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We aimed to detect the correlation between inflammatory mediators, such as Klotho (Klt), vitamin D (25(<jats:styled-content style=\"fixed-case\">OH<\/jats:styled-content>)D) and homocysteine (Hcy), and disease severity among <jats:styled-content style=\"fixed-case\">MSA<\/jats:styled-content> patients.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>A total of 53 <jats:styled-content style=\"fixed-case\">MSA<\/jats:styled-content> patients, 65 <jats:styled-content style=\"fixed-case\">PD<\/jats:styled-content> patients, and 62 normal subjects were recruited in our cross\u2010sectional study. Serum Klotho (Klt), vitamin D (25(<jats:styled-content style=\"fixed-case\">OH<\/jats:styled-content>)D), and homocysteine (Hcy) levels were measured. Several scales were undertaken to assess the motor\/nonmotor function and cognitive impairment of <jats:styled-content style=\"fixed-case\">MSA<\/jats:styled-content>.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Decreased Serum Klt and 25(<jats:styled-content style=\"fixed-case\">OH<\/jats:styled-content>)D levels and increased Hcy levels were found in patients with <jats:styled-content style=\"fixed-case\">MSA<\/jats:styled-content>, compared with healthy controls. These results were more pronounced in male patients. The three biomarkers also displayed differences between <jats:styled-content style=\"fixed-case\">MSA<\/jats:styled-content> and <jats:styled-content style=\"fixed-case\">PD<\/jats:styled-content> subgroups based on genders. Interestingly, Klt, 25(<jats:styled-content style=\"fixed-case\">OH<\/jats:styled-content>)D and Hcy levels associated with cognition impairment, motor dysfunction, mood\/cardiovascular disorder among <jats:styled-content style=\"fixed-case\">MSA<\/jats:styled-content> patients. In addition, the combination of Klt, 25(<jats:styled-content style=\"fixed-case\">OH<\/jats:styled-content>)D and Hcy had a better diagnostic ability\u00a0for distinguishing <jats:styled-content style=\"fixed-case\">MSA<\/jats:styled-content> patients from healthy subjects, as well as distinguishing male <jats:styled-content style=\"fixed-case\">MSA<\/jats:styled-content> patients from male <jats:styled-content style=\"fixed-case\">PD<\/jats:styled-content> patients.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>This study suggested that Klt, 25(<jats:styled-content style=\"fixed-case\">OH<\/jats:styled-content>)D and Hcy levels could be a potential predictor for <jats:styled-content style=\"fixed-case\">MSA<\/jats:styled-content> severity 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