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[HTT16-11] Carbon isotope ratios of proteinogenic amino acids in formalin-fixed fish from Lake Biwa
Keywords:amino acid, stable carbon isotope, formalin, Lake Biwa
Here, we examined formalin's effect on amino acid carbon isotope ratios. The study investigates the effects of formalin fixation on proteinogenic amino acids and ascertains whether the original amino acid 13C records could be extracted from the fixed samples. Biological archives from various global locations offer valuable biogeochemical and ecological insights. However, formalin-fixation, a widely used preservation method, poses challenges for carbon isotope studies.
The HPLC x nano-EA/IRMS method was employed to analyze formalin-fixed and unfixed fish samples from Lake Biwa: Freshwater minnow Opsariichthys platypus and Largemouth bass Micropterus nigricans. A quantity comparison of 16 amino acids extracted from the fixed and unfixed samples revealed a substantial decrease in certain amino acids, with some exhibiting a 10–50% reduction. Tyrosine levels were notably diminished, reaching a 70–100% reduction in the fixed samples. In contrast, the carbon isotopic ratios of the 15 amino acids from fixed and unfixed samples were consistent. This finding indicates that formalin-fixation exerts a particular effect on the amino acid composition of the sample but does not alter the carbon isotopic ratio of the amino acids. Within a fish, the carbon isotope ratios exhibited significant variability (up to 25‰) across amino acids. The amino acid most depleted in 13C was phenylalanine (-29.6‰ and -28.7‰, Minnow and Bass, respectively), and those most enriched in 13C were glycolytic amino acids, e.g., glycine (-14.0‰ and -7.9‰) and serine (-8.1‰ and -3.9‰). These patterns are similar to those previously obtained in marine organisms.
Our results demonstrated that the HPLC x nano-EA/IRMS method could be applied to reconstructing the original isotope ratio information even from samples fixed in formalin. This approach can also be a powerful tool for understanding amino acid dynamics in the Anthropocene.
The HPLC x nano-EA/IRMS method was employed to analyze formalin-fixed and unfixed fish samples from Lake Biwa: Freshwater minnow Opsariichthys platypus and Largemouth bass Micropterus nigricans. A quantity comparison of 16 amino acids extracted from the fixed and unfixed samples revealed a substantial decrease in certain amino acids, with some exhibiting a 10–50% reduction. Tyrosine levels were notably diminished, reaching a 70–100% reduction in the fixed samples. In contrast, the carbon isotopic ratios of the 15 amino acids from fixed and unfixed samples were consistent. This finding indicates that formalin-fixation exerts a particular effect on the amino acid composition of the sample but does not alter the carbon isotopic ratio of the amino acids. Within a fish, the carbon isotope ratios exhibited significant variability (up to 25‰) across amino acids. The amino acid most depleted in 13C was phenylalanine (-29.6‰ and -28.7‰, Minnow and Bass, respectively), and those most enriched in 13C were glycolytic amino acids, e.g., glycine (-14.0‰ and -7.9‰) and serine (-8.1‰ and -3.9‰). These patterns are similar to those previously obtained in marine organisms.
Our results demonstrated that the HPLC x nano-EA/IRMS method could be applied to reconstructing the original isotope ratio information even from samples fixed in formalin. This approach can also be a powerful tool for understanding amino acid dynamics in the Anthropocene.