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After two freeze-thaw cycles, 20 L of 5% 5-sulfosalicylic acid (SSA) was added, followed by centrifugation at 8,000g for 10 min

However, most melatonin is secreted outside the pineal gland (in the retina, skin, gastrointestinal tract, immune cells, mitochondria, etc.) and is not affected by circadian rhythms ( In mammals, melatonin (MLT) activates membrane-bound G protein-coupled receptor (GPCR) receptor binding (MT1, MT2) or by direct action (Jockers et al., 2016)

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