Evaluation of erythrocyte membrane oxidation due to their exposure to shear flow generated by extracorporeal blood pump.

Several similarities have been found between shear stress-induced erythrocyte damage and physiological aging of erythrocytes in terms of elevated mechanical fragility, increased erythrocyte aggregation, and decreased membrane surface charge. Accordingly, we hypothesized that blood pump circulation, which generates shear stress, would accelerate erythrocyte aging, manifesting as oxidation. Therefore, the purpose of this study was to investigate the effect of blood pump circulation on erythrocyte oxidation. Fresh porcine blood was acquired from a slaughterhouse and anticoagulated with sodium citrate. About 500 mL of anticoagulated whole blood was circulated for 180 min in an in vitro test circuit comprising a BP-80 blood pump with a pump speed and a pump pressure head of 100-120 mmHg. A blood sample was taken at the start of the circulation and 180 min afterward. The hemolysis level and oxidation amount of the erythrocyte membrane were analyzed and compared between samples. Hemolysis increased with the prolongation of shear exposure inside the pump circuit. After 180 min of blood pumping in circuit, the oxidation level of the erythrocyte membrane showed an increase of 0.1 nmol/mg protein. Moreover, the membrane oxidation levels of sheared erythrocytes were greater than those of control erythrocytes. These results suggest that blood pump circulation accelerates erythrocyte aging and give us a greater understanding of the effects of blood pump perfusion.

A Blended Vitamin Supplement Improves Spatial Cognitive and Short-Term Memory in Aged Mice.

Although many types of antioxidant supplements are available, the effect is greater if multiple types are taken simultaneously rather than one type. However, it is difficult to know which type and how much to take, as it is possible to take too many of some vitamins. As it is difficult for general consumers to make this choice, it is important to provide information based on scientific evidence. This study investigated the various effects of continuous administration of a blended supplement to aging mice. In 18-month-old C57BL/6 mice given a blended supplement ad libitum for 1 month, spatial cognition and short-term memory in the Morris water maze and Y-maze improved compared with the normal aged mice (spontaneous alternative ratio, normal aged mice, 49.5%, supplement-treated mice, 68.67%, p < 0.01). No significant differences in brain levels of secreted neurotrophic factors, such as nerve growth factor and brain-derived neurotrophic factor, were observed between these two groups. In treadmill durability tests before and after administration, the rate of increase in running distance after administration was significantly higher than that of the untreated group (increase rate, normal aged mice, 91.17%, supplement-treated aged mice, 111.4%, p < 0.04). However, training had no reinforcing effect, and post-mortem serum tests showed a significant decrease in aspartate aminotransferase, alanine aminotransferase, and total cholesterol values. These results suggest continuous intake of a blended supplement may improve cognitive function and suppress age-related muscle decline.

A mixed antioxidant supplement improves cognitive function, and coordination in aged mice.

Accumulation of oxidative damage increases the risk of several disorders. To prevent these diseases, people consume supplements. However, there is little evidence of the impact of supplement intake on cognitive function. Recently, frailty and sarcopenia have become serious issues, and these phenomena include a risk of mild cognitive impairment. In this study, aged mice were fed the combination supplement and cognitive and motor functions were measured. Following 1 month of treatment with the supplement, significant improvements in cognitive function and neuromuscular coordination were observed. Following 2 weeks of treadmill training, treatment with the supplement dramatically increased running distance compared to that in untreated normal aged mice. Serum indices such as triglyceride and total cholesterol were significantly decreased in the supplement-treated aged mice compared to untreated aged mice. These results indicate that the combination supplement may play a role in maintaining cognitive function, coordination ability and improving lipid metabolism.

Tocotrienols Prevent the Decline of Learning Ability in High-Fat, High-Sucrose Diet-Fed C57BL/6 Mice.

Obesity has been increasing worldwide and is well-known as a risk factor for cognitive decline. It has been reported that oxidative stress in the brain is deeply involved in cognitive dysfunction in rodent models. While there are many studies on oxidation in the liver and adipose tissue of obese mice, the relationship between obesity-induced cognitive dysfunction and brain oxidation has not been elucidated. Here, we show that obesity induced by a high-fat, high-sucrose diet (HFSD) alters cognitive function in C57BL/6 male mice, and it may involve the acceleration of brain oxidation. Tocotrienols (T3s), which are members of the vitamin E family, can prevent HFSD-induced cognitive changes. To elucidate these mechanisms, respiratory metabolism, locomotor activity, temperature around brown adipose tissue, and protein profiles in the cerebrum cortex were measured. Contrary to our expectation, respiratory metabolism was decreased, and temperature around brown adipose tissue was increased in the feeding of HFSD. The proteins that regulate redox balance did not significantly change, but 12 proteins, which were changed by HFSD feeding and not changed by T3s-treated HFSD compared to control mice, were identified. Our results indicated that HFSD-induced obesity decreases mouse learning ability and that T3s prevent its change. Additionally, feeding of HFSD significantly increased brain oxidation. However, further study is needed to elucidate the mechanisms of change in oxidative stress in the brain by obesity.

Impact of time from diagnosis to chemotherapy on prognosis in advanced pancreatic cancer.

BACKGROUND: Due to the aggressive nature and poor prognosis of advanced pancreatic cancer, prompt initiation of treatment is critical. We investigated the effect of the interval between cancer diagnosis and initiation of chemotherapy on survival in patients with advanced pancreatic cancer. METHODS: In this retrospective, single-centre study, consecutive patients with advanced pancreatic cancer between April 2013 and March 2022 were analyzed. Data were extracted from the electronic medical records of patients who received chemotherapy for metastatic, locally advanced or resectable pancreatic cancer or who received chemotherapy due to either being intolerant of or declining surgery. We compared overall survival between two groups: the early waiting time group (waiting time ≤30 days from diagnosis to chemotherapy initiation) and the elective waiting time group (waiting time ≥31 days). Prognostic factors, including biliary drainage, were considered. The impact of waiting time on survival was assessed by univariate and multivariate analyses with Cox proportional hazard models. A 1:1 propensity score matching approach was used to balance bias, accounting for significant poor prognosis factors, age and sex. RESULTS: The study involved 137 patients. Overall survival exhibited no statistically significant difference between the early and elective waiting time groups (207 and 261 days, P = 0.2518). Univariate and multivariate analyses identified poor performance status and metastasis presence as predictors of worse prognosis. This finding persisted post propensity score matching (275 and 222 days, P = 0.8223). CONCLUSIONS: Our study revealed that initiating chemotherapy ˃30 days later does not significantly affect treatment efficacy compared to within 30 days of diagnosis.

5-Aminosalicylic Acid-Induced Liver Injury in a Patient with Ulcerative Colitis: A Case Report.

Introduction: Drug-induced liver injury (DILI) associated with 5-aminosalicylic acid (5-ASA) is a rare but potentially life-threatening adverse event. Case Presentation: We report the case of a 58-year-old woman with ulcerative colitis who developed DILI after initiating maintenance therapy with the multimatrix system 5-ASA. The patient presented with grade 4 liver enzyme elevation on day 98 after initiating 5-ASA and was admitted to the hospital. Blood tests revealed the mixed liver injury, and imaging studies showed no abnormalities except for mild lymph node enlargement. Liver biopsy revealed acute lobular hepatitis with interfacial activity. The patient's score on the International Autoimmune Hepatitis Group 1999 revised scoring system was a total score of 10, causing a suspicion for the diagnosis of autoimmune hepatitis. The DDW-J 2004 scale calculated a total score of six, indicating a high probability of DILI. We suspected DILI due to 5-ASA, and the 5-ASA formulations were discontinued. The patient was treated with ursodeoxycholic acid and neominophagen C, and her liver function gradually improved without steroid treatment. Finally, we definitively diagnosed DILI based on the pathological findings and clinical course after discontinuation of 5-ASA. Conclusion: This case highlights the importance of monitoring liver function in patients receiving 5-ASA therapy.

Effects of cerium oxide nanoparticles on adenine-induced chronic kidney disease model rats.

AIM: Cerium oxide, particularly in nanoparticle form (nanoceria), has been investigated for biomedical applications as a promising new agent for treating several pathologies. The aim of the present study was to characterize the pharmacologic effects of nanoceria in an animal model of chronic kidney disease. METHODS: We created the chronic kidney disease animal model by feeding rats a 0.25% adenine diet. Male Wistar rats were divided into five groups: normal diet, 0.25% adenine diet, or adenine diet containing three different doses or durations of nanoceria treatment. Blood was collected weekly from the tail veins of each rat and analyzed for renal function markers. After 5 weeks, various biochemical markers in serum, plasma, and urine were also analyzed. RESULTS: In the adenine-treated group, body weight was significantly decreased, and the kidneys lost much of their healthy reddish color and became lumpy and white in appearance. In addition, levels of serum creatinine, blood urea nitrogen, and plasma uremic toxins were significantly increased in adenine-treated rats compared with controls. Renal functional and structural damage in adenine diet model rats tended to be ameliorated by nanoceria ingestion. The high-dose cerium-treated group maintained reddish areas in the kidneys, and the increases in biomarker levels of creatinine, blood urea nitrogen, and inorganic phosphorus were markedly reduced, regardless of treatment duration. CONCLUSIONS: Ingestion of nanoceria may be effective for improving or preventing renal damage caused by adenine. Geriatr Gerontol Int 2023; ••: ••-••.

Drug-induced Interstitial Nephritis in a Patient with Ulcerative Colitis Treated with 5-Aminosalicylic Acid: A Case Report.

This report describes the case of a 76-year-old man with ulcerative colitis who developed interstitial nephritis after starting 5-aminosalicylic acid (5-ASA) therapy. The patient experienced an initial improvement in symptoms, but developed fatigue, anorexia, and severe renal dysfunction 2.5 months later. Renal biopsy confirmed drug-induced interstitial nephritis, and conservative treatment with fluid replacement and the discontinuation of 5-ASA improved the patient's condition. Clinicians should monitor patients receiving 5-ASA therapy for potential adverse effects, particularly renal injury, and promptly investigate symptoms of renal dysfunction. Early recognition and discontinuation of the offending agent may prevent further damage and improve patient outcomes.

Impact of Initial Body Weight Loss on Prognosis in Advanced Pancreatic Cancer: Insights From a Single-Center Retrospective Study.

BACKGROUND: Pancreatic cancer (PC) has a poor prognosis, with body weight loss commonly observed at diagnosis. However, the impact on PC prognosis of weight loss at the time of diagnosis on PC prognosis is unknown. METHODS: This retrospective, single-center study enrolled consecutively patients diagnosed with metastatic or locally advanced PC or resectable PC who were intolerant of or refused surgery. Patients who had lost more than 5% of their body weight or more than 2% and had a body mass index (BMI) of less than 20 kg/m2 at diagnosis were classified as experiencing body weight loss. Patients were subclassified into 2 groups: patients with and without weight loss. The study evaluated patient-related and PC-related factors affecting prognosis. Cox proportional hazards models were used to assess factors affecting prognosis. The primary endpoint was overall survival. Additionally, 1:1 propensity score matching was performed to reduce bias. RESULTS: In total, 220 patients were included in the study. The median age of the patients was 74 years, and 49.1% were male. Weight loss at diagnosis was observed in 43.2% of patients. There were no significant differences in clinical factors, except for anthropometric parameters, between the groups. The median survival time did not differ between the weight loss and no weight loss groups (149 and 173 days, respectively, P = .669). After matching, no significant differences in survival times were observed between the 2 groups. CONCLUSIONS: This study found no association between weight loss at diagnosis and prognosis in patients with advanced PC treated with best supportive care or chemotherapy.

Elucidation of composition of chlorine compounds in acidic sodium chlorite solution using ion chromatography.

With the spread of coronavirus infections, the demand for disinfectants, such as a sodium chlorite solution, has increased worldwide. Sodium chlorite solution is a food additive and is used in a wide range of applications. There is evidence that chlorous acid or sodium chlorite is effective against various bacteria, but the actual mechanism is not well understood. One reason for this is that the composition of chlorine-based compounds contained in sodium chlorite solutions has not been clearly elucidated. The composition can vary greatly with pH. In addition, the conventional iodometric titration method, the N,N-diethyl-p-phenylenediamine sulfate (DPD) method and the absorption photometric method cannot clarify the composition. In this study, we attempted to elucidate the composition of a sodium chlorite solution using absorption spectrophotometry and ion chromatography (IC). IC is excellent for qualitative and quantitative analysis of trace ions. Through this, we aimed to develop an evaluation method that allows anyone to easily determine the bactericidal power of sodium chlorite. We found that commercially available sodium chlorite solution is 80% pure, with the remaining 20% potentially containing sodium hypochlorite solution. In addition, when sodium chlorite solution became acidified, its absorption spectrum exhibited a peak at 365 nm. Sodium chlorite solution is normally alkaline, and it cannot be measured by the DPD method, which is only applicable under acidic conditions. The presence of a peak at 365 nm indicates that the acidic sodium chlorite solution contains species with oxidizing power. On the other hand, the IC analysis showed a gradual decrease in chlorite ions in the acidic sodium chlorite solution. These results indicate that chlorite ions may not react with this DPD reagent, and other oxidizing species may be present in the acidic sodium chlorite solution. In summary, when a sodium chlorite solution becomes acidic, chlorine-based oxidizing species produce an absorption peak at 365 nm. Sodium hypochlorite and sodium chlorite solutions have completely different IC peak profiles. Although there are still many problems to be solved, we believe that the use of IC will facilitate the elucidation of the composition of sodium chlorite solution and its sterilization mechanism.

Oxidative stress induces tau hyperphosphorylation via MARK activation in neuroblastoma N1E-115 cells.

Reactive oxygen species are considered a cause of neuronal cell death in Alzheimer's disease (AD). Abnormal tau phosphorylation is a proven pathological hallmark of AD. Microtubule affinity-regulating kinases (MARKs) regulate tau-microtubule binding and play a crucial role in neuronal survival. In this study, we hypothesized that oxidative stress increases the phosphorylation of Ser262 of tau protein through activation of MARKs, which is the main reason for the development of AD. We investigated the relationship between tau hyperphosphorylation on Ser262 and MARKs in N1E-115 cells subjected to oxidative stress by exposure to a low concentration of hydrogen peroxide. This work builds on the observation that hyperphosphorylation of tau is significantly increased by oxidative stress. MARKs activation correlated with tau hyperphosphorylation at Ser262, a site that is essential to maintain microtubule stability and is the initial phosphorylation site in AD. These results indicated that MARKs inhibitors might serve a role as therapeutic tools for the treatment of AD.

Quenching effect of cerium oxide nanoparticles on singlet oxygen: validation of the potential for reaction with multiple reactive oxygen species.

Here we studied cerium oxide nanoparticles (nanoceria) as an agent for the future treatment of oxidative damage by validating and evaluating its scavenging activity towards reactive oxygen species (ROS) in vitro. Nanoceria has been shown to mimic the activities of superoxide dismutase and catalase, degrading superoxide (O2•-) and hydrogen peroxide (H2O2). We examined the antioxidative activity of nanoceria, focusing on its ability to quench singlet oxygen (1O2) in an aqueous solution. Electron paramagnetic resonance (EPR) was used to determine the rates of second-order reactions between nanoceria and three ROS (1O2, O2•-, and H2O2) in aqueous solution, and its antioxidative abilities were demonstrated. Nanoceria shows a wide range of ultraviolet-light absorption bands and thus 1O2 was produced directly in a nanoceria suspension using high-frequency ultrasound. The quenching or scavenging abilities of nanoceria for 1O2 and hypoxanthine-xanthine oxidase reaction-derived O2•- were examined by EPR spin-trapping methods, and the consumption of H2O2 was estimated by the EPR oximetry method. Our results indicated that nanoceria interact not only with two previously reported ROS but also with 1O2. Nanoceria were shown to degrade O2•- and H2O2, and their ability to quench 1O2 may be one mechanism by which they protect against oxidative damage such as inflammation.

A Drosophila model of diabetic neuropathy reveals a role of proteasome activity in the glia.

Diabetic peripheral neuropathy (DPN) is the most common chronic, progressive complication of diabetes mellitus. The main symptom is sensory loss; the molecular mechanisms are not fully understood. We found that Drosophila fed a high-sugar diet, which induces diabetes-like phenotypes, exhibit impairment of noxious heat avoidance. The impairment of heat avoidance was associated with shrinkage of the leg neurons expressing the Drosophila transient receptor potential channel Painless. Using a candidate genetic screening approach, we identified proteasome modulator 9 as one of the modulators of impairment of heat avoidance. We further showed that proteasome inhibition in the glia reversed the impairment of noxious heat avoidance, and heat-shock proteins and endolysosomal trafficking in the glia mediated the effect of proteasome inhibition. Our results establish Drosophila as a useful system for exploring molecular mechanisms of diet-induced peripheral neuropathy and propose that the glial proteasome is one of the candidate therapeutic targets for DPN.

Twendee X, a mixed antioxidant supplement, improves cognitive function, coordination, and neurotrophic factor expression in long-term vitamin E-deficient mice.

Oxidation products gradually accumulate during senescence, enhancing the risk of onset of many severe diseases. One such disease is dementia, and the number of cases of dementia, including Alzheimer's disease, has been increasing world-wide. These diseases can be prevented via attenuation of age-related physiological dysfunction; one preventive approach is the ingestion of antioxidants such as vitamin C and vitamin E. Many antioxidants are readily available commercially. Ingestion of mixed antioxidants is expected to provide further beneficial effects for human health. In this study, we used vitamin E-deficient mice as an animal model of increased oxidative stress and assessed the effects of dosing with mixed antioxidants. Administration of a commercial mixed antioxidant formula, Twendee X significantly improved cognitive function and coordination compared to untreated vitamin E-deficient animals. Furthermore, the levels of brain-derived neurotrophic factor and nerve growth factor were significantly increased in the cerebral cortex of Twendee X-dosed vitamin E-deficient mice compared to untreated animals. These results indicate that intake of a mixed antioxidant supplement may be beneficial to human health, even after oxidative stress has begun. In the next stage, it will be necessary to compare with other antioxidants and consider whether it is effective in the aged model.

Stratification Based on Risk Factors at Diagnosis Could Predict Progression in Patients with Pancreatic Cysts.

BACKGROUND: Predicting the risk of malignant transformation in pancreatic cyst patients is challenging. AIM: We retrospectively investigated the risk factors for malignant transformation in pancreatic cyst patients. METHODS: Patients with pancreatic cysts diagnosed using imaging tests were followed from November 2008 to December 2021. A significant change was defined as the additional development of high-risk stigmata (HRS), worrisome features (WFs), or pancreatic cancer during monitoring. RESULTS: In total, 479 patients were analyzed, with a median observation period of 50 months. Forty-four patients (9.2%) showed significant changes, and eight (1.7%) developed pancreatic cancer. The univariate analysis showed that the cyst diameter at diagnosis (≥ 14 mm), main pancreatic duct (MPD) diameter at diagnosis (≥ 3 mm), presence of multilocular cysts, and an inconsistent MPD caliber were significant predictive factors for a significant change. One point was assigned for each significant factor. We grouped the patients into three groups: the low-risk group (total score 0), medium-risk group (score 1-2), and high-risk group (score 3-4). The high-risk group had a higher risk of a significant change than the medium- and low-risk groups (age-adjusted HRs for the medium-risk and high-risk groups were 3.0 and 5.2 compared with the low-risk group). CONCLUSION: Stratification based on risk factors may help predict the development of significant changes in pancreatic cyst patients.

Predictive factors of collateral vessel development induced by oxaliplatin-based chemotherapy.

BACKGROUND AND AIM: Oxaliplatin can lead to hepatic sinusoidal injury, called hepatic sinusoidal obstruction syndrome (SOS), resulting in portal hypertension-related complications. This could worsen the clinical course of the patients treated with oxaliplatin. Early diagnosis is challenging. We explored predictive markers of oxaliplatin-induced collateral vessels. METHODS: Patients who received oxaliplatin-based chemotherapy were retrospectively screened. We evaluated their laboratory findings and spleen size on computed tomography immediately before oxaliplatin-based chemotherapy and after 6 months of treatment. The primary outcome was collateral vessel development, as a surrogate marker for oxaliplatin-induced SOS in patients who underwent oxaliplatin-based chemotherapy. The secondary outcome was the identification of factors that predicted the development of collateral vessels. RESULTS: We enrolled 161 patients who received oxaliplatin-based chemotherapy. They had a median age of 69 years, and 63.3% were men. Collateral vessels developed in nine (5.6%) patients during the study period. After oxaliplatin-based chemotherapy, the spleen size increased in 104 patients (64.6%), with a ≥ 30% increase in 19.4% of the patients. Univariate analysis showed that the Fibrosis-4 (FIB-4) index (≥ 1.76; OR 9.17), aspartate aminotransferase:platelet ratio index (APRI) (≥ 0.193; OR 9.62), cumulative dose of oxaliplatin (≥ 1000 mg; OR 8.43), and increase in spleen size (≥ 30%; OR 6.01) were significant risk factors for collateral vessel development. Multivariate analysis after stepwise selection revealed that the FIB-4 index and spleen size were significant independent predictive factors. CONCLUSION: A ≥ 1.76 increase in the FIB-4 index and a ≥ 30% increase in spleen size after 6 months of oxaliplatin-based chemotherapy were significant predictive markers for collateral vessel development.

Evaluation of cerium oxide as a phosphate binder using 5/6 nephrectomy model rat.

BACKGROUND: The number of chronic kidney disease (CKD) patients continues to increase worldwide. CKD patients need to take phosphate binders to manage serum phosphorus concentrations. Currently, several types of phosphate binder, including lanthanum carbonate, are used. However, they each have disadvantages. METHODS: In this study, we evaluated cerium oxide as a new phosphate binder in vitro and in vivo. First, cerium oxide was mixed with phosphoric acid at pH 2.5 or 7.0, and residual phosphoric acid was measured by absorption photometry using colorimetric reagent. Second, cerium oxide was fed to 5/6 nephrectomy model rats (5/6Nx), a well-known renal damage model. All rats were measured food intake, water intake, feces volume, and urine volume, and collected serum and urine were analyzed for biochemical markers. RESULTS: Cerium oxide can adsorb phosphate at acidic and neutral pH, while lanthanum carbonate, which is a one of popular phosphate binder, does not dissolve at neutral pH. Cerium oxide-treatment reduced serum phosphate concentrations of 5/6Nx rats without an increase in serum alanine transaminase levels that would indicate hepatotoxicity, and cerium oxide-treatment maintained serum creatinine and blood urea nitrogen levels, while those of normal 5/6Nx rats increased slightly. CONCLUSIONS: These results suggest that cerium oxide can be a potential phosphate binder. Decreased body weight gain and increased water intake and urine volume in 5/6Nx rats were thought to be an effect of nephrectomy because these changes did not occur in sham operation rats. Additional investigations are needed to evaluate the longer-term safety and possible accumulation of cerium oxide in the body.

Castration-resistant prostate cancer with metastasis to external auditory canal - Case report.

We report a case of castration-resistant prostate cancer metastasis to an external auditory canal. A 68-year-old man was diagnosed with prostate cancer (cT3aN1M1b GS5+5). Although abiraterone, docetaxel, and cabazitaxel were administered, and PSA decreased, liver metastasis appeared. Hearing loss in the left ear was also noted and the patient was referred to an otolaryngologist for an examination, which showed a neoplastic lesion in the external auditory canal. Biopsy findings resulted in a diagnosis of adenocarcinoma. Presented here are details of a rare case of prostate cancer with metastasis to an external auditory canal.

Importance of Locations of Iron Ions to Elicit Cytotoxicity Induced by a Fenton-Type Reaction.

The impact of the site of the Fenton reaction, i.e., hydroxyl radical (•OH) generation, on cytotoxicity was investigated by estimating cell lethality in rat thymocytes. Cells were incubated with ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2), or pre-incubated with FeSO4 and then H2O2 was added after medium was replaced to remove iron ions or after the medium was not replaced. Cell lethality in rat thymocytes was estimated by measuring cell sizes using flow cytometry. High extracellular concentrations of FeSO4 exerted protective effects against H2O2-induced cell death instead of enhancing cell lethality. The pre-incubation of cells with FeSO4 enhanced cell lethality induced by H2O2, whereas a pre-incubation with a high concentration of FeSO4 exerted protective effects. FeSO4 distributed extracellularly or on the surface of cells neutralized H2O2 outside cells. Cytotoxicity was only enhanced when the Fenton reaction, i.e., the generation of •OH, occurred inside cells. An assessment of plasmid DNA breakage showed that •OH induced by the Fenton reaction system did not break DNA. Therefore, the main target of intracellularly generated •OH does not appear to be DNA.

Simplifying quantitative measurement of free radical species using an X-band EPR spectrometer.

The quantitative measurement of free radicals in liquid using an X-band electron paramagnetic resonance (EPR) was systematized. Quantification of free radicals by EPR requires a standard sample that contains a known spin amount/concentration. When satisfactory reproducibility of the sample material, volume, shape, and positioning in the cavity for EPR measurements can be guaranteed, a sample tested and a standard can be directly compared and the process of quantification can be simplified. The purpose of this study was to simplify manual quantitative EPR measurement. A suitable sample volume for achieving a stable EPR intensity was estimated. The effects of different solvents on the EPR sensitivity were compared. The stability and reproducibility of the EPR intensity of standard nitroxyl radical solutions were compared among different types of sample tubes. When the sample tubes, sample volumes, and/or solvents were the same, the EPR intensity was reproduced with an error of 2% or less for µM samples. The quantified sample and the standard sample in the same solvent and the same volume drawn into the same sample tube was able to be directly compared. The standard sample for quantification should be measured just before or after every daily experiment.