Detection of Elevated Level of Tetrahydrobiopterin in Serum Samples of ME/CFS Patients with Orthostatic Intolerance: A Pilot Study

Abstract

Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) is a multisystem chronic illness characterized by severe muscle fatigue, pain, dizziness, and brain fog. Many patients with ME/CFS experience orthostatic intolerance (OI), which is characterized by frequent dizziness, light-headedness, and feeling faint while maintaining an upright posture. Despite intense investigation, the molecular mechanism of this debilitating condition is still unknown. OI is often manifested by cardiovascular alterations, such as reduced cerebral blood flow, reduced blood pressure, and diminished heart rate. The bioavailability of tetrahydrobiopterin (BH4), an essential cofactor of endothelial nitric oxide synthase (eNOS) enzyme, is tightly coupled with cardiovascular health and circulation. To explore the role of BH4 in ME/CFS, serum samples of CFS patients (n = 32), CFS patients with OI only (n = 10; CFS + OI), and CFS patients with both OI and small fiber polyneuropathy (n = 12; CFS + OI + SFN) were subjected to BH4 ELISA. Interestingly, our results revealed that the BH4 expression is significantly high in CFS, CFS + OI, and CFS + OI + SFN patients compared to age-/gender-matched controls. Finally, a ROS production assay in cultured microglial cells followed by Pearson correlation statistics indicated that the elevated BH4 in serum samples of CFS + OI patients might be associated with the oxidative stress response. These findings suggest that the regulation of BH4 metabolism could be a promising target for understanding the molecular mechanism of CFS and CFS with OI.

Keywords: ME/CFS; endothelial NOS; orthostatic intolerance; reactive oxygen species; small fiber polyneuropathy; tetrahydrobiopterin.

Conflict of interest statement

A.R., C.G.G., R.W. and D.P. are employees of Simmaron Research INC, a 501C non-profit research organization. The authors declare no conflict of interest.

Figures

Figure 1

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; t1,32 = 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; t1,27 = 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.

Figure 2

Comparison of BH4 levels between control and CFS + OI subjects. (A) A competitive BH4 ELISA assay in 1:2 diluted serum samples of n = 10 CFS + OI (red) and n = 10 age-/gender-matched control (n = 10) subjects. The significance of mean was tested by unpaired t-test between the two groups at * p < 0.05 (=0.0223). (B) A normality distribution of datapoints were analyzed by a Q-Q (Quantile-quantile) plot that showed all datapoints of both groups were normally distributed. (C) A scatter plot to evaluate pair-wise comparison of datasets (as summarized in Table 2) between control and CFS + OI subjects (** p < 0.01; = 0.004). (D) A scatter dot plot analysis was performed to test how many subjects had higher BH4 levels above the median BH4 concentration (99.63 ng/mL as shown by a solid black line). Green dots are controls (n = 10), and red dots are CFS + OI subjects (n = 10). OI = Orthostatic Intolerance. Results are the mean of three different experiments.

Figure 3

Assessment of difference in BH4 levels between control and CFS + OI + SFN subjects. (A) A competitive BH4 ELISA assay in 1:2 diluted serum samples of n = 12 CFS + OI + SFN (purple) and n = 12 age-/gender-matched control (n = 12) subjects. The significance of mean was tested by unpaired t-test between the two groups at * p < 0.05 (=0.0269). (B) Q-Q plot revealed normal distribution of all data points. (C) A gender- and age-based paired analysis of BH4 between control and CFS subjects. Paired t-test (* p < 0.05) confirmed that the CFS serum samples had significantly elevated BH4 compared with the control serum samples. (D) A scatter dot plot analysis was performed to test how many subjects had higher BH4 levels above the median BH4 concentration (81.83 ng/mL as indicated by a solid black line). Green dots are controls (n = 10), and purple dots are CFS + OI+SFN subjects (n = 12). SFN = small fiber polyneuropathy. Results are the mean of three different experiments.

Figure 4

Comparison of BH4 levels between CFS only and CFS + OI subjects. (A) Serum samples were 1:2 diluted by assay diluents and then assayed for BH4 by competitive ELISA method. n = 10 CFS only control and n = 14 CFS + OI subjects were included. The significance of mean was tested by unpaired t-test between the two groups at * p < 0.05 (=0.0166). (B) Q-Q plot represents the normal distribution of data points from both the groups. (C) A scatter dot plot analysis was performed to test how many subjects had higher BH4 levels above the median BH4 concentration (119.7 ng/mL as shown by a solid black line). Green dots are CFS only controls (n = 10), and red dots are CFS + OI subjects (n = 14). OI = Orthostatic Intolerance. Results are the mean of three different experiments.

Figure 5

Correlation between BH4 levels and ROS-inducing abilities in serum samples of CFS + OI subjects. (A) Control serum and CFS + OI serum-supplemented media was applied on DCFDA-transfected HMC3 human microglial cells for 90 min and then assayed for ROS using the fluorometric method [Ex:Em = 485 nm/535 nm]. n = 10 Control and n = 10 CFS + OI subjects were included. The significance of mean was tested by unpaired t-test between the two groups at * p < 0.05 (=0.0205). (B) Q-Q plot was derived to display normal distribution of data points. (C) A Pearson correlation analysis was performed to determine the relationship between ROS-inducing ability (as measured at fluorescence of 484 nm/535 nm) and serum BH4 levels of n = 20 subjects (Red = CFS + OI and green = control). Trendline was shown through the data points. (D) The potential role of BH4 in CFS was summarized in a sketch. BH4 induces the production of nitric oxide (NO) via catalytic activation of endothelial nitric oxide synthase (eNOS) enzyme. Endothelial NO stimulates vasodilation and facilitates hypotension. BH4 is prone to be oxidized in the presence of reactive nitrogen species (ONOO-) and induces oxidative stress. BH4 also inhibits electron transport chain (ETC) in complex-I and -IV (C-I and C-IV) steps and triggers mitochondrial toxicity. Another possibility is the activation of mTORC1 kinase complex and inhibition of autophagy.