ABSTRACT
Purpose: We herein describe the superficial preprostatic vein (SPV) anatomy and determine its relationship with the accessory pudendal artery (APA). Materials and Methods: We reviewed 500 patients with localized prostate cancer who underwent conventional robot-assisted radical prostatectomy between April 2019 and March 2023 at our institution. SPV was defined as "any vein coming from the space between the puboprostatic ligaments and running within the retropubic adipose tissue anterior to the prostate toward the vesical venous plexus or pelvic side wall." While APA was defined as "any artery located in the periprostatic region running parallel to the dorsal vascular complex and extending caudal toward the anterior perineum." The intraoperative anatomy of each SPV and APA was described. Results: SPVs had a prevalence rate of 88%. They were preserved in 252 men (58%) and classified as I-, reversed-Y (rY)-, Y-, or H-shaped (64%, 22%, 12%, and 2%, respectively) based on their intraoperative appearance. Overall, 214 APAs were found in 142 of the 252 men with preserved SPV (56%; 165 lateral and 50 apical APAs in 111 and 41 men, respectively). SPVs were pulsatile in 39% men perhaps due to an accompanying tiny artery functioning as a median APA. Pulsations seemed to be initially absent in most SPVs but become apparent late during surgery possibly due to increased arterial and venous blood flow after prostate removal. Pulsations were common in men with ≥1 APA. Conclusions: This study, which described the anatomical variations in arteries and veins around the prostrate and their preservation techniques, revealed that preserving this vasculature may help preserve postprostatectomy erection. ClinicalTrials: The Clinical Research Registration Number is 230523D.
Subject(s)
Arteries , Prostate , Prostatectomy , Robotic Surgical Procedures , Veins , Humans , Male , Prostatectomy/methods , Robotic Surgical Procedures/methods , Prostate/blood supply , Prostate/surgery , Middle Aged , Aged , Arteries/anatomy & histology , Veins/anatomy & histology , Veins/surgery , Prostatic Neoplasms/surgery , Prostatic Neoplasms/pathologyABSTRACT
OBJECTIVES: Patients with inguinal hernia (IH) may have voiding dysfunction and weak urethra-stabilizing periurethral fascial tissues, contributing to urinary incontinence. This study aimed to review the association between IH and urinary continence after robotic-assisted radical prostatectomy (RARP). METHODS: This single-institution retrospective study included 251 consecutive cases of RARP between April 2019 and June 2022. Patients with concurrent IH or a history of adult IH repair were examined. The urine loss rate (ULR), defined as 24-h urine loss volume divided by the total urine volume immediately after urinary catheter removal (i.e., 6 or 7 postoperative days), was compared between the groups with (n = 33) and without IH (n = 214). Possible contributing factors for ULR were assessed, including age, body mass index (BMI), after benign prostatic hyperplasia surgery, prostate weight, and nerve-sparing. ULR was compared intergroup after propensity score matching countering selection biases. RESULTS: Patients with IH were older (71.3 versus. 66.8 years, p < 0.01), had lower BMI (22.8 versus. 24.3, p < 0.01), and had higher ULR (14.5% versus. 5.1%, p < 0.01). In a multiple linear regression analysis (adjusted R2 = 0.084), IH (p < 0.01) was an independent contributing factor for ULR besides advancing age (p < 0.03). After propensity score matching adjusted for patient's age and nerve-sparing, patients with IH had higher ULR (14.1% versus. 5.7%, p < 0.03) as well. CONCLUSIONS: This study first reported that IH may be one of the risk factors of urinary incontinence after RARP.
Subject(s)
Hernia, Inguinal , Prostatic Neoplasms , Robotic Surgical Procedures , Robotics , Urinary Incontinence , Male , Adult , Humans , Prostate , Retrospective Studies , Robotic Surgical Procedures/methods , Hernia, Inguinal/surgery , Urinary Incontinence/etiology , Urinary Incontinence/surgery , Prostatectomy/adverse effects , Prostatic Neoplasms/surgeryABSTRACT
In the late 1970s, sulfane sulfur was defined as sulfur atoms covalently bound only to sulfur atoms. However, this definition was not generally accepted, as it was slightly vague and difficult to comprehend. Thus, in the early 1990s, it was defined as "bound sulfur," which easily converts to hydrogen sulfide upon reduction with a thiol-reducing agent. H2S-related bound sulfur species include persulfides (R-SSH), polysulfides (H2Sn, n ≥ 2 or R-S(S)nS-R, n ≥ 1), and protein-bound elemental sulfur (S0). Many of the biological effects currently associated with H2S may be attributed to persulfides and polysulfides. In the 20th century, quantitative determination of "sulfane sulfur" was conventionally performed using a reaction called cyanolysis. Several methods have been developed over the past 30 years. Current methods used for the detection of H2S and polysulfides include colorimetric assays for methylene blue formation, sulfide ion-selective or polarographic electrodes, gas chromatography with flame photometric or sulfur chemiluminescence detection, high-performance liquid chromatography analysis with fluorescent derivatization of sulfides, liquid chromatography with tandem mass spectrometry, the biotin switch technique, and the use of sulfide or polysulfide-sensitive fluorescent probes. In this review, we discuss the methods reported to date for measuring sulfane sulfur and the results obtained using these methods.
Subject(s)
Sulfides , Sulfur , Gas Chromatography-Mass Spectrometry , Sulfides/chemistry , Sulfur/chemistryABSTRACT
Advanced glycation end products (AGEs), which can have multiple structures, are formed at the sites where the carbonyl groups of reducing sugars bind to the free amino groups of proteins through the Maillard reaction. Some AGE structures exhibit fluorescence, and this fluorescence has been used to measure the formation and quantitative changes in carbonylated proteins. Recently, fluorescent AGEs have also been used as an index for the evaluation of compounds that inhibit protein glycation. However, the systems used to generate fluorescent AGEs from the reaction of reducing sugars and proteins used for the evaluation of antiglycation activity have not been determined through appropriate research; thus, problems remain regarding sensitivity, quantification, and precision. In the present study, using methylglyoxal (MGO), a reactive carbonyl compound to induce glycation, a comparative analysis of the mechanisms of formation of fluorescent substances from several types of proteins was conducted. The analysis identified hen egg lysozyme (HEL) as a protein that produces stronger fluorescent AGEs faster in the Maillard reaction with MGO. It was also found that the AGE structure produced in MGO-induced in HEL was argpyrimidine. By optimizing the reaction system, we developed a new evaluation method for compounds with antiglycation activity and established an efficient evaluation method (HEL-MGO assay) with greater sensitivity and accuracy than the conventional method, which requires high concentrations of bovine serum albumin and glucose. Furthermore, when compounds known to inhibit glycation were evaluated using this method, their antiglycation activities were clearly and significantly measured, demonstrating the practicality of this method.
ABSTRACT
In the present study, we evaluated the acute response of mice exposed to IQOS aerosol, a brand-name heated tobacco product (HTP), in the lung tissue. First, the thiobarbituric acid-reactive substances (TBA-RS) value was measured as an index to assess oxidative stress, and a significant increase was observed after exposure, followed by a significant increase in the total lung GSH concentration. The stress responses induced by IQOS aerosols was then analyzed by focusing on the changes in Nrf2 and ATF4, which are transcription factors that induce the expression of genes involved in GSH biosynthesis or metabolism. Although Nrf2 activation was not observed, significant accumulation of ATF4 in the nuclear fraction was noted three hours after exposure to IQOS aerosols. Upon an examination of changes in factors in the GSH biosynthetic system, a significant increase in cystine concentration in the lung tissue was measured, and an increase in xCT expression level was observed in the cell membrane fraction three-six hours after IQOS exposure. Furthermore, characteristic changes in HO-1, a stress-response protein regulated by ATF4, was discovered six hours after IQOS exposure. Moreover, analysis of the upstream ATF4 regulatory system revealed that phosphorylation of eIF2α was enhanced in the lung cytoplasmic fraction three hours after exposure to IQOS aerosols. These findings suggest that ER stress might be induced as an early response to IQOS aerosol exposure, accompanied by the activation of the eIF2α-ATF4 axis. These intracellular changes have also been reported after exposure to combustible cigarette smoke. Thus, the acute response found in the lungs of mice in the present study demonstrate that the inhalation of aerosols from IQOS elicits a biological response similar to that of combustible cigarette smoke. In conclusion, our results provide evidence that the biological effects of HTPs, such as IQOS, cannot be ignored in the lungs.
ABSTRACT
Peroxiredoxin (Prx) 2 in red blood cells (RBCs) reacts with various reactive oxygen species and changes to hyperoxidized Prx2 (Prx2-SO2/3). Therefore, Prx2 may serve as an indicator of oxidative stress in vivo. This study aimed to analyze Prx2-SO2/3 levels in clinical samples to examine whether the oxidation state of Prx2 in human RBCs reflects the pathological condition of oxidative stress diseases. We first focused on obstructive sleep apnea (OSA), a hypoxic stress-induced disease of the respiratory system, and investigated the levels of Prx2-SO2/3 accumulated in the RBCs of OSA patients. In measurements on a small number of OSA patients and healthy subjects, levels of Prx2-SO2/3 accumulation in patients with OSA were clearly increased compared to those in healthy subjects. Hence, we proceeded to validate these findings with more samples collected from patients with OSA. The results revealed significantly higher levels of erythrocytic Prx2-SO2/3 in patients with OSA than in healthy subjects, as well as a positive correlation between the severity of OSA and Prx2-SO2/3 levels in the RBCs. Moreover, we performed a chromatographic study to show the structural changes of Prx2 due to hyperoxidation. Our findings demonstrated that the Prx2-SO2/3 molecules in RBCs from patients with OSA were considerably more hydrophilic than the reduced form of Prx2. These results implicate Prx2-SO2/3 as a promising candidate biomarker for OSA.
ABSTRACT
In this study, we measured conjugated and unconjugated free bile acids (BAs) in plasma from patients with schizophrenia and healthy subjects to examine the possibility of BA as a biomarker for schizophrenia. Although the levels of each BA conjugate showed no significant differences, significant differences for three unconjugated bile acids were observed in the plasma between patients with schizophrenia and healthy subjects. Additionally, a more than three times difference between patients and healthy subjects was observed in the mean value of the total concentrations of primary BAs. These results indicate that cholic acid and chenodeoxycholic acid levels in plasma may be novel diagnostic markers for a sub-population of patients with schizophrenia. Thus, future studies should elucidate the relationship between this increase in BA levels and the pathology of schizophrenia and verify the potential of unconjugated BA in plasma as biomarkers for schizophrenia.
Subject(s)
Bile Acids and Salts , Schizophrenia , Humans , Schizophrenia/diagnosis , Chenodeoxycholic Acid , Biomarkers , PlasmaABSTRACT
Large adrenocortical adenomas have rarely been reported. We describe a case of a 26-year-old man who underwent an adrenalectomy for a large adrenocortical adenoma (8.6 × 7.7 cm). Although the lesion had typical malignant features on imaging, histopathological examination revealed an adrenocortical adenoma. This highlights that imaging alone may not be able to distinguish adrenocortical carcinomas from adrenal masses. In most cases, a resection should be performed for early diagnosis and management of large adrenal masses with malignant features on imaging. To our knowledge, this is the first report of a large adrenocortical adenoma diagnosed with multiple imaging investigations.
ABSTRACT
Methylglyoxal (MG) is a reactive and cytotoxic α-dicarbonyl byproduct of glycolysis. Our bodies have several bio-defense systems to detoxify MG, including an enzymatic system by glyoxalase (GLO) 1 and GLO2. We identified a subtype of schizophrenia patients with novel mutations in the GLO1 gene that results in reductions of enzymatic activity. Moreover, we found that vitamin B6 (VB6) levels in peripheral blood of the schizophrenia patients with GLO1 dysfunction are significantly lower than that of healthy controls. However, the effects of GLO1 dysfunction and VB6 deficiency on the pathophysiology of schizophrenia remains poorly understood. Here, we generated a novel mouse model for this subgroup of schizophrenia patients by feeding Glo1 knockout mice VB6-deficent diets (KO/VB6(-)) and evaluated the combined effects of GLO1 dysfunction and VB6 deficiency on brain function. KO/VB6(-) mice accumulated homocysteine in plasma and MG in the prefrontal cortex (PFC), hippocampus, and striatum, and displayed behavioral deficits, such as impairments of social interaction and cognitive memory and a sensorimotor deficit in the prepulse inhibition test. Furthermore, we found aberrant gene expression related to mitochondria function in the PFC of the KO/VB6(-) mice by RNA-sequencing and weighted gene co-expression network analysis (WGCNA). Finally, we demonstrated abnormal mitochondrial respiratory function and subsequently enhanced oxidative stress in the PFC of KO/VB6(-) mice in the PFC. These findings suggest that the combination of GLO1 dysfunction and VB6 deficiency may cause the observed behavioral deficits via mitochondrial dysfunction and oxidative stress in the PFC.
Subject(s)
Lactoylglutathione Lyase , Schizophrenia , Vitamin B 6 Deficiency , Animals , Humans , Lactoylglutathione Lyase/genetics , Lactoylglutathione Lyase/metabolism , Mice , Mitochondria/metabolism , Mitochondria/pathology , Prefrontal Cortex/metabolism , Schizophrenia/geneticsABSTRACT
Our previous studies have shown that glycerin, which is present at high concentrations in moisturizers and skin lotions, gradually oxidizes to produce methylglyoxal (MGO). In this study, we observed that MGO-treated porcine dermis type-I collagen was carbonylated in an MGO concentration- and time-dependent manner. Furthermore, we examined the structure of advanced glycation end products (AGEs) induced by MGO reacting with type-I collagen. Our findings demonstrate that the α chains of collagen reacted with MGO and easily transformed into a modified protein containing a methylglyoxal-derived hydroimidazolone (MG-H1) moiety in a concentration- and time-dependent manner. Moreover, porcine skin proteins underwent carbonylation when the skin section was treated with MGO for four weeks. Analysis of the structure of AGEs on the carbonylated proteins extracted from MGO-treated skin sections revealed that skin collagen had been converted to MG-H1-modified protein. These novel findings suggest that continuous application of MGO to the skin leads to carbonylation of proteins, which may cause prompt accumulation of MG-H1-modified dermis collagen, thereby resulting in morphological and functional changes of collagen in the skin.
Subject(s)
Collagen/metabolism , Protein Carbonylation , Pyruvaldehyde/metabolism , Skin/metabolism , Animals , Glycation End Products, Advanced/metabolism , Swine , Time FactorsABSTRACT
Recent studies have shown that carbonyl stress is a causative factor of schizophrenia, categorized as carbonyl stress-related schizophrenia (CS-SCZ). However, the correlation between carbonyl stress and the pathogenesis of this disease is not well established. In this study, glyoxalase 1(Glo1)-knockout and vitamin B6-deficient mice (KO/VB6 (-) mice), which are susceptible to methylglyoxal (MGO)-induced oxidative damages, were used as a CS-SCZ model to analyze MGO-modified protein and the carbonyl stress status in the brain. A comparison between Wild/VB6(+) mice and KO/VB6(-) mice for accumulated carbonyl proteins levels, with several advanced glycation end products (AGEs) in the brain, revealed that carbonyl protein levels with the Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl) ornithine (MG-H1) moiety were significantly increased in the hippocampus, prefrontal cortex, striatum, cerebral cortex, and brainstem regions of the brain in KO/VB6(-) mice. Moreover, two-dimensional electrophoresis and Liquid chromatography-tandem mass spectrometry analysis showed MG-H1-modified arginine residues in mitochondrial creatine kinase, beta-adrenergic receptor kinase 1, and T-complex protein in the hippocampus region of KO/VB6(-) mice, but not in Wild/VB6(+) mice. In particular, MG-H1 modification of mitochondrial creatine kinase was quite notable. These results suggest that further studies focusing on MG-H1-modified and accumulated proteins in the hippocampus may reveal the onset mechanism of CS-SCZ induced by MGO-induced oxidative damages.
ABSTRACT
Water transport in the brain is tightly controlled by blood-brain-barrier (BBB) composed of capillary endothelial cells expressing AQP1/AQP11 and glial foot processes expressing AQP4. Here we examined each AQP mRNA expression in acute hyponatremic and hypernatremic mouse models of wild type (WT) and AQP11 KO mice (KO). The expressions of AQP1, AQP4 and AQP11 mRNAs were quantified by real-time qRT-PCR analysis of whole brain RNA. Acute hyponatremia enhanced AQP4 expression without changing AQP1 expression in KO, whereas it did not change the expression of all AQPs in WT. On the other hand, acute hypernatremia increased AQP4 but decreased AQP1 expression by half in KO, whereas it decreased AQP1 and AQP11 by half without changing AQP4 expression in WT. Enhanced AQP4 expression by osmotic challenges with sodium in KO seems to be a compensation for the loss of AQP11. A stronger hypertonic stimulation with mannitol decreased all AQPs by 30-80% in WT. Since AQP4 plays an important role in the regulation of brain edema at BBB, the results suggest that AQP11 may also be involved in the osmotic regulation of the brain.
Subject(s)
Aquaporins/genetics , Brain/metabolism , Animals , Aquaporin 1/genetics , Aquaporin 4/genetics , Disease Models, Animal , Hypernatremia/metabolism , Hyponatremia/metabolism , Male , Mannitol/pharmacology , Mice, Inbred C57BL , Mice, Knockout , Osmolar Concentration , Sodium/pharmacologyABSTRACT
In this study, we developed an analytical method using LC-MS/MS for the simultaneous determination of five bile acids (BAs) that have been recently reported as candidate diagnostic biomarkers for Alzheimer's disease (AD) or AD related factors in the brain. The measurement of BAs in the brains of healthy mice led to the determination of candidate diagnostic markers for AD, such as cholic acid and deoxycholic acid, and other bile acids, such as chenodeoxycholic acid noted for the ameliorating effect on the symptoms of AD. Significant positive correlations were observed between the brain and plasma concentrations of four BAs in healthy young mice. These results indicate that the BA level in the brain may be estimated by the corresponding BA level in the plasma. Thus, our study suggested that the proposed method for the analysis of the five bile acids would aid in the diagnosis of AD or in studies that use AD model mice.
Subject(s)
Alzheimer Disease , Bile Acids and Salts , Alzheimer Disease/diagnosis , Animals , Biomarkers , Brain , Chromatography, Liquid , Mice , Tandem Mass SpectrometryABSTRACT
Methylglyoxal (MGO) is a reactive α-dicarbonyl compound that causes carbonylation of protein and DNA through the pathways of the Maillard reaction. It is known that MGO is physiologically involved in renal dysfunction, vascular disorders, and the acceleration of aging. In this study, we showed for the first time, that a trace amount of MGO was present as an impurity in glycerol preparations used as external medicines and intravenous infusions, when kept unused. The concentration of MGO in the glycerol solutions, diluted to a concentration of 20%, significantly increased after storage for one month when compared to the MGO concentration immediately after opening. Following storage for 6 months at 25°C, MGO concentration increased by about 300 times (approx. 170 µM), and at 40°C, it increased by about 600 times (approx. 350 µM). In the case of intravenous infusion preparations containing 10% glycerol, the MGO concentration increased by 4-15 times (approx. 70 µM) after 2 months of storage at 40°C, and reached over 200 µM after 6 months. Results from the present study showed that glycerol in pharmaceutical preparations is gradually oxidized to form MGO via autoxidation, depending on the temperature and dissolved oxygen content. Thus, we suggest that precautions should be taken when storing glycerol preparations in bottles or plastic containers, with respect to the storage temperature and sealability to prevent MGO formation due to oxidation of glycerol.
Subject(s)
Glycerol/chemistry , Pyruvaldehyde/chemistry , Antioxidants/chemistry , Drug Stability , Drug Storage , Edetic Acid/chemistry , Infusions, Intravenous , Oxidation-Reduction , Oxygen/chemistry , Sulfites/chemistry , TemperatureABSTRACT
Peroxiredoxins (Prxs) detoxify hydrogen peroxide (H2O2), peroxynitrite, and various organic hydroperoxides. However, the differential oxidative status of Prxs reacted with each peroxide remains unclear. In the present study, we focused on the oxidative alteration of Prxs and demonstrated that, in human red blood cells (RBCs), peroxiredoxin 2 (Prx2) is readily reactive with H2O2, forming disulfide dimers, but was not easily hyperoxidized. In contrast, Prx2 was highly sensitive to the relatively hydrophobic oxidants, such as tert-butyl hydroperoxide (t-BHP) and cumene hydroperoxide. These peroxides hyperoxidized Prx2 into oxidatively damaged forms in RBCs. The t-BHP treatment formed hyperoxidized Prx2 in a dose-dependent manner. When organic hydroperoxide-treated RBC lysates were subjected to reverse-phase high performance liquid chromatography, two peaks derived from hyperoxidized Prx2 appeared along with the decrease of that corresponding to native Prx2. Liquid chromatography-tandem mass spectrometry analysis clearly showed that hyperoxidation to sulfonic acid (-SO3H) at Cys-51 residue was more advanced in a newfound hyperoxidized Prx2 compared to another hydrophobic hyperoxidized form previously identified. These results indicate that irreversible hyperoxidation of the Prx2 monomer in RBCs was easily caused by organic hydroperoxide but not H2O2. Thus, it is important to detect the hyperoxidation of Prx2 into sulfinic or sulfonic acid derivates of Cys-51 because hyperoxidized Prx2 is a potential marker of oxidative injury caused by organic hydroperoxides in human RBCs.
Subject(s)
Erythrocytes/metabolism , Hydrogen Peroxide/metabolism , Peroxides/metabolism , Peroxiredoxins/metabolism , Adult , Chromatography, Reverse-Phase , Cysteine/chemistry , Cysteine/metabolism , Disulfides/chemistry , Disulfides/metabolism , Humans , Hydrogen Peroxide/chemistry , Middle Aged , Oxidants/chemistry , Oxidants/metabolism , Oxidation-Reduction , Peroxides/chemistry , Peroxiredoxins/chemistry , Sulfinic Acids/chemistry , Sulfinic Acids/metabolism , Sulfonic Acids/chemistry , Sulfonic Acids/metabolism , Young Adult , tert-Butylhydroperoxide/chemistry , tert-Butylhydroperoxide/metabolismABSTRACT
Methylglyoxal (MG) is an α-dicarbonyl compound that is naturally produced in vivo through glucose metabolism. In general, MG is metabolized by the glyoxalase 1(GLO1)/GLO2 system and aldose reductase (AR); however, excessive MG can react with proteins and nucleic acids to induce the accumulation of advanced glycation end products (AGEs). Recently, the accumulation of AGEs in the brain has been presumed to be related to neurodegenerative diseases such as Parkinson's and Alzheimer's disease, respectively. Research investigating the role of AGEs in such diseases is ongoing. However, the changes in MG concentration that occur in the brain during healthy ageing remain unclear. Therefore, we performed fractionation of the brains of aged and young mice, measured the MG concentration in each part of the brain, and then examined the distribution. We also investigated the expression levels of GLO1 and AR, the main metabolizing enzymes of MG, in various brain regions, across age groups. We show that MG concentration varies among different regions of the brain, and that MG concentration in aged mice is significantly lower than that in young mice across all regions of the brain, except the brain stem. In addition, although the expression level of the GLO1 protein in the brain did not change with ageing, the expression level of AR was higher in aged than in young mice. Moreover, although a significant positive correlation was observed between GLO1 expression and MG concentration in the brains of young mice, no significant correlations were observed in the brains of aged mice. Meanwhile, the production of protein carbonyls and the accumulation of AGEs were not observed in the brains of aged mice. These results suggest that the accumulation of MG in the brain, along with the carbonyl stress are suppressed and regionally controlled during healthy ageing. This finding is useful as the foundation for further studies to investigate the role and toxicity of MG in various age-related disease conditions.
Subject(s)
Age Factors , Glycation End Products, Advanced/metabolism , Pyruvaldehyde/metabolism , Aldehyde Reductase/metabolism , Animals , Brain/metabolism , Glucose/metabolism , Lactoylglutathione Lyase/metabolism , Male , Mice , Mice, Inbred C57BL , TranscriptomeABSTRACT
It has been known that hydrogen sulfide and/or polysulfides are produced from a (poly)sulfurated sulfur-acceptor substrate of 3-mercaptopyruvate sulfurtransferase (MST) via thioredoxin (Trx) reduction in vitro. In this study, we used thiosulfate as the donor substrate and the catalytic reaction was terminated on the formation of a persulfide or polysulfides. We can present alternative pathway of production of hydrogen sulfide and/or polysulfides from (poly)sulfurated catalytic-site cysteine of reaction intermediates of MST via Trx reduction. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometric analysis revealed that after prolonged incubation of MST with thiosulfate, a trisulfide adduct becomes predominant at the sulfurated catalytic-site cysteine. When these adducts were reduced by Trx with reducing system (MST:Escherichia coli Trx:E. coli Trx reductase:NADPHâ¯=â¯1:5:0.02:12.5 molar ratio), liquid chromatography with tandem mass spectrometric analysis for monobromobimane-derivatized H2Sn revealed that H2S2 first appeared, and then H2S and H2S3 did later. The results were confirmed by high-performance liquid chromatography-fluorescence analysis.
Subject(s)
Cysteine/metabolism , Hydrogen Sulfide/metabolism , Sulfides/metabolism , Sulfurtransferases/metabolism , Animals , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Oxidation-Reduction , Rats , Recombinant Proteins/metabolism , Thioredoxin-Disulfide Reductase/metabolism , Thioredoxins/metabolismABSTRACT
Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H2Sn), have various physiological functions in mammalian cells. Although H2Sn molecules have been considered as secondary metabolites derived from hydrogen sulfide (H2S) based on in vitro studies or predetermined reaction formula, the physiological form of BSS and their endogenous concentration remain unclear. In the present study, we aimed to improve the usual method using monobromobimane (mBB) followed by high performance liquid chromatographic (HPLC) analysis for HSï¼ for simultaneous determination of H2S, H2S2, H2S3 and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of H2S and H2Sn standards under alkaline conditions (pH 9.5) induced significant decreases in H2S2 and H2S3 levels and a significant increase in the H2S level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of H2S2 and H2S3 were stable under neutral conditions (pH 7.0), which is physiologically relevant. Therefore, we re-examined the method using mBB and applied an improved method for the evaluation of H2S, H2S2, and H2S3 in mouse brain under physiological pH conditions. The concentrations of H2S and H2S2 were 0.030 ± 0.004µmol/g protein and 0.026 ± 0.002µmol/g protein, respectively. Although the level of H2S3 was below the quantification limit of this method, H2S3 was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous H2S2 and H2S3 in mammalian brain tissues. H2S2 and H2S3 exert anti-oxidant activity and anti-carbonyl stress effects through the regulation of redox balance in neuronal cells. Thus, our observations provide novel insights into the physiological functions of BSS in the brain and into neuronal diseases involved in redox imbalance.
Subject(s)
Brain/metabolism , Cysteine/analogs & derivatives , Disulfides/isolation & purification , Hydrogen Sulfide/isolation & purification , Sulfides/isolation & purification , Animals , Brain Chemistry , Bridged Bicyclo Compounds/chemistry , Chromatography, High Pressure Liquid , Cysteine/isolation & purification , Cysteine/metabolism , Disulfides/metabolism , Hydrogen Sulfide/metabolism , Male , Mice , Mice, Inbred C57BL , Sulfhydryl Reagents/chemistry , Sulfides/metabolism , Tandem Mass SpectrometryABSTRACT
Cysteine-persulfide (Cys-SSH) is a cysteine whose sulfhydryl group is covalently bound to sulfur (sulfane sulfur). Cys-SSH and its glutathione (GSH) counterpart (GSSH) have been recognized as redox regulators, some of which were previously ascribed to cysteine and GSH. However, the production of Cys-SSH and GSSH is not well understood. Here, we show that 3-mercaptopyruvate sulfurtransferase (3MST) produces Cys-SSH and GSSH together with the potential signaling molecules hydrogen per- and tri-sulfide (H2S2 and H2S3). Cys-SSH and GSSH are produced in the brain of wild-type mice but not in those of 3MST-KO mice. The levels of total persulfurated species in the brain of 3MST-KO mice are less than 50% of that in the brain of wild-type mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST showed that Cys-SSH and GSSH were produced in the presence of physiological concentrations of cysteine and glutathione, while those with longer sulfur chains, Cys-SSnH and GSSnH, were produced in the presence of lower than physiological concentrations of cysteine and glutathione. The present study provides new insights into the production and physiological roles of these persulfurated species as well as the therapeutic targets for diseases in which these molecules are involved.
Subject(s)
Cysteine/analogs & derivatives , Glutathione/analogs & derivatives , Hydrogen Sulfide/metabolism , Oxidation-Reduction , Sulfurtransferases/metabolism , Animals , Brain/metabolism , Cell Line , Chromatography, Liquid , Cysteine/biosynthesis , Disulfides , Glutathione/biosynthesis , Glutathione/metabolism , Mice , Mice, Knockout , Signal Transduction , Sulfur/metabolism , Tandem Mass SpectrometryABSTRACT
Alzheimer's disease (AD) destroys brain function, especially in the hippocampus, and is a social problem worldwide. A major pathogenesis of AD is related to the accumulation of amyloid beta (Aß) peptides, resulting in neuronal cell death in the brain. Here, we isolated four saponins (1-4) and elucidated their structures from 1D and 2D NMR and HRFABMS spectral data. The structures of 1 and 2 were determined as new saponins which have cochalic acid as the aglycon, and 3 was determined as a new saponin with oleanolic acid as the aglycon. Compound 4 was confirmed as the known saponin chikusetsusaponin V (=ginsenoside R0). Isolated saponins (1-4) and six previously reported saponins (5-10) were tested for their inhibitory effects of Aß aggregation and their protective effects on SH-SY5Y cells against Aß-associated toxicity. As the results, compounds 3 and 4 showed inhibitory effect of Aß aggregation and compounds 5-8 exerted the protective effects on SH-SY5Y cells against Aß-associated toxicity.