Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics.

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.

Family Support for Medical Nutritional Therapy and Dietary Intake among Japanese with Type 2 Diabetes (JDDM 56).

The aim of this study was to investigate the association between habitual dietary intake for patients with diabetes and the content of family support for medical nutritional therapy (MNT). Analyzed were 289 Japanese with type 2 diabetes (men, 58.5%; mean age, 62.0 years; mean HbA1c, 53.4 mmol/mol) who completed the Food Frequency Questionnaire and Diabetes Family Behavior Checklist (DFBC). Relationships of mean values for food group intake to DFBC responses regarding MNT were examined using multivariate analysis of covariance. Positive response to "Praise for following diet" was associated with lower sweets intake (none: 60.1 g/day; ≥once monthly: 50.9 g/day, p = 0.038) and higher seasoning intake (none: 21.6 g/day, ≥once monthly: 24.1 g/day, p = 0.046). Energy intake was higher with positive responses to "Eat at the same time that you do" (none: 1636 kcal/day, ≥once monthly: 1818 kcal/day, p = 0.038). "Nags about not following diet" was associated with higher fish (none: 68.7 g/day, ≥once monthly: 78.7 g/day, p = 0.042) and salt intake (none: 8.3 g/day, ≥once monthly: 9.0 g/day, p = 0.014). Eating foods not part of the diabetic diet (none: 218.4 g/day, ≥once monthly: 246.9 g/day, p = 0.014) resulted in a higher vegetable intake. In females, significant differences in relationships in the overall analysis were reversed. Our results clarified relationships between types of family support of patients with type 2 diabetes and their dietary intake and the importance of sex differences for more effective MNT.

Widely-targeted quantitative lipidomics method by supercritical fluid chromatography triple quadrupole mass spectrometry.

Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylamine-bonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery.-Takeda, H., Y. Izumi, M. Takahashi, T. Paxton, S. Tamura, T. Koike, Y. Yu, N. Kato, K. Nagase, M. Shiomi, and T. Bamba.

[Current status of practice of providing dietary advice to pregnant women by members of the Japanese Midwives' Association. Knowledge and utilization of the "Dietary Guidelines for Pregnant and Lactating Women"].

OBJECTIVES: Midwives are actively involved in providing dietary advice to pregnant women. In order to describe the current status of prenatal dietary advice provided by midwives, we conducted a questionnaire survey inquiring about their knowledge and usage of the "Dietary Guidelines for Pregnant and Lactating Women" (Guidelines) issued by the Ministry of Health, Labour, and Welfare in 2006. METHODS: In total, 2000 members of the Japanese Midwives' Association were randomly selected for administration of the questionnaire. The recovery rate was 42.1%, and 785 responses were eligible for analyses. The questionnaire collected information on the respondent's age, years of work experience, current involvement in delivery practice, number of deliveries per year in the current affiliation, provision of dietary advice to pregnant women, and knowledge and usage of the Guidelines. RESULTS: The proportion of respondents in their 20 s was low (1.8%). About half of the respondents were practicing delivery. Further, 66.1% (519 midwives) reported they had knowledge of the Guidelines. Among those who had knowledge of the Guidelines and were currently providing dietary advice (426 midwives), 88.0% were using the Guidelines. The most frequently used item in the Guidelines was "Diet for preventing anemia" (75.8%). The least used item was "Shoku-iku (diet and nutrition education) for pregnant and lactating women" (58.5%). Midwives who were practicing delivery showed a significantly lower usage of the Guidelines than those who were not (84.9% vs. 92.6%, P = 0.02). Among midwives practicing delivery, the most common reason for not using the Guidelines was "using original educational material produced by oneself or the facility." CONCLUSION: Providing dietary advice to pregnant women constituted a large fraction of midwives' duties as well as delivery practice. About 90% of the midwives, who had knowledge of the Guidelines and provided dietary advice, were using the Guidelines. In order to increase the usage of individual items of the Guidelines, ready-to-use brochures should be developed for midwives.

Elevated maternal serum folate in the third trimester and reduced fetal growth: a longitudinal study.

This study aimed to examine the association of fetal growth and elevated third trimester maternal serum folate due to folic acid (FA) supplement intake. Dietary intake, use of FA supplements, weight, and blood biomarkers of B-vitamins (serum folate, pyridoxal, vitamin B(12), and plasma total homocysteine) were observed in 33 healthy pregnant women at the third trimester (average gestational age 35 wk). Birth outcomes were assessed through hospital birth records. Infant anthropometry and maternal blood biomarkers were followed up at 1 mo postpartum. Fourteen women were taking FA supplements at the third trimester. Dietary intake was similar among FA users and non-users, but serum folate and pyridoxal were significantly higher in users (11.6±6.7 vs. 6.1±3.2 ng/mL, and 13.8±21.7 vs. 3.2±1.4 ng/mL, respectively). Plasma total homocystein (tHcy) was higher in non-users compared to users, but not significantly. Nine FA users and eight non-users had low serum vitamin B(12) values (<203 pg/mL). Nine FA users and all non-users had low serum pyridoxal values (<7.0 ng/mL). Infant birthweight was significantly lower in users compared to non-users (2,894±318 vs. 3,154±230 g). At 1 mo postpartum, infant weight and length were similar between FA users and non-users, but infant weight gain was larger in users. Higher serum folate values due to FA use in the third trimester was related to reduced fetal size. Excess FA under low vitamin B(6) and B(12) status may affect fetal growth.

Iminosugars from Baphia nitida Lodd.

Chromatographic separation of the 50% aqueous EtOH extract of the leaves of the African medicinal tree Baphia nitida resulted in isolation of 10 iminosugars. The plant contained 2R,5R-dihydroxymethyl-3R,4R-dihydroxypyrrolidine (DMDP) as a major alkaloid. The structure of a new alkaloid was also elucidated by spectroscopic methods as the 1-O-beta-D-fructofuranoside of DMDP, and this plant produced 3-O-beta-D-glucopyranosyl-DMDP as well. DMDP is a potent inhibitor of beta-glucosidase and beta-galactosidase, whereas the other two derivatives lowered inhibition toward both of these enzymes and improved inhibitory activities toward rice alpha-glucosidase and rat intestinal maltase.

Isolation of glycosidase-inhibiting hyacinthacines and related alkaloids from Scilla socialis.

An examination of the bulbs of Scilla socialis has resulted in the isolation of 11 hyacinthacines, two pyrrolidines, and three piperidines. The structures of the new alkaloids were elucidated by spectroscopic methods as beta-1-C-ethyldeoxymannojirimycin (5), hyacinthacines B7 (10), C2 (11), C3 (12), C4 (13), and C5 (14), and alpha-5-C-(3-hydroxybutyl)hyacinthacine A2 (15). Although, beta-l-homofuconojirimycin (3) and alpha-7-deoxyhomonojirimycin (alpha-7-deoxy-HNJ, 4) are previously known alkaloids, this is the first report of their occurrence in the plant family Hyacinthaceae. Alkaloid 11 was found to be a good inhibitor of bacterial beta-glucosidase and human placenta alpha-l-fucosidase, with IC50 values of 13 and 17 microM, respectively, while alkaloid 12 showed no inhibitory activity toward alpha-l-fucosidase but was a more potent inhibitor of bovine liver beta-galactosidase (IC50 = 52 microM) than 11. Alkaloids 13 and 14 were shown to be inhibitory toward mammalian alpha-glucosidase (IC50 = 45 and 77 microM, respectively), and alkaloid 14 was demonstrated as a moderate inhibitor of bacterial beta-glucosidase (IC50 = 48 microM).