•Parallel UHPLC–MS/MS method for eight protease inhibitors in plasma and blood.•Integrated dried plasma or blood spot sample collection and preparation in tube-based device.•Accelerated sample spots ...drying by use of commercial microwave.
An assay was developed and validated for the quantification of eight protease inhibitors (indinavir (IDV), ritonavir (RTV), lopinavir (LPV), saquinavir (SQV), amprenavir (APV), nelfinavir (NFV), atazanavir (AZV) and darunavir (DRV)) in dried plasma spots using parallel ultra-high performance liquid chromatography and mass spectrometry detection in the multiple reaction monitoring mode. For each analyte an isotopically labeled internal standard was used and the assay based on liquid-solid extraction the area response ratio (analyte/IS) was found to be linear; from 0.025μg/ml to 20μg/ml for IDV, SQV, DRV, AZV, LPV, from 0.025μg/ml to 10μg/ml for NFV, APV and from 0.025μg/ml to 5μg/ml for RTV using 15μl of plasma spotted on filter paper placed in a sample tube. The total analysis time was of 4min and inter-assay accuracies and precisions were in the range of 87.7–109% and 2.5–11.8%, respectively. On dried plasma spots all analytes were found to be stable for at least 7 days. Practicability of the assay to blood was also demonstrated. The sample drying process could be reduced to 5min using a commercial microwave system without any analyte degradation. Together with quantification, confirmatory analysis was performed on representative clinical samples.
•We developed an automated precolumn derivatization amino acid analytical method.•This method achieved the separation of physiological amino acids within 8min.•We validated this method for 21 major ...types of free amino acids in human plasma.•Our developed method should be suitable for use in the clinical field.
The analysis of human plasma free amino acids is important for diagnosing the health of individuals, because their concentrations are known to vary with various diseases. The development of valid, reliable, and high-throughput analytical methods for amino acids analysis is an essential requirement in clinical applications. In the present study, we have developed an automated precolumn derivatization amino acid analytical method based on high-performance liquid chromatography/electrospray ionization mass spectrometry (so-called UF-Amino Station). This method enabled the separation of at least 38 types of physiological amino acids within 8min, and the interval time between injections was 12min. We also validated this method for 21 major types of free amino acids in human plasma samples. The results of the specificity, linearity, accuracy, repeatability, intermediate precision, reproducibility, limits of detections, lower limits of quantification, carry over, and sample solution stability were sufficient to allow for the measurement of amino acids in human plasma samples. Our developed method should be suitable for use in clinical fields.
Background/Aim: The enzyme-linked immunospot (ELISPOT) assay is a well-established method used to evaluate the strength of T cell-mediated immune activity, and accepted as a standard functional ...immunological assay. Cytokine activity is a novel parameter reflecting spot size and intensity, which has not been used in ELISPOT assay before. Materials and Methods: In the present study, from 113 ELISPOT assay data derived from previous clinical trials with dendritic cell vaccines, both spot number count and cytokine activity data for IFN-γ secretion were obtained using an ELISPOT reader. Comparing the new parameter cytokine activity with the existing parameter spot number, the feasibility of cytokine activity was investigated. Results: There were no significant differences in sensitivity and specificity between spot number and cytokine activity among ELISPOT assay data from CMVpp65 and other antigen peptide-stimulated cytotoxic T lymphocytes. Conclusion: Although cytokine activity is a novel parameter unreported so far, it did not show any advantages in the evaluation T cell immune responses compared to the existing spot number parameter.
The Edinburgh postnatal depression scale (EPDS) is commonly used in screening for major postpartum depression (PPD). We explored the clinical factors associated with score changes.
Mothers (n=1,287) ...who delivered a single live-born infant in Kokura Medical Center in Japan during 2018–2019 were analyzed. The EPDS-Japanese version was conducted at the first and fourth weeks after childbirth. Scores of ≥9 were considered to indicate an increased risk of PPD.
The scores improved during the four-week period (5.03±0.12 to 3.79±0.10). Primiparity, Cesarean section (CS), and a low Apgar score were identified as initial risk factors, however, primiparity remained in the multivariate analysis (aOR 2.02, 95% CI 1.37–2.97). Age ≥35 years was associated with worsened scores (aOR 1.88, 95%CI 1.01–3.51), but CS improved (aOR 0.38, 95%CI 0.21–0.70). Primiparity, CS, and neonatal respiratory support were the initial risk factors, while infant anomaly was a late risk factor in mothers whose infants were admitted to the neonatal intensive care unit (NICU) (aOR 3.35, 95%CI 1.31–8.56). In mothers of infants with an NICU stay of ≥4 weeks, infant anomaly was associated with worsened scores (aOR 6.61, 95%CI 1.11–39.3), while respiratory support was associated with improved scores (aOR 0.09, 95%CI 0.01–0.65). Twenty-six mothrs with worsened scores received psychiatric support; three developed PPD. Two of the three were ≥35 years of age, neither of their infants had anomalies.
Maternal aging and infant anomaly were risk factors for PPD. PPD occurred in mothers with worsened EPDS scores after mental care. Puerperants with worsening risk factors should be targeted to control PPD.
To clarify the relationship between arginine metabolism and hepatic injury, metabolomic analysis was performed in rats treated with 3 representative hepatotoxicants, monocrotaline (MCT), concanavalin ...A (ConA), and α-naphthyl isothiocyanate (ANIT); or a myotoxicant, tetramethyl-p-phenylenediamine (TMPD). A single dose of MCT, ConA, or ANIT dose-dependently induced hepatocellular necrosis accompanied by decreased blood arginine and increased blood alanine aminotransferase (ALT) and arginase. A close correlation was detected between arginine and ALT (r = -0.746, -0.795, -0.787 for MCT, ConA, ANIT, respectively) or between arginine and arginase (r = -0.605, -0.808, -0.672 for MCT, ConA, ANIT, respectively) in all three hepatic injury models. In contrast, neither hepatocellular necrosis nor alterations in arginine were found in the skeletal muscle injury model, although ALT was slightly increased. An in vitro assay revealed that blood samples obtained from ConA-treated rats transformed external arginine to ornithine, and the reaction was totally inhibited by an arginase inhibitor. These results suggest that blood arginase plays a crucial role in arginine metabolism associated with hepatic injury. In metabolomic analysis, nearly 450 endogenous metabolites were identified in blood obtained from all the models. Among the 13 metabolites involved in arginine metabolism, decreased arginine and increased ornithine occurred in common in the hepatic injury models, whereas citrulline and other metabolites were not altered. These results indicate that arginine metabolism, especially the arginine-to-ornithine pathway, is altered in association with acute hepatic injury. Furthermore, blood arginine and ornithine are possibly specific biomarkers for hepatic injury.
Solar flares trigger an increase in plasma density in the ionosphere including the D region, and cause the absorption of radio waves, especially in high-frequency (HF) ranges, called short-wave ...fadeout (SWF). To evaluate the SWF duration and absorption statistically, we analyze long-term (36 years) ionosonde data observed by the National Institute of Information and Communications Technology (NICT). The minimum reflection frequency,
f
min, is used to detect SWFs from 15-min-resolution ionosonde observations at Kokubunji, Tokyo, from 1981 to 2016. Since
f
min varies with local time (LT) and season, we refer to d
f
min, which is defined as
f
min subtracted by its 27-day running median at the same LT. We find that the occurrence of SWFs detected by three criteria, (i) d
f
min ≥ 2.5 MHz, (ii) d
f
min ≥ 3.5 MHz, and (iii) blackout, during daytime associated with any flare(s) greater than the C1 class is maximized at local noon and decreases with increasing solar zenith angle. We confirm that the d
f
min and duration of SWFs increase with the solar flare class. We estimate the absorption intensity from observations, which is comparable to an empirical relationship obtained from sudden cosmic noise absorption. A generalized empirical relationship for absorption from long-distance circuits shows quantitatively different dependences on solar flare flux, solar zenith angle, and frequency caused by different signal passes compared with that obtained from cosmic noise absorption. From our analysis and the empirical relationships, we estimate the duration of extreme events with occurrence probabilities of once per 10, 100, and 1000 years to be 1.8–3.6, 4.0–6.8, and 7.4–11.9 h, respectively. The longest duration of SWFs of about 12 h is comparable to the solar flare duration derived from an empirical relationship between the solar flare duration and the solar active area for the largest solar active region observed so far.
•A wide inter-individual variation of CYP activity was observed among 78 donors.•Diversity of CYP activity was partially correlated with genetic polymorphism.•Close correlation between some CYP ...activities and genotoxicity of cyclophosphamide.•Genotoxicity by cyclophosphamide was correlated with amount of the metabolites.•CYP2B6, 2C9, 2C19 and 3A4 are key determinants of genotoxicity by cyclophosphamide.
We investigated the relationship between metabolic activities of cytochrome P450 (CYP) isozymes present in microsomal fractions derived from the livers of 78 donors and micronucleus induction by cyclophosphamide (CPA). Consequently, a wide inter-individual variation in CYP activities was observed among the 78 donors. The CYP activities were partially correlated with the metabolic phenotypes predicted for the donors based on their single nucleotide polymorphisms. In addition, CPA induced micronucleus formation was seen for 47 out of 52 donors whose samples were tested with CPA doses ranging from 18.8 to 100 μg/mL. The CPA dose at which micronucleated cells were observed varied among the donors. Furthermore, a close correlation was identified between the catalytic activities of the CYP2B6, CYP2C9, CYP2C19, and CYP3A4 isozymes and micronucleus induction by CPA. To elucidate the mechanism underlying CPA-induced micronucleus formation in vitro tests were conducted on expression systems of CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Additionally, the metabolites of CPA generated by the expression systems were quantified by a liquid chromatography tandem mass spectrometer. Interestingly, several metabolites including the 4-hydroxyl form of CPA (4-OH-CPA) and phosphamide mustard were detected in the CYP2B6, CYP2C19, and CYP3A4 expression systems, but not in the CYP2C9 and CYP2D6 system. The presence of these metabolites was correlated with micronucleus induction by CPA. The absence of CPA metabolites in the CYP2C9 expression system might be associated with the lower 4-hydroxylase activity of this system.
The present results suggest that inter-individual variability in the metabolic capacity of each donor was associated with potential micronucleus induction due to CPA. Additionally, CPA metabolites like 4-OH-CPA and phosphamide mustard produced by human CYP2B6, CYP2C9, CYP2C19, and CYP3A4 are suggested to be major determinants of micronucleus induction by CPA.
We have investigated an M1.3 limb flare, which develops as a magnetic loop/arch that fans out from an X-ray jet. Using Hinode/EIS, we found that the temperature increases with height to a value of ...over 107 K at the loop top during the flare. The measured Doppler velocity (redshifts of 100-500 km s−1) and the nonthermal velocity (≥100 km s−1) from Fe xxiv also increase with loop height. The electron density increases from 0.3 × 109 cm−3 early in the flare rise to 1.3 × 109 cm−3 after the flare peak. The 3D structure of the loop derived with Solar TErrestrial RElations Observatory/EUV Imager indicates that the strong redshift in the loop-top region is due to upflowing plasma originating from the jet. Both hard X-ray and soft X-ray emission from the Reuven Ramaty High Energy Solar Spectroscopic Imager were only seen as footpoint brightenings during the impulsive phase of the flare, then, soft X-ray emission moved to the loop top in the decay phase. Based on the temperature and density measurements and theoretical cooling models, the temperature evolution of the flare arch is consistent with impulsive heating during the jet eruption followed by conductive cooling via evaporation and minor prolonged heating in the top of the fan loop. Investigating the magnetic field topology and squashing factor map from Solar Dynamics Observatory/HMI, we conclude that the observed magnetic-fan flaring arch is mostly heated from low atmospheric reconnection accompanying the jet ejection, instead of from reconnection above the arch as expected in the standard flare model.
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