Analysis of N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine/N1‐acetyl‐5‐methoxy‐kynuramine formation from melatonin in mice

Abstract: The interactions of melatonin, a potent endogenous antioxidant, with reactive oxygen species generate several products that include N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK) and N¹‐acetyl‐5‐methoxy‐kynuramine (AMK). The physiological or pathological significance of AFMK/AMK formation during the process of melatonin metabolism in mammals has not been clarified. Using a metabolomic approach in the current study, the AFMK/AMK pathway was thoroughly investigated both in mice and humans. Unexpectedly, AFMK and AMK were not identified in the urine of humans nor in the urine, feces or tissues (including liver, brain, and eyes) in mice under the current experimental conditions. Metabolomic analysis did identify novel metabolites of AMK, i.e. hydroxy‐AMK and glucuronide‐conjugated hydroxy‐AMK. These two newly identified metabolites were, however, not found in the urine of humans. In addition, oxidative stress induced by acetaminophen in the mouse model did not boost AFMK/AMK formation. These data suggest that AFMK/AMK formation is not a significant pathway of melatonin disposition in mice, even under conditions of oxidative stress.     

Rebaudioside A directly stimulates insulin secretion from pancreatic beta cells: a glucose-dependent action via inhibition of ATP-sensitive K-channels

Recently, we showed that rebaudioside A potently stimulates the insulin secretion from isolated mouse islets in a dose-, glucose- and Ca(2+)-dependent manner. Little is known about the mechanisms underlying the insulinotropic action of rebaudioside A. The aim of this study was to define the signalling system by which, rebaudioside A acts. Isolated mouse islets were used in the cAMP[(125)I] scintillation proximity assay to measure total cAMP level, and in a luminometric method to measure intracellular ATP and ADP concentrations. Conventional and permeabilized whole-cell configuration of the patch-clamp technique was used to verify the effect of rebaudioside A on ATP-sensitive K(+)-channels from dispersed single beta cells from isolated mouse islets. Insulin was measured by radioimmunoassay from insulinoma MIN6 cells. In the presence of 16.7 mM glucose, the addition of the maximally effective concentration of rebaudioside A (10(-9) M) increased the ATP/ADP ratio significantly, while it did not change the intracellular cAMP level. Rebaudioside A (10(-9) M) and stevioside (10(-6) M) reduced the ATP-sensitive potassium channel (K(ATP)) conductance in a glucose-dependent manner. Moreover, rebaudioside A stimulated the insulin secretion from MIN6 cells in a dose- and glucose-dependent manner. In conclusion, the insulinotropic effect of rebaudioside A is mediated via inhibition of ATP-sensitive K(+)-channels and requires the presence of high glucose. The inhibition of ATP-sensitive K(+)-channels is probably induced by changes in the ATP/ADP ratio. The results indicate that rebaudioside A may offer a distinct therapeutic advantage over sulphonylureas because of less risk of causing hypoglycaemia.     

On the free radical scavenging activities of melatonin's metabolites,AFMK and AMK

The reactions of N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK) and N¹‐acetyl‐5‐methoxykynuramine (AMK) with ^?OH, ^?OOH, and ?OOCCl₃ radicals have been studied using the density functional theory. Three mechanisms of reaction have been considered: radical adduct formation (RAF), hydrogen transfer (HT), and single electron transfer (SET). Their relative importance for the free radical scavenging activity of AFMK and AMK has been assessed. It was found that AFMK and AMK react with ?OH at diffusion‐limited rates, regardless of the polarity of the environment, which supports their excellent ?OH radical scavenging activity. Both compounds were found to be also very efficient for scavenging ?OOCCl₃, but rather ineffective for scavenging ?OOH. Regarding their relative activity, it was found that AFMK systematically is a poorer scavenger than AMK and melatonin. In aqueous solution, AMK was found to react faster than melatonin with all the studied free radicals, while in nonpolar environments, the relative efficiency of AMK and melatonin as free radical scavengers depends on the radical with which they are reacting. Under such conditions, melatonin is predicted to be a better ?OOH and ?OOCCl₃ scavenger than AMK, while AMK is predicted to be slightly better than melatonin for scavenging ?OH. Accordingly it seems that melatonin and its metabolite AMK constitute an efficient team of scavengers able of deactivating a wide variety of reactive oxygen species, under different conditions. Thus, the presented results support the continuous protection exerted by melatonin, through the free radical scavenging cascade.     

Metabolism of melatonin by cytochrome P450s in rat liver mitochondria and microsomes

Abstract: In the present study we provide direct evidence for the involvement of rat microsomal cytochrome P450s in melatonin O‐demethylation and hydroxylation at two different positions: 2 and 6, as well as generation of N¹‐acetyl‐N²‐formyl‐5‐methoxy‐kynuramine (AFMK) and two unknown products. Moreover, we found that mitochondrial cytochrome P450s also converts melatonin into AFMK, N‐acetylserotonin, 2‐hydroxymelatonin, 6‐hydroxymelatonin and the same two unknown products. Eadie–Hofstee plots for 6‐hydroxylation and O‐demethylation reactions were curvilinear for all tested fractions, suggestive of involvement of at least two components, one with a high affinity and low capacity, and another with a low affinity and high capacity. Mitochondrial cytochrome P450s exhibited higher affinity (suggesting lower K_m value) and higher V_max for melatonin 6‐hydroxylation and O‐demethylation for both high‐affinity and low‐affinity components as compared with microsomal enzymes. The intrinsic clearance for melatonin hydroxylation by high‐ and low‐affinity components displayed the highest values in all tested fractions, indicating that both mitochondrial and microsomal cytochrome P450s metabolize melatonin principally by 6‐hydroxylation, with O‐demethylation representing a minor metabolic pathway.     

Usefulness of Technetium-99 m-2-methoxy-isobutyl-isonitrile liver scintigraphy for evaluating disease activity of non-alcoholic fatty liver disease

Aim: Non‐alcoholic steatohepatitis (NASH) is a progressive form of non‐alcoholic fatty liver disease (NAFLD). Therefore, it is important to evaluate disease activity and distinguish NASH from simple steatosis in NAFLD. Technetium‐99 m‐2‐methoxy‐isobutyl‐isonitrile (^99mTc‐MIBI) is a lipophilic cation designed for myocardial perfusion scintigraphy in the diagnosis of ischemic heart diseases, and its retention reflects mitochondrial function. It was reported that hepatic mitochondrial abnormalities would be an important predictive factor for NASH disease progression. The aim of this study was to examine the clinical usefulness of ^99mTc‐MIBI liver scintigraphy for evaluating disease activity of NAFLD and distinguishing NASH from simple steatosis in patients with NAFLD. Methods: Twenty‐six patients with biopsy‐proven NAFLD were enrolled. Clinicolaboratory tests and ^99mTc‐MIBI liver scintigraphy were performed. To evaluate hepatic uptake, regions of interest were set at the liver and heart, and the uptake ratio of the liver to heart (liver/heart ratio) was calculated. Results: All patients with NAFLD were classified into three groups according to the NAFLD activity score: non‐NASH (simple steatosis) (n = 4), borderline NASH (n = 11), and NASH (n = 11). Liver/heart ratios were significantly lower in NASH than in simple steatosis (P < 0.05). Moreover, liver/heart ratios were significantly correlated with NAFLD activity scores among the patients (r = ?0.413, P < 0.05). Conclusions: The present study indicates that ^99mTc‐MIBI liver scintigraphy would be a useful non‐invasive functional imaging method with which to evaluate disease activity of NAFLD and distinguish NASH from simple steatosis.     

Initial clinical experience of selective coronary angiography using one prolonged injection and a 180 degrees rotational trajectory.

OBJECTIVE: Evaluate the safety of prolonged coronary injections during a rotational acquisition covering 180 degrees. BACKGROUND: Rotational angiography has been adapted to coronary angiography and shown to reduce radiation and contrast exposure. Three-dimensional (3D) reconstructions and other advanced applications require imaging over a 180 degrees -arc with a single but longer injection of larger contrast volumes. METHODS: Thirty patients referred for angiography were enrolled. Blood pressure (BP), heart rate (HR), symptoms, and ectopy were recorded before-and-after injections. RESULTS: Pre and post-injection HRs for the LCA/RCA were not statistically different (LCA-pre-injection 63+/-13 bpm vs. LCA-post-injection 62+/-11 bpm, P=0.54 and RCA-pre-injection 65+/-12 bpm vs. RCA-post-injection 65+/-10, P=0.88). Central aortic pressure values were not statistically different for the RCA injections (RCA-systolic-pre-injection 118+/-14 mm Hg vs. RCA-systolic-post-injection 112+/-25 mm Hg, P=0.15, and RCA diastolic-pre-injection 69+/-9 mm Hg vs. RCA-diastolic-post-injection 60+/-10 mm Hg, P=0.88) but were statistically significant for the LCA injections (LCA systolic-pre-injection 122+/-19 mm Hg vs. LCA-systolic-post-injection 116+/-17 mm Hg, P=0.0004, and LCA-diastolic-pre-injection 69+/-10 mm Hg vs. LCA-diastolic-post-injection 65+/-9 mm Hg, P=0.0007). There were no symptoms or electrical events documented during or immediately post-injection. CONCLUSION: This study demonstrates the feasibility and safety of longer coronary injections. There were no significant HR changes, clinically insignificant pressure changes, and no adverse reactions. Additional studies will be needed to assure its safety in a larger and clinically more varied patient population.     

Development of reduced-intensity hematopoietic stem cell transplantation in the National Cancer Center Hospital,Japan

Reduced-intensity hematopoietic stem cell transplantation (RIST) is a new approach of stem cell transplantation, which has shown promising features as reported in multiple phase I and II studies. Elderly patients, who are not eligible for conventional myeloablative hematopoietic stem cell transplantation (HSCT), are now treatable with RIST. It has also reduced regimen-related toxicity and provided better prognosis in short-term follow-up than that of conventional HSCT. Favorable results have been reported particularly in hematological malignancies, such as chronic myelocytic leukemia and malignant lymphoma. Among solid tumors, metastatic renal cell carcinoma was found to respond well to RIST. Clinical studies are currently being conducted to evaluate the efficacy of RIST in other types of solid tumors. However, the mechanism of graft-versus-host disease and graft-versus-tumor remains unclear. More knowledge on the mechanism is crucial to enhance antitumor effect and to further improve the prognosis.     

Role of intra-arterial steroid administration in the management of steroid-refractory acute gastrointestinal graft-versus-host disease

We report here a case of severe steroid-refractory gastrointestinal graft-versus-host disease treated with intra-arterial administration of corticosteroids. A 53-year-old female with non-Hodgkin's lymphoma received peripheral blood hematopoietic stem cell transplant from her HLA-matched sibling. She developed grade II skin and grade IV gastrointestinal graft-versus-host disease with no hepatic involvement. Therapy with oral prednisone easily controlled her skin rash but she had profuse diarrhea that did not respond to high dose intravenous corticosteroids and denileukin diftitox. Infusion of methyl-prednisolone into superior and inferior mesenteric arteries produced dramatic improvement of diarrhea, with complete resolution of gastrointestinal graft-versus-host disease.     

Controversies on the prognostic value of interim FDG‐PET in advanced‐stage Hodgkin lymphoma

Hodgkin lymphoma, even in advanced‐stage, is a highly curable malignancy, but treatment is associated with short‐term toxicity and long‐term side effects. Early predictive markers are required to identify those patients who do not require the full‐length standard therapy (and thus qualify for therapy de‐escalation) and those patients who will not be cured by standard therapy (and thus qualify for therapy escalation). Multiple trials have assessed the value of ¹⁸F‐fluoro‐2‐deoxy‐d ‐glucose positron emission tomography (FDG‐PET) after a few cycles of chemotherapy (also known as ‘interim FDG‐PET’) in predicting outcome in advanced‐stage Hodgkin lymphoma. Furthermore, multiple interim FDG‐PET‐adapted trials, in which patients with positive interim FDG‐PET scans are assigned to escalated therapies, and patients with negative interim FDG‐PET scans are assigned to de‐escalated therapies, have recently been published or are currently ongoing, with generally heterogeneous results. The present article reports the currently available evidence (and controversies) on the prognostic value of interim FDG‐PET in advanced‐stage Hodgkin lymphoma in patients with positive and negative interim FDG‐PET findings following continuation of standard chemotherapy or escalated/de‐escalated therapy.     

Mitochondria and chloroplasts as the original sites of melatonin synthesis: a hypothesis related to melatonin's primary function and evolution in eukaryotes

Mitochondria and chloroplasts are major sources of free radical generation in living organisms. Because of this, these organelles require strong protection from free radicals and associated oxidative stress. Melatonin is a potent free radical scavenger and antioxidant. It meets the criteria as a mitochondrial and chloroplast antioxidant. Evidence has emerged to show that both mitochondria and chloroplasts may have the capacity to synthesize and metabolize melatonin. The activity of arylalkylamine N‐acetyltransferase (AANAT), the reported rate‐limiting enzyme in melatonin synthesis, has been identified in mitochondria, and high levels of melatonin have also been found in this organelle. From an evolutionary point of view, the precursor of mitochondria probably is the purple nonsulfur bacterium, particularly, Rhodospirillum rubrum, and chloroplasts are probably the descendents of cyanobacteria. These bacterial species were endosymbionts of host proto‐eukaryotes and gradually transformed into cellular organelles, that is, mitochondria and chloroplasts, respectively, thereby giving rise to eukaryotic cells. Of special importance, both purple nonsulfur bacteria (R. rubrum) and cyanobacteria synthesize melatonin. The enzyme activities required for melatonin synthesis have also been detected in these primitive species. It is our hypothesis that mitochondria and chloroplasts are the original sites of melatonin synthesis in the early stage of endosymbiotic organisms; this synthetic capacity was carried into host eukaryotes by the above‐mentioned bacteria. Moreover, their melatonin biosynthetic capacities have been preserved during evolution. In most, if not in all cells, mitochondria and chloroplasts may continue to be the primary sites of melatonin generation. Melatonin production in other cellular compartments may have derived from mitochondria and chloroplasts. On the basis of this hypothesis, it is also possible to explain why plants typically have higher melatonin levels than do animals. In plants, both chloroplasts and mitochondria likely synthesize melatonin, while animal cells contain only mitochondria. The high levels of melatonin produced by mitochondria and chloroplasts are used to protect these important cellular organelles against oxidative stress and preserve their physiological functions. The superior beneficial effects of melatonin in both mitochondria and chloroplasts have been frequently reported.     

Successful liver transplantation despite donor‐transmitted ESBL‐producing Klebsiella pneumoniae infection: Case report and review of the literature

Donor‐derived bacterial infection is a recognized complication of solid organ transplantation. Patients admitted to the intensive care unit are increasingly exposed to infection with multidrug‐resistant microorganisms. However, no specific recommendations are available about their suitability as donors. We report a case of donor‐transmitted extended‐spectrum beta‐lactamase‐producing Klebsiella pneumoniae infection in a liver recipient, and review the related literature.