Three structurally and functionally distinct ß-glucuronidases from the human gut microbe Bacteroides uniformis.

The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct ß-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid-containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.

Saccharolactone: The History, the Myth, and the Practice.

BACKGROUND: Over the past two decades, saccharolactone has been routinely used in in vitro microsomal incubations, and sometimes in incubations with recombinant Uridine diphosphoglucuronosyl transferases (UGT) while investigating glucuronidation reactions. The addition of saccharolactone is aimed at completely inhibiting ß-glucuronidases that may be present in the microsomes, in the anticipation of accurate identification and quantification of the formed glucuronide metabolites. Recent research has demonstrated that saccharolatone may not serve the intended objective, and may even lead to inhibition of certain UGTs. OBJECTIVE: This report investigates the historic evidence in the practice of saccharolactone addition in relation to ß- glucuronidases and UGTs. The chemical nature and inhibition potency of saccharolactone are explored in an attempt to unravel the myth in its application. Finally, the collective evidence is discussed in an effort to provide guidance to drug metabolism scientists on the utilization of saccharolactone. CONCLUSION: In-depth evaluation of the experimental evidence in the literature points toward a weak rationale for general in vitro application of saccharolactone. Furthermore, inhibition of recombinant or microsomal UGTs by saccharolactone may be model dependent. Overall, the integrated data suggests that saccharolactone should not be utilized in in vitro microsomal incubations with the objective of inhibiting ß-glucuronidases.

Metabonomics of d-glucaro-1,4-lactone in preventing diethylnitrosamine-induced liver cancer in rats.

CONTEXT: d-Glucaro-1,4-lactone (1,4-GL) exists in many vegetables and fruits. Metabonomics has not been used to investigate the role of 1,4-GL in preventing liver cancer. OBJECTIVE: The pharmacological effects and metabolite alterations of 1,4-GL on the prevention of diethylnitrosamine (DEN)-induced liver cancer were investigated. MATERIALS AND METHODS: Ten healthy Sprague-Dawley rats served as control and 46 were used to establish rat liver cancer model. 1HNMR-based metabonomics was used to compare the effects of oral 1,4-GL (50 mg/kg) in liver cancer rats (n = 26) after 10 consecutive weeks of intervention. The amino acids in rat serum were quantified by HPLC-UV, and the changes in Fischer's ratio were calculated. RESULTS: The 20-week survival rate of DEN-induced liver cancer rats administered with oral 1,4-GL was increased from 45.0 to 70.0% with reduced carcinogenesis of the liver and significantly lowered serum α-fetoprotein level (14.28 ± 2.89 ng/mL vs. 18.56 ± 4.65 ng/mL, p = 0.012). The serum levels of leucine, valine, 3-hydroxybutyrate, lactate, acetate and glutamine in the DEN + 1,4-GL group returned to normal levels compared with those of the DEN group on week 20. Fischer's ratio in the rat serum of DEN group was 1.62 ± 0.21, which was significantly lower than that in healthy rats (2.3 ± 0.12). However, Fischer's ratio increased to 1.89 ± 0.22 in the DEN + 1,4-GL group. DISCUSSION AND CONCLUSIONS: 1,4-GL exerted positive effects on liver carcinogenesis in rats by pathological examination and metabonomic analysis. Its mechanism may be related to the restoration of amino acid and energy metabolism.

Involvement of bilitranslocase and beta-glucuronidase in the vascular protection by hydroxytyrosol and its glucuronide metabolites in oxidative stress conditions.

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because it is extensively metabolized into glucuronides (GCs). The aim of this study was to test HT and GC vasculoprotective effects and the underlying mechanisms using aorta rings from 8-week-old male Wistar rats. In the absence of oxidative stress, incubation with 100 µM HT or GC for 5 min did not exert any vasorelaxing effect and did not influence the vascular function. Conversely, in condition of oxidative stress [upon incubation with 500 µM tert-butylhydroperoxide (t-BHP) for 30 min], preincubation with HT or GC improved acetylcholine-induced vasorelaxation compared with untreated samples (no t-BHP). This protective effect was lost for GC, but not for HT, when a washing step (15 min) was introduced between preincubation with HT or GC and t-BHP addition, suggesting that only HT enters the cells. In agreement, bilitranslocase inhibition with 100 µM phenylmethanesulfonyl fluoride for 20 min reduced significantly HT, but not GC, effect on the vascular function upon stress induction. Moreover, GC protective effect (improvement of endothelium-dependent relaxation in response to acetylcholine) in oxidative stress conditions was reduced by preincubation of aorta rings with 300 µM D-saccharolactone to inhibit ß-glucuronidase, which can deconjugate polyphenols. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that, in conditions of oxidative stress, GC can be deconjugated into HT that is transported through the cell membrane by bilitranslocase to protect vascular function.

Determination of D-glucaric acid and/or D-glucaro-1,4-lacton in different apple varieties through hydrophilic interaction chromatography.

d-Glucaric acid (GA) derivatives exhibit anti-cancerogenic properties in vivo in apples, but quantitative information about these derivatives is limited. Hydrophilic interaction-based HPLC with ultraviolet detection or mass spectrometry was developed to quantify GA and/or D-glucaro-1,4-lacton (1,4-GL) in apples. Although the formation of 1,4-GL from GA could be the prerequisite to exert biological effects in vivo, only a small portion of GA (<5%) was identified and converted to 1,4-GL in the rat stomach. The 1,4-GL content in apples ranged from 0.3 mg/g to 0.9 mg/g, and this amount can substantiate health claims associated with apples. The amount of 1,4-GL was 1.5 times higher in Gala and the ratio of 1,4-GL to GA was lower in Green Delicious apples than those in the other varieties. Our findings suggested that the variety and maturity of apples at harvest are factors that determine 1,4-GL content.

Triple Recycling Processes Impact Systemic and Local Bioavailability of Orally Administered Flavonoids.

Triple recycling (i.e., enterohepatic, enteric and local recycling) plays a central role in governing the disposition of phenolics such as flavonoids, resulting in low systemic bioavailability but higher gut bioavailability and longer than expected apparent half-life. The present study aims to investigate the coexistence of these recycling schemes using model bioactive flavonoid tilianin and a four-site perfused rat intestinal model in the presence or absence of a lactase phlorizin hydrolase (LPH) inhibitor gluconolactone and/or a glucuronidase inhibitor saccharolactone. The result showed that tilianin could be metabolized into tilianin glucuronide, acacetin, and acacetin glucuronide, which are excreted into the bile and luminal perfusate (highest in the duodenum and lowest in the colon). Gluconolactone (20 mM) significantly reduced the absorption of tilianin and the enteric and biliary excretion of acacetin glucuronide. Saccharolactone (0.1 mM) alone or in combination of gluconolactone also remarkably reduced the biliary and intestinal excretion of acacetin glucuronide. Acacetin glucuronides from bile or perfusate were rapidly hydrolyzed by bacterial ß-glucuronidases to acacetin, enabling enterohepatic and enteric recycling. Moreover, saccharolactone-sensitive tilianin disposition and glucuronide deconjugation, which was more active in the small intestine than the colon, points to the small intestinal origin of the deconjugation enzyme and supports the presence of local recycling scheme. In conclusion, our studies have demonstrated triple recycling of a bioactive phenolic (i.e., a model flavonoid), and this recycling may have an impact on the site and duration of polyphenols pharmacokinetics in vivo.

Application of metabonomic strategy to discover an unreported active ingredient in LiuWeiDiHuang pills suppressing beta-glucuronidase.

Identification of the bioactive ingredient from traditional Chinese medicine (TCM) remains a challenging task by traditional approach that focuses on chemical isolation coupled with biological activity screening. Here, we present a metabonomics-based approach for bioactive ingredient discovery in LiuWeiDiHuang pills (LWPs). First, a non-targeted high-performance liquid chromatography ultraviolet (HPLC-UV) profiling of rat urine was used to discriminate urinary profiling intervened by LWPs. Orthogonal partial least-squares discriminant analysis (OPLS-DA) revealed that eight chromatographic peaks made a significant contribution to the classification of the LWPs group and the control group. Five of these chromatographic peaks were successfully isolated and identified as hippurate, genistein (GT), daidzein (DZ), and glucuronide conjugate of GT and that of DZ by mass spectroscopy (MS). Subsequently, we found that LWPs significantly decreased the activity of intestinal ß-glucuronidase by 18 % and exerted a dose-dependent inhibitory effect on rat liver lysosomal fraction, suggesting that LWPs were a ß-glucuronidase inhibitor. In the end, by inhibiting ß-glucuronidase-guided isolation, D-glucaro-1,4-lactone, a previously unreported ingredient of LWPs, was identified by MS, MS/MS, and nuclear magnetic resonance spectroscopy. Our findings indicated that metabonomics might increase research productivity toward the drug targets and/or bioactive compounds from TCM.

Alteration of gut bacteria and metabolomes after glucaro-1,4-lactone treatment contributes to the prevention of hypercholesterolemia.

D-Glucaro-1,4-lactone (1,4-GL) has been shown to have a hypocholesterolemic effect in rats and human subjects. However, little information is known concerning the alteration of metabolome associated with the effect. Here, we show that 1,4-GL delays the development of hypercholesterolemia with the coadministration of a high-fat, high-cholesterol diet (HFHC) in rats. Metabonomic results based on proton nuclear magnetic resonance indicate that urinary trimethylamine N-oxide, trimethylamine, lactate, acetate, formate, and creatinine are significantly altered after 1,4-GL and HFHC treatments. Colonic flora test results reveal that the quantity of Bifidobacterium and Lactobacillus in the intestines respectively increase by about 1.7- and 4.2-fold in rats treated with 1,4-GL compared with those in the control group. Rats that were coadministered with HFHC and 1,4-GL exhibit normal levels of lactate and acetate in serum and display urinary excretions of lactate and acetate that are 2 to 3 times higher compared with those treated with HFHC alone. The results imply that the increased probiotic quantities and urinary excretion of breakdown products of fat/cholesterol after 1,4-GL treatment contribute to the prevention of hypercholesterolemia. Our study offers insights into the model of action for 1,4-GL in preventing hypercholesterolemia.

Is [(99m)Tc]glucarate uptake mediated by fructose transporter GLUT-5?

PURPOSE: There is growing interest in the ability of [(99m)Tc]Glucarate ([(99m)Tc]GLA) to accumulate in viable tumor cells. Recent vivo studies suggest that [(99m)Tc]Glucarate could be helpful for tumor detection. Fructose transport is thought to be implicated. It is clearly established that facilitated fructose transport in tumor cells is related to the GLUT-5 transporter. This study therefore investigated whether [(99m)Tc]GLA uptake is mediated by GLUT-5 transporter. METHODS: Different tumor cell lines were used. Modulation of GLUT-5 expression was assessed with and without antisense oligonucleotides directed against GLUT-5. GLUT-5 expression was assessed by indirect cell ELISA. To correlate GLUT-5 expression with tracer accumulation, [(99m)Tc]GLA uptake was determined after antisense treatment. A competition with fructose was also monitored. RESULTS: Inhibition of GLUT-5 expression by antisense oligonucleotides directed against GLUT-5 was effective after 24 h. An optimal of 10µM antisense oligonucleotides directed against GLUT-5 produced a 30%-40% decrease in protein expression. Modulation of [(99m)Tc]GLA uptake was monitored either by use of specific antisense oligonucleotides or by competition with fructose. Both of them produced a significant decrease of [(99m)Tc]GLA accumulation in all tested cell lines. CONCLUSION: Our results clearly demonstrate that [(99m)Tc]GLA uptake is related to GLUT-5 transporter expression and transport. In tumor imaging, [(99m)Tc]GLA may be a useful tool for non-invasive detection of malignant tumors expressing high levels of GLUT-5 transporter as, for example, breast cancers.

A novel local recycling mechanism that enhances enteric bioavailability of flavonoids and prolongs their residence time in the gut.

Recycling in the gastrointestinal tract is important for endogenous substances such as bile acids and for xenobiotics such as flavonoids. Although both enterohepatic and enteric recycling mechanisms are well recognized, no one has discussed the third recycling mechanism for glucuronides: local recycling. The intestinal absorption and metabolism of wogonin and wogonoside (wogonin-7-glucuronide) was characterized by using a four-site perfused rat intestinal model, and hydrolysis of wogonoside was measured in various enzyme preparations. In the perfusion model, the wogonoside and wogonin were interconverted in all four perfused segments. Absorption of wogonoside and conversion to its aglycon at the upper small intestine was inhibited in the presence of a glucuronidase inhibitor (saccharolactone) but was not inhibited by lactase phlorizin hydrolase (LPH) inhibitor gluconolactone or antibiotics. Further investigation indicated that hydrolysis of wogonoside in the blank intestinal perfusate was not correlated with bacterial counts. Kinetic studies indicated that K(m) values from blank duodenal and jejunal perfusate were essentially identical to the K(m) values from intestinal S9 fraction but were much higher (>2-fold) than those from the microbial enzyme extract. Lastly, jejunal perfusate and S9 fraction share the same optimal pH, which was different from those of fecal extract. In conclusion, local recycling of wogonin and wogonoside is the first demonstrated example that this novel mechanism is functional in the upper small intestine without significant contribution from bacteria ß-glucuronidase.

Detection of myocardial ischemia-reperfusion injury using a fluorescent near-infrared zinc(II)-dipicolylamine probe and 99mTc glucarate.

A fluorescent zinc 2,2'-dipicolylamine coordination complex PSVue®794 (probe 1) is known to selectively bind to phosphatidylserine exposed on the surface of apoptotic and necrotic cells. In this study, we investigated the cell death targeting properties of probe 1 in myocardial ischemia-reperfusion injury. A rat heart model of ischemia-reperfusion was used. Probe 1, control dye, or 99mTc glucarate was intravenously injected in rats subjected to 30-minute and 5-minute myocardial ischemia followed by 2-hour reperfusion. At 90 minutes or 20 hours postinjection, myocardial uptake was evaluated ex vivo by fluorescence imaging and autoradiography. Hematoxylin-eosin and cleaved caspase-3 staining was performed on myocardial sections to demonstrate the presence of ischemia-reperfusion injury and apoptosis. Selective accumulation of probe 1 could be detected in the area at risk up to 20 hours postinjection. Similar topography and extent of uptake of probe 1 and 99mTc glucarate were observed at 90 minutes postinjection. Histologic analysis demonstrated the presence of necrosis, but only a few apoptotic cells could be detected. Probe 1 selectively accumulates in myocardial ischemia-reperfusion injury and is a promising cell death imaging tool.

Vascular deconjugation of quercetin glucuronide: the flavonoid paradox revealed?

SCOPE: The dietary flavonoid quercetin exerts protective cardiovascular effects. Because quercetin is rapidly metabolized into less active or inactive glucuronidated metabolites and the plasma concentrations of free quercetin are very low, a huge amount of scientific data generated along decades with the unconjugated compounds in vitro has been questioned. We aimed to determine whether glucuronidated quercetin can deconjugate in situ and whether deconjugation leads to a biological effect. METHODS AND RESULTS: Quercetin and quercetin-3-O-glucuronide (Q3GA) were perfused through the isolated rat mesenteric vascular bed. Quercetin was rapidly metabolized in the mesentery. In contrast, the decay of Q3GA was slower and was accompanied by a progressive increase of quercetin in the perfusate and in the tissue over 6 h, which was prevented by the ß-glucuronidase inhibitor saccharolactone. Incubation of mesenteric arterial rings mounted in a wire myograph with Q3GA for ≥1 h resulted in a significant inhibition of the contractile response which was also prevented by saccharolactone. Moreover, the intravenous administration of Q3GA resulted in a slow onset and sustained blood pressure lowering effect, demonstrating for the first time that Q3GA has effects in vivo. CONCLUSION: We propose that Q3GA behaves as a quercetin carrier in plasma, which deconjugates in situ releasing the aglycone which is the final effector.

A brief evaluation of tumor imaging in mice with 99mTc-glucarate including a comparison with 18F-FDG.

OBJECTIVE: Recently 99mTc-glucarate, a radiolabeled glucose analogue, has been considered as a SPECT alternative to 18F-FDG and PET for non-invasive detection of certain tumors. Thus far there have been few studies on (99m) Tcglucarate for tumor imaging and fewer, if any, studies comparing (99m)Tc-glucarate with 18F-FDG. As a preliminary indication of the properties of (99m)Tc-glucarate as a possible substitute for 18F-FDG in animal studies, we have imaged mice bearing xenografts of four tumor types with (99m)Tc-glucarate and have compared in two mice with one of these tumor types the 99mTc and 18F biodistributions. METHODS: Two mice bearing SUM190 breast cancer xenografts received 1 mCi of (99m)Tc-glucarate and were imaged on a NanoSPECT/CT small animal camera. One day later, the same animals received 1 mCi of 18F-FDG and were imaged on a MosaicHP PET small animal camera. In addition, 0.5-1 mCi of (99m)Tc-glucarate only was administered to mice bearing xenografts induced by BxPC3 pancreatic cancer cells, HEK-293 renal cell carcinomas cells or HCT-116 colorectal tumor cells. NanoSPECT/CT acquisitions were performed in these mice to evaluate tumor accumulations. RESULTS: In the SUM190 xenografted mice, the average tumor accumulation was 1.4 % (ID%/cm3) for (99m)Tc-glucarate and 2.1 % (ID%/cm3) for 18F-FDG. While slightly higher than (99m)Tc-glucarate, the tumor accumulation of 18F-FDG was accompanied by higher bone marrow and muscle accumulations at levels that could interfere with the tumor image depending upon location. The whole body clearance of (99m)Tc-glucarate was faster than that of 18F-FDG. Tumor accumulation of (99m)Tc-glucarate varied among tumor types but the tumors were readily visible in all images. CONCLUSION: In a direct comparison in the same two SUM190 tumored animals, SPECT images obtained with (99m)Tcglucarate compared favorably with PET images obtained with 18F-FDG. Tumor images with 99mTc-glucarate were also positive in three additional tumor mouse models. While further comparison studies are necessary, we conclude that (99m)Tcglucarate may be a more convenient and less expensive alternative to 18F-FDG for tumored mouse studies.

D-saccharic acid-1,4-lactone ameliorates alloxan-induced diabetes mellitus and oxidative stress in rats through inhibiting pancreatic ß-cells from apoptosis via mitochondrial dependent pathway.

Oxidative stress plays a vital role in diabetic complications. To suppress the oxidative stress mediated damage in diabetic pathophysiology, a special focus has been given on naturally occurring antioxidants present in normal diet. D-saccharic acid 1,4-lactone (DSL), a derivative of D-glucaric acid, is present in many dietary plants and is known for its detoxifying and antioxidant properties. The aim of the present study was to evaluate the beneficial role of DSL against alloxan (ALX) induced diabetes in the pancreas tissue of Swiss albino rats. A dose-dependent study for DSL (20-120 mg/kg body weight) was carried out to find the effective dose of the compound in ALX-induced diabetic rats. ALX exposure elevated the blood glucose, glycosylated Hb, decreased the plasma insulin and disturbed the intra-cellular antioxidant machineries whereas oral administration of DSL at a dose of 80 mg/kg body weight restored these alterations close to normal. Investigating the mechanism of the protective activity of DSL we observed that it prevented the pancreatic ß-cell apoptosis via mitochondria-dependent pathway. Results showed decreased mitochondrial membrane potential, enhanced cytochrome c release in the cytosol and reciprocal regulation of Bcl-2 family proteins in the diabetic rats. These events were also found to be associated with increased level of Apaf-1, caspase 9, and caspase 3 that ultimately led to pancreatic ß-cell apoptosis. DSL treatment, however, counteracted these changes. In conclusion, DSL possesses the capability of ameliorating the oxidative stress in ALX-induced diabetes and thus could be a promising approach in lessening diabetic complications.

Modulation of biomarkers related to tumor initiation and promotion in mouse skin by a natural ß-glucuronidase inhibitor and its precursors.

Carcinogen-mediated labilization of lysosomal enzymes such as ß-glucuronidase (ßG) is often associated with the general process of inflammation. Therefore, the primary goal of this study was to demonstrate that exposing the skin of SENCAR mice to the natural ßG inhibitor D-glucaro-1,4-lactone (1,4-GL) and its precursor D-glucuronic acid-γ-lactone (GUL), prior to and during 7,12-dimethylbenz[α]anthracene (DMBA) treatment inhibits not only epidermal hyperplasia but also inflammation in the mouse skin complete carcinogenesis model, i.e., the 4-week inflammatory-hyperplasia assay. Topical administration of 1,4-GL or GUL prior to repetitive, high-dose DMBA treatment markedly and in a dose-related manner inhibited DMBA-induced epidermal hyperplasia (i.e., up to 57%). DMBA-mediated Ha-ras mutations in codon 61 were reduced by up to 78% by 1,4-GL. DMBA-induced inflammation, as measured by dermal leukocyte counts and immunologically, was inhibited by up to 37% by topical 1,4-GL but not by GUL. The inhibition of cellular proliferation and inflammation coincided with the inhibition of ßG expression. Thus, the present study suggests that in the DMBA-induced complete skin carcinogenesis model, 1,4-GL when applied topically had both anti-proliferative properties as well as anti-inflammatory properties, whereas GUL had only anti-proliferative when applied topically. However, the number of inflammatory cells in the dermal portion of the skin of mice was significantly reduced by dietary treatment of GUL, whereas both topical and dietary treatments with 1,4-GL were very effective.

Evaluation of 99mTc-glucarate as a breast cancer imaging agent in a xenograft animal model.

INTRODUCTION: The use of [(99m)Tc]glucarate has been reported as an infarct-avid agent with the potential for very early detection of myocardial infarction. [(99m)Tc]Glucarate has also been postulated as an agent for non-invasive detection of tumors. The aim of our study was to develop a Glucarate kit and evaluate [(99m)Tc]glucarate as a potential cancer imaging agent in female SCID mice bearing human MDA-MB-435 breast tumors. METHODS: Glucarate in a kit formulation was labeled with (99m)Tc and evaluated for radiolabelling efficiency and radiochemical purity. The Glucarate kit stability was assessed by monthly quality controls. The pharmacokinetics of [(99m)Tc]glucarate were determined in female SCID mice bearing MDA-MB-435 human breast carcinoma tumors at 0.5, 1, 2, 4 and 24 h. Nuclear imaging studies were performed with a micro-single photon emission tomography (SPECT)/computed tomography (CT) system at 2 h post injection, while magnetic resonance imaging (MRI) was employed for tumor morphology analysis and metastatic deposit localization. RESULTS: The Glucarate kits exhibited a stable shelf life of 6 months. [(99m)Tc]Glucarate was obtained with radiochemical purity greater than 95%. Biodistribution studies demonstrated moderate tumor uptake coupled with high renal clearance. Tumor-to-muscle ratios were 4.85 and 5.14 at 1 and 4 h post injection. MRI analysis showed tumors with dense cellular growth and moderate central necrosis. [(99m)Tc]Glucarate uptake in the primary MDA-MB-435 shoulder tumors and metastatic lesions were clearly visualized with micro-SPECT/CT imaging. CONCLUSIONS: Selective tumor uptake and rapid clearance from nontarget organs makes [(99m)Tc]glucarate a potential agent for breast cancer imaging that awaits validation in a clinical trial.

99mTc-glucarate kinetics differentiate normal, stunned, hibernating, and nonviable myocardium in a perfused rat heart model.

PURPOSE: (99m)Tc-glucarate is an infarct-avid imaging agent. However, patients may have mixtures of normal, irreversibly injured, stunned, and hibernating myocardium. The purposes were to determine (99m)Tc-glucarate uptake and clearance kinetics in these four conditions, and its ability to determine the extent of injury. METHODS: Twenty-two perfused rat hearts were studied: controls (n = 5), stunned (n = 5; 20-min no-flow followed by 5-min reflow), hibernating (n = 6; 120-min low flow at 4 ml/min), and ischemic-reperfused (n = 6; 120-min no-flow followed by reflow). (99m)Tc-glucarate was then infused. Tracer activity was monitored using a NaI scintillation detector and a multichannel analyzer. Creatine kinase, electron microscopy, and triphenyltetrazolium chloride determined viability. RESULTS: (99m)Tc-glucarate 10-min myocardial uptake was significantly greater in ischemic-reperfused (2.50 +/- 0.09) (cpm, SEM) than in control (1.74 +/- 0.07), stunned (1.68 +/- 0.11), and hibernating (1.59 +/- 0.11) (p < 0.05). Tracer retention curves for ischemic-reperfused were elevated at all time points as compared with the other groups. (99m)Tc-glucarate 60-min myocardial uptake was significantly greater in ischemic-reperfused (7.60 +/- 0.63) than in control (1.98 +/- 0.15), stunned (1.79 +/- 0.08), and hibernating (2.33 +/- 0.15) (p < 0.05). The 60-min well-counted tracer activity ratio of ischemic-reperfused to control was 9:1 and corroborated the NaI detector results. Creatine kinase, triphenyltetrazolium chloride, and electron microscopy all demonstrated significantly greater injury in ischemic-reperfused compared to the other groups. An excellent correlation was observed between viability markers and tracer activity (r = 0.99 triphenyltetrazolium chloride; r = 0.90 creatine kinase). CONCLUSION: (99m)Tc-glucarate activity continually and progressively increased in irreversibly injured myocardium. (99m)Tc-glucarate uptake was strongly correlated with myocardial necrosis as determined by three independent assessments of viability. There were minimal and similar (99m)Tc-glucarate uptakes in control, stunned, and hibernating myocardium.

Evaluation of patients with head and neck cancer by means of 99mTc-glucarate.

UNLABELLED: Preliminary findings have suggested that (99m)Tc-glucarate has tumor-seeking properties. The purpose of this study was to explore the potential of this tracer to evaluate malignant head and neck tumors by means of SPECT/CT software fusion imaging. METHODS: Eleven male patients with advanced head and neck carcinoma were included in the study: 9 with locally advanced disease and 2 with clinical suspicion of local relapse. Scanning started 3-6 h after the injection of 1,110 MBq of (99m)Tc-glucarate. Planar and SPECT images of the head, neck, and thorax were acquired. Three-dimensional images were also coregistered with CT. RESULTS: We found (99m)Tc-glucarate uptake in all suspected lesions. SPECT/CT fusion imaging was helpful in all cases for topographically localizing the tracer foci. CONCLUSION: (99m)Tc-glucarate can be considered a potential tracer for the evaluation of patients with head and neck tumors.

Convenient large-scale synthesis of D-glucaro-1,4:6,3-dilactone.

Calcium D-glucarate was converted into D-glucaro-1,4:6,3-dilactone on 32-g, 1-kg, and 22-kg scale, using azeotropic distillation with methyl isobutyl ketone to drive the dehydration. The crystalline product was > or = 99.5% pure by GC and NMR, and overall yield was as high as 72%.

Molecular imaging of cell death.

Apoptosis (programmed cell death) and necrosis (uncontrolled cell death) are two distinct processes of cell death that have been described. Non-invasive molecular imaging of these two processes can have several clinical applications and has various approaches in pre-clinical research. Apoptosis imaging enables a specific and early measurement of response in cancer patients. In case of acute myocardial infarction (AMI) and cerebral stroke the degree of both apoptosis and necrosis is abundant. Imaging of both types of cell death is crucial for diagnosis and could differentiate between "real" and "rescuable" cell damage. In a pre-clinical setting cell death imaging offers the possibility for dynamic study protocols and repeated measurements of cell death in the same animal. This review provides an overview of the radiopharmaceutical development and in vivo evaluation of apoptosis and necrosis detecting radioligands that have emerged so far. Some apoptosis radiopharmaceuticals have made it to clinical trials ((99m)Tc-labeled Anx and (18)F-ML-10) while others need further optimization and evaluation (e.g., (18)F-WC-II-89). (99m)Tc-glucarate has been widely used in patients to image necrosis, but this radiopharmaceutical only works early after the onset of necrosis. Other necrosis avid probes like (123)I labeled hypericin and its monocarboxylic acid derivative and (99m)Tc(CO)(3)-bis-hydrazide-bis-DTPA pamoic acid need further evaluation but show already promising results for imaging of necrosis. As a general conclusion molecular imaging of both apoptosis and necrosis is necessary to understand the cell death process in several pathologies.