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. 2023 May 13;24(10):8713. doi: 10.3390/ijms24108713

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© 2023 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Quantitative analysis of BH4 in serum samples of control and CFS subjects. (A) Serum samples were 1:2 diluted by assay diluents and then assayed for BH4 by competitive ELISA method. n = 30 control and n = 32 CFS subjects were included. The significance of mean was tested by a Mann–Whitney U test between two groups at * p < 0.05. (B) A non-parametric Spearman correlation analysis was performed to determine the relationship between age and serum BH4 levels of n = 62 subjects (Red = CFS and green = control). Trendline was shown through the data points. (C) A scatter dot plot analysis compares BH4 levels in control males (n = 16; light green), control females (n = 18; deep green), CFS males (n = 13; light red), and CFS females (n = 19; deep red). A two-way ANOVA analysis (effectors are gender and disease) showed that irrespective of gender difference, the BH4 level is always higher in CFS compared to control. To compare the significance of mean between groups, a normality distribution test was performed, which indicates the first three groups were normally distributed. However, CFS female group failed the normality test. As a result, both parametric (unpaired t-test) and non-parametric tests (Mann-Whiney U test) were performed. No significance was observed neither between control males and control females [unpaired t-test; t_1,32 = 1.867; p > 0.05 (=0.07)], nor between CFS males and CFS females [MWU test; p > 0.05 (=0.9654); U = 123]. However, both CFS males and CFS females had significantly higher levels of BH4 compared to control males (unpaired t-test; t_1,27 = 1.008; * p < 0.05; =0.0384) and females (MWU test; * p < 0.05; =0.0494), respectively. Ns = no significance. (D) A pair-wise comparison of BH4 between age- and gender-matched subjects (n = 15 pairs). The age and gender of each pair (Control/CFS) were as follows: 82M/76M, 70F/72F, 69M/67M, 64F/64F, 67F/67F, 52F/54F, 33M/32M, 74M/76M, 38M/41M, 22F/26F, 43F/45F, 44M/49M, 70F/69F, 57 F/56F, and 44F/49F. M = male and F = female. Significance was tested by Wilcoxon matched-pairs signed rank test ** p < 0.01 (=0.0015) versus control.