A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D₃BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D₃BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.     

Ultrasonographic assessment of regional radial and longitudinal systolic function in healthy awake dogs

BACKGROUND: Strain (St) and strain rate (SR) imaging are new ultrasound modalities based on tissue Doppler imaging (TDI) that allow quantitative assessment of segmental myocardial contraction or stretching and rate of deformation, respectively. HYPOTHESIS: Regional peak systolic St and SR could allow repeatable and reproducible assessment of systolic function of the right (RVW) and left (LVFW) myocardial walls in dogs. ANIMALS: Six healthy Beagle dogs were used to determine the repeatability and reproducibility of regional peak systolic St and SR in the RVW and LVFW (Study 1). These variables were also assessed in 30 healthy dogs of several breeds (Study 2). METHODS: Longitudinal peak systolic St and SR were recorded in 2 segments (basal and apical) of the RVW and LVFW. Radial peak systolic St and SR of the LVFW were also assessed. RESULTS: All within- and most (7/10) between-day coefficients of variation were <15%. Absolute values of the longitudinal deformation indices were significantly higher (P < .001) in the RVW (St = -39.5 +/- 5.5% and SR = -5.2 +/- 0.8 s(-1) at the base; St = -36.3 +/- 4.3% and SR = -4.7 +/- 1.1 s(-1) at the apex) than in the LVFW. Absolute values were also higher for the radial (St = 62.9 +/- 10.4% and SR = 5.8 +/- 1.1 s(-1), P < .001) than for the longitudinal LFVW motions. CONCLUSIONS AND CLINICAL IMPORTANCE: St and SR imaging is a repeatable and reproducible method for assessing systolic myocardial function. The combination of these indices with conventional echocardiographic variables may be useful for screening canine myocardial diseases.     

Quantitative assessment of velocities of the annulus of the left atrioventricular valve and left ventricular free wall in healthy cats by use of two-dimensional color tissue Doppler imaging

OBJECTIVE: To analyze velocities of the annulus of the left atrioventricular valve and left ventricular free wall (LVFW) in a large population of healthy cats by use of 2-dimensional color tissue Doppler imaging (TDI). ANIMALS: 100 healthy cats (0.3 to 12.0 years old; weighing 1.0 to 8.0 kg) of 6 breeds. PROCEDURE: Radial myocardial velocities were recorded in an endocardial and epicardial segment, and longitudinal velocities were recorded in 2 LVFW segments (basal and apical) and in the annulus of the left atrioventricular valve. RESULTS: LVFW velocities were significantly higher in the endocardial than epicardial layers and significantly higher in the basal than apical segments. For systole, early diastole, and late diastole, mean +/- SD radial myocardial velocity gradient (MVG), which was defined as the difference between endocardial and epicardial velocities, was 2.2 +/- 0.7, 3.3 +/- 1.3, and 1.8 +/- 0.7 cm/s, respectively, and longitudinal MVG, which was defined as the difference between basal and apical velocities, was 2.7 +/- 0.8, 3.1 +/- 1.4, and 2.1 +/- 0.9 cm/s, respectively. A breed effect was documented for several TDI variables; therefore, reference intervals for the TDI variables were determined for the 2 predominant breeds represented (Maine Coon and domestic shorthair cats). CONCLUSIONS AND CLINICAL RELEVANCE: LVFW velocities in healthy cats decrease from the endocardium to the epicardium and from the base to apex, thus defining radial and longitudinal MVG. These indices could complement conventional analysis of left ventricular function and contribute to the early accurate detection of cardiomyopathy in cats.     

A preliminary study of the genetic diversity of Bolivian oca (<Emphasis Type="Italic">Oxalis tuberosa</Emphasis> Mol.) varieties maintained<Emphasis Type="Italic"> in situ</Emphasis> and<Emphasis Type="Italic"> ex situ</Emphasis> through the utilization of ISSR molecular markers

ISSR molecular markers have been used to investigate genetic diversity of oca (Oxalis tuberosa Mol.), an Andean neglected tuber crop species. Sampling procedure allowed a preliminary study of the genetic diversity at the intra- and intervarietal levels. Twenty tuber lots conserved in situ in the microcentre of Candelaria and ex situ in the Toralapa Centre (Bolivia) were identified. Four ISSR primers amplified a total of 25 fragments of which 17 (68%) were polymorphic. These experiments show that the structure of oca varieties is mainly based upon vernacular names with a greater differentiation among tuber lots than within them, supporting agromorphological data. ISSR technique enlightened the existence of heterogeneous varieties in oca and divergence between in situ and ex situ conservation strategies. These observations are potentially linked to the different ways of management of tubers in these two conservation systems.     

Differential protein expression of the inhibitory effects of a standardized extract from Scutellariae radix in liver cancer cell lines using liquid chromatography and tandem mass spectrometry

The purpose of this study is to verify the inhibitory effect of a chemically standardized extract from Scutellariae radix in liver cancer cell lines (HepG2). The botanical extract was prepared using pressurized liquid extraction (PLE). A method using proteolytic digest with single dimensional and two-dimensional liquid chromatography with tandem mass spectrometry was used to characterize differential protein expression in mammalian cells in response to the botanical extract. The whole cell lysates were digested with trypsin, and the peptides were separated by one-dimensional (reversed phase) or by two-dimensional (cation exchange and reversed phase) solid-phase extraction (SPE) cleanup and separated by liquid chromatography with UV detection and mass spectrometry. In the presence of the botanical extracts, drug-induced apoptosis was not observed, and a number of proteins that played an important role in the metabolic pathways in HepG2 cell line had been affected. The data, as presented, suggest that the inhibitory effects of the standardized extracts from Scutellariae radix resulted from expression of heat shock protein and other proteins related to energy metabolism. The proposed platform had the potential to provide significant information about the particular proteome such as human hepatoma HepG2. At the molecular level, it was possible to study the proteins and how their levels and modifications change in response to the effects of the botanical extract.     

Comparison of dietary variety and ethnic food consumption among Chinese,Chinese-American,and white American women

The study's purpose was to estimate the variety of foods consumed within standard and ethnic food categories by three groups of women between 18 and 35 years of age. Foreign-born Chinese women [N = 21], Chinese-American women [N = 20] and white American women [N = 23] kept 4-day food records, after instruction. Analysis of variance showed that the mean number of different foods consumed by the foreign-born Chinese was significantly [p < 0.05] lower than those eaten by the other two groups for breads/cereals, dairy products, ethnic foods, fats, meats, sweets/sugars, and vegetable categories. White American women consumed significantly more dairy products and legumes/nuts than either of the groups of Chinese women. Diets of the Chinese-American women were more nutrient dense than those of the women in the other groups, containing significantly more energy, riboflavin, iron, folacin, and calcium than those of the foreign-born Chinese women. The percentage of kilocalories derived from fat [33–34%] did not differ among groups. 95% of foreign-born and 85% of Chinese-American women affirmed their ethnic identification by consuming foods belonging to Chinese cuisine; whereas, the percentage [30%] of white American women selecting Chinese food was significantly lower. Mexican and Italian foods were selected by significantly more white and Chinese-American women than by foreign-born Chinese women. Approximately 20% of both groups of Chinese women, but 49% of white American subjects, ate Japanese food. Chinese-American women retained a preference for Chinese food, similar to that of the foreign-born Chinese women. However, the Chinese-American women consumed cheeses, legumes, raw vegetables in salads, and foods from Italian and Mexican cuisines, indicating their acceptance of foods commonly consumed by white American women. Dietary acculturation among the Chinese-American women improved their diets over those of the foreign-born Chinese women and these results support the theory that consumption of a greater variety of foods increases nutrient density of diets.     

Clinical pharmacokinetics and outcomes of oral fluconazole therapy in dogs and cats with naturally occurring fungal disease

This multi-institutional study was designed to determine the clinical pharmacokinetics of fluconazole and outcomes in client-owned dogs (n = 37) and cats (n = 35) with fungal disease. Fluconazole serum concentrations were measured. Pharmacokinetic analysis was limited to animals at steady state (≥72 hr of treatment). The mean (range) body weight in 31 dogs was 25.6 (2.8–58.2) kg and in 31 cats was 3.9 (2.4–6.1) kg included in pharmacokinetic analyses. The dose, average steady-state serum concentrations (C_SS), and oral clearance in dogs were 14.2 (4.5–21.3) mg/kg/d, 26.8 (3.8–61.5) µg/mL, and 0.63 ml min⁻¹ kg⁻¹, respectively, and in cats were 18.6 (8.2–40.0) mg/kg/d, 32.1 (1.9–103.5) µg/mL, and 0.61 ml min⁻¹ kg⁻¹, respectively. Random inter-animal pharmacokinetic variability was high in both species. Two dogs had near twofold increases in serum fluconazole when generic formulations were changed, suggesting lack of bioequivalence. Median C_SS for dogs and cats achieving clinical remission was 19.4 and 35.8 µg/ml, respectively. Starting oral doses of 10 mg/kg q12h in dogs and 50–100 mg total daily dose in cats are recommended to achieve median C_SS associated with clinical remission. Due to the large pharmacokinetic variability, individualized dose adjustments based on C_SS (therapeutic drug monitoring) and treatment failure should be considered.