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The sugar model: catalytic flow reactor dynamics of pyruvaldehyde synthesis from triose catalyzed by poly-l-lysine contained in a dialyzer.

The formation of pyruvaldehyde from triose sugars was catalyzed by poly-l-lysine contained in a small dialyzer with a 100 molecular weight cut off (100 MWCO) suspended in a much larger triose substrate reservoir at pH 5.5 and 40 degrees C. The polylysine confined in the dialyzer functioned as a catalytic flow reactor that constantly brought in triose from the substrate reservoir by diffusion to offset the drop in triose concentration within the reactor caused by its conversion to pyruvaldehyde. The catalytic polylysine solution (400 mM, 0.35 mL) within the dialyzer generated pyruvaldehyde with a synthetic intensity (rate/volume) that was 3400 times greater than that of the triose substrate solution (12 mM, 120 mL) outside the dialyzer. Under the given conditions the final yield of pyruvaldehyde was greater than twice the weight of the polylysine catalyst. During the reaction the polylysine catalyst was poisoned presumably by reaction of its amino groups with aldehyde reactants and products. Similar results were obtained using a dialyzer with a 500 MWCO. The dialyzer method of catalyst containment was selected because it provides a simple and easily manipulated experimental system for studying the dynamics and evolutionary development of confined autocatalytic processes related to the origin of life under anaerobic conditions.

Catalysis↗

A new intermediate of the aldolase reaction, the pyruvaldehyde-aldolase-orthophosphate complex.

Fructose 1,6-bisphosphate aldolase from rabbit muscle forms by reaction with dihydroxyacetone phosphate a pyruvaldehyde-aldolase-orthophosphate complex that is in equilibrium with the eneamine intermediate. The new intermediate accumulates in two phases. The first one is practically complete in 40ms, and the second occurs with an apparent first-order rate constant of 4.6 +/- 0.5s-1. The new intermediate breaks down slowly with the release into the medium of pyruvaldehyde and Pi. The rate of the spontaneous release is higher at acidic than at neutral pH.

Aldehydes↗

Methylglyoxal synthetase, enol-pyruvaldehyde, glutathione and the glyoxalase system.

enol-Pyruvaldehyde (ePY or 2-hydroxypropenal, O=C(H)-C(OH)=CH(2)) a transient intermediate in the alkaline decomposition of the triosephosphates to methylglyoxal is now observed by UV and (1)H NMR spectroscopy as the immediate product of the methylglyoxal synthetase (MGS) reaction: dihydroxyacetone-P --> P(i) + ePY --> methylglyoxal (MG). Analysis of ePY formed from 1-(13)C- and (1R, 3S) -[1,3-(2)H]-DHAP establishes the stereochemical course of its formation by MGS. Its rate of ketonization is much too slow to be in the sequence required for the assay of MGS by coupling of the MG produced to glyoxalase I (Glx I): MG + glutathione (GSH) --> (S)-lactylglutathione (D-LG). Instead, ketonization occurs by way of the hemithioacetal (HTA) formed between ePY and GSH, and could be either an enzymatic function of Glx I or occur nonenzymatically at an activated rate. Enzymatic ketonization was ruled out because the methyl group of D-LG formed from specifically labeled ePY is achiral. Chemical ketonization of ePY is activated by general bases, such as acetate, and by thiols such as GSH and 2-mercaptoethanol, which disrupt its stabilizing double bond conjugation as hemithioacetal (HTA) adducts. 2-Mercaptoacetate combines both functions, acting as the HTA adduct of ePY with the appended carboxylate group presumably positioned to promote abstraction of the enol proton and protonation of the enolate carbon at an accelerated rate. In the MGS-Glx I system (dihydroxyacetone-P --> ePY, ePY + GSH --> GS-ePY, GS-ePY --> GS-MG, GS-MG --> D-LG), the nonenzymatic 2nd and 3rd steps describe the catalytic role of GSH in the critical ketonization process and set the stage for its participation in the glyoxalase system.

Buffers↗

Evaluation of copper(II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) for tissue blood flow measurement using a trapped tracer model.

Copper(II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) (Cu-PTSM) labelled with 62,64Cu is a promising radiotracer for the study of blood flow using positron emission tomography (PET). We have investigated the application of a simple trapped tracer model to measurements of tissue 64Cu-PTSM uptake combined with continuous arterial sampling. A dual-tracer method was used to compare blood flow estimated by 64Cu-PTSM with values derived from measurements using cobalt-57 microspheres in the rat. Prolonged retention of 64Cu-PTSM following intravenous administration was initially confirmed in both normal tissues and tumours. After intraventricular 64Cu-PTSM infusion, cumulative arterial 64Cu activity increased progressively, and after extraction in n-octanol was found to plateau to levels corresponding with those reached following administration of 57Co microspheres. Rapid and species-dependent rates of 64Cu-PTSM decomposition to non-extractable 64Cu complexes were found in rat and human blood in vitro (70% +/- 6% and 43 +/- 5% respectively at 16 min), demonstrating the need for immediate processing of arterial samples. Close agreement was found between blood flow estimated by 64Cu-PTSM and 57Co microsphere methods in tissues of low to moderate flow: muscle (0.01, 0.08, 0.07 ml/min per gram; mean difference, mean 64Cu, mean 57Co), brain (0.09, 0.52, 0.43 ml/min per gram) and kidney (-0.16, 2.29, 2.45 ml/min per gram). Estimates of cardiac output also compared favourably between the two methods (5.7, 59.8, 54.1 ml/min). We conclude that a simple tissue trapping model may be suitable for the derivation of blood flow estimates using 62,64Cu-PTSM, PET imaging and continuous arterial blood sampling.

Animals↗

Cytosolic/microsomal redox pathway: a reductive retention mechanism of a PET-oncology tracer, cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (cu-PTSM).

OBJECTIVE: To clarify the retention mechanism of a PET imaging agent Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-62-PTSM) in tumor cells, reductive metabolism of non-radioactive Cu-PTSM in five cultured tumor cell lines, a tumor specimen and non-tumor tissues in vitro was evaluated by electron spin resonance spectrometry (ESR). RESULTS: In the brain, mitochondrial electron transport enzyme reduced Cu-PTSM specifically. On the other hand, Cu-PTSM was not reduced in tumor mitochondria. The mitochondrial electron transport enzyme in tumor cells was not damaged, but NADH was considered to be depleted. In compensation for that, the tumor cells acquired complementary reduction activity in the microsome/cytosol. The reduction was enzymatic and NADH-dependent, possibly similar to the activation mechanism of bioreductive anticancer drugs. CONCLUSION: Cu-PTSM and its derivatives are considered to be used as a marker for microsome/cytosol redox ability in PET oncology, although the physiological role of the redox enzyme system in tumor cells has not been clarified. The change in electron (NADH) flow in tumor cells might be a mechanism supporting aerobic glycolysis in tumor cells.

Animals↗

A potential copper radiopharmaceutical for imaging the heart and brain: copper-labeled pyruvaldehyde bis(N4-methylthiosemicarbazone).

An investigation of the biodistribution of lipophilic copper-64 (half-life = 12.7 h) compounds has been initiated in order to screen potential tracers that could be used to measure regional cerebral and/or myocardial blood flow when labeled with generator-produced 62Cu. The 64Cu complex of pyruvaldehyde bis(N4-methylthiosemicarbazone), [64Cu]Cu-PTSM, was prepared and found to be lipophlic (octanol/saline partition coefficient, log p = 1.97 +/- 0.03). Biodistribution studies following i.v. injection of [64Cu]Cu-PTSM into rats show tracer uptake by the brain and heart. At 1, 5, and 15 min post-injection 3.3, 3.0 and 2.7% of the injected dose was found in the brain. Corresponding brain to blood ratios (per gram) were 3.2, 3.6 and 4.0 respectively. Heart to blood ratios of 7.6, 7.6 and 7.3 were observed at these same time points.

Animals↗

Structural characterization of a metal-based perfusion tracer: copper(II) pyruvaldehyde bis(N4-methylthiosemicarbazone).

Copper(II) pyruvaldehyde bis(N4-methylthiosemicarbazone), Cu(PTSM), has been obtained as a dark red crystalline solid from EtOH-DMSO solvent mixture and structurally characterized by x-ray crystallography. The molecule possesses the expected pseudo-square planar N2S2 metal coordination sphere; however, the copper center also interacts through its axial coordination site with the sulfur atom of an adjacent Cu(PTSM) molecule in the crystal lattice. The structure of this compound is compared with the structures of other metal complexes that have been proposed in the nuclear medicine literature as perfusion tracers.

Crystallography↗

Copper-62-pyruvaldehyde bis(N-methyl-thiosemicarbazone) PET imaging in the detection of coronary artery disease in humans.

BACKGROUND: Copper-62 (II)-pyruvaldehyde bis(N(4)-methyl-thiosemicarbazone) (PTSM) has been proposed for cardiac imaging with positron emission tomography (PET). This study evaluated the agreement between Cu-62-PTSM and coronary angiography in the detection of occlusive coronary artery disease. The normalcy rate for Cu-62-PTSM PET in a group of healthy volunteers was also assessed. METHODS AND RESULTS: Forty-five subjects completed the study. Twenty-eight patients underwent stress technetium-99m sestamibi single photon emission computed tomography (SPECT) imaging and cardiac catheterization followed by Cu-62-PTSM rest/dipyridamole stress PET scans, and 17 volunteers underwent Cu-62-PTSM rest/dipyridamole stress PET scans. Cu-62-PTSM myocardial perfusion defects were identified in 100% of patients with 3-vessel disease (n = 8), 100% of patients with 2-vessel disease (n = 9), and 67% of patients with single-vessel disease (n = 6). When considering individual vessels, Cu-62-PTSM perfusion defects were seen in 72% of patients with occlusive disease in the left anterior descending artery territory, 67% in the left circumflex artery territory, and 60% in the right coronary artery territory, respectively. All 17 healthy volunteers had Cu-62-PTSM scans interpreted as normal, for a normalcy rate of 100%. CONCLUSIONS: Perfusion abnormalities are demonstrated by means of Cu-62-PTSM PET in 91% of patients with occlusive coronary artery disease seen at the time of cardiac catheterization, and it shows an excellent normalcy rate of 100%.

Adult↗

Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography.

We have used copper-64-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) to radiolabel cells ex vivo for in vivo positron-emission tomography (PET) imaging studies of cell trafficking in mice and for eventual application in patients. 2-[18F]-Fluoro-2-deoxy-d-glucose (FDG) cell labeling also was evaluated for comparison. 64Cu-PTSM uptake by C6 rat glioma (C6) cells increased for 180 min and then stabilized. The labeling efficiency was directly proportional to 64Cu-PTSM concentration and influenced negatively by serum. Label uptake per cell was greater with 64Cu-PTSM than with FDG. However, both 64Cu-PTSM- and FDG-labeled cells showed efflux of cell activity into supernatant. The 64Cu-PTSM labeling procedure did not interfere significantly with C6 cell viability and proliferation rate. MicroPET images of living mice indicate that tail-vein-injected labeled C6 cells traffic to the lungs and liver. In addition, transient splenic accumulation of radioactivity was clearly detectable in a mouse scanned at 3.33 h postinfusion of 64Cu-PTSM-labeled lymphocytes. In contrast, the liver was the principal organ of tracer localization after tail-vein administration of 64Cu-PTSM alone. These results indicate that in vivo imaging of cell trafficking is possible with 64Cu-PTSM-labeled cells. Given the longer t(1/2) of 64Cu (12.7 h) relative to 18F (110 min), longer cell-tracking periods (up to 24-36 h) should be possible now with PET.

Animals↗

Mitochondria-selective reduction of 62Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (62Cu-PTSM) in the murine brain; a novel radiopharmaceutical for brain positron emission tomography (PET) imaging.

The retention mechanism of 62Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (62Cu-PTSM) in the murine brain was evaluated. For this purpose, stable Cu-PTSM was subjected to electron spin resonance spectrometry (ESR) and high performance liquid chromatography (HPLC) analysis to determine the valence state, coordination structure and tissue metabolism. In murine brain homogenate, ESR and HPLC analysis indicated the reduction and cleavage of Cu(II)-PTSM to Cu(I). This virtually irreversible reduction was specifically initiated by the mitochondrial enzymatic system in the murine brain.

Animals↗

Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM), a metal complex with selective NADH-dependent reduction by complex I in brain mitochondria: a potential radiopharmaceutical for mitochondria-functional imaging with positron emission tomography (PET).

The reductive retention mechanism of copper(II)-pyruvaldehyde-bis (N4-methylthiosemicarbazone) (Cu-PTSM), a generator-produced positron-emitting 62Cu-labeled radiopharmaceutical, was studied with non-radioactive and radioactive copper. Changes in the chemical form of Cu-PTSM were detected by electron spin resonance spectrometry (ESR) with cold copper. The effects of electron transport chain inhibitors on the reduction of Cu-PTSM were also examined. Rotenone and antimycin A activated the reduction of Cu-PTSM in the brain mitochondria by 1.6- and 1.4-fold, respectively, compared with untreated controls, while thenoyltrifluoroacetone (TTFA) had no effect on the reduction. These results were confirmed with radioactive copper. Furthermore, this reduction of Cu-PTSM was dependent on the protein concentration of mouse brain submitochondrial particle (SMP) with 1 mM NADH (0 mg-protein/ml: 1.8 +/- 2.5%, 8 mg-protein/ml: 69.0 +/- 5.5%, each value was % of reduced Cu). Similarly, this reduction depended on NADH concentration at a fixed concentration of SMP (8 mg-protein/ml). These results indicated that the electron transport chain, especially complex I, participated in the reduction of Cu-PTSM in brain mitochondria, and this suggested that Cu-PTSM has the potential to act as a functional imaging agent for diagnosis of the electron transport chain.

Animals↗

Copper-64-pyruvaldehyde-bis(N(4)-methylthiosemicarbazone) for the prevention of tumor growth at wound sites following laparoscopic surgery: monitoring therapy response with microPET and magnetic resonance imaging.

Laparoscopic colectomy for curable colon cancer may result in the development of abdominal wall implants because of disseminated disease and the favorable environment of the wound site for cell implantation. Injection of disaggregated human GW39 colon cancer cells into the hamster peritoneum represents a model of tumor spillage that may occur during dissection, manipulation, resection, and extraction of tumor during surgery in the clinical setting. Using this well-established animal model, we tested the efficacy of (64)Cu-pyruvaldehyde-bis(N(4)-methylthiosemicarbazone) ((64)Cu-PTSM) in inhibiting tumor cell implantation in trocar wound sites. Anesthetized hamsters had four 5-mm trocars inserted through the anterior abdominal wall. GW39 cells ( approximately 3.2 x 10(4) cells in 0.5 ml) were injected into the peritoneum through a midline incision. Ten min later, hamsters were randomized to receive 5, 3, or 1 mCi of (64)Cu-PTSM through the same midline incision. High-resolution magnetic resonance imaging and microPET were used to monitor tumor volume and morphology after surgery. After 7 weeks, animals were sacrificed, and trocar and midline wounds were harvested for macroscopic and histological analysis. No macroscopic tumor was found in any of the group treated with 5 mCi of (64)Cu-PTSM, whereas 96% of the wound sites in the group treated with saline had macroscopic tumor growth (P < 0.001). This study demonstrates the therapeutic potential of (64)Cu-PTSM in inhibiting cancer cell implantation and growth at doses well below the maximum tolerated dose, with no signs of toxicity to the hamsters.

Abdominal Muscles↗

Copper-62-labeled pyruvaldehyde bis(N4-methylthiosemicarbazonato)copper(II): synthesis and evaluation as a positron emission tomography tracer for cerebral and myocardial perfusion.

Generator produced positron-emitting radionuclides could potentially expand the application of positron emission tomography (PET) to centers that do not have access to a local cyclotron. The zinc-62/copper-62 radionuclide generator system could serve as a source of positron-emitting copper-62 (62Cu) (t1/2 = 9.74 min) for physiologic imaging. Accordingly, we have prepared zinc-62/copper-62 generators capable of high output (greater than 300 mCi) and used the no-carrier-added eluate in a rapid high yield synthesis of [62Cu] Cu(PTSM) that provides the radiopharmaceutical in a form suitable for intravenous injection (where Cu(PTSM) = pyruvaldehyde bis(N4-methylthiosemicarbazonato) copper(II]. We then demonstrated in pilot studies that [62Cu]Cu(PTSM) provides high quality brain and heart images with PET, accurately delineating cerebral and myocardial perfusion in both experimental animals and in humans (corroborating results of previous experimental studies utilizing longer-lived copper isotopes). The results of this work demonstrate that 62Cu can be conveniently obtained from high-level generators and, when used to label Cu(PTSM), provides a generator-produced radiopharmaceutical capable of providing estimates of cerebral and myocardial perfusion independent of cyclotron-produced radionuclides.

Animals↗

Tissue blood flow estimation with copper(II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) and PET.

Copper (II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) (Cu-PTSM) labelled with 62Cu or 64Cu is currently under investigation as a radiotracer for imaging the distribution of blood flow with positron emission tomography (PET). The application of a simple trapped tracer model in conjunction with tissue uptake and continuous arterial sampling to estimate blood flow has been compared with the 57Co-microsphere method in the rat. After intraventricular injection the cumulative arterial function for 64Cu increased progressively due to the presence of circulating non lipophilic complexes. The cumulative function for lipophilic 64Cu-PTSM extracted in n-octanol plateaued at levels corresponding to those reached by 57Co-microspheres. No consistent disagreement was found between cardiac output and blood flow estimated by 64Cu-PTSM and 57Co-microspheres in low to moderate flow tissues: muscle (0.08, 0.07 mL/min/g; 64Cu mean, 57Co mean), brain (0.52, 0.43 mL/min/g) and kidney (2.29, 2.45 mL/min/g). However, 64Cu-PTSM underestimated blood flow measured by 57Co-microspheres in myocardium (4.09, 6.55 mL/min/g). A simple tissue trapping model may therefore be suitable for the derivation of blood flow estimates in low to moderate flow tissues using 62,64Cu-PTSM, PET imaging and continuous arterial sampling with n-octanol extraction.

Animals↗

Acute toxicity and mutagenicity of the copper complex of pyruvaldehyde-bis (N-4-methylthiosemicarbazone), Cu-PTSM.

Cu-PTSM is a potential imaging agent for the heart and brain when labeled with either 64Cu or 62Cu. Unlabeled Cu-PTSM was evaluated for its acute toxicity and mutagenicity. Cu-PTSM had an i.v. LD50 of 26 mg kg-1 in the rat and 2 mg kg-1 in the rabbit. At necropsy, rats exhibited severely hemorrhagic lungs, histological findings of acute pulmonary congestion, hemorrhage and edema, and mild congestion in kidney, liver and brain. The rabbit displayed marked polymorphonuclear infiltration in alveoli, peribronchial and periarterial areas with marked macrophage hyperplasia, congestion and mild hemorrhage into alveolar spaces. No effects were found in kidney, liver, testes or brain. Administration of 2.16 micrograms kg-1 day-1 for 5 days per week for 2 weeks resulted in no changes in histopathology, hematology or clinical chemistry parameters. This daily dose is at least 300 times the diagnostic dose intended for use in man. Cu-PTSM was not mutagenic when tested in the absence of S9 supernatant, but elicited a weakly mutagenic response in the presence of S9. Since acute effects in the lung occur at doses approaching 300,000 times the diagnostic dose, it is highly unlikely that the clinical use of Cu-PTSM would result in any acute adverse effects.

Animals↗

Differential mechanism of retention of Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM) by brain and tumor: a novel radiopharmaceutical for positron emission tomography imaging.

The reductive retention of 62Cu-PTSM was comparatively studied in the brain and Ehrlich ascites tumor cells by electron spin resonance spectrometry and nonradioactive Cu-PTSM. In the brain, only the mitochondrial fraction showed the ability to reduce Cu-PTSM, and the other subcellular fractions did not. In contrast, the cytosolic fraction of Ehrlich ascites tumor cells was the specific site of Cu-PTSM reduction. It was therefore considered that the retention of Cu-PTSM in the brain is closely related to mitochondrial reduction, most probably involving the mitochondrial electron transport system.

Animals↗