Poly(ADP-ribose) Polymerase (PARP) is Critically Involved in Liver Ischemia/Reperfusion-injury.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) is a DNA-repairing enzyme activated by extreme genomic stress, and therefore is potently activated in the remnant liver suffering from ischemia after surgical resection. However, the impact of PARP on post-ischemic liver injury has not been elucidated yet. MATERIALS AND METHODS: We investigated the impact of PARP on murine hepatocyte/liver injury induced by hypoxia/ischemia, respectively. RESULTS: PJ34, a specific inhibitor of PARP, markedly protected against hypoxia/reoxygenation (H/R)-induced cell death, though z-VAD-fmk, a pan-caspase inhibitor similarly showed the protective effect. PJ34 did not affect H/R-induced caspase activity or caspase-mediated cell death. z-VAD-fmk also did not affect the production of PAR (i.e., PARP activity). Therefore, PARP- and caspase-mediated cell death occurred in a mechanism independent of each other in H/R. H/R immediately induced activation of PARP and cell death afterwards, both of which were suppressed by PJ34 or Trolox, an antioxidant. This suggests that H/R-induced cell death occurred redox-dependently through PARP activation. H/R and OS induced nuclear translocation of apoptosis inducing factor (AIF, a marker of parthanatos) and RIP1-RIP3 interaction (a marker of necroptosis), both of which were suppressed by PJ34. H/R induced PARP-mediated parthanatos and necroptosis redox-dependently. In mouse experiments, PJ34 significantly reduced serum levels of AST, ALT & LDH and areas of hepatic necrosis after liver ischemia/reperfusion, similar to z-VAD-fmk or Trolox. CONCLUSION: PARP, activated by ischemic damage and/or oxidative stress, may play a critical role in post-ischemic liver injury by inducing programmed necrosis (parthanatos and necroptosis). PARP inhibition may be one of the promising strategies against post-ischemic liver injury.

Detection of catechins using a fluorescent molecule and its application toward the evaluation of astringent intensity.

To evaluate sensory intensities of tastes, astringency, or pungency with ease and high reproducibility, the present study proposes a method using a fluorescent substance as a sensor. Unlike conventional taste sensors that each require a specialized system, this approach has the advantage in that it can be conducted using common analytical equipment such as a fluorescence spectrometer. In this report, a method to detect catechins and its application toward astringent evaluation is described. The binding of a sensor molecule with eight catechins revealed that the sensor molecule has higher affinity for catechins with stronger astringency. The complexation with the more astringent catechins showed larger fluorescence quenching. The change in the fluorescence intensity of a sample solution with the sensor molecule based on a reference solution was defined as the astringent intensity. Evaluation tests of green tea astringency demonstrated that the outputs of the sensor molecule correlate highly with the results by the human sense.

Extracts of bilberry (Vaccinium myrtillus L.) fruits improve liver steatosis and injury in mice by preventing lipid accumulation and cell death.

Bilberry has been reported to have anti-oxidant and anti-inflammatory properties. We studied the effect of bilberry (Vaccinium myrtillus L.) fruits extracts (BEs) on the pathogenesis caused by lipid accumulation in fatty liver and non-alcoholic steatohepatitis (NASH). 5 µg/ml of BEs was enough to suppress lipid accumulation in the fatty liver model of the mouse hepatic AML12 cells. BEs increased cell viability and anti-oxidant capacity, presumably by activating (phosphorylating) Akt/STAT3 and inducing MnSOD/catalase. BEs also significantly reduced Rubicon and induced p62/SQSTM1, possibly contributing to reduce cellular lipids (lipophagy). When the mice were fed supplemented with BEs (5% or 10%, w/w), hepatic steatosis, injury, and hypercholesterolemia/hyperglycemia were significantly improved. Furthermore, histological and cytokine studies indicated that BEs possibly suppress hepatic inflammation (hepatitis) and fibrosis. Therefore, BEs improved liver steatosis and injury, and potentially suppress fibrosis by suppressing inflammatory response, which therefore may prevent the progression of fatty liver to NASH.

Semiautomated Segmentation and Measurement of Cytoplasmic Vacuoles in a Neutrophil With General-Purpose Image Analysis Software.

BACKGROUND: Morphological observation of blood or marrow film is still described nonquantitatively. We developed a semiautomatic method for segmenting vacuoles from the cytoplasm using Photoshop (PS) and Image-J (IJ), called PS-IJ, and measured the relative entire cell area (rECA) and relative areas of vacuoles (rAV) in the cytoplasm of neutrophil with PS-IJ. METHODS: Whole-blood samples were stored at 4°C with ethylenediaminetetraacetate and in two different preserving manners (P1 and P2). Color-tone intensity levels of neutrophil images were semiautomatically compensated using PS, and then vacuole portions were automatically segmented by IJ. The rAV and rECA were measured by counting pixels by IJ. For evaluating the accuracy in segmentations of vacuoles with PS-IJ, the rAV/rECA ratios calculated with results from PS-IJ were compared with those calculated with human eye and IJ (HE-IJ). RESULTS: The rECA and rAV/ in P1 significantly (P < 0.05, P < 0.05) were enlarged and increased, but did not significantly (P = 0.46, P = 0.21) change in P2. The rAV/rECA ratios by PS-IJ were significantly correlated (r = 0.90, P < 0.01) with those by HE-IJ. CONCLUSION: PS-IJ method can successfully segment vacuoles and measure the rAV and rECA, becoming a useful tool for quantitative description of morphological observation of blood and marrow film.

[Differential diagnosis of primary aldosteronism by measurement of hybrid steroids using mass spectrometry].

Primary aldosteronism (PA), characterized by the autonomous hypersecretion of aldosterone, is the most common cause of secondary hypertension. Patients with PA have a higher risk of cardiovascular morbidity than essential hypertension. The two common subtypes of PA, aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA), should be differentiated, because the former is an indication for adrenalectomy, and the latter is treated by medication. 18-Hydroxycortisol and 18-oxocortisol, known as hybrid steroids, have been recognized as markers for the differentiation of aldosterone-producing adenoma and rare glucocorticoid remediable hyperaldosteronism from other subtypes of PA. Hybrid steroids have been measured using immunoassays such as enzyme-linked immunoassays; however, immunoassays for hybrid steroids are not widely used. Recently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) for hybrid steroids was developed. The ability to measure hybrid steroids using LC-MS/MS will be useful for the differential diagnosis of subtypes of PA.

Postmortem contrast-enhanced computed tomography via direct large-vessel puncture.

The aim of this study was to assess the usefulness of postmortem contrast-enhanced CT (PMeCT) performed via direct large-vessel puncture when routine postmortem CT suggests a vascular lesion as the cause of death. PMeCT was performed in 9 cases (4 male, 5 female) with a mean age of 76 years (range 52-92) at the time of death. The mean time elapsed since death was 29.1 h (12.0-72.0). The location of the target vessel for puncture was determined based on the CT table position and a grid placed on the body surface. An 18-G spinal needle was advanced to the puncture site, and the needle tip was confirmed to have reached the intended blood vessel. Using negative pressure with a 20-ml syringe, the needle tip was advanced until reverse bleeding was confirmed. Diluted contrast medium was injected slowly to ensure its dispersion within the blood vessels. Following confirmation of no extravasation, additional doses of diluted contrast agent were injected in 3-4 divided doses, with CT scans obtained at each step to track the distribution of contrast agent over time. PMeCT was successful in all cases, revealing cardiac tamponade in 7 (ascending aortic dissection, n = 6; cardiac rupture, n = 1), thoracic aortic aneurysm rupture, n = 1, and iliac artery aneurysm rupture, n = 1. There were no cases of procedure-related extravasation (pseudo-lesions). When postmortem CT reveals pericardial hematoma or bleeding in the thoracic or abdominal cavity, PMeCT can identify the source of bleeding.

Analysis of triacylglycerol hydroperoxides in human lipoproteins by Orbitrap mass spectrometer.

Herein, we represent a simple method for the detection and characterization of molecular species of triacylglycerol monohydroperoxides (TGOOH) in biological samples by use of reversed-phase liquid chromatography with a LTQ Orbitrap XL mass spectrometer (LC/LTQ Orbitrap) via an electrospray ionization source. Data were acquired using high-resolution, high-mass accuracy in Fourier-transform mode. Platform performance, related to the identification of TGOOH in human lipoproteins and plasma, was estimated using extracted ion chromatograms with mass tolerance windows of 5 ppm. Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy donor were oxidized by CuSO4 to generate oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No TGOOH molecular species were detected in the nLDL and nHDL, whereas 11 species of TGOOH molecules were detected in the oxLDL and oxHDL. In positive-ion mode, TGOOH was found as [M + NH4](+). In negative-ion mode, TGOOH was observed as [M + CH3COO](-). TGOOH was more easily ionized in positive-ion mode than in negative-ion mode. The LC/LTQ Orbitrap method was applied to human plasma and three molecular species of TGOOH were detected. The limit of detection is 0.1 pmol (S/N = 10:1) for each synthesized TGOOH.

Objective evaluation of chest findings in infants by postmortem computed tomography.

PURPOSE: The purpose of this study was to review the findings of computed tomography (CT) performed early postmortem on infants and to clarify the postmortem CT lung findings that occur in the absence of abnormal histopathological findings. MATERIALS AND METHODS: From July 2016 to March 2022, 72 infants were autopsied with postmortem CT (41 boys 31 girls, aged 0-36 (mean 8.2) months). Autopsy and postmortem CT lung findings were compared with the causes of death identified by the autopsies, namely sudden infant death syndrome (n = 37), acute circulatory system disease (18), drowning (7), asphyxia (5), and dehydration/undernutrition (5). RESULTS: The %aerated lung volume (-700 HU or less) ranged from 0 % to 33 % (mean 1.5 %, median 0 %), being <1 % in 61 cases (84.7 %) and >3 % in 3/5 (60 %) of the dehydration/undernutrition group. The dehydration/undernutrition group showed significant preservation of lung field air content compared with the other causes of death groups (p < 0.05). Receiver characteristic curve analysis showed a cut off value of 0.8 % and area under the curve of 0.88806. The drowning group had significantly greater pleural cavity fluid retention than the other causes of death groups (p < 0.05). No correlation was found between postmortem interval and pleural cavity fluid retention. However, resuscitation time and pleural cavity fluid retention were correlated. CONCLUSION: Evaluation of CT values on postmortem lung fields of infants usually reveals a marked decrease in air content. When air content exceeds 0.8% on infant postmortem CT, dehydration/undernutrition should be considered in the differential diagnosis.

Objective Evaluation of Oral and Pharyngeal Areas in Autopsy Cases of Obstructive Sleep Apnea Syndrome via Postmortem CT.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) can cause sudden death during sleep. Previous findings have suggested that OSAS development is related to maxillofacial morphology. Evaluation of facial morphology can determine the risk of developing the disease, and establishing an objective method to assess the underlying etiology of OSAS-related death would be advantageous. OBJECTIVE: The objective of this study is to determine the key features of obstructive sleep apnea syndrome (OSAS) using postmortem oral and pharyngeal computed tomography (CT). METHODS: We retrospectively assessed autopsy cases of patients with (n=25) and without (n=25) OSAS-related death. We used oral and pharyngeal CT images to compare the oral and pharyngeal cavity volume (OPCV), oral and pharyngeal soft tissue volume (OPSV), oral and pharyngeal air space volume (OPAV), and OPAV to OPCV ratio (%air). Receiver operating curve (ROC) analysis was used to determine the accuracy of OSAS prediction. We assessed participants with body mass index (BMI) values within the normal range. RESULTS: Among the 50 subjects, we observed significant between-group differences in OPSV, OPAV, and % air, whereas there were significant between-group differences in OPSV and %air among 28 subjects with normal BMI values. Both comparisons suggested that OSAS-related death was associated with low %air and high OPSV values. CONCLUSION: The %air and OPSV are useful for assessing postmortem oropharyngeal CT images. OSAS-related sudden death is likely when %air and OPSV values are ≤20.1% and ≥127.2 ml, respectively. Among those with normal BMI values, % air and OPSV values of ≤22.8% and ≥111.5 ml, respectively, predict OSAS-related sudden death.

Removal of acidic or basic α-amino acids in water by poorly water soluble scandium complexes.

To recognize α-amino acids with highly polar side chains in water, poorly water soluble scandium complexes with both Lewis acidic and basic portions were synthesized as artificial receptors. A suspension of some of these receptor molecules in an α-amino acid solution could remove acidic and basic α-amino acids from the solution. The compound most efficient at preferentially removing basic α-amino acids (arginine, histidine, and lysine) was the receptor with 7,7'-[1,3-phenylenebis(carbonylimino)]bis(2-naphthalenesulfonate) as the ligand. The neutral α-amino acids were barely removed by these receptors. Removal experiments using a mixed amino acid solution generally gave results similar to those obtained using solutions containing a single amino acid. The results demonstrated that the scandium complex receptors were useful for binding acidic and basic α-amino acids.

Detection and characterization of cholesteryl ester hydroperoxides in oxidized LDL and oxidized HDL by use of an Orbitrap mass spectrometer.

Oxidation of cholesteryl esters in lipoproteins by reactive oxygen species yields cholesteryl ester hydroperoxides (CEOOH). In this study, we developed a novel method for identification and characterization of CEOOH molecules in human lipoproteins by use of reversed-phase liquid chromatography with an hybrid linear ion trap-Orbitrap mass spectrometer (LC-LTQ Orbitrap). Electrospray ionization tandem mass spectrometric analysis was performed in both positive-ion and negative-ion modes. Identification of CEOOH molecules was completed by use of high-mass-accuracy (MA) mass spectrometric data obtained by using the spectrometer in Fourier-transform (FT) mode. Native low-density lipoproteins (nLDL) and native high-density lipoproteins (nHDL) from a healthy donor were oxidized by CuSO(4), furnishing oxidized LDL (oxLDL) and oxidized HDL (oxHDL). No CEOOH molecules were detected in the nLDL and the nHDL, whereas six CEOOH molecules were detected in the oxLDL and the oxHDL. In positive-ion mode, CEOOH was detected as [M + NH(4)](+) and [M + Na](+) ions. In negative-ion mode, CEOOH was detected as [M + CH(3)COO](-) ions. CEOOH were more easily ionized in positive-ion mode than in negative-ion mode. The LC-LTQ Orbitrap method was applied to human plasma and six species of CEOOH were detected. The limit of detection was 0.1 pmol (S/N = 5:1) for synthesized CEOOH.

Quantitative determination of phosphatidylcholine hydroperoxides during copper oxidation of LDL and HDL by liquid chromatography/mass spectrometry.

1-Palmitoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 16:0/18:2-OOH) and 1-stearoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 18:0/18:2-OOH) were measured by liquid chromatography/mass spectrometry (LC/MS) using nonendogenous 1-palmitoyl-2-heptadecenoylphosphatidylcholine monohydroperoxide as an internal standard. The calibration curves for synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, which were obtained by direct injection of the internal standard into the LC/MS system, were linear throughout the calibration range (0.8-12.8 pmol). Within-day and between-day coefficients of variation were less than 10%, and the recoveries were between 86% and 105%. The limit of detection (LOD) and the limit of quantification (LOQ) were determined using synthetic standards. The LOD (signal-to-noise ratio 3:1) was 0.01 pmol, and the LOQ (signal-to-noise ratio 6:1) was 0.08 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. With use of this method, the concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH in the lipoprotein fractions during copper-mediated oxidation were determined. We prepared oxLDL and oxHDL by incubating native LDL and native HDL from human plasma (n = 10) with CuSO(4) for up to 4 h. The time course of the PC 16:0/18:2-OOH and PC 18:0/18:2-OOH levels during oxidation consisted of three phases. For oxidized LDL, both compounds exhibited a slow lag phase and a subsequent rapidly increasing propagation phase, followed by a gradually decreasing degradation phase. In contrast, for oxidized HDL, both compounds initially exhibited a prompt propagation phase with a subsequent plateau phase, followed by a rapid degradation phase. The analytical LC/MS method for phosphatidylcholine hydroperoxides might be useful for the analysis of biological samples.

Objective skull fracture evaluation by using the postmortem 3D-CT skull fracture score in fatal falls.

PURPOSE: We evaluated the usefulness of skull fracture analysis using three-dimensional computed tomography skull fracture scores (3DCT-SFs) in cases of fatal falls. MATERIALS AND METHODS: From April 2016 to September 2020, 46 cases of fatal falls from great heights (33 males, 13 females; mean age: 52.7 (range: 18-89) years) were examined using routine postmortem CT. The 3DCT-SFs were determined as the sum of the fracture line lengths measured on a volume rendering image. Skull fracture severity was classified into four stages according to the 3DCT-SFs. These stages were compared by macroscopic evaluation of skull fracture severity (injury level 0: no fracture; injury level I: fracture without deviation; injury level II: fracture with deviation; injury level III: comminuted open skull fracture). The relationship between 3DCT-SFs values, the fall distance, and the hardness of the landing surface was also examined. RESULTS: Skull fractures occurred in 26 cases (56.5%). The mean 3DCT-SFs of the cases that were classified as stages I, Ⅱ, and III were 86.6 (5.0-187.0), 832.0 (235.1-1865.8), and 3582.5 (2171.6-4787.6), respectively. Upon macroscopic evaluation of fracture severity, there were 8, 10, and 8 cases of injury levels I, II, and III, respectively. The 3DCT-SFs-based stages correlated significantly with the macroscopic skull fracture severity levels (R2 = 0.936). Solid-surface fall points resulted in significantly higher 3DCT-SFs than soft surfaces. Comminuted open fracture of the skull (stage III) occurred with fall distances ≥ 24 m. CONCLUSION: The 3DCT-SFs correlate well with macroscopic findings and are useful as an objective skull fracture index.

The usefulness of measuring n-butyric acid concentration as a new indicator of blood decomposition in forensic autopsy.

In forensic medicine, although various alcohols have been reported as indicators of decomposition in collected blood, no studies have examined short-chain fatty acids as indicators. In this study, the blood n-butyric acid concentration was quantified, and the association between n-butyric acid and decomposition was investigated to determine whether the detection of n-butyric acid could be a new indicator of decomposition. Among the forensic autopsies performed from 2016 to 2018 in our laboratory, the cases were divided into decomposed (n = 20) and non-decomposed (n = 20) groups based on macroscopic findings. Blood samples collected at the time of autopsy were derivatized with 3-nitrophenylhydrazine hydrochloride after solid-phase extraction. The n-butyric acid concentration was measured using liquid chromatography-tandem mass spectrometry. In addition, ethanol and n-propanol were measured using a gas chromatography-flame ionization detector. There was a significant difference (p < 0.01) in the concentrations of n-butyric acid between the decomposed and non-decomposed groups (0.343 ± 0.259 [0.030-0.973] and 0.003 ± 0.002 [0.001-0.007] mg/mL, respectively). In the decomposed group, n-butyric acid was detected at high concentrations, even in cases where n-propanol was low. These results suggest that n-butyric acid is more likely to be an indicator of blood decomposition than n-propanol.

New microbial mannan catabolic pathway that involves a novel mannosylglucose phosphorylase.

The consecutive genes BF0771-BF0774 in the genome of Bacteroides fragilis NCTC 9343 were found to constitute an operon. The functional analysis of BF0772 showed that the gene encoded a novel enzyme, mannosylglucose phosphorylase that catalyzes the reaction, 4-O-ß-d-mannopyranosyl-d-glucose+Pi→mannose-1-phosphate+glucose. Here we propose a new mannan catabolic pathway in the anaerobe, which involves 1,4-ß-mannanase (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772), finally progressing to glycolysis. This pathway is distributed in microbes such as Bacteroides, Parabacteroides, Flavobacterium, and Cellvibrio.

Comparison of anthocyanin distribution in berries of Haskap (Lonicera caerulea subsp. edulis (Turcz. ex. Herder) Hultén), Miyama-uguisukagura (Lonicera gracilipes Miq.), and their interspecific hybrid using imaging mass spectrometry.

Haskap (Lonicera caerulea subsp. edulis), a shrub with violet-blue fruits, is distributed mainly in Hokkaido, Japan. Miyama-uguisukagura (Lonicera gracilipes), a species related to Haskap, produces red fruits. Interspecific hybridization of Miyama-uguisukagura and Haskap was performed to introduce novel characteristics in the resulting hybrids. The shape and color of the interspecific hybrid fruits differed from those of the parent fruits. A comparison of anthocyanin distribution among these three fruit types by imaging mass spectrometry (IMS) revealed the presence of five different anthocyanins. The average cyanidin 3,5-diglucoside and peonidin 3,5-diglucoside intensities in the interspecific hybrid fruit were higher than those of the parent fruits, whereas the average pelargonidin 3-glucoside, cyanidin 3-glucoside, and peonidin 3-glucoside intensities were the highest in Haskap. All anthocyanins were mainly accumulated in the inner and outer skins of Haskap and interspecific hybrid fruits, and in the skin of Miyama-uguisukagura fruits. The order of signal intensities of all anthocyanins among the three fruits was unchanged in different regions. Additionally, a comparison of IMS and LC/MS data from our previous study confirmed the possibility of comparing multiple fruits in the same plate by IMS. Thus, we elucidated anthocyanin distribution patterns of the interspecific hybrid and parent fruits by IMS.

Analysis of steryl glucosides in rice bran-based fermented food by LC/ESI-MS/MS.

Steryl glucosides (SGs) and acylated steryl glucosides (ASGs) are phytochemicals found in plant-based foods and are known as bioactive compounds with potential health benefits. These include anti-inflammatory properties, anti-diabetic effects, and modulation of immunoregulatory functions as well as having cholesterol lowering effects. In this study, three major SGs, i.e., glucosides of ß-sitosterol, stigmasterol, and campesterol, were synthesized and used as standards for measurement of their contents in rice bran (RB)-based fermented food (FBRA) utilizing Aspergillus oryzae and raw material (RM). The compounds were quantified using liquid chromatography/electrospray ionization-tandem mass spectrometry. It was found that ß-sitosteryl glucoside was most abundant among the analyzed glucosides in both samples, and the contents of each SG in FBRA decreased about 35% from those of RM. In contrast to SGs, the contents of ASGs in FBRA increased 1.5-fold during the fermentation process as evidenced by an alkaline hydrolysis. The present results suggest that the FBRA might have greater beneficial effects than the RM, since ASGs have shown to have more potent cholesterol lowering effects and stronger anti-diabetic properties than SGs.