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Monday 29 April 2013

Can Spirulina Produce a Sustained Virological Response in Chronic Hep C? More Studies Here Please.

I was going to give the whole algae as immune stimulants theme a rest, until Silvia Hinojosa-Price sent me this paper.
Spirulina Platensis versus Silymarin in the treatment of chronic hepatitis C virus infection. A pilot randomized, comparative clinical trial.

I've been reading these Hep C natural therapy papers for many years now and this is the first to record SVR. Essentially, this means that the treatment (Spirulina, a simple dried extract at 500mg 3x daily) was associated with undetectable virus levels after 6 months treatment. Only in 13.3% of the subjects (4 of 30, with another 2, or 6.7% having a significant drop of over 2 log) but still - this NEVER happens in clinical trials of "natural" or "alternative" therapies. The best will often lower viral load, but not to undetectable levels. HCV researchers never use the word "cure", but having no virus is as close as it gets. There are a few caveats I'll add later, but this is an impressive paper. I don't often see papers of this sort so well written or with the data so well presented. There's not much left to the imagination, which is how it should be. The authors are coming from a conventional, standard of care background and have had their interest piqued by patients reporting beneficial effects from Spirulina supplements; so they ran this clinical trial. 

The motivation to conduct this study, "apart from the theoretically convincing background", was the unintended data coming from some of our patients who took Spirulina as nutritional supplement and reported to us marked improvement of the general well being and sexual activity. From this probing experience, as well as from the results of Danoff A, et al. [44] and Soykan A, et al. [45] who reported an association of chronic HCV with depressed sexual functions independent of depression, we opted to compare the effect of both treatments on sexual functions beside the other efficacy parameters in such patients. We assumed that improvement in sexual appetite; frequency and performance are logical indicators for the improvement in the overall wellbeing. Our results went in agreement with this assumption.
This is how it should be done, respecting the patients' anecdote as a hypothesis generator, yet these kinds of trials often end in disappointment. But not this time. Yakoot and Salem were smart enough to allow the trial to last 6 months. They made the logical assumption that the time needed for any response to Spirulina would be at least as long as that needed for a response to Interferon-based therapies.
We hypothesized that there must be a time needed to establish and solidify the immune mechanisms behind the activation, release and action of endogenous interferons and other interplaying cells and mediators. Even the parenterally administered high dose of interferon alpha took some months to manifest its maximal virological response, that is why we wait at least 3 months to predict the sustained virological response through the early complete or even partial virological response.

Silymarin was used as an "active placebo". In other words, Spirulina was compared to extract of Milk Thistle seeds (140mg 3x daily), the supplement most likely to be used by chronic Hep C sufferers, but one that has only a small objectively measurable effect (at least, in this basic dosage form: but you can see here that other, more bioavailable forms might be worth testing).
Lo and behold, one of the Silymarin group (1 of 29) had an SVR. This reduced the significance of the 4 Spirulina SVRs and doesn't seem to be commented on. For what it's worth, Silymarin does have inhibitory effects on HCV replication, but these should be mild at the levels attained in a 140mg 3x daily dosing, and it's the first I've ever heard of SVR from Silymarin. However, there is a natural rate of spontaneous clearance in chronic Hep C, estimated at 1% per year, though no-one really knows and most clinicians would rather not follow up such cases. Lazarus was a disappointment to his doctor.
The patients with the lowest viral loads were the most likely to have an SVR in response to Spirulina. In those patients with viral loads below 100,000 the difference between Spirulina and Silymarin was statistically significant.
Our results showed significantly greater effects of Spirulina than Silymarin on most studied parameters including the significantly greater reduction of serum ALT and the greater improvement in both disease specific health related quality of life and sexual functions scores. Though the virological response rates were not statistically significantly different between the 2 treatment groups, yet it reached the level of significance with the one sided Z test for proportions in those who presented with low or intermediate baseline viremia.

One limitation was the absence of post-treatment follow-up. 
It is the main limitation of our study that we did not follow up patients for one year treatment followed by 6 months off-treatment period as the case in the protocols for the study of interferon alpha based therapy. We designed this relatively short term pilot study to answer a simple research question; is there any therapeutically feasible potential for Spirulina in chronic HCV patients, worthy to conduct a larger study with longer follow up period.
I would expect that some of the SVR cases would relapse once they stopped taking Spirulina; after all, this happens with conventional drugs. But why should anyone stop taking Spirulina? It would be no imposition at all to keep taking it for the rest of one's life, indeed it probably be a good idea even if the sustained viral response didn't depend on it.
This study was mainly focusing to help a considerable percentage of chronic HCV patients who are facing the situation of contraindication, intolerability or non-response to the current gold standard therapy. They usually become feeling hopeless and unsecure with deterioration in their overall wellbeing, functional status and quality of life. If further studies confirm our results with reproducibility, this could be an alternative treatment in such situations if at least it can improve quality of life, physical activity and performance. We did not focus on the luxury of sustained virological response at this exploratory stage, but it will be our objective in the coming planned study.

Where to from here?

The raised issues from the already discussed in-vitro and preclinical data about the potential immune stimulation and virus entry blocking also urged us to plan to test a new hypothesis; could the complementary therapy with Spirulina improve the response to the current gold standard antiviral therapy?. This will be tried to answer in our next study; through testing the effect of combining Spirulina with the current gold standard therapy, or the effect of offering a lead-in course for those who have high baseline virus load.
I would like to see a longer study of constant treatment, similar to the long-running Japanese zinc carnosinate (Polaprezinc) study. And there is no reason why the dose couldn't be increased. The nutrients that support Interferon - B12, D3 and retinol - could be optimized. And so on.

About the Physician Authors; AA Salem has authored 420 medical papers (well not all of those, possibly a common name, but certainly including 5 based on clinical experience of HCV cases. M Yakoot has authored 13 papers, mostly clinical trials of dietary supplements or reviews of OTC drugs. He appeared on Egyptian TV during the swine flu outbreak there.)
Dr Yakoot presents the results of the Spirulina paper in the last 2 minutes of this video, which also features some cool animations of HCV, LDL and LDL-R interaction:

The competing interests: 
Beovita-Safe Pharma, a Joint German Egyptian Company, Katzbachstr. 29, D-10965 Berlin, had supplied the drugs and partly the costs of the laboratory tests.
There seems to be nothing unique or patentable about Beovita-Safe Pharma's Spirulina or Silymarin products that would prevent the results being replicated with another company's products. I suspect Beovita-Safe Pharma's motivation was to have the use of their products accepted as safe and justified by the clinicians treating their customers. I wonder what Beovita-Safe Pharma will do with this additional information. They'll certainly be motivated to support other trials.
We opted to use the whole herbal extract and not any one of the fractionated bioactive molecules presuming that the whole natural multi-components as previously discussed in introduction could offer not only antiviral activity but also other immune enhancing activities that might be summated together to produce therapeutic effects on this state of chronic viral infection that evades the immune system.

Update: I looked at Spirulina products while shopping today. Most are whole dried Spirulina 500mg tablets, but I found one product that was a "herbal extract" of 2,300mg Spirulina in an approximately 500mg cap (I suspect) together with 50mcg selenium from selenomethionine. It had been made in New Zealand for the Japanese market.
This may be close to the Beovita product. I hope to find out more - including, what is the iron content of the Beovita tabs? 1g of Spirulina can supply 3mg iron and a standard dose of whole dried spirulina is 9g daily.


Tuesday 16 April 2013

Spirulina and LPS; and, Maybe Trans Fats are Not the Devil?

One of the very minor controversies in Paleo-land is whether algal foods or supplements are healthful. For what it's worth, pond algae appeared in the Paleolithic menu (according to the 2008 book Feast: why humans share food by Martin Jones). Spirulina and Chlorella supplements have both been used in the treatment of Hepatitis C with interesting results:
(music - Free Kim Dotcom by The Puddle featuring Matthew Bannister)

Efficacy and safety of Chlorella supplementation in adults with chronic hepatitis C virus infection

Eighteen adults with chronic infection by HCV genotype 1 received daily oral supplementation of Chlorella for 12 wk. Changes in the RNA levels of HCV, as well as those of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were evaluated following this treatment period. Paired t tests were conducted to compare the means of the different variables at the beginning and end of the study. Side effects and quality of life aspects were also compared between weeks 0 and 12 of the study period.

RESULTS: A majority 84.61% of the patients had a significant decrease in their ALT levels from week 0 to week 12. Evaluation of side effects showed that Chlorella was well tolerated. Quality of life assessment showed that 76.9 of the participants reported an improvement in their energy levels and 46.1% reported an improvement in their perception of general health. Although 69.23% also showed a decrease in their AST levels, this was not statistically significant. Most patients that exhibited an improvement in their ALT and AST levels also showed a tendency toward a decreased HCV viral load. The HCV RNA levels showed a decrease in 69.23% of the patients, which along with changes in AST/ALT ratios from week 0 to week 12, these results were not statistically significant.

One very interesting outcome, not captured in the abstract was that those subjects who had not undergone previous treatment with interferon almost all (5/6) saw significant drops in viral load, whereas the 7 subjects that had previously used, and failed to respond to, interferon had no such reaction (see Table 1). 
What effect of algae could account for these results? Both spirulina and chlorella express PAMPs (Braun polysaccharides in the case of spirulina, and glycoprotein in the case of chlorella) that possess activity at TLR2. TLR2 is interesting because, as well as its immune system signalling role, it also promotes the intestinal detoxification of benzopyrene carcinogens. But that curious fact need not concern us here.

[One hundred years ago the birth of immunology was made official by the Nobel Prize award to Elie Metchnikoff and Paul Ehrlich. Metchnikoff discovered phagocytosis by macrophages and microphages as a critical host-defense mechanism and thus is considered the father of cellular innate immunity. He also was interested in the impact of normal flora on well-being and in pre- and probiotic diet and their influence on the normal flora. Ehrlich described the side-chain theory of antibody formation and the mechanisms of how antibodies neutralize toxins and induce bacterial lysis with the help of complement and thus is considered one of the fathers of humoral adaptive immunity.]

TLR2 activation by algal ligands activates elements of the innate immune system; natural killer T cells (NKT) and interferon (PDF). This will be responsible for the drops in viral load (in people whose immune systems are not already either unresponsive to interferons, or damaged by exposure to the supraphysiological levels of interferon-alpha used in HCV therapy). 
HCV core protein is a pro-inflammatory TLR2 ligand; this allows the immune system to recognise the virus and respond appropriately. Most HCV exposures (antibody+) will be cleared in the first 6 months, but chronic infection means that the virus has managed to subvert these responses. So supplying algal TLR2 ligands is a way of switching back on (or turning up) responses that the virus has managed to dim.
But TLR2 is also expressed on Treg cells (regulatory T Cells), able to induce immune tolerance (less Th17 inflammation, lower antibody production) to specific immune triggers, so HCV core protein's interaction with Treg TLR2 could be a way of numbing the immune system to the presence of HCV. This can be a good thing in some ways; sometimes "fighting the dragon" aggressively is just too destructive a strategy to be sustainable.
Another way in which HCV is able to reduce the host immune response is by the binding of HCV core protein to the C1q complement receptor; this inhibits complement activation, resulting in depletion of the C3 complement factor, and inhibits proliferation of T cells. 
Strikingly, this inhibitory effect of core on lymphocyte proliferation was observed at a concentration of core protein as low as 1.3 nM. 
This (I hypothesise) may result in compensatory increase in uptake of, and sensitivity to, LPS, as activation of both TLR4 and the alternate complement pathway by LPS are default ways for the immune system to maintain adequate activity (uptake of LPS from the intestine is not normally an accident, but a regulated and pseudo-hormonal activity). Thus restoring immune activation through TLR2 may decrease the LPS sensitivity which drives liver inflammation, as well as improving immune surveillance of and response to the ongoing HCV infection. As well as spirulina, probiotic bacteria such as Lactobacillus Rhamnosus also have TLR2 activity (PDF). 

My own suggestion would be to combine live Rhamnosus and/or Del-immune V with sprirulina or chlorella. The quantity of chlorella consumed in that paper seems rather daunting, and spirulina is a rich source of iron, so it will be good if lower intakes are effective. My own experience is that spirulina and probiotics go well together. Spirulina is also a very good source of mixed carotenoids, and high carotenoid intakes are (independently of retinol) associated with lower rates of hepatocellular cancer in chronic hep C populations; it is also a source of vitamin K2. (It is important to buy algal products from reputable suppliers who will test them for hepatotoxic contaminants).
Fatty liver, or steatosis, is a metabolic phenomenon and mainly diet driven and is a virtual precondition for hepatic fibrosis. Fibrosis itself, on the other hand, seems to be an immunological phenomenon, with the aspects of diet that have most influence being concerned with the microbiota (probiotics, prebiotics, parasites, pathogens, spices, and active foods like algae), the movement of LPS into the liver (polyunsaturated vs saturated fats), and the immune response to these (vitamins A and D, niacinamide and herbal medicines), as well as glucose and insulin regulation. 

(Note: in this neural stem cell paper "the diet supplemented with spirulina was able to negate acute systemic inflammatory insult of lipopolysaccharide")

(More Music: Sketches of Spain by Miles Davis)

Maybe Trans Fats are Not the Devil
Trans-palmitoleic acid isn't a special trans-fat like CLA (which everyone agrees is good). It's found in both dairy fat and partially hydrogenated vegetable oil. So why do people with the highest levels of TPA have a halved risk of developing diabetes?

Am J Clin Nutr. 2013 Apr;97(4):854-61. doi: 10.3945/ajcn.112.045468. Epub 2013 Feb 13.

trans-Palmitoleic acid, other dairy fat biomarkers, and incident diabetes: the Multi-Ethnic Study of Atherosclerosis (MESA).

Mozaffarian Dde Oliveira Otto MCLemaitre RNFretts AMHotamisligil GTsai MYSiscovick DSNettleton JA.


Division of Cardiovascular Medicine and Channing Laboratory, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, Harvard School of Public Health, Boston, MA.



Dairy consumption is linked to a lower risk of type 2 diabetes, but constituents responsible for this relation are not established. Emerging evidence suggests that trans-palmitoleate (trans 16:1n-7), a fatty acid in dairy and also partially hydrogenated oils, may be associated with a more favorable metabolic profile and less incident diabetes.


We investigated the association of trans-palmitoleate with metabolic risk and incident diabetes in a multiethnic US cohort.


Phospholipid fatty acids and metabolic risk factors were measured in 2000-2002 among 2617 adults in the Multi-Ethnic Study of Atherosclerosis (MESA), a cohort of white, black, Hispanic, and Chinese Americans. In 2281 participants free of baseline diabetes, we also prospectively assessed the risk of new-onset diabetes (205 cases) from baseline to 2005-2007.


trans-Palmitoleate concentrations correlated positively with self-reported consumption of whole-fat dairy, butter, margarine, and baked desserts and with other circulating biomarkers of both dairy fat and partially hydrogenated oil consumption, which suggested mixed dietary sources. After multivariable adjustment, trans-palmitoleate concentrations were associated with higher LDL cholesterol (quintile 5 compared with quintile 1: +6.4%; P-trend = 0.005), lower triglycerides (-19.1%; P-trend < 0.001), lower fasting insulin (-9.1%; P-trend = 0.002), and lower systolic blood pressure (-2.4 mm Hg; P-trend = 0.01). In prospective analyses, trans-palmitoleate was independently associated with lower incident diabetes (P-trend = 0.02), including a 48% lower risk in quintile 5 compared with quintile 1 (HR: 0.52; 95% CI: 0.32, 0.85). All findings were similar between men and women and between different race-ethnic subgroups.


Circulating trans-palmitoleate is associated with higher LDL cholesterol but also with lower triglycerides, fasting insulin, blood pressure, and incident diabetes in a multiethnic US cohort. Our findings support the need for further experimental and dietary intervention studies that target circulating trans-palmitoleate. The MESA trial was registered at as NCT000054
Trans-palmitoleate was associated with substantially lower incidence of diabetes, with multivariable-hazard-ratios=0.41 (95%CI=0.27–0.64) and 0.38 (95%CI=0.24–0.62) in quintile-4 and quintile-5, versus quintile-1.

One might surmise that in this case either the health benefits of increased fat intake override any negatives from hydrogenated vegetable oils (backed up by the finding that palmitoleate from DNL is associated with negative effects that are the inverse of those associated with trans-palmitoleate), or the health benefits of dairy fat are so profound that the negative effects of hydrogenated oils are swallowed up by them.

P.S. A few thoughts on the MESA trans-palmitoleate and palmitoleate studies.
I've looked at some of the other MESA research (there is a great deal of it) and this group's epidemiological work stands out for these reasons:

1) They are measuring an accurately quantifiable factor that is diet-dependent, not relying on food frequency questionaires.
2) They have verified that that factor does correlate with reported dietary intake
3) They are using an actual disease diagnosis (DM2) as the end-point, as well as differences in serum markers.
4) These are prospective studies.
5) Given the short follow-up period (5 years) the odds ratios for DM2 incidence are striking. 
6) There is little in the way of a priori assumptions to colour the interpretation of the data.