Kari Åkerman

Metabolism, cellular actions, and cytotoxicity of selenomethionine in cultured cells

Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized toS-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 μM levels. Yet, low selenomethionine levels (10 μM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes inS-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way.

High-performance liquid chromatography method for analyzing citalopram and desmethylcitalopram from human serum.

This report describes a sensitive and specific method for analyzing a serotonin reuptake blocker, citalopram, and its active metabolite, desmethylcitalopram, in human serum. For high-performance liquid chromatography (HPLC) analysis, samples and standards are prepared with ASPEC automatic sample preparator using 100 mg Bond-Elut C-18 solid-phase extraction columns. The method is an isocratic HPLC method with a mobile phase of acetonitrile:methanol:50 mM dipotassium hydrogenphosphate, pH 4.7 (40:100). Detection is performed with diode array detector at 220 nm and the peak purity analyses at 210 to 365 nm. The intraassay coefficient of variation ranges from 3.7% to 7.3%, and the interassay coefficient of variation ranges from 6.9% to 9.9% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method is suitable for therapeutic drug monitoring in a clinical laboratory. A clear correlation, r = 0.72 (y = 0.36x + 17.94), between citalopram and its metabolite levels is observed in routine therapeutic drug monitoring service. A linear correlation between serum concentration and daily dose of citalopram in patient groups is also observed.

Radiolabeling and in-vivo distribution of nanobacteria in rabbits

Nanobacteria are minute bacteria recently isolated from mammalian blood. They encapsulate themselves with apatite mineral. Cultured nanobacteria were radiolabeled with 99mTc, using a method which has been previously used for labeling red blood cells with 99mTc, and in vivo distribution of nanobacteria was followed with Single Photon Emission Computed Tomography (SPECT) imaging. The labeling yield was over 30%. Two rabbits were studied using dynamic planar imaging performed in the AP-position immediately after injection. Serial SPECT scans were acquired up to 24 h and one planar image was taken at 45 h. A control study was performed administering a similar dose of [99mTc] labeled albumin nanocolloids. Regional nanobacteria-to- nanocolloid ratios were calculated along with time and tissues (45 h) were analyzed for radioactivity and for nanobacteria. The main finding was that radiolabeled nanobacteria remained intact and showed a tissue specific distribution with a high accumulation in the kidneys and also in urine. Spleen, stomach, heart and intestine also showed increased uptake. Excretion into urine started 10 - 15 min after injection. These were live nanobacteria in the urine, which had better capabilities to penetrate into cells in vitro. The nanobacteria accessed the urine via tubular cells since nanobacteria were found in their cytoplasm and tubular surfaces. The results suggest that nanobacteria utilize endocytic transport of tubular cells and may be involved in the pathogenesis of mineral formation in mammalian kidney stones.

Iodine-123 labelledN-(2-fluoroethyl)-2β-carbomethoxy-3β-(4-iodophenyl)nortropane for dopamine transporter imaging in the living human brain

There are several cocaine analogs which have potential for imaging the dopamine transporters (DAT). Earlier studies have shown that iodine-123 labelled 2β-carbomethoxy-3β-(4-iodophenyl)tropane ([123I]β-CIT) andN-(3-fluoropropyl)-2β-carbomethoxy-3β-(4-iodo-phenyl)nortropane ([123I]β-CIT-FP) are promising DAT imaging agents in the living human brain with single-photon emission tomography (SPET). Here we report a pilot comparison of [123I]β-CIT and [123I]β-CIT-FP with a new tropane derivative, [123I]N-(2-fluoroethyl)-2β-carbomethoxy-3β-(4-iodophenyl)nortropane ([123I]β-CITFE), using SPET imaging in four healthy male subjects. Peak uptake of [123I]β-CIT-FE into the basal ganglia occurred very rapidly (0.5 h after injection of tracer), after which the striatal washout obeyed a bi-exponential form. The specific DAT binding of [[123I]β-CIT FE into the basal ganglia was somewhat less (0.785 ± 0.117) than that of [123I]β-CIT (0.922 ± 0.004) or [123I]β-CIT-FP (0.813 ± 0.047). All these tracers have excellent imaging quality in healthy control subjects. However, the relatively fast washout of [123I]β-CIT-FE and low temporal resolution of older SPET cameras may limit the use of this tracer to the measurement of the DAT density.

Striatal and extrastriatal imaging of dopamine D2 receptors in the living human brain with [123I]epidepride single-photon emission tomography

The iodine-123 labelled ligand benzamide epidepride was evaluated as a probe for in vivo imaging of striatal and extrastriatal dopamine D2 receptor sites in the human brain. Four healthy males were imaged with a high-resolution single-photon emission tomography scanner. Striatal radioactivity peaked at 3 h after injection. The specific binding in the striatum was 0.91±0.03 at 3 h and this ratio steadily increased with time. Extrastriatal radioactivity was highest in the thalamus, in the midbrain and in the temporal cortex, and peaked at 45–60 min after injection of tracer. A smaller amount of radioactivity was found in the parietal, frontal and occipital cortices. Two radioactive metabolites were observed, of which one was more lipophilic than the parent compound. The radiation burden to the patient was 0.035 mSv/MBq (effective dose equivalent). The preliminary results showed that [123I]epidepride can be used for imaging striatal and extrastriatal dopamine D2 receptor sites in the living human brain.

Characterization of C-11 or I-123 Labelled β-CIT-FP and β-CIT-FE Metabolism Measured in Monkey and Human Plasma. Identification of Two Labelled Metabolites with HPLC

β‐CIT‐FP and β‐CIT‐FE which are novel fluoroalkyl derivatives of 2β‐carbomethoxy‐3β‐(4‐iodophenyl)tropane (β‐CIT), have recently been labelled with11C or123I and used for imaging the dopamine transporter with positron emission tomography (PET) or single photon emission tomography (SPET). The metabolite pattern of these novel tracers was studied with HPLC in plasma in monkeys and in urine and plasma in humans. Four labelled metabolites were found of which two were identified, the123I‐labelled free acid and11C‐ or123I‐labelled nor‐β‐CIT. The latter was detected only in small amounts (<4 per cent). Furthermore, a polar metabolite and an unknown lipophilic11C‐ or123I‐labelled metabolite were found. The amount of this unidentified labelled metabolite of [11C]β‐CIT‐FP or [11C]β‐CIT‐FE in monkey plasma was 21±8 per cent and 16±9 per cent at 60 min after injection, respectively. In conclusion, four types of labelled metabolites were found after injection of11C‐ or123I‐labelled β‐CIT‐FP and β‐CIT‐FE in monkeys and humans of which one may hamper the quantitation of the dopamine transporter by PET or SPET.

Fentanyl decreases beta-CIT binding to the dopamine transporter.

Evidence from animal studies suggest that centrally acting opiates increase synaptic dopamine (DA) concentration. However, the interaction between μ‐opioid receptors and the DA system is unclear. We report here an effect of fentanyl on striatal [123I]β‐CIT binding to the DA transporter in a patient and in rats. A female patient underwent [123I]β‐CIT single‐photon emission tomography (SPET) study after intrathecal injection of fentanyl for her back pain. After a 2‐week drug‐free period, the SPET study was repeated. In the experimental study, male Wistar rats were treated with fentanyl either acutely (50 μg/kg, i.p.) before imaging study or subacutely for 4 days (10 μg/kg, twice a day, i.p.). Brain planar imaging was performed at 3.5 hours after an intravenous injection of [123I]β‐CIT with gamma camera with a pinhole collimator. In a female patient, [123I]β‐CIT binding in the basal ganglia was decreased by 37% during fentanyl as compared to the binding after 2‐week drug‐free period. Similarly in rats, acute fentanyl treatment decreased [123I]β‐CIT binding to the striatum by 30% as compared to that of with the control rats. After subacute administration of fentanyl, no significant difference was observed compared to the control group. According to the present data, fentanyl decreases [123I]β‐CIT binding in the basal ganglia both in human and rats, suggesting that opiates possibly directly affect DA reuptake. Synapse 29:413–415, 1998.

Methionine adenosyltransferase activity in cultured cells and in human tissues

We have investigated methionine adenosyltransferase activity (MAT) in extracts of a variety of normal and malignant human tissues and cultured cell lines. MAT activity assayed from 17 different cultured cell lines varied to a great extent. Ramos (human, Burkitts lymphoma) and EL4 (mouse, T cell lymphoma) cells showed MAT activity near 300 pmol/mg per min. Daudi (human, Burkitts lymphoma) and almost all monolayer cells had MAT activity below 100 pmol/mg per min. Human peripheral blood lymphocytes had MAT activity of 36 pmol/mg per min. The MAT activity of the cell lines can be related to doubling time: cell lines with short doubling times have much higher MAT activity than other cell lines. A large variation in MAT activity in different human tissues was observed. In autopsy samples MAT activity was highest in the brain and in the colon. Malignant tissue samples gave much higher MAT activity than normal tissues. Lung cancer (carcinoma squamocellulare pulmonis) had MAT activity of 30.7 pmol/mg per min, while in normal lung it was 2.4 pmol/mg per min.

Initial human studies with single-photon emission tomography using iodine-123 labelled 3-(5-cyclopropyl-1,2,4-oxadiazo-3-yl)-7-iodo-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]-benzodiazepine (NNC 13-8241)

The iodine-123 labelled ligand 3-(5-cyclopropyl-1,2,4-oxadiazo-3-yl)-7-iodo-5,6-dihydro-5-methyl-6oxo-4H-imidazo[1,5-a][1,4]-benzodiazepine ([123I]NNC 13-8241) was evaluated as a probe for in vivo imaging of benzodiazepine receptor sites in the human brain. Four healthy volunteers were imaged with a high-resolution single-photon emission tomography (SPET) scanner. The metabolism of [123I]NNC 13-8241 in plasma was slow. The total brain uptake was about 1.5-fold higher than that of [123I]iomazenil. The specific binding in the cortical areas was high and less intense in the thalamus. The most intense uptake was seen in the occipital cortex. The peak cortical uptake of [123I]NNC 13-8241 was observed 6–10 h after the injection of tracer. The radiation burden to the patient was moderate, being 2.5·10−2 mSv/MBq (effective dose equivalent). A slow metabolism together with favourable kinetics indicates that [123I]NNC 13-8241 is a specific and promising SPET ligand for imaging benzodiazepine receptor sites in the living human brain.

Metabolism of [123I]epidepride may affect brain dopamine D2 receptor imaging with single-photon emission tomography

Abstract.Iodine-123 labelled epidepride is a novel radiopharmaceutical for the study of cerebral dopamine D2 receptors using single-photon emission tomography (SPET). A lipophilic labelled metabolite of [123I]epidepride which may enter the brain and hamper the quantitation of receptors has been observed in human plasma. In the present study, gradient high-performance liquid chromatography (HPLC) was used to investigate the plasma concentration of the lipophilic labelled metabolite and its correlation to SPET imaging of striatal dopamine D2 receptors. A linear regression fit showed a negative correlation between the amount of the lipophilic labelled metabolite and the striatum to cerebellum ratio (n=16, R=–0.58, P<0.02), suggesting that plasma metabolite analysis is essential when imaging dopamine D2 receptors with SPET using [123I]epidepride.