Dear Doctor, Pharmacist, Health Practitioner,         December 4^th   2001         

On September 15, 2001 an article “Supplementation with Vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise” appeared in “Free Radic Biol Med” 2001 Sep 15, 31(6): 745-53. 

 

The basis of this article was that supplementing with Vitamin C and N-acetyl-cysteine, post eccentric exercise, increased muscle damage.  Many markers were used to measure this damage including LDH and CPK, various inflammatory markers (one of which actually decreased with supplementation) and lipid peroxidation.  Serum iron levels were also found to be elevated in the supplemented group.

 

A subjective analysis of pain during recovery was also made by the participants in this controlled study.  Additionally, measures of muscle flexion were quantitatively measured.  Interestingly, there was no difference in these parameters between the control group and the supplemented group.  A more pertinent measure of muscle damage, and just as importantly as the general biochemical indicators of muscle repair, may have been to perform tissue biopsies.  It may well be that the supplemented group underwent more rapid and complete tissue repair. More rapid tissue repair may well have meant transiently increased biochemical markers compared with the control. However, no biopsies were performed.

 

Some increases in cell damage as measured by LDH and CPK and lipid peroxidation were noted in the supplemented group.  As already mentioned one of the measures of inflammation actually decreased.

 

Taken at face value these results may indicate, to those not wishing to dig deeper, that taking supplementation of Vitamin C and N-acetyl-cysteine may not be advisable immediately after performing eccentric exercise.  There are many different avenues that one could go down in analysing the multiple deficiencies in this paper.

 

Firstly, the statistics leave a lot to be desired.  The authors make it clear that for all times that blood samples were collected only duplicate or at the best triplicate results were obtained.  Results were then expressed as mean +/- SEM (Standard Error of the Mean).  As we have no knowledge whether most of the points on the graph were only represented by duplicate results, such an analysis may well be misleading.  Differences between the control and the supplemented groups have been accentuated by plotting means ( +/- SEM).  The differences have been “neatened” by plotting mean +/- SEM rather than plotting the scatter of duplicate? results. We are then asked to consider the results from the point of view of comparing the means. Clearly, the scatter is quite large and if one observes the closest differences between the error bars for the control and supplemented groups, quite often there is virtually no difference.

 

Additionally, LDH values are often artificially increased unless special care is taken to separate plasma from blood cells immediately post phlebotomy.  No mention is made whether such action was taken, and we must repeat here that the statistical representation of the results leaves a lot to be desired.

 

 As already mentioned, the measures of inflammation were far from conclusive and in fact by day 7 quite clearly the levels of interleukin-6 were considerably diminished in the supplemented group.  It is interesting to note that there were considerable differences at day 0 (prior to supplementation) in the levels of the inflammatory markers myeloperoxidase and interleukin-6 in the control group and supplemented group.  As these differences were so great (as much as 20% for myeloperoxidase and 50% for interleukin-6) it must cast some doubt on the validity of these results.

 

 Although there was apparently some increase in lipid peroxidation this is inherently difficult to measure and is subject to a great deal of invitro effects.  Unless extremely careful quality control measures are exercised, blood samples are prone to invitro oxidation.  Patients who have been supplemented with antioxidants are going to have increased levels of antioxidants in their blood stream at the time of blood collection.  Immediately this blood is collected the increased levels of antioxidants may be oxidised themselves through contact with oxygen and exacerbate any increases in invitro lipid peroxidation.  There is no evidence in this study that any effort was made to measure the potential invitro effects of antioxidants in blood once it has been collected.

 

The level of iron was shown to be increased in the supplemented group but numerous experiments and published papers by Professor Frei (Academic Head of the Linus Pauling Institute) and many others have shown that increased intake of Vitamin C does not cause increased lipid peroxidation invivo, even in the presence of increased iron levels, as proposed by some antagonists of Vitamin C therapy.          

The following is communication that I recently had with Professor Frei and he has 

allowed me to publish his response on the Biological Therapies website:

 

Dear Professor Frei,                                  28^th October  2001

There appears to be something fundamentally flawed in the research paper that I just E-mailed you about (“ Supplementation with Vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induce by eccentric exercise”). You may well answer this paper from the point of view for example:

? handling of blood specimens post phlebotomy Higher levels of vitamin C in the plasma may well induce extra lipid peroxidation ex vivo in the presence of increased levels of iron unless the blood specimens are handled correctly.

Patients who have megadoses of vitamin C simply don't fall over in a rusty heap THEY GET BETTER. Surely, we need to rationalise our experiments based on these decades and decades of positive clinical observations rather than on a few negative in vitro observations??

Once again my very best wishes to you

 

Ian Dettman
 

 

In reply Professor Frei wrote:

 

 

Dear Dr. Dettman:                                             28^th November  2001

Thank you for your inquiries Re:  “Supplementation with vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise" Free Radic Biol Med 2001 Sep 15,31(6): 745-53).

I think your points below are very well taken. One has to be extremely careful in the interpretation of these kinds of studies and critically evaluate experimental procedures, methodology, and ex vivo oxidation artifacts before jumping to conclusions (as so many authors do in this field). For example, once a plasma sample is extracted with acid or an organic solvent, iron may be released from its natural binding proteins (transferrin, ferritin, etc.), and together with exposure of the sample to ambient oxygen (20%), one can create all kinds of ex vivo artifacts. In addition, many authors use non-specific markers of lipid peroxidation, in particular TBARS, which can be derived from a variety of reactions, not just lipid peroxidation, and are easily formed ex vivo.

As you indicated, we have done quite a few studies on the possible interactions between vitamin C and iron under physiological conditions and in vivo, using specific and sensitive assays to measure lipid peroxidation.
We always found anti-, rather than pro-oxidant effects of vitamin C under these conditions. I am currently working on another manuscript where we "pushed" the oxidation conditions even further, i.e., in addition to iron
also added hydrogen peroxide to human plasma (the so-called complete Udenfried system), and again found no pro-oxidant effects of ascorbate towards lipids as well as proteins under these conditions.

My hunch is that N-acetyl cysteine may be responsible for the adverse effects observed. Note that they didn't use a vitamin C group only, so can't really tell whether it's vitamin C, NAC or both that exert the observed effects. Not a very clean experiment!

The bottom line is, as you said, people who take vitamin C don't get rusty, but they usually get better. I believe the totality of evidence clearly shows that vitamin C does not act as a pro-oxidant in vivo and under physiological conditions, and has multiple health benefits, even in people with iron overload.

I hope this is helpful. Thank you for your patience!

With best wishes,

Balz Frei, Ph.D.

 

I would like to make some final comments about the plethora of antagonistic Vitamin C papers that are appearing in the literature at the present time.

 

1. These papers are often composed by people who do not have a broad medical perspective of the Vitamin C literature.  However, the composers often make quite unsubstantiated claims about the negative clinical effects of Vitamin C supplementation.
   
    
2. The papers are quite clearly NOT evaluated by people who would be considered to be experts in the field of Vitamin C. Such experts include  Professor Frei, the Head of the Linus Pauling Institute (at Oregon State University, USA).
   
    
3. The clinical (and public) use of mega doses of Vitamin C has now been happening for well over 60 years.  The benefits are many and have been published by many reputable scientists and physicians, yet still some researchers, clearly with a limited understanding of the enormous volume of literature on Vitamin C, continue to publish negative papers that do not give a balanced point of view of the medical and scientific literature.
   
    
4. I commend all physicians and health practitioners to continue to use appropriate levels of Vitamin C supplementation.

 

Dr Ian Dettman

Managing Director,  Biological Therapies.

 

 

Response by Robert Cathcart.