Direct chiral separation of abscisic acid by high-performance liquid chromatography with a phenyl column and a mobile phase containing γ-cyclodextrin.

Abscisic acid (2-cis,4-trans-abscisic acid) is a plant hormone that has an asymmetric carbon atom. We tried to separate the enantiomers of native abscisic acid by HPLC using a phenyl column and a chiral mobile phase containing γ-cyclodextrin. The optimum mobile phase conditions were found to be 0.8% (w/v) γ-cyclodextrin, 4% (v/v) acetonitrile, and 20 mM phosphate buffer (pH 6.0). It was found that (R)-abscisic acid was earlier detected than (S)-abscisic acid. Since γ-cyclodextrin is hardly retained on a phenyl column, it was suggested that (R)-abscisic acid formed a more stable complex with γ-cyclodextrin than the (S)-abscisic acid. Abscisic acid in an acacia honey sample was successfully enantioseparated with the proposed method and only (S)-abscisic acid was detected. A biologically inactive 2-trans,4-trans-abscisic acid, which was prepared by irradiation of abscisic acid with a light-emitting diode lamp at 365 nm, was partially enantioseparated by the proposed method. Since the irradiation of (S)-abscisic acid-induced cis-to-trans isomerization to produce one 2-trans,4-trans-abscisic acid enantiomer, it is reasonable that racemization did not proceed during the cis-to-trans isomerization. (S)-Abscisic acid and probably (S)-2-trans,4-trans-abscisic acid were detected in a honey sample, where the peak area of (S)-abscisic acid was 7 times larger than that of (S)-2-trans,4-trans-abscisic acid.

Enantioseparation of phenethylamines by using high-performance liquid chromatography column permanently coated with methylated ß-cyclodextrin.

Cyclodextrins and their derivatives have been used for chiral high-performance liquid chromatography selectors, while they are costly to use as mobile phase additives in high-performance liquid chromatography. Here, we report application of phenyl column coated permanently with methylated ß-cyclodextrin for chiral high-performance liquid chromatography. A 0.1% (v/v) phosphoric acid solution containing 1 M NaCl and 0.5% (w/v) methylated ß-cyclodextrin was subjected to a phenyl column at a flow rate of 0.5 mL/min at 30°C for 2 h. Using the precoating phenyl column, all the enantiomers of the four phenethylamines (norepinephrine, epinephrine, octopamine, and synephrine) were successfully separated simultaneously by high-performance liquid chromatography with a mobile phase without methylated ß-cyclodextrin at a flow rate of 0.2 mL/min at 30°C. The enantioseparation ability was retained for successive analyses during 1 week. It is suggested that inclusion complex of methylated ß-cyclodextrin with a phenyl group on the surface of the stationary phase could be formed and that the inclusion complex could form the ternary complex with the injected analytes. The longer retention time of (S)-enantiomers of analytes than corresponding (R)-enantiomers for high-performance liquid chromatography could be explained from the higher stability of the methylated ß-cyclodextrin complexes with (S)-enantiomers, which were confirmed by capillary electrophoresis and 1 H NMR spectroscopy experiments.

Direct enantioseparation of mandelic acid by high-performance liquid chromatography using a phenyl column precoated with a small amount of cyclodextrin additive in a mobile phase.

Direct enantioseparation of mandelic acid by high-performance liquid chromatography (HPLC) with a reversed phase column and a mobile phase containing a small amount of hydroxylpropyl-ß-cyclodextrin (HP-ß-CD) was studied as an efficient method for saving consumption of the CD additive. As a result, it was proposed that racemic mandelic acid can be analyzed with a phenyl column by using a mobile phase composed of 10 mM ammonium acetate buffer (pH 4.2) and 0.02% (w/v) HP-ß-CD at a flow rate of 1.0 mL/min at 40°C after the passage of 10 mM ammonium acetate buffer (pH 4.2) containing 0.1% (w/v) HP-ß-CD as a precoating mobile phase for 60 min. It is suggested that HP-ß-CD is bound with a phenyl group on the surface of the stationary phase to allow a phenyl column to act as a transient chiral column, and injected mandelic acid can form the ternary complex with the adsorbed HP-ß-CD. The longer retention time of D-mandelic acid than the L-isomer for HPLC can be explained from the higher stability of the HP-ß-CD complex with D-mandelic acid, which was confirmed by CE experiment with HP-ß-CD as a selector. The efficiency of a phenyl column compared with other stationary phases was also discussed.

Chiral separation of isoxanthohumol and 8-prenylnaringenin in beer, hop pellets and hops by HPLC with chiral columns.

Xanthohumol, isoxanthohumol, and 8-prenylnaringenin in beer, hop and hop pellet samples were analyzed by HPLC using an InertSustain phenyl column and the mobile phase containing 40% methanol and 12% 2-propanol. Fractions of isoxanthohumol and 8-prenylnaringenin obtained by the above HPLC were separately collected. Isoxanthohumol and 8-prenylnaringenin were enantioseparated by HPLC using a Chiralcel OD-H column with a mobile phase composed of hexane-ethanol (90:10, v/v) and a Chiralpak AD-RH column with a mobile phase composed of methanol-2-propanol-water (40:20:40, v/v/v), respectively. Isoxanthohumol and 8-prenylnaringenin from beer, hop and hop pellet samples were found to be present in a racemic mixture. This can be explained by the fact that the two analytes were produced by a nonenzymatic process. The effects of boiling conditions on the conversion of xanthohumol into isoxanthohumol were also studied. A higher concentration of ethanol in heating solvent resulted in a decrease in the conversion ratio and the conversion was stopped by addition of ethanol at >50% (v/v). The isomerization was significantly affected pH (2-10) and the boiling medium at pH 5 was minimum for the conversion. Therefore, it was suggested that xanthohumol was relatively difficult to convert to isoxanthohumol in wort (pH 5-5.5) during boiling.

Simultaneous determination of alcohols including diols and triols by HPLC with ultraviolet detection based on the formation of a copper(II) complex.

We developed a reversed-phase high-performance liquid chromatography method with ultraviolet detection using on-line complexation with Cu(II) ion for analysis of five alcohols including diols and triol (methanol, ethanol, 1,2-propanediol, 1,3-propanediol, and glycerol). The Cu(II) ion concentration in the mobile phase had a great influence on the peak areas of these alcohols, but not on their retention times. Column temperature (25-40°C) and pH of the mobile phase did not affect the separation of analytes. The optimum separation conditions were determined as 5 mM CuSO4 , 3 mM H2 SO4 , and 3 mM NaOH at 30°C. The ratio of the peak areas for three alcohols (methanol, 1,2-propanediol, and glycerol) was in good agreement with that calculated from the obtained stability constants, molar absorption coefficients for the 1:1 Cu(II) complexes with the three alcohols, and the injected molar quantities. This fact strongly suggests that the observed high-performance liquid chromatography signals resulted from formation of the 1:1 Cu(II)-alcohol complexes. Using the proposed method, these five alcohols in spirit, liquid for electronic cigarette, mouthwash, and nail enamel remover samples were successfully analyzed with only a simple pretreatment.

Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl-γ-cyclodextrin-modified micellar electrokinetic chromatography.

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl-γ-cyclodextrin (HP-γ-CD)-modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate-15 mM borate buffer (pH 9.0) containing 30 mM HP-γ-CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)- and (R)-enantiomers of regioisomeric 8-, 11-, 12-, and 15-HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8- and 12-HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8-HETE was found to be a mixture of 98% (R)-enantiomer and 2% (S)-enantiomer, while the 12-HETE was a mixture of 98% (S)-enantiomer and 2% (R)-enantiomer. The present study demonstrates that the HP-γ-CD-modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs.

Simultaneous Enantioseparation of Aldohexoses and Aldopentoses Derivatized With L-Tryptophanamide by Reversed Phase HPLC Using Butylboronic Acid as a Complexation Reagent of Monosaccharides.

Three aldohexoses, glucose, galactose, and mannose, and three aldopentoses, arabinose, xylose, and ribose, were derivatized with L-tryptophanamide (L-TrpNH2 ) under alkaline conditions. Using a basic mobile phase (pH 9.2), the three aldohexoses or the three aldopentoses were simultaneously enantioseparated, respectively, but all the six monosaccharides could not be simultaneously enantioseparated. A large amount of nonreacted L-TrpNH2 was detected after the derivatized monosaccharides. In order to widen the separation window, a large portion of nonreacted L-TrpNH2 could be eliminated by liquid-liquid extraction with ethylacetate, and elution order of the derivatized monosaccharides and nonreacted L-TrpNH2 was found to be reversed using a neutral mobile phase. All of the six monosaccharides were simultaneously enantioseparated by reversed phase high-performance liquid chromatography (HPLC) using InertSustainSwift C18 column (4.6 mm i.d. × 150 mm) and a mobile phase containing 180 mM phosphate buffer (pH 7.6), 1.5 mM butylboronic acid, and 5% acetonitrile at 40 °C. Nomenclature of D and L for monosaccharides is based on the configurations of the asymmetric C4 center for aldopentoses and C5 center for aldohexoses. It was found that the enantiomer elution order of these six monosaccharides and fucose in the proposed method conformed to be the absolute configuration of the C2 center.

Separation of inorganic anions on reversed-phase C18 columns with a phosphomolybdate mobile phase.

Reversed-phase high performance liquid chromatography (RP-HPLC) is the most widely used chromatographic method. In addition to hydrophobic interactions, additional interactions such as electrostatic interactions may participate in the retention behaviour of an analyte. This makes it possible to use RP-HPLC for many types of analyte. We describe a simple method for separating inorganic anions on a C18 column, in which retention of inorganic anions is almost entirely due to electrostatic interactions. This leads to rapid separations as well as higher theoretical plate numbers. We used 2 mM phosphoric acid containing a low concentration of disodium molybdate as the mobile phase, which allows UV detection of non-UV-absorbing anions. With this method, we determined eight inorganic anions including several non-UV-absorbing anions photometrically at 220 nm. The detection limits of the examined eight inorganic anions calculated at a signal-to-noise ratio of 3 were between 0.3 and 10 µM. The detector response was linear over three orders of magnitude of inorganic anion concentration. The proposed RP-HPLC/UV method was successfully applied to determine inorganic anions in some water samples.

Impact of immune-related adverse events on survival outcomes in extensive-stage small cell lung cancer patients treated with immune checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors have recently become the standard of care in the first-line treatment of extensive-stage small cell lung cancer. Although immune-related adverse events have been reported to influence prognosis in non-small cell lung cancer patients, few studies have investigated the prognostic value of immune-related adverse events in small cell lung cancer patients. In this study, we evaluated the prognosis of patients who developed immune-related adverse events after first-line treatment with immune checkpoint inhibitor-based chemotherapy for extensive-stage small cell lung cancer. METHODS: We enrolled 90 patients with extensive-stage small cell lung cancer who received immune checkpoint inhibitor-based chemotherapy as first-line treatment from September 2019 to December 2022 in six hospitals in Japan. The patients were categorized into groups with and without immune-related adverse events. RESULTS: There were 23 patients with and 67 without immune-related adverse events. Seventeen patients had grade 1-2 immune-related adverse events, and nine (including overlapping cases) had grade ≥3. The most frequent immune-related adverse event was a skin rash. The median survival time was 22 months in patients with immune-related adverse events and 9.3 months in patients without immune-related adverse events. The hazard ratio was 0.40 (95% confidence interval: 0.19-0.83, p = 0.013). CONCLUSIONS: The results of this study show that immune-related adverse events are associated with improved survival outcomes in patients with extensive-stage small cell lung cancer.

Determination of α-hydroxy acids and their enantiomers in fruit juices by ligand exchange CE with a dual central metal ion system.

The content of α-hydroxy acids and their enantiomers can be used to distinguish authentic and adulterated fruit juices. Here, we investigated the use of ligand exchange CE with two kinds of central metal ion in a BGE for the simultaneous determination of enantiomers of dl-malic, dl-tartaric and dl-isocitric acids, and citric acid. Ligand exchange CE with 100 mM d-quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl-tartaric acid but not dl-malic acid or dl-isocitric acid. Addition of 1.8 mM Sc(III) ion to the BGE with 10 mM Cu(II) ion to create a dual central metal ion system permitted the simultaneous determination of these α-hydroxy acid enantiomers and citric acid. The proposed ligand exchange CE was thus well suited for detecting adulteration of fruit juices.

Predictive prognostic biomarkers in patients with COVID­19 infection.

The present study aimed to identify useful biomarkers to predict deterioration in patients with coronavirus disease 2019 (COVID­19). A total of 201 COVID­19 patients were classified according to their disease severity into non­severe (n=125) and severe (n=76) groups, and the behavior of laboratory biomarkers was examined according to the prognosis. Neutrophil count, aspartate aminotransferase (AST), alanine aminotransferase, lactate dehydrogenase (LDH), C­reactive protein (CRP), sialylated carbohydrate antigen KL­6 (KL­6), procalcitonin (PCT), presepsin (PSP) and D­dimer levels were significantly higher, and lymphocyte count and platelet count were significantly lower in the non­severe group compared with the severe group. In the non­severe group, ROC analysis demonstrated that only four biomarkers, CRP, PSP, AST and LDH were useful for differentiating the prognosis between improvement and deterioration subgroups. No strong correlation was revealed for any of the markers. Multivariate analysis identified CRP as a significant prognostic factor in non­severe cases (odds ratio, 41.45; 95% confidence interval, 4.91­349.24; P<0.001). However, there were no blood biomarkers that could predict the outcome of patients in the severe group. Overall, several blood markers changed significantly according to disease severity in the course of COVID­19 infection. Among them, CRP, PSP, LDH and AST were the most reliable markers for predicting the patient's prognosis in non­severe COVID­19 cases.

Mechanism of change in enantiomer migration order of enantioseparation of tartaric acid by ligand exchange capillary electrophoresis with Cu(II) and Ni(II)-D-quinic acid systems.

The mechanism of change in the enantiomer migration order (EMO) of tartarate on ligand exchange CE with Cu(II)- and Ni(II)-D-quinic acid systems was investigated thoroughly by circular dichroism (CD) spectropolarimetry. The (13) C NMR spectra of solutions containing D-quinate (pH 5.0) with Cu(II) or Ni(II) revealed the coordination of carboxylate and hydroxyl groups on D-quinate. The D-quinic acid concentration dependence of the CD spectra at a fixed Cu(II) concentration at pH 5.0 indicates that the 1:1, 1:2 and 1:3 Cu(II)-D-quinate complexes were formed with an increase in the concentration of D-quinic acid. The CD spectral behavior revealed that D-tartarate is selectively coordinated to the 1:1 complex to give the 1:1:1 Cu(II)-D-quinate-D-tartarate ternary complex while L-tartarate is selectively bound to the 1:2 and 1:3 complexes to form the 1:2:1 ternary complex. In the Ni(II)-D-quinic acid system, it became apparent that the 1:2 Ni(II)-D-quinate complex is mainly formed in the wide range of D-quinic acid concentration at pH 5.0 and D-tartarate is selectively coordinated to the 1:2 complex to form the 1:2:1 ternary complex. The change in EMO of tartarate on ligand exchange CE was explainable by the change in coordination selectivity for D- and L-tartarates in the Cu(II)- and Ni(II)-D-quinic acid systems depending on the compositions of the complexes formed in BGE.

Direct enantioseparation of lipoic acid in dietary supplements by capillary electrophoresis using trimethyl-ß-cyclodextrin as a chiral selector.

Lipoic acid, an antioxidant, naturally occurs as the (R)-enantiomer, while synthetic lipoic acid is racemic. It is thus of interest to know the (R)-enantiomer content of lipoic acid supplements. Here, we used capillary electrophoresis to directly enantioseparate lipoic acid in dietary supplements by using a sulfonated capillary with an effective voltage of +18 kV and direct detection at 200 nm. Factors affecting migration time and resolution of lipoic acid were investigated. The optimum background electrolyte was found to be 100 mM phosphate buffer (pH 7.0) containing 8 mM trimethyl-ß-cyclodextrin as a chiral selector at 20°C. Under the proposed conditions, direct chiral resolution of lipoic acid in dietary supplements was conducted successfully.

Enantioseparation of α-hydroxy acids by chiral ligand exchange CE with a dual central metal ion system.

Using two kinds of central metal ions in a background electrolyte, ligand exchange CE was investigated for the simultaneous enantioseparation of dl-malic, dl-tartaric, and dl-isocitric acids. Ligand exchange CE with 100 mM d-quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl-tartaric acid but not dl-malic acid or dl-isocitric acid. A dual central metal ion system containing 0.5 mM Al(III) ion in addition to 10 mM Cu(II) ion in the background electrolyte enabled the simultaneous enantioseparation of the three α-hydroxy acids. These results suggest that the use of a dual central metal ion system can be useful for enantioseparation by ligand exchange CE.

Radiation Therapy for Malignant Lumbosacral Plexopathy: A Case Series.

Background Malignant lumbosacral plexopathy is caused by a direct extension of an intrapelvic malignancy to involve the plexus nerves. In this report, we describe the effect of radiotherapy on patients with malignant lumbosacral plexopathy. Patients and methods We performed a retrospective review of the medical records of patients who underwent radiation therapy for pain caused by malignant lumbosacral plexopathy between 2017 and 2020 at our institution. The pain was measured using a numeric rating scale (0-10) at initiation and completion of radiotherapy or at the time when the maximum response was observed. Results A total of 12 tumor sites in 11 patients were included. Eight of the tumors invaded the iliopsoas muscle, and the remaining four invaded or abutted the piriformis muscle. The mean duration of follow-up was 215 days (31-675 days). All patients achieved pain relief at the end of radiotherapy, with complete resolution of pain in nine patients. The maximum effect was seen at a mean of three weeks (1-12 weeks) after the initiation of radiotherapy. Toxicities related to radiotherapy included grade 1 diarrhea in four patients and grade 1 frequent urination in one patient. Two patients experienced a relapse of pain at one and two months, respectively, after achieving their maximal response. Conclusion Radiotherapy provides significant pain relief for patients with the malignant lumbosacral syndrome. The recognition and diagnosis of this syndrome, and the use of radiation therapy as a therapeutic option, are important. Patients should be offered all possible therapies, regardless of curative or palliative intent.

Chiral separation of catechin and epicatechin by reversed phase high-performance liquid chromatography with ß-cyclodextrin stepwise and linear gradient elution modes.

Catechin and epicatechin were enantioseparated by high-performance liquid chromatography (HPLC) with a phenyl column and aqueous mobile phases containing 0.05% (w/v) and 0.6% (w/v) of ß-cyclodextrin for catechin and epicatechin, respectively. ß-Cyclodextrin was found to be scarcely retained on a phenyl column. Consequently, it was suggested that catechin, which was eluted earlier than epicatechin, formed more stable inclusion complex with ß-cyclodextrin than epicatechin and earlier eluted enantiomers, (-)-catechin and (+)-epicatechin, formed more stable diastereomer complexes with ß-cyclodextrin than the respective enantiomers. This was confirmed by ß-cyclodextrin-modified micellar electrokinetic chromatography and Benesi-Hildebrand plots by fluorescence spectrophotometry. Effect of sugars (D-sucrose, D-glucose, and D-fructose) on the epimerization of (+)-catechin and (+)-epicatechin by heating was investigated by HPLC with a ß-cyclodextrin stepwise elution mode, in which two kinds of aqueous eluents containing different concentrations of ß-cyclodextrin were used by turns. The epimerization of the two enantiomers was suppressed only when D-fructose was added. Separation of ten kinds of catechins including catechin and epicatechin enantiomers was investigated by a ß-cyclodextrin linear gradient HPLC elution mode without using organic solvents, where two kinds of aqueous eluents containing different concentrations of ß-cyclodextrin were used with changing their ratio gradually. These catechins in a green tea infusion could be separated successfully by this method.

Non-aqueous bonding of leuprorelin to ochratoxin A for peptide-based solid-phase extraction.

The anticancer therapeutic leuprorelin was found to have excellent affinity to the carcinogen ochratoxin A (OTA), with an equilibrium constant of 2.2 × 108 M-1 at 273 K (dissociation constant Kd = 4.5 nM) when functionalized into a mesoporous polymer. Binding between the surface-bound leuprorelin and mycotoxin was corroborated with DFT calculations, and it was extended to the extraction of OTA from the heavily fatty matrices of coffee, achieving 95% recovery with improved cyclability as compared with immunoaffinity. This work presents the potential of peptide-mycotoxin interactions for durable non-aqueous extraction.

Efficacy and Safety of Amrubicin in Small Cell Carcinoma Previously Treated with Immune Checkpoint Inhibitors and Chemotherapy.

Adding an immune checkpoint inhibitor to chemotherapy to treat extensive-stage small cell lung cancer is effective. However, there are no reports of an effective second-line treatment in patients previously treated with chemotherapy and immune checkpoint inhibitors as a first-line treatment. Here, we assessed the efficacy and safety of amrubicin as a second-line treatment for extensive-stage small cell lung cancer after chemotherapy and immune checkpoint inhibitor combination therapy. The study enrolled 150 patients with extensive-stage small cell lung cancer. The efficacy and the incidence of adverse events were compared between patients previously treated with immune checkpoint inhibitors and patients without previous immune checkpoint inhibitor treatment. One hundred and twenty-three patients were eligible. There was no difference in objective response rate, time-to-treatment failure, progression-free survival, and overall survival between both groups. The incidence of adverse events was similar in both treatment groups. Pretreatment with immune checkpoint inhibitors was not associated with an increase in amrubicin-related adverse events. This study shows that the efficacy of amrubicin in extensive-stage small cell lung cancer remains unchanged irrespective of previous treatment with immune checkpoint inhibitors. Amrubicin-related adverse events did not increase in patients previously treated with immune checkpoint inhibitors.

A label-free, direct solid-phase fluorimetric analysis of ochratoxin A in agricultural products with monoclonal antibody-immobilized monolith.

We established a method for directly measuring mycotoxin ochratoxin A (OTA) in foods by solid-phase fluorescence of monolith-immobilized antibodies. The antibody was introduced onto only one side of an 8 mm-diameter, 3 mm-thick monolith via covalently immobilized protein G. 4 µg (2.7 × 10-11 mol) of antibody was immobilized per one monolith. A maximum of 10 µg (2.4 × 10-11 mol) OTA adsorbed to the activated side of each monolith. The amount of OTA adsorbed and the fluorescence intensity showed good linearity in the range of 0.5-3 ng OTA. Loading the sample solution onto the non-antibody side on the monolith blocked the hydrophobic fluorescent matrices from reaching the immobilized surface of the antibody. The proposed method was able to detect 1 ng OTA/g in solid samples with complex matrices. Mean recoveries obtained at spiked concentration of 3 ng g-1 OTA/g were 78-90% with relative standard deviations of <7.9%.

Evaluation of Biomarkers of Severity in Patients with COVID-19 Infection.

OBJECT: Although many Japanese patients infected with coronavirus disease 2019 (COVID-19) only experience mild symptoms, in some cases a patient's condition deteriorates, resulting in a poor outcome. This study examines the behavior of biomarkers in patients with mild to severe COVID-19. METHODS: The disease severity of 152 COVID-19 patients was classified into mild, moderate I, moderate II, and severe, and the behavior of laboratory biomarkers was examined across these four disease stages. RESULTS: The median age and male/female ratio increased with severity. The mortality rate was 12.5% in both moderate II and severe stages. Underlying diseases, which were not observed in 45% of mild stage patients, increased with severity. An ROC analysis showed that C-reactive protein (CRP), ferritin, procalcitonin (PCT), hemoglobin (Hb) A1c, albumin, and lactate dehydrogenase (LDH) levels were significantly useful for the differential diagnosis of mild/moderate I stage and moderate II/severe stage. In the severe stage, Hb levels, coagulation time, total protein, and albumin were significantly different on the day of worsening from those observed on the day of admission. The frequency of hemostatic biomarker abnormalities was high in the severe disease stage. CONCLUSION: The evaluation of severity is valuable, as the mortality rate was high in the moderate II and severe stages. The levels of CRP, ferritin, PCT, albumin, and LDH were useful markers of severity, and hemostatic abnormalities were frequently observed in patients in the severe disease stage.