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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.


Bill said...

Hi George,

The trans vs cis 16:1 stuff is interesting - both seem "OK?" trans from dairy fat, and cis from DNL - I didn't think trans 16:1 was too high in PHVO, which should [I think] have more of the 18C varieties (?). I have a feeling the trans story is still in its infancy.

Puddleg said...

Yes, I think the cis-palmitoleate here is symptomatic of a high-carb diet in people predisposed to DM2 for whatever reason. I don't believe that the metabolic effects of these fats is very productive of the different metabolic profiles - I really think this just shows - in quite a striking way - that higher fat diets beat low fat diets for preventing DM2.
Previous theories of trans fat toxicity seem to have been based in part on effects on lipid hypothesis markers. Also, ALA is preferentially targeted over LA in hydrogenation reactions, so partial hydrogenation is a way of skewing the 6:3 balance even further when canola or soy oil are used.
I suspect you're right that PHVO didn't contribute much trans-16:1.
Low-carb science does tend to show that macronutrient ratios trump fat quality, and early low-carb diets included margarine.
Trans-16:1 might actually be easier to metabolize than cis-16:1 as it is half-way to saturation. Such trans fats that are merely intermediates of normal dietary unsaturate oxidation may, in theory, be innocent.

Puddleg said...

This is an interesting attempt to try to prove a mechanism for trans-fat harms.
These results suggest that dietary trans fats cause atherosclerosis, at least in part, by suppressing TGF-β responsiveness.

Bill said...

These cats attribute the athero to increased CETP:

Puddleg said...

"Cholesterol esters can be transferred among lipoprotein classes, a process mediated by an exchange carrier protein present in normal human plasma and in the plasma of most animals (absent in the rat)"

Best and Taylor's Physiological Basis of Medical Practice 11th edn.

What is this IS a cause of heart disease in humans, and the affected protein is "absent in the rat"?
All these CETP studies seem to use primates/humans.

This monkey study compares eliadic acid with palmitic acid; surprise surprise, palmitic acid lowered cholesterol.

This goat milk fat study gives a comparison of eliadic acid and trans-palmitoleic levels; 0.16% vs. 2.12%. Whereas PHVO trans fat is mostly eliadic.

Puddleg said...

I must remember to transcribe ALL of Best and Taylor's notes on the differences between human and rodent lipoprotein transport sometime.
There are a fair few.
I have never noticed mention of these differences in a paper about a rat or mouse model of anything.

Wout Mertens said...

If I just keep reading your blog long enough I'll eventually understand it, at least I hope so ;-)

Very interesting studies! Your hypothesis around the low carb effect on trans fat sounds plausible... I wonder how they thought to research that specific trans fat, perhaps any would have yielded the same results?

Puddleg said...

I don't understand it myself Wout. I think I do, but writing it out helps me understand that I don't. This post started out saying something different but I realised that it was mostly wrong while I was writing it and found something better.
About the trans fats, I think Bill is right and there is not much good about eliadic acid (the omega-9 trans fat). The trans-palmitoleic acid does mostly come from dairy and the 2010 study specifies this. It must have been pointed out to the authors after that that there is also some TPA in PHVO, and so this is included in the 2013 study.
But this doesn't mean that these were a significant source. I believe there is also TPA in ruminant body fat such as tallow, I don't see why there wouldn't be.

Look at this: this is the paper that started it all
"Based on our experimental work,(1) we hypothesized that non-hepatic sources of palmitoleate may suppress hepatic fat synthesis and produce metabolic benefits."
So trans-palmitoleate was initially seen by the authors as a marker for dietary palmitoleate (as opposed to endogenous palmitoleate from de novo lipogenesis). And I think this should still be the default interpretation of this study; that eating ruminant fat reduces the risk of diabetes.

Puddleg said...

Mozaffarian, an author of the TPA studies, gets together with Walter Willet on this review:

"The limited data suggest that the experimental effects of ruminant and industrial TFA are similar when consumed in similar quantities, but very few persons consume such high levels of ruminant TFA, and observational studies do not support adverse CHD effects of ruminant TFA in amounts actually consumed."

The dose makes the poison, then...
CLA at least is somewhat cytotoxic, and this is a good thing in small doses.

sweetlady said...

Hmmm. I don't think organic spirulina may cause any harm.

flipper said...

So, in maintaining a good HCV diet, I think that I understand that goose fat is not so good? I would be unhappy to have that confirmed.

Puddleg said...

I don't see anything wrong with goose fat if it's not the only fat. Alternated say with butter it should be fine.

flipper said...

Thanks so much for the reply, and since I usually use the goose fat as a spread on a small slice of hard cheese (less than 3 oz total for both as a snack), I now feel less concerned. Very interesting, very large amount of info here, which I appreciate so much.

Unknown said...

I arrived here looking for more clues about the LPS in Chlorella. There are some articles suggesting it would be detrimental to stimulate humoural immunity in this way, especially if dealing with autoimmunity. They suggest increased Parkinson's risk. But some studies suggest other peptides would negate this effect and suggest an overall anti-inflammatory effect. I'd love to know what you think? Thanks!

Puddleg said...

Hi Joe,

as far as I could tell algal cell walls stimulate mostly TLR2, whereas gram -ve bacteria LPS stimulate TLR4, TLR2 activation seems to improve gut integrity, and should protect against TLR4 activation. Whether this is important in Parkinson's I don't know. Paleolithic diets often included freshwater algae, and large amounts are still eaten in Japan as seaweed.

Unknown said...

Thanks George - that's very useful. It looks like, as is often the case, the whole food contains factors that balance each other out. Do you prefer spirulina or chlorella? All the best, Joe

Puddleg said...

Hi Joe,

I prefer spirulina because there is more relevant research into it and it's cheaper and more widely available here. It's available as a food, whereas chlorella is mainly a low dose pill.