Dear Doctor, Pharmacist, Health
Practitioner, December 4th 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, 28th 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: 28th 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.
Dr Ian Dettman
Managing Director, Biological Therapies.
Response by Robert Cathcart.
Dear Ian,
Another factor occurs to me. I do not know what article is being referred to
however. I would be interested in the amount of ascorbate used where they say
there is no immediate effect on pain and therefore inflammation. I would like to
point out that where there is serious injury, the mitochondria are damaged and
not only cannot provide electrons to fuel the ascorbate but the damaged
mitochondria leak the massive amounts of free radicals in injury. Small amounts
of ascorbate must wait for the recovery of the mitochondria before they are
refuelled. With the massive amounts of ascorbate where we are throwing away the
vitamin C for the electrons carried will eliminate pain and inflammation before
the mitochondria recover.
I have had the personal experience that in the case of a corneal transplant and
with a crush laceration of the eyebrow involving the supra orbital nerve that 12
grams of ascorbic acid every half hour for a couple of hours and then 12 grams
of ascorbic acid every hour until going to sleep and then on awakening a couple
of times during the night, completely relieved to pain by the time the local
anesthetic wore off.
The smaller amounts of ascorbate can be expected to be helpful only when the
mitochondria recover some. I have not doubt that the "buddy system" of all the
antioxidants and nutrients help the recovery of the mitochondria so that they
can provide the large amounts of electrons necessary to neutralize the
free radicals of a serious injury but this takes some time. Sufficient massive
amounts of electrons provided by massive doses of ascorbate will immediately
shut down the inflammatory free radical cascade.
About this iron thing. It is my experience that ascorbate increase the
absorption of iron when the patient needs more iron but it increases
the excretion of iron when the patient has an excessive amount like with
hemochromatosis. The body is more able to regulate the optimum amount of iron
with the presence of adequate amounts of ascorbate.
It is possible that dehyroascorbate might show up in drawn samples that are not
protected from oxygen, however, the DHA created in the body is created when
ascorbate neutralizes free radicals with a greater oxidative redox potential
than the DHA formed so the net result is to reduce the oxidative redox
potential. Massive amounts of ascorbate that maintain the DHA/AA redox couple on
the reducing side in vivo would be of only benefit and would reduce almost all
the free radicals in the body. It takes massive amounts of ascorbate to force a
reducing redox potential on an injured area where the mitochondria are injured.
Robert F. Cathcart, M.D.
12/07/01