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MDA in cell lysate reacts with thiobarbituric acid (TBA) solution to generate MDA-TBA adduct, which is colorimetric (OD = 532 nm) and is proportional to the MDA present

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The groups at highest risk include indoor workers with limited midday sun exposure, people living in northern latitudes (roughly above Atlanta or Los Angeles in the U.S.), individuals with darker skin tones (melanin reduces UVB-driven D3 synthesis), people carrying significant excess body fat (D3 gets trapped in adipose tissue, reducing circulating levels), older adults (skin becomes less efficient at producing D3 with age), anyone on a GLP-1 weight loss protocol where reduced food intake and rapid fat loss can both affect fat-soluble vitamin status, and people with GI conditions that impair fat absorption