A Protocol for Fish Lipid Analysis Using Nuclear Magnetic Resonance Spectroscopy

Abstract

This article reports on an easy-to-follow methodology for fish lipids analysis, including the biological sample collection, lipid extraction, sample preparation, nuclear magnetic resonance (NMR) spectroscopy analysis and statistical data analysis, with details on parameters used for the NMR analysis and the care needed in each step execution. As an example of the biological matrix of lipids, for the entire procedure, we have used two Amazonian fish samples. Phenotype factor was taken into account when lipid contents of the Amazonian fish samples were evaluated, such as fish-eating habits in distinct Amazon seasonal periods, the flood and the drought. Results demonstrated the applicability of NMR as a powerful and useful tool for fish oil analysis. The omnivorous (T. elongatus) and the piscivorous (C. monoculus) Amazonian fish differ in composition and distribution of lipids, which show differences not only because of the different eating habits between the two species, but also by the availability and quantity of food along the year, i.e., during the flood and drought Amazon periods.

NMR in Analysis of the Nutritional Value of Lipids from Muscles and Livers of Wild Amazonian Fishes with Different Eating Habits over Seasonal Variation

“…Lipids were assigned according to reported NMR data. [37,30,38,39] The assigned peaks are displayed in Table 1. [40].…”

“…The dorsal muscles and livers tissues were submitted to protocols for lipids extraction as described in literature. [28,29,30] Total lipids' extracts were fractionated into three classes of lipids with different polarities as described elsewhere. [29,30] We have extracted 224 lipid samples from 9 fish species (170 samples of total lipids from livers and muscles and 54 samples of lipids classes from muscles).…”

“…MestReNova38 (Mestrelab Research Sl) and saved as the data matrix. Principal component analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) //www.metaboanalyst.ca/faces/home.xhtml), applying the parameters used by Mor et al [30]. …”

NMR in Analysis of the Nutritional Value of Lipids from Muscles and Livers of Wild Amazonian Fishes with Different Eating Habits over Seasonal Variation

“…The dorsal muscles and livers tissues were submitted to protocols for total lipids extraction as described in the literature. 28,29 Total lipids' extracts were fractionated into three classes of lipids with different polarities as described elsewhere. 29,30 We have extracted 224 lipid samples from 9 fish species (170 samples of total lipids from livers and muscles and 54 samples of lipids classes from muscles).…”

“…The usual parameters (pulse sequence, pulse power, data points, number of scans) of 1 H NMR were the same of the qualitative analysis, the specific parameters were: an acquisition time of 8.19 s, a spectral width of 9.9955 Hz, a recycle delay of 40 s with a relaxation delay of 48.19 s (higher than 5 times T1) based on estimated longitudinal relaxation time (T1). 29 To estimate T1 initially the pulse of 90° was calibrated, then it was used the sequence of inversion and recovery pulses; resulting in the T1 obtained for the slowest signal of interest as 8.69 s. Considering the estimated time multiplied by 5 times in this experiment and the acquisition time (at) equal to 8.19 s, the established recycling time was 35.286 s (d1) with a relaxation delay of 43.476 s (higher than 5 times T1).…”