vitro but continuously accumulated in hypoxic cells; 2) ceased to accumulate when hypoxic cells were exposed to air; 3) showed 40% retention upon washing the cells; 4) showed selective hypoxic accumulation only at 37°C; 5) accumulation could be modulated by addition of electron-affinic compounds; and 6) exhibited higher accumulation in cells which overexpress cytochrome P450 reductase. Both BRU59-21 and ^99mTc-BnAO share properties making them suitable for hypoxia imaging.     

The labelling of Nanocoll with [In] for dual-isotope scanning

Visualization and biopsy of sentinel lymph nodes play an important role in planning and controlling the therapy of breast cancer. Hitherto two methods—scintigraphy or gamma probe detection after injection of [^99mTc]-nanocolloids and visual detection after injection of patent blue dye—are used routinely. There are no conclusive publications elucidating such important parameters as injection site, injection method and colloidal parameters. The present work aims to label Nanocoll^® with [¹¹¹In] to provide an alternative method, a simultanous one-compound dual-isotope application.

Methods: [¹¹¹In]-Indiumchloride was buffered with acetate and transferred to the nanocolloid. The colloid labelling reaction was complete after 30 min and filtrated through 100 nm Nuclepore^® filters.

Results: Incorporation yield of [¹¹¹In]-Indium into the nanocolloid was nearly quantitative, the step associated with the major loss of activity was the particle sizing with a mean yield of 55%.

Conclusion: The presented method allows for the routine supply of [¹¹¹In]-nanocolloids. Size-filtered [¹¹¹In]-Nanocoll^® shows the same particle size range as [^99mTc]-Nanocoll^®.     

An improved (99m)Tc-aprotinin kit formulation: quality control analysis of radiotracer stability and cold kit shelf life

^99mTc-aprotinin scintigraphy has been demonstrated to be a useful noninvasive imaging technique for amyloid deposits located in extraabdominal regions of patients. The aim of this study was to develop an improved aprotinin cold kit formulation, to validate the kit for long-term stability, as well as to assess the radiotracer stability by novel quality control methods. The aprotinin cold kit formulation of Trasylol, pyrophosphate (PYP)-chelated stannous reductant and an alkaline buffer, was dispensed into nitrogen-filled vials and aliquots frozen at −20°C. After 0, 1, 2, 3 and 6 months of storage, three samples were reconstituted with 750–850 MBq of ^99mTc-pertechnetate, followed by quality control analyses by paper chromatography methods at 25, 85 and 265 min postreconstitution (pr). Cation-exchange cartridge quality control methods were also investigated. The cold kits proved to be stable to long-term storage for up to 6 months, and the radiotracer was stable for at least 4 h pr. ^99mTc-aprotinin was formed at greater than 95% efficiency at all time points tested with ^99mTcO₂ present as the major impurity (1–4%) and ^99mTc-pertechnetate and ^99mTc-PYP present in trace amounts. An alternative, rapid, safe and reliable method was found in Oasis MCX–BSA-treated cartridges using saline as the eluting solution to assay for ^99mTc-aprotinin.     

Synthesis of 1-[c]methylpiperidin-4-yl propionate ([c]pmp) for in vivo measurements of acetylcholinesterase activity

Synthesis of 1-[¹¹C]methylpiperidin-4-yl propionate ([¹¹C]PMP), an in vivo substrate for acetylcholinesterase, is reported. An improved preparation of 4-piperidinyl propionate (PHP), the immediate precursor for radiolabeling, was accomplished in three steps from 4-hydroxypiperidine by (a) protection of the amine as the benzyl carbamate, (b) acylation with propionyl chloride, and (c) deprotection of the carbamate by catalytic hydrogenation. The final product was obtained in an overall 82% yield. Reaction of the free base form of PHP with [¹¹C]methyl trifluoromethanesulfonate at room temperature in N,N-dimethylformamide, followed by high performance liquid chromatography (HPLC) purification, provided [¹¹C]PMP in 57% radiochemical yield, >99% radiochemical purity, and >1500 Ci/mmol at the end of synthesis. The total synthesis time from end-of-bombardment was 35 min. [¹¹C]PMP can thus be reliably prepared for routine clinical studies of acetylcholinesterase in human brain using positron emission tomography.     

Preparation and in-vivo evaluation of Tc-IOTIDA for cholescintigraphy

The synthesis, radiolabeling and in vivo evaluation of ^99mTc-IOIDA(3-iodo 2,4,6-trimethylpheyl carbamoylmethyl iminodiacetic acid) for the assessment of hepatocytic function and the functional status of the cystic duct and the gallbladder are described.

For a scintigraphic imaging comparison, three different ^99mTc-IDA derivatives, ^99mTc-DISIDA, ^99mTc-mebrofenin and ^99mTc-IOTIDA, were prepared and evaluated for their in vivo pharmacokinetic behavior through animal studies. Serial static image scans of rabbits injected with ^99mTc-IOTIDA revealed that none of the tissues except the hepatobiliary system showed radioactivity concentrations.

A scintigraphic study in a healthy volunteer showed that most of the administrated radioactivity accumulated in the liver and was rapidly excreted through the hepatobiliary system, visualizing the gallbladder within 15 min.

In conclusion, ^99mTc-IOTIDA is a potential hepatobiliary imaging agent for the evaluation of the functional status of hepatocytes and the patency of the biliary duct.     

Use of 99mTc-mononuclear leukocyte scintigraphy in nosocomial fever

Purpose: To determine the overall diagnostic accuracy of mononuclear leukocyte- ^99mTc scintigraphy in the routine detection of infectious lesions and fever of unknown origin (FUO) in inpatients.

Material and Methods: The use of mononuclear leukocyte ^99mTc scintigraphy is presented in 87 patients who fulfilled the Durack and Street diagnostic criteria of nosocomial FUO; 66 patients were suspected of having infectious lesions (myocarditis, endocarditis, infected catheters, diabetic foot, and osteomyelitis) and 21 patients presented with unknown causes of FUO. Scans were carried out 1, 3, and 24 h after injection of labeled leukocytes.

Results: In three cases (3/27) where scintigraphs were negative, biopsies were positive. There were two (2/87) false-positive scintigrams. We found a 95.8% sensitivity and 92.3% specificity. PPV was 93.8%, PPN 94.7%, and accuracy 94.2%.

Conclusion: Mononuclear leukocyte ^99mTc scintigraphy showed high sensitivity, specificity, positive and negative predictive values in patients with nosocomial FUO. These results suggest an important role for nuclear medicine in the management of patients with infection/inflammation.     

N-[C]methylpiperidine esters as acetylcholinesterase substrates: an in vivo structure–reactivity study

A series of simple esters incorporating the N-[¹¹C]methylpiperidine structure were examined as in vivo substrates for acetylcholinesterase in mouse brain. 4-N-[¹¹C]Methylpiperidinyl esters, including the acetate, propionate and isobutyrate esters, are good in vivo substrates for mammalian cholinesterases. Introduction of a methyl group at the 4-position of the 4-piperidinol esters, to form the ester of a teritary alcohol, effectively blocks enzymatic action. Methylation of 4- N-[¹¹C]methylpiperidinyl propionate at the 3-position gives a derivative with increased in vivo reactivity toward acetylcholinesterase. Esters of piperidinecarboxylic acids (nipecotic, isonipecotic and pipecolinic acid ethyl esters) are not hydrolyzed by acetylcholinesterase in vivo, nor do they act as in vivo inhibitors of the enzyme. This study has identified simple methods to both increase and decrease the in vivo reactivity of piperidinyl esters toward acetylcholinesterase.     

Influence of a TcN core on the biological and physicochemical behavior of Tc complexes of L,L-EC and L,L-ECD

^99mTc-nitrido complexes of L,L-ethylene dicysteine (^99mTcN-L,L-EC) and ^99mTcN-L,L-ethylene dicysteine diethylester (^99mTcN-L,L-ECD) were prepared and their characteristics compared to those of the respective ^99mTc-oxo complexes. ^99mTcN-L,L-EC and ^99mTcO-L,L-EC migrate to similar extents during electrophoresis at pH 12, but, at pH 6, ^99mTcN-L,L-EC migrates further than ^99mTcO-L,L-EC. Renal excretion of ^99mTcN-L,L-EC is inferior to that of ^99mTcO-L,L-EC, indicating that the TcN-glycine sequence has lower affinity for the renal tubular system. Both ^99mTcO-L,L-ECD and ^99mTcN-L,L-ECD are neutral, but ^99mTcN-L,L-ECD is hydrophilic and shows minimal brain uptake in both mice and the baboon.     

Dose-dependent biodistribution of [(99m)Tc]DTPA-mannosyl-dextran for breast cancer sentinel lymph node mapping

[^99mTc]DTPA-mannosyl-dextran is a receptor-binding radiopharmaceutical specifically designed for sentinel lymph node mapping. The purpose of this study was to test the biodistribution and safety of [^99mTc]DTPA-mannosyl-Dextran at different molar doses. Twenty-four female breast cancer patients participated in this study. Four groups of 6 patients received an injection of 0.2, 1.0, or 5.0 nmol of [^99mTc]DTPA-mannosyl-Dextran or filtered [^99mTc]sulfur colloid. The injection site clearance was monitored by dynamic imaging for three hours. Whole body scans were acquired at 2.5 and 12, and lymph nodes were assayed for radioactivity after gamma-guided sentinel lymph node biopsy. Injection site clearance of [^99mTc]DTPA-mannosyl-Dextran was not statistically different in a dose-dependent manner. Dose-dependent sentinel node uptake was observed (p = 0.03). There were no clinically significant alterations in laboratory parameters among all dose levels at 4 h or 24 h post injection compared to preoperative levels. Radiation absorbed doses did not differ among the three dose levels, but were lower than filtered [^99mTc]sulfur colloid.     

Role of scintigraphy with technetium-99m depreotide in the diagnosis and management of patients with suspected lung cancer

Background: In Sweden, there are over 3000 new lung cancer cases every year. There are still numerous patients with undetermined lesions after routine diagnostic evaluation by clinical examination, chest radiography, computed tomography (CT) of the thorax, and bronchoscopy. An appropriate method for further diagnostic workup is therefore needed.

Purpose: To evaluate the diagnostic value of the somatostatin analogue depreotide in patients with suspected lung cancer, and to determine in which clinical settings it would be beneficial to use ^99mTc-depreotide scintigraphy.

Material and Methods: We included 99 consecutive patients referred to our hospital with suspected lung cancer. A clinical examination, bronchoscopy, chest radiography, CT of the thorax and upper abdomen, and scintigraphy were done. Scintigraphy was performed after injection of 740 MBq ^99mTc depreotide with tomographical imaging of the thorax and whole-body scanning. The diagnostic outcome of the scintigrams was compared to CT, using morphology or clinical outcome as the endpoint.

Results: ^99mTc-depreotide uptake was found in 62 out of 66 malignancies, including 57 of 58 primary lung cancer cases. Two cases of lung metastasis (one from a colon cancer and one from an adenoid cystic carcinoma originating in the palate) and one rib chondrosarcoma did not show depreotide uptake. There were 33 patients with benign lesions, of whom 16 displayed false-positive ^99mTc-depreotide uptake, whereof 11 were pneumonias. Tc-99m-depreotide uptake was absent in 17 patients with benign lesions, including all 10 hamartomas. The sensitivity in detecting malignancy was 94%, and in detecting lung cancer 98%. The specificity was calculated based on two sets of data. When all cases were used, the specificity was 52%. If the 12 pneumonias are excluded, the specificity was 77%.

Conclusion: ^99mTc-depreotide scintigraphy has a high sensitivity in detecting lung cancer. The method is useful in decision-making with respect to surgery.     

Understanding the radiolabelling mechanism of Tc–antimony sulphide colloid

The chemistry of antimony trisulphide colloid (ATC) was examined to elucidate the radiolabelling mechanism with ^99mTcO₄⁻. Ion exchange chromatography and atomic absorption spectrophotometry techniques determined ATC to be resistant to hydrolysis in 0.1 M hydrochloric acid (HCl) at 25°C or 100°C (>97% recovery, Sb³⁺ absent). Hydrogen sulphide gas detected did not participate in the mechanism, where antimony trisulphide and ^99mTcO₄⁻ in HCl/100°C yielded 96% ^99mTc-product from a K₂S-free formulation (versus 98% when K₂S was present). ^99mTcO₄⁻ was reduced >90% by DMSA or dithiothreitol under the same conditions, identifying involvement of thiol groups. Infrared analysis of Re-ATC showed S=O bonds, indicating excess thiol groups at the colloid surface were oxidised at the expense of ^99mTcO₄⁻ reduction.