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Abstract
A series of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients have been studied to test two previous predictions: (1) Sauna therapy acts, at least in part, by raising the availability of tetrahydrobiopterin (BH4); and (2) ME/CFS is caused by a biochemical vicious cycle mechanism known as the NO/ONOO− cycle. Sauna therapy is shown here to increase BH4 synthesis, acting by raising the rate-limiting enzyme in BH4 synthesis, GPCH. This confirms, then, the first prediction. ME/CFS patients have very high levels of peroxynitrite as measured by the peroxynitrite marker 3-nitrotyrosine, averaging 5.43 times the levels of healthy controls with no overlap with those controls, and also have low levels of BH4, confirming predictions of the NO/ONOO− cycle mechanism. Sauna treatment, presumably acting via increased BH4 availability, lowers peroxynitrite levels, again in agreement with prediction. Sauna treatment also lowers the very high levels of neopterin found in these patients, possibly by stimulating the signaling of the IL-1b cytokine. Several important conclusions can be drawn here. First, sauna therapy does not act solely via detoxification, as has been widely assumed, but acts, at least in part, by raising BH4 levels. Second, three important predictions of the NO/ONOO− cycle mechanism are confirmed for the first time in ME/CFS patients, providing support for this mechanism as the etiologic mechanism of this disease. Third, three biochemical parameters are partially normalized by sauna treatment, BH4 levels, peroxynitrite levels and neopterin levels, suggesting that ME/CFS is fundamentally biochemical in nature. The very high, nonoverlapping levels of 3-nitrotyrosine (marker for peroxynitrite) in ME/CFS patients, as compared with healthy controls, suggest that this may be a very useful objective test for severity in ME/CFS patients.
Introduction
This article focuses on two interconnected series of predictions. The first is that sauna therapy works, at least in part, by raising the levels of tetrahydrobiopterin (BH4).1 The second is that in studying the effect of sauna therapy on the biochemistry of human patients, we can test not only this first prediction but also a series of other biochemical changes that may support a biochemical vicious cycle mechanism thought to cause many chronic inflammatory diseases, called the NO/ONOO− cycle.2-7
Let's consider these briefly. Sauna therapy has often been assumed to act entirely by a process of detoxification, acting to lower the levels of stored toxicants in the body.8-18 While there is some published evidence that sauna therapy does lead to increased excretion of toxicants and therefore some detoxification over a period of weeks, to our knowledge there is no evidence that this is the sole or even primary mechanism leading to improvement in symptoms in response to sauna therapy.14,18
In contrast to this view, it has been proposed that sauna therapy may act, to a substantial extent, by raising the levels of BH4.1 The evidence supporting this view (reviewed earlier) is that:
1. Sauna therapy is reported to be helpful in the treatment of several diseases proposed to involve, among other things, BH4 depletion; namely, multiple chemical sensitivity, fibromyalgia, and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and also several diseases wherein BH4 depletion has been shown to play an important causal role; namely, hypertension, vascular endothelial dysfunction, and heart failure.
2. Sauna therapy can act via two distinct mechanisms to raise the levels of BH4, acting in both cases by raising the levels of the enzyme GTP cyclohydrolase I (GPCH), the first and rate-limiting enzyme in the de novo synthesis pathway for BH4. These two mechanisms are the stabilization of GPCH by the heat shock protein Hsp90 and the induction of GPCH produced by increased peripheral blood flow.
3. Both of the mechanisms listed above have been shown to lower uncoupling of eNOS nitric oxide synthase, and the only known mechanism for such lowered uncoupling is to raise BH4 availability.
These two views of the action of sauna therapy, detoxification vs. raising BH4 levels, are not mutually exclusive and are not presented as such here – both may have roles.
In this study of a series of ME/CFS patients, we show that:
1. Sauna therapy acts to raise the biosynthesis of BH4 by raising the levels of GPCH, but that much of this BH4 can be subsequently oxidized to BH2 and biopterin, showing that there is still a lot of oxidative stress going on in these patients following sauna therapy.
2. Two NO/ONOO− cycle elements that have not previously been measured in ME/CFS patients are both elevated in these patients, notably peroxynitrite (measured through the marker 3-nitrotyrosine [3-NT]) and the depletion of BH4.
3. Sauna therapy, acting in part via raising of BH4 levels, can act to partially normalize the biochemistry in ME/CFS patients, raising BH4 levels, lowering 3-NT levels, and also greatly lowering the levels of neopterin.
4. This partial normalization of the biochemistry following sauna therapy suggests that the etiology of ME/CFS is biochemical in nature and specifically provides confirmation for certain parts of what is called the NO/ONOO− cycle.
Materials and Methods
17 patients (12 female and 5 male) who each met the Fukuda criteria for myalgic encephalomyelitis/chronic fatigue syndrome were studied before and after 4 sauna treatments, given at 2-day intervals, with fasting, venous blood samples, and urine samples taken before the first and immediately after the last sauna treatment.19 Sauna treatments were for 50 minutes each, 122 to 125 ºF in a far infrared sauna. Of these 17 patients, 15 had been previously been diagnosed as having Lyme disease, but had been treated with antibiotics and had been apparently borrelia free for the preceding 6 months at the time of study. They still suffered from ME/CFS, as indicated above.
Blood for biochemical analysis was obtained from fasting venous samples. For analysis of plasma levels of biopterin, dihydrobiopterin (BH2) and tetrahydrobiopterin (BH4), we used reversed phase HPLC with electrochemical detection and fluorescence detection. Detailed methods were previously described by Cai et al.20
For the 3-NT assay, a cold vacutainer tube (7 ml) containing EDTA, PMSF, and a proprietary stabilizer obtained from Health Diagnostics Research Institute (South Amboy, NJ) was used to draw blood and after mixing was placed on ice immediately. After centrifugation the plasma was used for analysis. Samples were derivatized by the method of Crowley et al. and then assayed for 3-NT as described by Schwedhelm et al.21,22
GTP cyclohydrolase I (GPCH), in lymphocytes isolated from blood, was assayed by the method of Werner et al.23
Each of these assays, as well as assays for neopterin, are available from Health Diagnostics Research Institute for clinical measurements and for experimental studies.
Table 1: Biochemical Parameters Before and After Sauna Treatments (.pdf)
Table 2. Normal Reference Range for Biochemical Parameters
Total biopterin in plasma (ng/ml).................................................................1.84-3.70
Dihydrobiopterin (BH2) in plasma (ng/ml)...................................................1.16-2.49
Tetrahydrobiopterin (BH4) in plasma (ng/ml)...............................................1.24-2.93
BH4/BH2 ratio...........................................................................................1.05-1.31
Biopterin (urine, mg/mg creatinine)...............................................................0.05-1.54
Neopterin (urine, ng/ml)...............................................................................0.08-2.70
3-nitrotyrosine (3-NT) in plasma (ng/ml)......................................................1.1-6.88
Results and Conclusions
The levels of various biochemical parameters were measured before and after sauna therapy in ME/CFS patients, as shown in Table 1 (p. 61), including that of biopterin, (Biop in the urine, columns 2 & 3), dihydrobiopterin (BH2, columns 4 & 5), tetrahydrobiopterin (BH4, columns 6 & 7), the enzyme GTP cyclohydrolase I in lymphocytes (GPCH, columns 8 & 9), 3-nitrotyrosine (3-NT, columns 10 & 11), and neopterin in the urine (Neo, columns 12 & 13). Let's examine columns 2 through 9 first. It can be seen that Biop, BH2, BH4, and GPCH all show highly statistically significant increases following sauna therapy (all p < 0.0001, using a paired t-test for statistical analysis [Table 1]). To analyze these further, it is important to consider the specific treatment protocol used. As noted above in the preceding section, it involved four sauna treatments at 2-day intervals with blood and urine samples taken before the first sauna treatment and immediately after the last sauna treatment. However, since the last sauna treatment had just occurred immediately before the final samples were taken, it is unlikely that it had time to have much impact on these final parameters. It follows that there would have been 2 to 6 days between the previous three treatments and the taking of the final samples. This 2- to 6-day period means that if a lot of oxidative stress is still occurring in these patients, there will be much time to oxidize the end product of the pathway (BH4) to its oxidation products, BH2 and biopterin. It seems clear that this is what happened in this study such that there are even larger percent increases in BH2 levels and in biopterin levels than there in BH4 levels after sauna treatment. Because BH4 is the end product of the biosynthetic pathway, it must be the case that the increased BH2 and biopterin reflect an increase in BH4 biosynthesis. This interpretation is also supported by the increase in GPCH following sauna treatment, the first and rate-limiting enzymatic step in the de novo pathway for BH4 synthesis (p < 0.0001).
It should be useful to compare the values of BH4 and 3-NT before sauna therapy in these ME/CFS patients with those of healthy controls. Here we use values of 111 healthy controls, of widely variable ages, ranging from 18 to 73. The BH4 levels of healthy controls were 1.82 +/− 0.51 as compared values with the ME/CFS patient levels of 1.29 +/− 0.192, (p < 0.0001, nonpaired t-test; data presented as mean +/− standard deviation). The values from 3-NT for healthy controls were 4.29 +/− 1.72 as compared with 23.3 +/− for the ME/CFS patients (5.43 times higher). The levels of 3-NT for the 111 healthy controls were completely nonoverlapping, with the highest value for the 111 healthy controls 6.88 as compared with the lowest value for the ME/CFS patients being 15.6 or over 2¼ times as high. This is quite a striking difference. Because the probability of each ME/CFS 3-NT value being above the total range of healthy controls by chance being less than 1/100, the probability of all 17 of these being above the health control range simply by chance can be estimated as p < 10−34.
In summary, then, these data confirm three important predictions of the NO/ONOO− cycle mechanism: s proposed, it is the central etiologic mechanism for ME/CFS: BH4 levels are predicted to be low in ME/CFS patients, peroxynitrite levels are predicted to be high, and a treatment that raises BH4 levels, notably sauna treatment, lowers peroxynitrite levels. However, it seems clear that peroxynitrite levels are still highly elevated after sauna treatment, and this is presumably a key part of the mechanism causing increased BH4 synthesis to show up mainly as the oxidation products BH2 and biopterin. They also support the theory that this cycle is the central etiologic mechanism of ME/CFS.
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