The dried trapped particles were removed from the filter and homogenized by grinding in an agate mortar and pestle to measure the organic components. To analyze the organic carbon content and isotope ratio, approximately 20 mg of the dried samples were fumigated with hydrochloric acid for 12 h to remove particulate carbonate and dried in a vacuum oven. The organic carbon and nitrogen contents of trapped particles, particulate organic carbon and nitrogen concentrations of suspended particles, and isotopic compositions (δ13Corg and δ15N) of trapped and suspended particles were determined using a continuous flow isotope mass spectrometer (Delta PLUS, Thermo Fisher Scientific, USA) fitted with an elemental analyzer (NC-2000, CE Instruments, UK) with a ConFlo II (Thermo Fisher Scientific, USA). The analytical precision based on the replicate analyses of δ13Corg and δ15N was ± 0.2‰. For the analysis of organic components, we used different machines (Finnigan MAT 252, Thermo Fisher Scientific, USA and another NC-2000, CE Instruments, UK) in the previous study (Sukigara and Saino 2005 (link)), but used the same standards to confirm isotope ratios and masses of organic carbon and nitrogen.
Finnigan mat 252
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Finnigan MAT 252 is a high-performance dual-inlet isotope ratio mass spectrometer designed for precise and accurate analysis of stable isotope ratios.
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2 protocols using finnigan mat 252
1
Organic Content and Isotope Analysis
2
Plasma Metabolite Analyses Protocol
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Plasma glucose (Uni Kit III, 07367204; Roche) concentrations were analyzed with a COBAS-FARA semiautomatic analyzer (Roche). Insulin was analyzed by using a radioimmunoassay (Insulin RIA kit; LINCO Research Inc). Plasma (100 mL) for amino acid analyses was deproteinized on ice with 10 mg dry 5-sulphosalicylic acid, mixed, and the clear supernatant fluid was collected after centrifugation. Plasma amino acid concentrations were determined by using HPLC after precolumn derivatization with o-phthaldialdehyde [23 (link)]. For plasma enrichment measurements, plasma Phe and Tyr were derivatized to their t-butyldimethylsilyl derivatives and analyzed by using gas chromatography–mass spectrometry (GC-MS) (Agilent 6890N GC/5973N MSD; Agilent) by using selected ion monitoring of masses 336 and 341 for unlabeled and labeled (ring-2H5) Phe, respectively; and masses 466, 468, and 470 for unlabeled and labeled (ring-2H2 and ring-2H4) Tyr, respectively [24 ]. Thereafter, ratios of labeled: unlabeled derivatives were analyzed by using gas chromatography–combustion isotope ratio mass spectrometry (FinniganMAT 252; ThermoFisher Scientific). Standard regression curves were applied in all isotopic enrichment analyses to assess the linearity of the mass spectrometer and to control for the loss of tracer.
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