[Implementation and Evaluation of Risk Communication Regarding Residual Pesticides].

In this study, a public seminar on risk communication methods was conducted to raise awareness and disseminate accurate knowledge about residual pesticides to consumers. Additionally, surveys on consumer awareness were conducted on the attendees before and after the seminar to evaluate its effectiveness. Responses were obtained from 84 participants. The paired t-test was used to analyze the changes in awareness before and after the seminar. The results showed significant improvements in "trust in the government" and "understanding of residual pesticides." Furthermore, step-wise multiple regression analysis was performed to explore the factors influencing satisfaction with the risk communication seminar, and the item "understanding of the safety of residual pesticides in food" was extracted. Understanding food safety is a crucial concern in daily life for consumers. To enable consumers to have an accurate understanding of food risks and make appropriate judgments, it is essential to continue implementing risk communication and conveying information about food safety and security in the future.

A reliable enzyme-linked immunosorbent assay for the determining of sesame proteins in raw food ingredients and in processed foods.

Sesame is a frequent cause of adverse food reactions in allergic patients. We developed a novel sandwich enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies and a unique extraction buffer for the detection and quantification of sesame proteins in processed foods and in raw food ingredients to clarify the validity of sesame labeling and for precautionary allergen labeling. The developed sandwich ELISA method is highly specific for sesame proteins. The limit of detection (LOD) and limit of quantification (LOQ) are 0.013 µg/g and 0.025 µg/g, respectively. The recoveries for incurred food samples, such as dressing, breads, sauce and pudding, ranged from 67 % to 81 %, while the repeatability and reproducibility coefficients of variation were less than 4.7 % and 4.5 %, respectively. The developed method has applicability for food products and is a reliable tool for the detection of hidden sesame proteins in raw food ingredients and in processed foods.

An improved Simon reaction method to discriminate between methamphetamine and false-positive substances.

The conventional methamphetamine (MA) detection method using the Simon reaction can be affected by false positives owing to compounds similar to aliphatic secondary amines. In this study, we examined the new Simon reaction to improve the qualitative accuracy of MA detection to discriminate substances that give false positives in a conventional Simon reaction. After the conventional Simon reaction for MA and false positives (N-isopropylbenzylamine (NIP-BA), N-methylbenzylamine (NMe-BA), L-proline (Pro), and L-hydroxyproline (HYP)), which are colored blue, di-tert-butyl dicarbonate (t-Boc) reagent was added, and color tone changes were observed. When t-Boc was added to the false positives (NIP-BA, NMe-BA, Pro, and HYP), the colors of MA, Pro, and HYP changed to purple; NIP-BA changed to blue; and NMe-BA changed to light pink after 3 min. These results suggested that MA can be differentiated from NIP-BA and NMe-BA. Furthermore, the solid-phase chromogenic method was examined, and it was confirmed that MA could be differentiated from Pro and HYP. The method developed in this study should increase the accuracy of MA appraisal at crime scenes and contribute to the reduction of misclassifications arising from false-positive substances.

Myosin Light Chain Phosphatase Plays an Important Role in Cardiac Fibrosis in a Model of Mineralocorticoid Receptor-Associated Hypertension.

BACKGROUND: Myosin phosphatase targeting subunit 2 (MYPT2) is an important subunit of cardiac MLC (myosin light chain) phosphatase, which plays a crucial role in regulating the phosphorylation of MLC to phospho-MLC (p-MLC). A recent study demonstrated mineralocorticoid receptor-related hypertension is associated with RhoA/Rho-associated kinase/MYPT1 signaling upregulation in smooth muscle cells. Our purpose is to investigate the effect of MYPT2 on cardiac function and fibrosis in mineralocorticoid receptor-related hypertension. METHODS AND RESULTS: HL-1 murine cardiomyocytes were incubated with different concentrations or durations of aldosterone. After 24-hour stimulation, aldosterone increased CTGF (connective tissue growth factor) and MYPT2 and decreased p-MLC in a dose-dependent manner. MYPT2 knockdown decreased CTGF. Cardiac-specific MYPT2-knockout (c-MYPT2-/-) mice exhibited decreased type 1 phosphatase catalytic subunit ß and increased p-MLC. A disease model of mouse was induced by subcutaneous aldosterone and 8% NaCl food for 4 weeks after uninephrectomy. Blood pressure elevation and left ventricular hypertrophy were observed in both c-MYPT2-/- and MYPT2+/+ mice, with no difference in heart weights or nuclear localization of mineralocorticoid receptor in cardiomyocytes. However, c-MYPT2-/- mice had higher ejection fraction and fractional shortening on echocardiography after aldosterone treatment. Histopathology revealed less fibrosis, reduced CTGF, and increased p-MLC in c-MYPT2-/- mice. Basal global radial strain and global longitudinal strain were higher in c-MYPT2-/- than in MYPT2+/+ mice. After aldosterone treatment, both global radial strain and global longitudinal strain remained higher in c-MYPT2-/- mice compared with MYPT2+/+ mice. CONCLUSIONS: Cardiac-specific MYPT2 knockout leads to decreased myosin light chain phosphatase and increased p-MLC. MYPT2 deletion prevented cardiac fibrosis and dysfunction in a model of mineralocorticoid receptor-associated hypertension.

Elucidation of Degradation Behavior of Nitrazepam, a Benzodiazepine Drug, under Basic Conditions: Study on Degradability of Drugs in Stomach (IV).

This study investigates the stability of nitrazepam (NZP), a benzodiazepine drug, under basic conditions, since alkaline putrefactive amines and ammonia are produced once bodies are left to decompose for a long period postmortem after a murder involving NZP or an accidental overdose of NZP. The degradation of NZP in an aqueous alkaline solution was investigated by LC/photodiode array detector (PDA) where the NZP degradation product was isolated and purified by solid-phase extraction using Oasis® MCX, and its chemical structure was determined by LC/time-of-flight (TOF)-MS, NMR spectroscopy, and single-crystal X-ray crystallography. The results revealed that NZP was immediately degraded under basic conditions with 2-amino-5-nitrobenzophenone being an intermediate which further degraded to provide 2-hydroxy-5-nitrobenzophenone as the final degradation product. These results are expected to be useful in clinical chemistry and forensic science, such as the detection of drugs during postmortem examination and suspected addiction.

[Severe neuropsychiatric disorder developing during busulfan-containing regimen for stem cell transplantation].

Here we describe two patients that required interruption of a busulfan (BU) containing conditioning regimen due to severe mental disorder before stem cell transplantation. The first patient was a 66-year-old man scheduled for unrelated peripheral blood stem cell transplantation with fludarabine/BU conditioning for myelodysplastic syndrome. He received 9.6 mg/kg BU and developed hallucinations that worsened the next day. BU was stopped on the final day, but the patient became comatose (grade 4). He recovered the next day. The second patient was a 69-year-old man scheduled for autologous peripheral blood stem cell transplantation with thiotepa (TT)/BU conditioning for cerebral nervous system relapse of mantle cell lymphoma. He received 12.8 mg/kg BU and developed hallucinations. His mental symptoms worsened on the next day, and thus administration was stopped on the second day of TT. His symptoms improved the next day. Both patients were over 65 years old, and their psychiatric symptoms worsened 1-2 days after the final dose of BU. Our findings suggest that BU may cause psychiatric disorders in elderly patients. When performing BU conditioning, it may be necessary to avoid azole antifungal medication and acetaminophen and to reduce the dose or perform therapeutic dose monitoring for elderly patients.

Identification of animal species by nucleic acid chromatography of hair samples and investigation of its applicability to human biological samples.

GeneFields®-Hair is a simple analysis kit that uses nucleic acid chromatography and polymerase chain reaction (PCR) to identify animal species of hair-like food contaminants. In this study, we evaluated GeneFields®-Hair as a simple and rapid method for identifying animal species from hair-like materials collected in forensic science, such as at crime scenes. The use of this kit with other human biological materials (whole blood, head dandruff, nails, saliva, oral mucosa, sebum, and urine) was also investigated. Animal body hair samples were pretreated by grinding in a buffer solution, centrifuged, and the supernatant was used for PCR. Nucleic acid chromatography of the PCR products allowed the identification of the animal species by the presence or absence of coloration on the decision line. For human biological materials, nucleic acid chromatography was performed after the appropriate pretreatment like body hair material. The determination of some animal species was difficult, even if they had a dedicated DNA Strip determination line. Furthermore, animals from the same family but different genera were sometimes detected on the same determination line. All the human biological samples were correctly identified. Smartphone photographs of the coloration of the judgment line were processed using the ImageJ software for quantitative determination.

Optimization of QuEChERS Extraction for Determination of Carotenoids, Polyphenols, and Sterols in Orange Juice Using Design of Experiments and Response Surface Methodology.

Several compounds with different physical properties are present in foods, biological components, and environmental samples, and there are cases in which these must be analyzed simultaneously. However, it is difficult to extract compounds with different physical properties from the same sample using a single method. In the present study, we examined the optimal conditions for the QuEChERS extraction of several kinds of compounds from orange juice using design of experiments (DoE) and response surface methodology (RSM) to determine the optimal ratio of organic solvent to sodium chloride. We determined the optimal extraction conditions, which were within the design space, using 100% tetrahydrofuran (THF) as the extraction organic solvent and NaCl:MgSO4 = 75:25 as the salt. The developed LC/MS/MS method using QuEChERS extraction achieved specific detection and precise quantification. Finally, we measured the polyphenols, sterols, and carotenoids in citrus juice using the optimized QuEChERS extraction method before LC/MS/MS analysis. Most of the analytes were quantifiable in orange juice. The optimized method achieved ease of operation, the extraction of analytes from food samples in a short time (within 30 min), minimization of analytical residues, and reliability. The DoE and RSM approach may contribute to better optimization of the extraction conditions for the lowest number of experiments.

Development of a Solid-Phase Dispersive Extraction Method for Molecularly Imprinted Polymers and LC-MS/MS for Analysis of Clenbuterol Residues in Swine Livers and Kidneys.

BACKGROUND: Clenbuterol (CLB) is approved as a veterinary drug because of its tracheal smooth muscle and uterine relaxant effects. However, if improperly administered for the purpose of fattening livestock, CLB can remain in the organs, which may pose a health hazard to humans. OBJECTIVE: We aimed to examine the combination of molecularly imprinted polymer (MIP) and solid-phase dispersive extraction (SPDE) as a pretreatment method for swine liver and kidney, which contain more coexisting impurities than muscle tissue, and attempted to construct an analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: Swine livers and kidneys were homogenized and extracted using liquid-liquid partitioning with an ethyl acetate-n-hexane (1 + 1) mixture, followed by SPDE using an MIP gel, and measured using LC-MS/MS. For LC-MS/MS, either an absolute calibration method or isotope dilution mass spectrometry (IDMS) was used. For method validation, a recovery test (additive concentrations: 0.05 and 0.5 ng/g) was conducted, and the data were analyzed using one-way analysis of variance (ANOVA). RESULTS: The recoveries (trueness), repeatability, and intermediate precision obtained using absolute calibration were similar to those obtained using IDMS. CONCLUSION: Using MIP-SPDE as a pretreatment method for CLB in swine liver and kidney samples yielded comparable results for absolute calibration and IDMS in LC-MS/MS analysis. HIGHLIGHTS: MIP-SPDE can be used as a pretreatment method to analyze CLB in swine organs with high accuracy.

Determination of Cyanide and Cyanoglycosides in Sweetened Bean Paste by HPLC with Fluorescence Detection.

It is necessary to evaluate the efficiency of reduction for cyanide and cyanoglycosides during the manufacturing process from raw material beans to sweetened bean paste in a food hygiene control system from the viewpoint of food safety. Analytical methods for cyanide and cyanoglycoside determination in sweetened bean paste by HPLC with fluorescence detection were developed. In analysis of collection time of free cyanide in the free cyanide assay, the recovery was improved by extending the collection time, the recovery rate was >80% by 2 h. The accuracy, repeatability and intra-laboratory precision of the free cyanide assay were 82.3, 2.0, and 2.4%, respectively. The method for cyanoglycoside analysis was evaluated by 5 repeated spiked recovery experiments at a concentration of 10 ppm. The accuracy, repeatability and intra-laboratory precision of the cyanoglycoside method were 82.2, 1.9, and 3.4%, respectively. These analytical methods will enable the analysis of cyanide and cyanoglycosides in sweetened bean paste without using steam distillation method in the pretreatment.

Elucidation of Degradation Behavior of Hydroxy Group-Containing Benzodiazepines in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (III).

The degradation behavior of three benzodiazepines (BZPs)-lormetazepam (LMZ), lorazepam, and oxazepam-with hydroxy groups on the diazepine ring in artificial gastric juice and the effect of storage pH conditions on drug degradability were monitored using an LC/photodiode array detector (PDA) to estimate their pharmacokinetics in the stomach. Although the three BZPs degraded in artificial gastric juice, none could be restored, despite increasing the storage pH, implying that the degradation reaction was irreversible. As for LMZ, we discussed the physicochemical parameters, such as the activation energy and activation entropy involved in the degradation reaction as well as the reaction kinetics; one of the degradation products was isolated and purified for structural analysis. In the LMZ degradation experiment, peaks corresponding to degradation products, (A) and (B), were detected through the LC/PDA measurements. Regarding the degradation behavior, we hypothesized that LMZ was degraded into (B) via (A), where (A) was an intermediate and (B) was the final product. Although the isolation of degradation product (A) was challenging, degradation product (B) could be isolated and was confirmed to be "methanone, [5-chloro-2-(methylamino)phenyl](2-chlorophenyl)-" based on structure determination using various instrumental analyses. The compound exhibited axis asymmetry as determined using single-crystal X-ray structure analysis. Because the formation of degradation product (B) was irreversible, it would be prudent to target the final degradation product (B) and LMZ for identification when detecting LMZ in human stomach contents, such as during forensic dissection.

End-of-Life Care of Acute Myeloid Leukemia Compared with Aggressive lymphoma in Patients Who Are Eligible for Intensive Chemotherapy: An Observational Study in a Japanese Community Hospital.

Background: Patients with hematological malignancies (HMs) are reported to receive more aggressive care at the end of life (EOL) than patients with solid tumors. However, the reasons behind this occurrence are not fully understood. Objectives: To examine whether the care at EOL for HMs is mainly because of the disease characteristics or hematologists' attitudes and systems of care, we compared the EOL care of patients with acute myeloid leukemia (AML) and diffuse large B cell lymphoma (DLBCL). Design: We retrospectively analyzed the EOL care of patients with AML and DLBCL younger than 80 years who were receiving combination chemotherapy at a city hospital in Japan. Results: Fifty-nine patients with AML and 65 with DLBCL were included. Those with AML received chemotherapy more often within their last 30 days (48% vs. 19%, p < 0.001) and 14 days (37% vs. 1.5%, p < 0.001) of life, and consulted the palliative team less frequently (5.3% vs. 29%, p < 0.001). In the last 3 years, the mortality rate in hematological wards decreased from 74% to 29% in the DLBCL group, but only from 95% to 90% in the AML group. In multivariate analysis, AML (odds ratio [OR] 0.065) and death before 2018 (OR, 0.077) were significant factors associated with reduced referrals to specialized palliative teams. Conclusion: Patients with AML tend to have lesser access to specialized palliative care and fewer options for their place of death than those with DLBCL. Detailed EOL care plans are needed for these patients, considering the characteristics of the disease.

Simultaneous determination of glyphosate, glufosinate, and their metabolites in honey using liquid chromatography-tandem mass spectrometry and solid-phase extraction.

Here, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of residual glyphosate, glufosinate, and their metabolites N-acetylglyphosate (Gly-A), 3-methylphosphinicopropionic acid (MPPA) and N-acetylglufosinate (Glu-A) in honey using a mixed mode column of reversed-phase and anion exchange without derivatization. The target analytes were extracted from honey samples using water, cleaned up on a reverse phase C18 cartridge column and an anion exchange NH2 cartridge column, and quantified using LC-MS/MS. Glyphosate, Glu-A, Gly-A, and MPPA were detected in negative ion mode based on deprotonation, whereas glufosinate was detected in positive ion mode. The coefficients of determination (R2) of the calibration curve, calculated in the range of 1-20 µg/kg for glufosinate, Glu-A, and MPPA, and 5-100 µg/kg for glyphosate and Gly-A, were higher than 0.993. The developed method was evaluated using honey samples spiked with glyphosate and Gly-A at 25 µg/kg and glufosinate, and MPPA and Glu-A at 5 µg/kg, based on the maximum residue levels. The validation results show good recoveries (86-106%) and precision (< 10%) for all target compounds. The limit of quantification of the developed method is 5 µg/kg for glyphosate, 2 µg/kg for Gly-A, and 1 µg/kg for glufosinate, MPPA and Glu-A. These results suggest that the developed method is applicable for quantifying residual glyphosate, glufosinate, and their metabolites in honey in compliance with Japanese maximum residue levels. Moreover, the proposed method was applied to the analysis of honey samples and glyphosate, glufosinate, and Glu-A were detected in some samples. The proposed method will be a useful tool for the regulatory monitoring of residual glyphosate, glufosinate, and their metabolites in honey.

Development of a simple and reliable LC-MS/MS method to simultaneously detect walnut and almond as specified in food allergen labelling regulations in processed foods.

We developed a simple and reliable analytical method using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously detect walnut and almond as specified in regulations for food allergen labelling in processed foods. Five specific target peptides derived from walnut 2S albumin and 7S globulin and three target peptides from almond 11S globulin were selected by analysing several varieties of walnut and almond, eight kinds of other nuts, and ten kinds of major allergen ingredients or cereals. The limit of detection for the walnut 2S albumin peptide GEEMEEMVQSAR (m/z 698.3 [precursor] > 316.1 [product]) was 0.22 ± 0.02 µg/g, and that for almond 11S globulin peptide GNLDFVQPPR (m/z 571.8 [precursor] > 369.2 [product]) was 0.08 ± 0.02 µg/g when extracted walnut and almond protein were spiked into butter cookie chocolate ice cream. These peptides had good linearity (R2 > 0.999) for each calibration curve with a range of 0.1-50 µg/mL protein concentration in the sample solutions, and sufficient recovery rates (90.4-101.5%) from the spiked samples. The developed analytical approach is applicable to a wide variety of processed foods for food allergen labelling.

Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression.

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression. NBRC 10746 showed the best performance as evaluated by Cypridina noctiluca luciferase expression. Subsequently, clone #5-30 named tat06213, which was obtained by single-colony isolation from NBRC 10746, was established as a promising host for heterologous protein expression. To deepen our understanding of the characteristics of O.minuta var. minuta strains, sequence analysis of the D1/D2 domain of large subunit rRNA was conducted and the resulting phylogenetic tree derived from the D1/D2 domain showed that NBRC 10402 and NBRC 10746 were grouped into a different cluster far from NBRC 0975. Furthermore, a chromosome structure topology with electrophoretic karyotype and AOX1 loci analyzed by pulsed-field gel electrophoresis with Southern blotting showed different chromosome patterns and AOX1-hybridization loci among the strains. Additionally, the sequences of the promoter regions of the cloned AOX1 genes were not identical among the three strains. These findings might explain the differences in heterologous protein expression among the tested O. minuta var. minuta strains.

Analysis of Methamphetamine in Urine by HPLC with Solid-Phase Dispersive Extraction and Solid-Phase Fluorescence Derivatization.

We have developed a fluorescence detection-liquid chromatography (HPLC-FL) method that involves sample pretreatment by solid-phase dispersive extraction (SPDE) and solid-phase fluorescence derivatization for the simple and rapid analysis of methamphetamine (MA) in urine. This method uses a reversed-phase polymeric solid-phase gel to clean up analytes in SPDE, followed by fluorescence derivatization with 9-fluorenylmethyl chloroformate (FMOC) in the solid-phase. The optimal conditions for SPDE and solid-phase fluorescence derivatization were obtained when J-SPEC PEP was used as the solid-phase gel and 0.5 mmol/L FMOC in 50 mmol/L borate buffer solution (pH 10) was used as the fluorescence derivatization reagent. The recovery experiment of MA in urine yielded a clean chromatogram with no interfering peaks, demonstrating the validity of our method; the recoveries were 83.6% when spiked at a low concentration level (100 ng/mL) and 80.7% when spiked at a high concentration level (1000 ng/mL). Compared with the conventional liquid-phase method, the reaction product (FMOC-MA) of solid-phase fluorescence derivatization had higher stability. Reaction rate constants were calculated by changing the temperature conditions, and physicochemical parameters, including activation energy and activation entropy involved in the degradation reaction, were obtained from the Arrhenius plot and analyzed thermodynamically. Taken together, our results suggest that the HPLC-FL method with SPDE and solid-phase fluorescence derivatization for sample pretreatment provides a simple and rapid means of analyzing MA in urine samples.

Development of the ultra-weak chemiluminescence method based on luminol reaction for use in the detection of ultra-trace levels of blood.

In this study, we focused on ultra-weak chemiluminescence (uwCL) measurements and aimed to develop a blood detection method at trace levels, that are difficult to observe using a conventional luminol reaction method (visual observation). Furthermore, we investigated sampling methods that could detect trace bloodstains from the field in our laboratory settings. To achieve these highly sensitive detection levels in the uwCL measurements, the optimal measurement conditions were established as follows: the luminol reaction solution chosen was a mixture of 6.0 × 10-3% luminol/1.5 × 10-2% sodium hydroxide solution and 1.5 × 10-2% hydrogen peroxide water (6:1); the temperature in the sample chamber was set to 20 °C; the sample chamber was filled with an oxygen displacement atmosphere; the sample chamber was filled with 3 mL of 0.01% sodium hydroxide solution prior to the experiment, and the measurement wavelength was set to 460 nm. Using the developed method, blood diluted to 12.5 million-fold (8.0 ng equivalent of the absolute weight of whole blood) was detectable, and high linearity (r = 0.9986) between uwCL intensity and whole blood equivalent was observed in the range of 8.0-100 ng. In contrast, the detection limit of the conventional method was 1.0 µg of the whole blood equivalent. Thus, the uwCL method was approximately 125 times more sensitive than the conventional method. In addition, we demonstrated that the sampling method of wiping with a melamine sponge soaked in a 10% sodium dodecyl sulfate solution is effective for sampling evidence materials at an appraisal site suspected of having traces of adhered blood.

Simultaneous detection of sibutramine and phenolphthalein in a diet jelly health food product that caused health problems.

The Public Health Center in Chiba Prefecture, Japan, received a consultation from a resident of Chiba Prefecture who consumed a diet jelly health food product and experienced health problems. To investigate the cause of the health problems, we examined the two food products for the presence of pharmaceutical ingredients. A screening analysis using ultra-high-performance liquid chromatography with a photodiode array detector (UPLC-PDA) indicated the presence of sibutramine and phenolphthalein in the food product. Analysis using an ultra-high-performance liquid chromatography-quadrupole-Kingdon trap mass spectrometer (UHPLC-Q-Kingdon trap MS) confirmed the presence of sibutramine and phenolphthalein. Quantitative analysis using UPLC-PDA showed that sibutramine and phenolphthalein were present at 15 and 16 mg/bag and 2.4 and 2.6 mg/bag, respectively. According to the drug insert for sibutramine capsules in the United States, the recommended medicinal dose of sibutramine should not exceed 15 mg/day, and the amount ingested in the present case exceeded that value. The present study results indicated that ingestion of the jelly health food product may cause health problems.

Serum-Induced Expression of Brain Natriuretic Peptide Contributes to Its Increase in Patients with HFpEF.

Brain natriuretic peptide (BNP) levels are increased in both patients with heart failure with preserved (HFpEF) and reduced ejection fraction (HFrEF), but the reasons for this remain unclear. Our purpose was to examine whether serum-induced BNP (iBNP) expression partly contributes to increased BNP in patients with HFpEF. BNP reporter cardiomyocytes from pBNP-luc-KI mice were stimulated with serum from patients with HFpEF or HFrEF (n = 114 and n = 82, respectively). Luciferase activity was examined as iBNP and the iBNP-to-BNP ratio was evaluated. Patient characteristics and clinical parameters were compared, and multivariate regression analysis was performed to determine independent predictors of the iBNP-to-BNP ratio. Female sex and frequencies of atrial fibrillation, hypertension and the use of a calcium channel blocker (CCB) were higher in HFpEF. The iBNP-to-BNP ratio was significantly higher in HFpEF (26.9) than in HFrEF (16.1, p < 0.001). Multivariate regression analysis identified the existence of HFpEF as an independent predictor of the iBNP-to-BNP ratio after adjusting for all other measurements (ß = 0.154, p = 0.032). Age, hemoglobin, CCB usage and deceleration time were also independent predictors (ß = 0.167, p = 0.025; ß = 0.203, p = 0.006; ß = 0.138, p = 0.049; and ß = 0.143, p = 0.049, respectively). These results indicate that the elevated BNP in patients with HFpEF is partly due to iBNP from the heart.