Origin determination of the Eastern oyster (Crassostrea virginica) using a combination of whole-body compound-specific isotope analysis and heavy metal analysis.

Various samples of the Eastern oyster, Crassostrea virginica, were collected from five harvest bay areas in the Gulf of Mexico coastal waters of Florida (FL), Louisiana (LA) and Texas (TX). Cadmium and lead concentrations from the extracted whole-body soft tissues were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS), and bulk δ13C and δ15N isotope ratios and amino-acid-specific δ13C values were analyzed via isotope ratio mass-spectrometry (IRMS). The combined data was subjected to multivariate statistical analysis to assess whether oysters could be linked to their harvest area. Results indicate that discriminant analysis using the δ13C values of five amino acids-serine, glycine, valine, lysine and phenylalanine-could discriminate oysters from two adjacent harvesting in Florida with 90% success rate, using leave-one-out cross validation. The combination of trace elements and isotope ratios could also predict geographic provenance of oysters with a success rate superior to the isolated use of each technique. The combinatory approach proposed in this study is a proof-of-concept that compound specific stable isotope analysis is a potential tool for oyster fisheries managers, wildlife, and food safety enforcement officers, as well as to forensics and ecology research areas, although significantly more work would need to be completed to fully validate the approach and achieve more reliable statistical results.

Investigation into the Concentrations and Sources of Nitrates and Nitrites in Milk and Plant-Based Powders.

Milk powders in the United States (US) may contain nitrates and nitrites from several potential sources. These sources include the ingestion of nitrates and nitrites by dairy cows during grazing and drinking, nitric acid used during the sanitization of dairy equipment, and the production of nitrous oxides in directly heated spray dryers. Recently, milk powders manufactured in the US have been rejected during import to other countries because nitrite concentrations were greater than 2 mg/kg (ppm). To date, the concentrations of nitrates and nitrites in milk and plant-based powders in the US are unknown. In this study the nitrate and nitrite concentrations present in diverse milk powders were investigated including 81 milk powders from local and online retailers from 2015 to 2018. In addition, 71 commercial milk powders were obtained from blinded production facilities. Nitrate and nitrite concentrations were determined using ion chromatography with conductivity and UV detection. A subset of samples was analyzed for N-nitrosodimethylamine using gas chromatography-mass spectrometry. Carbon and nitrogen bulk isotope ratios analyzed using isotope ratio mass spectrometry were used to obtain some insights into the production method (organic vs conventional) and geographic source of the milk powder samples. Background nitrate concentrations in US-produced milk powder samples averaged 17 ± 12 mg/kg. Nitrite was detected at concentrations greater than 2 mg/kg in 5 out of 39 different brands of retail milk and plant-based powders. Of these brands, two were plant-based (soy and coconut) powders and the other three had consistently high nitrites. The analysis of milk powders using stable isotope analysis revealed further information about the cow's diet.

Compound-Specific Isotope Analysis of Human Hair: Predicting Behaviors and Biometrics beyond Dietary Factors.

This study describes the use of amino acid quantitation and amino-acid-specific isotope ratio analysis of scalp hair of American individuals to predict soft biometric traits about the donors. The scalp hair of each donor was washed, dried, homogenized and acid hydrolyzed before analysis using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). A variety of statistical tests examined the relationships between the amino acid variables and biometric questionnaire responses, the latter of which could be assessed as continuous variables-in the case of age or body mass index (BMI)-or as categorical variables in the case of sex and alcohol consumption. Correlations between biometric factors and amino acid δ13C values were more significant after controlling for the extent of 13C in the subjects' diets. Multivariate analysis revealed that the sex of a donor could be correctly predicted with cross-validated accuracies of 80% using the isotope ratios and 89% using amino acid quantities. Using amino acid δ13C values or quantities, the age of a subject could only be predicted with an accuracy of ±27 years (95% CI). Hair treatments, such as chemical straitening and dying, did not have any measurable effect on the isotope ratios or quantities of amino acids in the hair. Unexpectedly, the δ13C values of sulfur-containing amino acids were significantly different between teetotalers and subjects who consumed alcohol daily. Further refinement of this study, including larger cohorts with controlled behavioral states or genetic factors, could provide helpful investigative leads in forensic casework.

Analysis of the 13C isotope ratios of amino acids in the larvae, pupae and adult stages of Calliphora vicina blow flies and their carrion food sources.

Adult blow flies are one of the first necrophagous insects to colonize fresh carcasses. The eggs they lay hatch into larvae, which then feed on the decomposing body. Like all organisms, blow flies "are what they eat," meaning that the isotopic composition of their body tissues reflects their diet. This manuscript combines ecology with a forensic application by using isotope ratio mass spectrometry (IRMS) to understand the relationship between the δ13C of amino acids in different carrion sources and the blow fly that feed on them. We also measure the amino acid-level fractionation that occurs at each major life stage of the blow flies. Adult blow flies from a commercial strain of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) oviposited on raw pork muscle, beef muscle, or chicken liver. Larvae, pupae, and adult blow flies from each carrion were selected for amino acid compound-specific isotope analysis. Canonical discriminant analysis showed that flies were correctly classified to specific carrion types in 100% (original rules) and 96.8% (leave-one-out cross-validation [LOOCV]) of cases. Regarding life stages, we obtained 100% and 71% of correct classification in original rules and LOOCV, respectively. The isotope ratios of most of the essential amino acids did not significantly change between life stages (at 95% CI). However, some non-essential amino acids (Ala, Ser, and Glu) and some conditionally essential amino acids (Gly and Pro) were isotopically depleted in the adult stage. Except for the essential amino acids, the amino acids in larvae and pupae were enriched in 13C, and adult blow flies were depleted in 13C relative to the carrion on which they fed. These results make it possible to exclude potential sources of carrion as larval food. Amino acid-specific IRMS could help inform entomologists whether a fly has just arrived from another location to feed on a corpse or has emerged from a pupa whose feedstock was the corpse. Such insight could enhance the significance of blow flies for post-mortem interval determinations. The analytical ability to link organisms from one trophic level to another through the use of compound-specific isotope analysis of amino acids could have wide-reaching consequences in a variety of disciplines. Graphical abstract ᅟ.

Characterization of the Spectral Accuracy of an Orbitrap Mass Analyzer Using Isotope Ratio Mass Spectrometry.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source coupled to the Q Exactive Plus has been extensively used in untargeted mass spectrometry imaging (MSI) analyses of biological tissue sections. Although the Orbitrap is a high-resolution and accurate-mass (HRAM) mass analyzer, these attributes alone cannot be used for the reliable identification of unknown analytes observed in complex biological matrices. Spectral accuracy (SA) is the ability of the mass spectrometer to accurately measure the isotopic distributions which, when used with high mass measurement accuracy (MMA), can facilitate the elucidation of a single elemental composition. To investigate the effects of different ion populations on an Orbitrap's SA and MMA, a solution of caffeine, the tetrapeptide MRFA, and ultramark was analyzed using a Q Exactive Plus across eight distinct automatic gain control (AGC) targets. The same compounds from the same lot numbers were also individually analyzed using isotope ratio mass spectrometry (IRMS) to accurately determine the isotopic abundance of 13C, 15N, and 34S. We demonstrated that at optimum absolute ion abundances the Orbitrap can be used to accurately count carbons, nitrogens, and sulfurs in samples with varying masses. Additionally, absolute monoisotopic ion abundances required for high SA were empirically determined by using the expected (IRMS) and experimental (Orbitrap) isotopic distributions to calculate the Pearson chi-square test. These thresholds for absolute ion abundances can be used in untargeted MSI studies to shorten an identification list by rapidly screening for isotopic distributions whose absolute ion abundances are high enough to accurately estimate the number of atoms.

Comparison of immunomodulatory properties of mannose-binding lectins from Canavalia brasiliensis and Cratylia argentea in a mice model of Salmonella infection.

The immunomodulatory properties of mannose-binding lectins ConBr (Canavalia brasiliensis) and CFL (Cratylia argentea) were investigated comparatively in a model of Salmonella infection. The lectins were intraperitoneally (i.p.) administered to mice daily for three days before the bacterial challenge with Salmonella enterica Ser. Typhimurium (0.2 mL i.p.; 10(7) CFU/mL). In vivo assays have shown that both lectins induced a significant leukocyte infiltration into the peritoneal cavity of uninfected mice, which was higher in the CFL group 3 days post-infection. Total and differential cell counts in the bloodstreams have shown uninfected animals pretreated with ConBr and CFL exhibited accentuated lymphopenia. Conversely, there was an increasing population of lymphocytes following 3 days post-infection in mice pretreated with both lectins. In addition, the bacterial burden was significantly reduced into the peritoneal cavity, bloodstreams, spleen and the liver in these mice. The lectins did not induce the release of pro- or anti-inflammatory cytokines into the peritoneal fluid of uninfected animals. However, following infection, the release of TNF-α and IL-10 in the peritoneal fluid were down-regulated in mice pretreated with both lectins whereas IL-1 was only reduced in mice pretreated with ConBr. Uninfected animals pretreated with CFL exhibited high nitric oxide (NO) content in the peritoneal fluid, which was decreased after infection in comparison to ConBr group. The lectins did not alter the serum levels of NO in uninfected mice but treatments with ConBr significantly reduced the NO content in infected animals in comparison to CFL group 24h after the bacterial challenge. Survival experiments have shown survival rates ranging from 70% to 100% in mice that received CFL or ConBr. On the other hand, untreated mice (PBS group) died 1-6 days after infection. We conclude that ConBr and CFL are prospective phytotherapeutics capable of modulate the cascade of pro-inflammatory plus regulatory cytokines and nitric oxide release derived from systemic infections.

Protective effect of proteins derived from the latex of Calotropis procera against inflammatory hyperalgesia in monoarthritic rats.

Calotropis procera (family: Apocynaceae) is a plant growing in the wild and has been used in the traditional medicinal system for the treatment of various diseases. The plant produces milky latex that possesses potent antiinflammatory and analgesic properties. In present study the non-dialysable protein fraction isolated from the latex (LP) of this plant was evaluated for its efficacy against inflammation in rats where paw edema was induced by sub-plantar injection of carrageenin or monoarthritis was induced by intra-articular injection of Freund's complete adjuvant (FCA). The effect of LP was evaluated on edema volume in the paw model and on joint diameter, stair climbing ability, motility, dorsal flexion pain, levels of oxidative stress markers and joint histology in arthritis model. The protection afforded by LP was compared with that of standard antiinflammatory drug, diclofenac (5 mg/kg). LP exhibited a dose-dependent antiinflammatory effect and produced 32% and 60% inhibition of paw edema at 10 and 25 mg/kg doses and 12% and 36% inhibition of joint inflammation at 50 and 150 mg/kg doses. The protective effect of LP was associated with normalization of joint functions, histology and levels of oxidative stress markers in joint tissue. The findings of this study suggest that the protein fraction of latex of Calotropis procera has the potential to relieve inflammation and pain associated with various arthritic conditions.