The miracle of safflower oil (4) Soybean oil is just as good

I picked this paper up from George Henderson via twitter

Effects of dietary fat on gut microbiota and faecal metabolites, and their relationship with cardiometabolic risk factors: a 6-month randomised controlled-feeding trial

which is part of the same study as this one

Effects of Macronutrient Distribution on Weight and Related Cardiometabolic Profile in Healthy Non-Obese Chinese: A 6-month, Randomized Controlled-Feeding Trial

The study is amazing. The researchers provided all of the food to all of the participants for six months. The only fat source was soybean oil. It could almost be a rodent study. This is what the study says:

"The three diets were isocaloric, the primary distinguishing feature being their fat and carbohydrate content (Table 1). By replacing a proportion of energy derived from carbohydrates (white rice and wheat flour, the most consumed carbohydrate sources in China contributing to 70% and 17% total carbohydrate respectively) with fats (soybean oil, the most consumed edible oil in China rich in unsaturated fatty acids)..."

Just a few things to point out: The diets were isocaloric. No one was allowed to select their calorie intake. Blokes got just over 2000kcal/day, women got 1700kcal/day. Some supplementary fruit was allowed, to be recorded as and when eaten.

Here are the baseline diets for each of the intervention groups:

Here is what the intervention diets looked like:

Particularly note the exact match of calories normally eaten at baseline compared with that supplied by the study intervention, which was utterly accurately measured out in the study kitchen.

What happened to the weights during the intervention? This did:

The yellow line is 24% PUFA, red is 18% PUFA and blue is 11% PUFA, all by energy intake.

The first thing to say is that all participants lost weight. I guess that has something deep to say about the accuracy of three day dietary records!

However the weight loss was far from uniform and was clearly inversely related to the level of PUFA in the diet. Less PUFA, greater weight loss, 2kg in eight weeks for the lowest PUFA group. This cannot be explained by reduced food intake because all food calories were provided, in exact amounts, by the study kitchens. Diets were isocaloric...

Protons would suggest that the higher the PUFA content the more dietary fat was "lost" in to adipocytes then not subsequently released. From the cico-tard point of view just over a kilo of fat was "not released" from adipocytes over eight weeks of weight loss in the high PUFA vs the low PUFA group. That's 1000g over 56 days or just under 20g/day. This would have to be countered by a decrease in metabolic rate, in NEAT or specific activity. Or more cheating, recorded or not recorded. The study authors assure us there was no cheating.

After eight to 12 weeks all subjects started to regain weight. The regain slope suggests about a kilo per year on a rigidly fixed calorie, weight reducing food intake.

These people were 23 years old and weighed 60kg. If they stuck with this diet over 30 years they might end up at 90kg. Of course this wouldn't happen if they really stuck with the diet. As weight gain tried to continued on a fixed calorie intake, hunger would increase.

You cannot argue with hunger for 30 years. The subjects would break the diet and eat more. This manifest lack of "willpower" or gross "gluttony" would take the blame for the increased weight gain, which would be blamed on the increased calorie intake.

But the weight regain was already there on a rigidly fixed calorie intake. A calorie intake which gave eight weeks of initial weight loss. This still gave a progressive weight gain with no declared increase in caloric intake.

The beauty of PUFA and the Protons concept.

Peter

NB It is difficult to emphasise how good this study is. Just ignore anything the authors have to say.

The miracle of safflower oil (3)

TLDR: PUFA in a mixed diet are obesogenic. PUFA under hypoinsulinaemic conditions are not. I doubt they get a free pass long term.



There have been some interesting snippets on Twitter recently, triggered by Diet Doctor's discussion about vegetable oils here. Perhaps the most controversial quote is this one:

"Disclaimer: Vegetable oils are routinely recommended as “heart healthy.” There is high-quality evidence demonstrating that replacing saturated fat with vegetable oils reduces LDL cholesterol levels. But at this point, there is inconsistent evidence whether this translates into fewer heart events or lower rates of cardiovascular mortality".

This is absolutely incorrect for people with pre existing cardiovascular disease as it was found, in a randomised control trial using safflower oil, that increasing vegetable oil for bulk calories will increase all cause mortality (p = 0.05), cardiovascular disease mortality (p = 0.04) and coronary heart disease mortality (p = 0.04). Mortality is an utterly hard end point and particularly the all cause mortality is an end point which cannot be argued with.

Let's rephrase that: in the context of a mixed diet in people with established heart disease vegetable oil (from safflower seeds) is going to increase you risk of death, especially from cardiovascular disease.

The main issue is to ask whether this still applies under low carbohydrate eating conditions. Given the role of insulin in CVD this is far from certain. But context will be crucial here and who would like to be the guinea pig?

The interesting twitter conversation goes like this:

Dr Westman: "In 20 yrs of clinical research and practice using LCHF/keto, I’ve never even mentioned reducing omega 6s, and it works wonderfully. Just cutting carbs gets the job done!"

Tucker: "I disagree, but @drericwestman is an excellent physician who does great work. This is more about determining ultimate causation so we can address people who can't just go low-carb, which is most of the planet".

I think both people are correct. I came to LC because it works. Over decades I've read studies where it works pretty much invariably on a group basis and studies from the mainstream usually advise progressively increasing carbs if they want to knock low carb and secure future funding. You have to pay the mortgage.

I am perfectly willing to accept that consuming carbohydrate in a rapidly absorbable form will overwhelm the liver's ability to protect the systemic circulation from hyperglycaemia so will require systemic hyperinsulinaemia to control that systemic hyperglycaemia. In particular hyperinsulinaemia comes with its attendant problems (ie most of medicine) but obesity only occurs when hyperinsulaemia is marked enough to overcome insulin-induced insulin resistance. I have no doubt this can occur without PUFA but I think it is massively easier in the presence of PUFA, which delay normal insulin-induced insulin resistance in the immediate post prandial period.

The role of polyunsaturated fatty acids is to stop adipocytes developing insulin resistance by limiting ROS generation. Combining hyperinsulinaemia with hypersensitive somatic cells is a recipe for maximising lipid storage in adipocytes and simultaneous packing lipid in to muscles, pancreas and anywhere else you care to imagine that sprouts an insulin receptor (most brain cells excepted).

Eating a low carbohydrate diet side-steps the problem by reducing absolute levels of systemic insulin. Down a set of unrelated rabbit holes I'm looking at what might control hunger under LC eating and PUFA may have some influence on this, but it is clearly a small effect when compared to the same dose of PUFA combined with an insulogenic diet.

Ultimately at low levels of insulin it doesn't matter how well or badly adipocytes respond to/resist insulin. There is so little insulin about that FFAs and ketones are able supply the body's energy needs, given some excess fat (especially visceral fat) available to be utilised.

Back to long term speculation: Do PUFA matter for non-insulin reasons on a low carb diet? Recall that López-Domínguez et al used a low calorie semi-starvation model (which is a partial mimic of low carb eating) to look at longevity in rodents (post is here). It certainly matters under their study conditions but the effect is small enough that I doubt it would show in any way for someone at 40 years of age under a year or two's exposure to a high PUFA but low carbohydrate diet. For those of us in this for the long haul it's much easier not to be the test case and PUFA avoidance seems prudent to me.

And I am undoubtedly still a low carb eater.

Peter

The miracle of safflower oil (2)

Just a brief mention of this one:

Use of dietary linoleic acid for secondary prevention of coronary heart disease and death: evaluation of recovered data from the Sydney Diet Heart Study and updated meta-analysis

merely because they used the same miraculous safflower oil as featured in the last post. This was a secondary prevention trial and increased safflower oil derived linoleic acid to around 15% of calories, again with no attempt to remove the ubiquitous industrial trans fatty acids from the control diet.

Here is the all cause mortality over 5 years

It's interesting that the increased death rate kicked in almost immediately, ie there is a case to be made for direct toxicity rather than the rather abstract concept of accelerated ageing that I've speculated about previously.

Ramsden published these recovered data in 2013. I guess seven years might be a little too soon for it to have filtered down to the World Health Organisation or Public Health England.

Talk about blood on their hands.

Peter

The miracle of safflower oil

TLDR: Increasing insulin sensitivity makes you fat.

This study is a bit of a mess because there are no control groups. People either got the safflower intervention or the conjugated linoleic acid intervention, then they were crossed over:

Comparison of dietary conjugated linoleic acid with safflower oil on body composition in obese postmenopausal women with type 2 diabetes mellitus

I'm going to ignore all of the CLA/post CLA data and look at the subjects who got just safflower oil, a total of eight capsules per day, two with each meal plus two at bedtime, eight grams a day of the oil for the first 16 weeks of the study. The safflower oil was 78% linoleic acid, regularly checked by gas chromatography.

Looking at Table 3 there was no change in total fat mass (and subjects didn't gain any weight on the scales) by DEXA scan while there was a loss of 1.2kg of "truncal" adipose tissue. With a PUFA supplement. It appears that DEXA scanning cannot differentiate between visceral and subcutaneous fat in the trunk area. The authors can't quite claim that there was selective loss of visceral fat but I think it is very likely that this did happen.

Throw in a fall in fasting glucose and a downward trend in fasting insulin levels coupled with a rise in adiponectin, some muscle gain and well, that's pretty impressive. You can, absolutely, see why people might have the idea that PUFA could be very positive for metabolic health.


How might one view this from a Protons perspective, other than reaching for a bottle of safflower oil?

I think the first thing to consider is the (probable) loss of visceral fat. Visceral fat, in my opinion, is utterly harmless. It contains the most insulin sensitive adipocytes in the body. If you are chronically hyperinsulinaemic, especially overnight, your insulin may never drop low enough to release any significant lipid from your visceral fat. So visceral fat is a surrogate for nocturnal hyperinsulinaemia, which is what is actually bad for you.

We have values for 10h fasting insulin; at enrolment it was 19.9microU/ml and this dropped to 18.2microU/ml over the first 16 weeks of the study. I would not expect 19.9microU/ml to maintain visceral fat and 18.2microU/ml to melt it away. I think it is much more likely that the gross hyperinsulinaemia induced by the sort of evening meal recommended by the ADA for diabetic people might well have resolved faster with safflower oil supplementation than it did without safflower oil, ie the duration of the period of gross hyperinsulinaemia through the night was reduced. Fasting levels were unchanged but the time spent above this ought to have been reduced.

We just have to revisit the Spanish study to see why:














This graph is over eight hours, 10 hours would be similar. These are healthy volunteers, the hyperinsulinaemia would be worse in DMT2 patients eating a high carbohydrate meal. Black squares are butter, white triangles are a high PUFA seed oil. The higher the PUFA content of the meal, the faster insulin level drops. Adding PUFA a mixed meal should allow insulin to drop faster and sooner than saturated fats. This happens because PUFA fail to generate the ROS needed to maintain the physiological insulin resistance which ought to occur post prandially to limit calorie ingress in to cells, adipocytes included. This leaves glucose and fatty acids available to signal satiety to the brain. Also noted in the Spanish study was that PUFA induced more rapid clearance of chylomicrons and more rapid drop in FFAs compared to saturated fats. As I wondered at the time, where do the FFAs and chylomicrons go to?

They go in to adipocytes, because the adipocytes cannot say "no" if PUFA generate too little ROS.

So this drug (safflower oil) allows increased insulin sensitivity (reminiscent of the "glitazones") or, rather, it fails to generate the ROS needed to limit the over expansion of adipocytes, which shows as increased insulin sensitivity during peak insulin exposure. This increased insulin sensitivity puts calories in to adipocytes rapidly so reduces the need for sustained hyperinsulinaemia. All adipocytes gain fat, but the faster fall in insulin allows an increase in the time window where visceral fat can actually release at least some FFAs to the systemic circulation via the portal vein and liver. Visceral fat shrinks, non-visceral fat expands.

The "benefit" of reducing visceral fat in this way during fasting is paid for by increasing the non-visceral fat depots in the immediate post prandial period. The extra fat in non-visceral adipose tissue will come primarily from the diet and the lost fat from visceral adipocytes will be used to provide fasting calories. In this particular study, the amount gained by non-visceral adipocytes was roughly equal to that lost by visceral adipocytes, it's probably random chance that the numbers balanced. And DEXA seems a pretty crude technology to use to work in small numbers of grams of adipose tissue, just looking at the non-balancing cited changes in fat and lean tissue mass in the results.

These processes can continue until non-visceral fat mass eventually become high enough that the loss of FFAs due to adipocyte distension over rides the insulin sensitising effect of the safflower oil. At this point overall insulin exposure will increase and visceral fat will return, on top of a higher mass of non-visceral adipose tissue. It will take longer than 16 weeks.

If you are an obese diabetic taking part in a study like this you should see a prompt but transient improvement in insulin sensitivity. This enhanced sensitivity should allow more non-visceral fat gain until you convert to being a somewhat more obese diabetic. It nicely illustrates that extra PUFA convert you from being established "obese" to being "pre-more-obese". Time is all that is needed to convert you from being "pre-more-obese" to simply"more-obese".

But your lab numbers will improve transiently in the first part of the intervention.

Peter

Double Bond Index and longevity in humans

Preamble: I've had this post written for some time (there are a fair few in this category) but this tweet from the World Health Organisation has prompted me to hit the publish button. In particular this piece of advice begs the question of incompetence vs malicious intent (I doubt the latter):

Here's the post:

I thought I would revisit the idea of trans fatty acids because the late Fred Kummerow got an honourable mention on twitter recently. He is largely responsible for the removal of industrial trans fatty acids from the food chain. No one would argue that that was not a Good Thing.

Back in the 1970s a study was completed which applied a diet from which saturated fats were largely removed and linoleic acid, mostly from corn oil, was increased to about 13% of calories. In a "control" diet saturated fats were as unchanged as practical and linoleic acid limited to just under 5% of calories. That should be a pretty good test of the miraculous benefits of dietary PUFA for blood cholesterol lowering.

However the "control" diet just happened to be specifically increased in industrial trans fatty acids from commercial margarine (1960s style USA margarine), though no one knows by how much, ie there was no genuine "control" diet.

This is what the diets looked like

Cholesterol lowering diet:

"Liquid corn oil was used in place of the usual hospital cooking fats (including hydrogenated oils) and was also added to numerous food items (for example, salad dressings, filled beef (lean ground beef with added oil), filled milk, and filled cheeses). Soft corn oil polyunsaturated margarine was used in place of butter. This intervention produced a mean reduction in dietary saturated fat by about 50% (from 18.5% to 9.2% of calories) and increased linoleic acid intake by more than 280% (from about 3.4% to 13.2% of calories)".

"Control" diet:

"It was designed to appear similar to the experimental diet. Notably, free surplus USDA food commodities including common margarines and shortenings were key components of the control diet, making the daily per participant allocation from the state of Minnesota adequate to cover the full costs. As common margarines and shortenings of this period were rich sources of industrially produced trans fatty acids, the control diet contained substantial quantities of trans fat. Compared with the pre-randomization hospital diet, the control diet did not change saturated fat intake but did substantially increase linoleic acid intake (by about 38%, from 3.4% to 4.7% of calories)".

You have to wonder about the inclusion of trans fats in the control diet. Did Ancel Keys (co-principal non-author) realise, even as long ago as the mid 1960s, that trans fats were bad? A little stacking of the deck has never been been considered an issue when it might help support the lipid hypothesis.

The experiment, designed to confirm the benefits of PUFA, failed completely. There was zero benefit from cholesterol lowering using dietary linoleic acid. Keys never published the results, hence my use of the term "co-principal non-author" because non published study results cannot have any authors. Happily enough data were excavated by Ramsden et al 40 years later to be published in 2016 as

Re-evaluation of the traditional diet-heart hypothesis: analysis of recovered data from Minnesota Coronary Experiment (1968-73)

There was, overall, no effect on total mortality when comparing the two interventions. To rephrase that: Increasing dietary linoleic acid was no worse than increasing trans fats, overall.

OK. So we could stop there with nothing more insightful than an observation of the moral and scientific bankruptcy of the architects of the lipid hypothesis. Nothing new there.




But what we actually have here is a study comparing two diets, one with a marked increase in the double bond index (DBI) of the lipids vs one with a modest increase in DBI, if we ignore the problems of trans fats.

We have something resembling the CRON mouse study in which lard as the lipid source gave a greater longevity benefit compared to fish oil or soya oil. We can view the present study as an intervention which altered mitochondrial membrane lipid composition in a direction of enhanced ageing based on increased DBI of those membrane lipids. But this time in humans, and with no calorie restriction.

Rather than looking at specific diseases we can ask whether increasing the DBI of your mitochondrial lipids might simply make you biologically older than your chronological age. This should show in the all cause mortality data, irrespective of the cause of death (ignoring "One flew over the cuckoo's nest" scenarios, even though this was a mental hospital study). However this would be hard to isolate in younger people because they are far enough, chronologically, away from death that ageing them by 10 years (a totally fictitious value, merely used for illustrative purposes) wouldn't show up much in all cause mortality. Assuming the risk of death at 40 years of age is similar to that at 50 years of age, nothing will show.

But, if you are 65 years of age and eating 13% of your calories as corn oil derived polyunsaturated fats makes you behave biologically as if you are 75 years of age, this just might show as increased all cause mortality.

Ramsden provides us with these graphs. In people under 65 years of age nothing shows:

but in people over 65 years of age there is visibly increased all cause mortality in the corn oil subjects:

Because the raw data for these graphs could not be recovered it is impossible to perform any sort of statistical analysis but it looks to me like there might be some indication that basing your diet around corn oil PUFA might be worse than eating trans fats, late in life. Given the raw data I suspect it might be possible to calculate how much linoleic acid might shorten your lifespan and by implication I would expect it might also shorten your healthspan, which could actually be worse.

Trans fats come out unexpectedly well. You have to wonder how much more benefit removing linoleic acid might provide, especially if you are an elderly person trying to avoid ARDS in the ITU.

Peter

ARDS isoprostanes and isofurans

This study from 2012 uses a model and it's based on mice. No one, ever, develops ARDS by inhaling reagent grade lipopolysaccharide following an intra tracheal injection of said LPS. So some caution.

Low levels of tissue factor lead to alveolar hemorrhage, potentiating murine acute lung injury and oxidative stress

I was interested, not because of the tissue factor knockout, but in the control group because I wanted quantification of how much haemorrhage occurred in to the alveoli during the progression of ARDS. I was modestly interested in the concept that pulmonary haemorrhage might reduce systemic haemoglobin value, elevate bilirubin, elevate ferritin and induce oxidative stress due to free haemoglobin or its derivatives. It can do some of these things but what caught my eye was the section on inflammatory markers within the small "human" arm of the study.  This is what they found in real people with real ARDS:

"Patients with diffuse alveolar hemorrhage had progressively increasing BAL isoprostanes and isofurans as their sequential BAL aliquots become more bloody. These findings suggest that liberation of free hemoglobin into the airspace and intraalveolar lipid peroxidation may be important mechanisms of clinical acute lung injury".

I would just comment that while both isoprostanes and isofurans measured in the study are arachidonic acid non-enzymic derived peroxidation products I can see no reason why the toxic derivatives of linoleic acid peroxidation would not also be formed, though these weren't measured in the study.

This would fit well with the likelihood of who is most likely to develop ARDS being predicted by the proportion of polyunsaturated fatty acids in their plasma free fatty acid pool on admission to the ITU.

The role in ARDS of pulmonary haemorrhage, intra-alveolar red cell lysis, free haemoglobin and severe oxidative stress in the airspaces is fascinating and is likely to be a routine feature of all types of ARDS. I got here by trying to decide whether the widely discussed concept that SARS-CoV-2 might be doing something special based on viral protein modelling suggesting the displacement of the Fe atoms from haemoglobin could be causal of whole body damage via systemic hypoxia. Looking at the clinical data coming out of Wuhan and what we have known about ARDS for decades makes me doubtful that we need the "iron hypothesis". I'd not say that it is incorrect, just that an awful lot of the clinical data fit with a severe viral infection induced ARDS including pulmonary haemorrhage in patients loaded with polyunsaturated fatty acids.

Peter



I wrote this post some time ago and felt it wasn't really interesting enough to put up, but the iron/haemoglobin idea keeps resurfacing. Loss of Fe from Hb will undoubtedly impair oxygen delivery but it would not cause arterial oxygen desaturation without concomitant lung dysfunction. With normal lung function whatever undamaged haemoglobin remains would saturate perfectly well. A pulse oximeter would still render a reading of 100% saturated even if little functional haemoglobin remained and tissue oxygen delivery was very low, assuming the colour of the haemoglobin derivatives did not interfere with pulse oximeter function. This is the situation in "normal" profound anaemia. In one of the Wuhan reports clinical anaemia on presentation was undoubtedly associated with decreased survival (though this was not related to mean haemoglobin levels) but equally so were decreased platelet count and decreased albumin levels. These would be compatible with serious problems from multiple tissue haemorrhage, lungs included, as part of the advanced stages of SIRS (systemic inflammatory response syndrome) where DIC (disseminated intravascular coagulation, better known as Death Is Coming) becomes one of the terminal features.

The discussion of mechanical ventilation techniques in causing/avoiding pressure injury to the lungs is almost as old as the eternal discussion of colloid vs crystalloid for volume resuscitation. Personally I prefer crystalloids and would favour IPPV techniques which avoid barotrauma to the lungs if at all possible. But that's just me.

Look after your lysosomes: Hydroxychloroquine

This fascinating:

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

Forty two patients were enrolled. Sixteen were not given or refused hydroxychloroquine and none of these were admitted to the ITU, none walked out of the hospital and none were nauseated enough to withdraw from the trial.

Of the 26 patients given hydroxychloroquine three were subsequently excluded from the study because they were admitted to the ITU. Oops. One died (not one of those admitted to the ITU). One walked out of the hospital and never came back. Another was too nauseated on the drug to continue in the study.

Exclude these six patients and in the remaining 20 patients hydroxychloroquine was reported as successful at clearing the virus on nasopharyngeal swabbing.

You have to wonder if the poor patient who actually died while taking hydroxychloroquine ended up with high pH lysosomes which leaked enough cysteine to extract the FeS clusters from the nearby complex I FeS chains. Or simply died of pneumonia before he/she could be admitted to the ITU. We'll never know. I hope the patients "lost" in to the ITU made a full recovery.

Perhaps the patients who accepted hydroxychloroquine were simply iller in the first place and were so more willing to accept an experimental drug. I do hope so but I feel that the initial data on hydroxychloroquine are not looking too promising.

Peter

Edit: On the plus side there might be an effect if given early. However only time and trials will tell if there really is an effect or whether Turkey happens to have done several other things correctly in addition to using hydroxychloroquine. At least they are not reporting toxicity. End edit.

Coronavirus is possibly everywhere

This tweet

https://twitter.com/Yascha_Mounk/status/1246240181440540672

links to this news report

https://www.lastampa.it/topnews/primo-piano/2020/04/02/news/coronavirus-castiglione-d-adda-e-un-caso-di-studio-il-70-dei-donatori-di-sangue-e-positivo-1.38666481

which should be headline news everywhere.

In Lombardy 40 out of 60 blood donations from healthy donors with no history of coronavirus illness are seropositive. They have been exposed, infected but were never ill.

In comments to a pervious post I suggested that the best advice re coronavirus was a) try not to be elderly and b) try not to be diabetic.

Obviously assessment a) was incorrect.

Being elderly is probably only a problem if you drop in to category b) as well. ie age is a surrogate for risk of metabolic syndrome or as Kraft would have described it "diabetes in-situ". There is a simple solution to diabetes in-situ.

Testing of UK blood donations, if it duplicates Italy, should allow some return to normality with continued or increased protection for those most at risk while we get their metabolic syndrome under control. That should take a few weeks of LC eating and might have to be maintained long term. Damn, bacon and eggs for breakfast every day and steak with broccoli and 'shrooms for supper. Cheese and olives for lunch if you're hungry. Sounds awful I know but sacrifices will have to be made.

Peter

Look after your lysosomes

I was a little cautious about the efficacy vs toxicity of chloroquine and its derivatives in my last post.

George Henderson just retweeted this snippet;

https://twitter.com/UnitedRda/status/1244299325963829248

Sadly the narrow line between the degree of raising lysosomal pH to blunt viral replication and that which might release sufficient cysteine to strip the FeS clusters out from complex I can be crossed quite easily, so it appears.

Worryingly Dr Barman appears to have been one of those people with some degree of metabolic syndrome and who might have been someone most likely to benefit from prophylaxis against coronavirus replication.

My own observation during my very rare trips to our local hospital is that medical professionals are far from immune to metabolic syndrome. Couple that with extreme stress, high viral load exposure, severe sleep deprivation and the sort of food/snacks available in hospitals and you have to worry for the health of these people.

None of them want to have metabolic syndrome, a problem which is built in to our public health guidelines. These people are laying their lives on the line to support the lipid hypothesis. Most of their patients are in hospital secondary to the lipid hypothesis. Those developing ARDS in the ITU do so in a large part as a result of the lipid hypothesis.

Just my rather sad view from the sidelines.

Peter

From Yeasts to Chloroquine

This paper is from Hughes and Gottschling

An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast

It got a mention in the blog back in 2012 when it was freshly published. The group have gone on to study yeasts, ageing and the lysosome-like vacuole of yeasts. Their core finding is that vacuolar pH controls mitochondrial "health" which controls ageing, at least in their model.

The group has been very busy and earlier this year this paper was published from Hughes' lab:

Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis

The paper describes a very long series (way too many to detail here) of experiments aimed at adjusting vacuolar pH upwards and downwards and observing the effect on the survival of mother yeast cells through repeated cell divisions (replicative age rather than chronological age, there are arguments about which matters most).

Bottom line: Acidifying vacuolar pH extends lifespan, reducing its acidity shortens it.

Why should that be?

Their next series of experiments demonstrated that cysteine toxicity was the driver of early mitochondrial functional decline secondary to loss of vacuolar acidity. Cysteine is normally harmless and essential for life. Your cells love it, just so long as it is within the vacuole (or lysosome in humans), not in the cytoplasm. It's kept there by a vacuolar amino acid transporter driven by the vacuole proton gradient. The pH gradient is generated using a vacuolar vATP-ase to pump protons from the cytoplasm in to the vacuole, using ATP. It's related to the mitochondrial ATP synthase but normally runs in reverse.

If, on a long term basis, vacuolar pH rises (ie the vATP-ase fails), cysteine is released from the vacuole in to the cytoplasm where it auto-oxidises, generating much too much hydrogen peroxide. This reacts with the iron-sulphur clusters of complex I and many other crucial enzymes in the mitochondria. In old age cysteine becomes toxic through vacuolar failure.

I've been interested in this for some time because Barja and Sinclair have both intimated that they are tending to avoid animal proteins in favour of low cysteine/methionine plant proteins. Cysteine is the cellular executioner when vacuole pH rises during the old age of yeasts or lysosomal pH rises in ageing mammalian cells. It's interesting because methionine restriction (which reduces cysteine levels) appears to core to the longevity promotion seen with caloric restriction or protein restriction in mice fed on crapinabag.

You have to wonder whether we are looking at this the wrong way round. What if crapinanbag, based on starch and sucrose, causes early onset lysosomal failure which can be ameliorated by removing the cysteine, which is the cellular execution mechanism?

This would make methionine restriction's longevity extension rather specific to glucose based metabolism. My biases would tend to favour this point of view. There are no data, yet.



As an aside:

Now, I have speculated that both influenza and corona viruses need anabolic processes generated by mTOR activation. This requires acute acidification of the lysosome. Blocking acute lysosomal acidification is one technique currently being investigated for treating the life threatening pneumonia which develops in susceptible individuals during the current COVID-19 pandemic. There are suggestions that chloroquine, a suppressor of lysosomal acidification, might be an effective treatment. My guess is because it blocks anabolism.

There is probably a fine line between suppressing anabolism and releasing a mitochondrial-executing concentration of cysteine.

Neither Hughes nor Gottschling were considering therapeutic inhibition of vacuolar acidification as a stratagem for anything. They were more interested in avoiding long term loss of vacuolar acidity to delay mitochondrial function decline. But blunting anabolism without causing catastrophic cysteine release is a current anti-viral/anti-neoplastic therapeutic target.

You can see that the drug chloroquine a) might work and b) might be very toxic in overdose.

It does currently appear that it might work but we should never forget that "clinical experience is no guarantee of therapeutic efficacy".

However it would be great if it really did work.

Peter

ARDS and linoleic acid

Adult/Acute Respiratory Distress Syndrome is topical at the moment. In the comments to the last post I wondered whether omega six fatty acids, especially linoleic acid, might be a driver of ARDS, which is one of the most intractable ITU problems in response to major infection/trauma/inflammatory insults.

Tucker came up with this abstract

Plasma fatty acid changes and increased lipid peroxidation in patients with adult respiratory distress syndrome

and I peeked at the related papers to find this gem:

An increase in serum C18 unsaturated free fatty acids as a predictor of the development of acute respiratory distress syndrome

Again, only an abstract and mostly describing a pilot study. But here is the critical statement:

"Increases in unsaturated serum acyl chain ratios differentiate between healthy and seriously iII patients, and identify those patients likely to develop ARDS".

That is, the more linoleic (and oleic) acid you have as FFAs in your bloodstream, relative to my beloved palmitic acid, the more likely you are to develop ARDS. Which carries a high risk of death.

That was 1996.  The work will have been done before that, so we have known that linoileic acid is bad news for well over 20 years.

If you are a Standard American on the Standard American Diet, or anyone else in the world poisoned by a cardiologist-promoted PUFA based diet, any weight loss through illness will release significant amounts of linoleic acid from your adipocytes. That might just trigger ARDS in the aftermath of a viral pneumonia.

There's a lot of it about.

Peter

BTW Steve Cooksey has a rather nice post up citing a lot of the refs featuring how to maintain an effective innate immune system, so as to avoid the viral pneumonia in the first place. It's a good read.

Cell surface oxygen consumption (4) Influenza

This press release, from 2013, surfaced on twitter (embarrassingly I have again lost the tweeter due a hat tip for this. Mea culpa. Found him, it was resurfaced/retweeted by Guðmundur Jóhannsson).

Glucose: Potential new target for combating annual seasonal flu

which summarises this paper:

Glycolytic control of vacuolar-type ATPase activity: a mechanism to regulate influenza viral infection.

Over the last few weeks I happen to have been immersed in vacuoles/lysosomes, cysteine toxicity, longevity and yeasts. Oh, and mTORC1, which is deeply associated with lysosomes. So I'm in a mindset of how lysosomes/mTOR control longevity/anabolism.

Anyhoo. Influenza A virus uses lysosomes to maximise its survival. My prediction is that it activates mTOR to induce a marked anabolic state and hijacks that anabolic state to generate lots and lots of influenza A virus particles. It will do that, much as a cancer cell might, by aerobic glycolysis working on the basis that glycolysis, while inefficient, is very, very fast at generating ATP compared to OxPhos. This would suggest that the free availability of glucose secondary to hyperglycaemia (or increased access of glucose to the cytoplasm secondary to hyperinsulinaemia) will increase the success of the influenza virus, as found in Kohio's paper.

Which brings us to anabolism and glycolysis. Not only does aerobic glycolysis supply ATP for anabolism faster than OxPhos can but it also supplies phosphoenolpyruvate for amino acid synthesis, plus other anabolic substrates come from glucose via assorted pathways.

However for every glucose molecule which generates a pair of 1-3 bisphosphoglycerate molecules two NAD+ are consumed. If these glycerate molecules are used for anabolism via phosphoenolpyruvate they will not restore the NAD+ balance by converting to lactate. The basic story is in

Cell surface oxygen consumption (2)

and

Cell surface oxygen consumption (3)

with an introduction to the concept in

Cell surface oxygen consumption (1)

The glycerophosphate shuttle won't do the job because this too is limited to the speed of OxPhos. Cell surface oxygen consumption does fit the bill for rapid restoration of NAD+.

So. Does influenza virus drive cell surface oxygen consumption to facilitate anabolism at a speed fast enough to keep it one step ahead of the innate immune system?

I don't know.

But another standard (primarily rodent) model RNA virus certainly does.

Oxygen uptake associated with Sendai-virus-stimulated chemiluminescence in rat thymocytes contains a significant non-mitochondrial component

I think this will be a basic feature of rapid anabolism, be that viral or neoplasia related.

Will hyperglycaemia and/or hyperinsulinaemia facilitate viral directed anabolism under infection by another, more topical novel human RNA virus?

Personally, I'm not planning on finding out the hard way when I get around to catching the current bug.

Peter

Coffee

It came up in conversation with Ally as part of the Paleo Canteen podcast that I like coffee but that it doesn't like me.

Over the years before LC my coffee ingestion had stabilised at around 7 or 8 mugs per day. That's quite a lot. At the time I started on LC I did Atkins induction and cold turkey-ed from all methyl xanthines. The headache was tolerable, especially as I knew exactly why it was there and that it would be gone by about seven days in, which it was. The need for an evening stimulant also disappeared because I no longer fell asleep during the hyperinsulinaemic phase of the post prandial period.

For which I was infamous.

Over the years I have reintroduced coffee a couple of times but  stopped it again due to either minor lower GI upsets or worsening of either low back pain or finger arthritis.

I had done a desultory Pubmed search to see if there was any evidence for clear cut, lectin induced GI damage from coffee which might explain my own signs. When the penny dropped that coffee "beans" were actually seeds rather than legume-like beans I sort of gave up hunting.

So I was avoiding coffee and expected to do so long term. My issue was that I quite like the jittery restlessness which comes from an acute large dose.

In the aftermath of chatting to Ally I received an e-mail for Mason about Dr Paul Mason, his local Dr in Sydney. I have a lot of time for Dr Mason and I really enjoyed his lecture from the 2019 Carnivory.com conference.

It turns out that Dr Mason is pretty sure there is a lectin in coffee. Not only that but the lectin is heat labile.

If you boil your coffee for 10 minutes you appear to pretty well destroy the lectin.

So....

I can boil down a double strength cafetiere of coffee to the volume and bitterness of a double espresso in 10 minutes.

The caffeine is still there and absolutely produces the desired pharmacological effect.

For myself, drinking two or three double espressos per day produces tachyphilaxis to the caffeine within a week or two. Withdrawal is mild and sensitivity is pretty well restored within about 4-5 days. I have no interest in using caffeine to blunt caffeine withdrawal, so coffee is probably a weekend treat.

Plant poison, undoubtedly. Contains disgusting antioxidants too, no doubt. At the moment I feel that there is an acceptable trade-off.

Peter

For those who enjoy confirmation bias and worm studies:

Lifespan Extension Induced by Caffeine in Caenorhabditis elegans is Partially Dependent on Adenosine Signaling

Lard makes hungry mice live longest

Over the past few weeks I've been looking for papers where Barja's group might have run longevity experiments. This does not seem to have been their forte. They have done lots of observational comparative studies looking at long vs short lived species and lots of interventions to modify mitochondrial membrane lipid composition but no hard-core lifespan measuring studies that I can find.

So Barja threw in the rather off comment about avoiding "excessive intake of animal proteins and fats typical of western diets" in his review without obvious direct testing of these variables on lifespan.

I have to leave the mechanism of calorie restriction, aka protein restriction, aka methionine restriction for another day.

What we can do today is to look at Barja's dreaded animal fats. Like lard.

The data are, sadly, only available from CRON fed mice. This is the study:

The Influence of Dietary Fat Source on Life Span in Calorie Restricted Mice

Diets had their fat source modified thus and also had their calories restricted by 40%:

"The modified AIN-93G diets (% of total kcal) each contained 20.3% protein, 63.8% carbohydrate, and 15.9% fat. Soybean oil was the dietary fat in the control group (standard AIN-93G diet). The dietary fats for the CR groups were soybean oil (high in n-6 fatty acids, 55% linoleic acid, Super Store Industries, Lathrop, CA), lard (high in monounsaturated and saturated fatty acids, ConAgra Foods, Omaha, NE) and fish oil (high in n-3 PUFAs, 18% eicosapentaenoic acid, 12% docosahexaenoic acid, Jedwards International, Inc., Quincy, MA). To meet linoleic acid requirements, the fish oil diet contained 1% (w/w) soybean oil".

Here are the survival curves:

The left hand curve of green circles is from (nearly) ad-lib feeding of crapinabag. The yellow squares showing best survival are from feeding the dreaded animal fats from lard, combined with CRON. The fish oil group, full of EPA and DHA, did worst of the three CRON groups with soy oil being intermediate.

I think beef dripping would have done better than lard and beef suet even better still, but then I would think that.

Peter, saturophile.

Insulin sensitivity makes you fat: growth hormone receptor deletion

TLDR: Excessive insulin sensitivity sets you up to become obese.

I have to apologise for citing Valter Fastingbar Longo, sometimes you have little choice. This paper

GH Receptor Deficiency in Ecuadorian Adults Is Associated With Obesity and Enhanced Insulin Sensitivity

documents the physiology of humans who are homozygous for a large growth hormone receptor gene defect. They make their GH, lots of it. It does absolutely nothing, having no receptor. GH normally works in opposition to insulin on adipocytes, causing both lipolysis and systemic insulin resistance.

Also, in the absence of GH signalling, these people make no IGF-1 so are of dwarf stature. They are exquisitely insulin sensitive. As in here are the OGTT results. Dark lines are the GHR deficient people:

Plasma glucose is comparable to that of controls throughout, matched for BMI (and lots of other things). But just look at that insulin level, peaking at 25microIU/ml vs 80microIU/ml in controls. The dwarves are very, very insulin sensitive.

And very fat.

Despite having a mean BMI of 27.6 (controls are higher at 29.4) the dwarves have 48% of their weight as fat mass compared to 41% in the controls.

Let's put this in to context: The GHr deficient people are fat because they are insulin sensitive. There is no paradox. We are not thinking that their obesity should have caused insulin resistance, it's that their failure to generate one type of physiological insulin resistance has allowed pathological insulin sensitivity to prevail, hence obesity.

Oh, and leptin:

Leptin in the dwarves with 48% body fat is 7.32ng/ml. Leptin in controls with 41% body fat is 10.36ng/ml, p is just over 0.02 if you are wondering or care.

It looks to me as if these excessively insulin sensitive individuals have yet to reach their "ideal" metabolic level of obesity to counteract their lack of GH signalling. Interesting to wonder what determines the level of adiposity at a given age in the absence of GH signalling. That's not simple.

We have no data on RER under fasting or post prandially. But we can be fairly confident that the fasting RER will be low, reflecting high basal lipolysis from distended adipocytes and post prandial RER will be high as insulin action facilitates glucose metabolism and locks lipids in to adipocytes.

A bit like those insulin sensitive pre-obese humans a couple of posts ago. But these dwarves will have to become very, very obese to behave like normal overweight insulin resistant people.

Peter

Addendum, not worth a post in its own right but on-topic:

Does Weight Gain Associated with Thiazolidinedione Use Negatively Affect Cardiometabolic Health?

Epic quote of failed perception:

"This review paper discussed the mechanism of action of TZDs on weight gain and the so-called “glitazone paradox”, the phenomenon that TZD-associated weight gain improves rather than exacerbates insulin resistance".

There is no paradox. Insulin signalling improves with glitazones, this makes you fat.

CPT1aL479 resurfaces nicely

Originally from Erik Arnesen, via a retweet by Miki Ben-dor:

Inuit metabolism revisited: what drove the selective sweep of CPT1a L479?

as in

Coconuts and Cornstarch in the Arctic?

The P479L gene for CPT-1a and fatty acid oxidation

The abstract looks very nice, I can't wait to get hold of the full text!

Peter

Edit: The paper is long and somewhat repetitive. There is a much neater paper from Amber which people might enjoy:

Evidence on chronic ketosis in traditional Arctic populations

End edit.

Insulin sensitivity makes you fat

TLDR: Excessive insulin sensitivity sets you up to become obese. Becoming obese makes you insulin resistant. Eventually excessive adipocyte size will induce systemic insulin resistance. Further weight gain is still possible given a diet which induces systemic hyperglycaemia combined with a pancreas of steel. Here we go.


I picked this paper up from Pubmed while looking for something else:

Insulin sensitivity is increased and fat oxidation after a high-fat meal is reduced in normal-weight healthy men with strong familial predisposition to overweight

It's very interesting.

Over the years I have collected various models, mostly mouse/rat models, which generate obese, insulin resistant rodents.

These mostly involve damaging the hypothalamus in some way and letting the mice eat ad lib until they reach the desired level of obesity, with the associated insulin resistance. There is the ventromedial hypothalamic injury model

Molecular and metabolic changes in white adipose tissue of the rat during development of ventromedial hypothalamic obesity

The MSG injury model:

Decreased lipolysis and enhanced glycerol and glucose utilization by adipose tissue prior to development of obesity in monosodium glutamate (MSG) treated-rats

Late effects of postnatal administration of monosodium glutamate on insulin action in adult rats

The gold thioglucose injury model:

Adiponectin expression is paradoxically increased in gold-thioglucose-induced obesity

What they all have in common is that the models are always more insulin sensitive in the first weeks after injury compared to the non-injured controls. This excess sensitivity persists until a certain level of obesity is achieved. As obesity increases so does systemic insulin resistance increase (a separate mechanism) until it overwhelms the excess insulin sensitivity and rate of weight gain markedly reduces. The model is now insulin resistant.

Inappropriate insulin sensitivity is what generates the obesity. Insulin resistance limits its progression.

Insulin resistance in adipocytes can, undoubtedly, occur but this is not a feature of the adipocytes in the early stages of obesity. They are insulin sensitive. Insulin acts easily. Adipocytes distend.

Back to the paper. It enrolled young, male, non-obese offspring of obese parents. Let's call them pre-obese. Sadly the paper is from 2004, it's now 2020, I would expect the "pre" prefix might nowadays be redundant. Here are the subject characteristics:

To me it is interesting that the pre-obese chaps were carrying more fat mass than the controls. There is a 1.7kg excess, statistically ns but the trend is there. You have to wonder how close to 0.05 the p value might have been.

Here are the fasting metabolic parameters for both groups:

Notice that the fasting insulin is lower in the group with higher fat mass, provided they have obese parents. It's also interesting that their fasting FFAs are higher than those of the folks with slim parents. This difference is also ns but the numbers after the +/- sign are standard deviations, not standard errors, so my guess these too are close to significance (for what that is worth). I also like the ns elevated trigs, I suspect related to repackaging the elevated fasting FFAs. Which are elevated due to increased adipocyte size allowing increased basal lipolysis. All speculation.

Next we have the insulin response to a quite pleasant sounding, mixed macro, highish fat meal:

The fasting insulin is the one from Table 2, p being 0.007 and for a large percentage of the post-meal eight hour period insulin stays significantly lower in the pre-obese group than in the normal-weight parent group. The pre-obese subjects are consistently more insulin sensitive.

Here is the FFA graph for the same eight hours:

Converting the FFA levels to real money terms it appears that the lean parent group had FFAs of 280micromol/l and the pre-obese people had 390micromol/l. I've already speculated that the elevated FFAs in the pre-obese group are from increased basal lipolysis, not insulin resistance. As soon as insulin is released after the meal FFA levels become identical for eight hours. I've not copied the trigs graph but the trend is for chylomicrons to be the same between groups for 4 hours and then lower in the pre-obese as insulin sequesters fat in adipocytes.

Which group will be metabolising most fat under hypoinsulinaemic, near-basal lipolytic conditions? Pre-obese have elevated fasting FFAs and they're oxidising more fat, 1150 vs 740mg/kg FFM/d,  ns but you can see the trend:

However, as soon as insulin rises fat oxidation drops because insulin sequesters fat in to adipocytes at levels way below those which translocate GLUT4s. It will also divert intracellular FFAs in to intracellular triglycerides. Lipid oxidation under insulin drops to 90mg/kg FFM x 8h compared to 163mg/kg FFM x 8h in the more normal individuals. Giving p less than 0.007.

BTW FFAs stay high in both groups because the meal was around 50% fat. I would predict that a high carbohydrate, low fat meal would have produced a marked drop in FFAs and a rise in RER, both more pronounced in the people with obese parents. No data on that one.

I do not think these pre-obese people have an injury to their hypothalamus. It is more likely the problem is with their adipocytes causing the excess insulin sensitivity.

I think we can ignore discussion comments about the influence of medium chain acyl CoA dehydrogenase variation as a red herring because the pre-obese folks are oxidising more fat under fasting conditions, ie when more lipid is available. The leptin receptor comment is lovely because we know that in mice with a complete leptin receptor deficiency that providing less than 5% of calories from PUFA is highly protective against obesity while providing 15% PUFA in the diet is grossly obesogenic (first link in the blog post). Clearly dietary fatty acid composition trumps even gross leptin signalling deficiency.

What were the diets like in the pre-obese participants? All we know from this study is that the ratio of PUFA:SFA was higher in the pre-obese people:

"The polyunsaturated to saturated (P/S) ratio was 0.34+/-0.06 in the group with overweight parents and 0.31+/-0.09 in the control group".

However you try to reverse engineer the limited data from the results it's hardly 5% vs 15% PUFA, but these people have taken around 25 years of eating a slightly heart-healthier PUFA rich-er diet to gain an excess of 1.7kg of fat mass. My biases are willing to accept this as real.

Maybe it is, maybe not. I'm not exactly a bias free source of opinion.

Peter

BTW leptin is consistently lower in the pre-obese group carrying excess fat mass. My suspicion is that their fat cells "feel" empty, so are refusing to signal their true state of fullness. Once the adipocytes become full enough then leptin will increase to give a more accurate representation of the absolute fat mass. This will be associated with the onset  of the more expected insulin resistance of obesity.

Looking in to the future of Low Energy Diets

I think I picked this up from Jan Vyjidak on Faceache but it's done the rounds on twitter too.

Low-energy total diet replacement intervention in patients with type 2 diabetes mellitus and obesity treated with insulin: a randomized trial

"At randomization, participants commenced a 12-week TDR [total diet replacement] formula LED [low energy diet]... followed by 12 weeks of structured food reintroduction and then ongoing followup in combination with an energy deficit diet at 3-month intervals until 12 months. For the first 12 weeks, all meals were replaced with four formula LED products per day (800–820 kcal/day, 57%
carbohydrate, 14% fat, 26% protein and 3% fiber) in addition to at least 2.25 liters of energy-free beverages. A fiber supplement was recommended, if required, to avoid constipation, a common side effect of using a TDR".

For three months patients were starved on 800kcal per day. At 56% carbohydrate that makes carbs come out at around 100g/d. Oddly enough, restricting carbs to this level allowed a drop in insulin usage. Indeed, there was such a marked drop in insulin usage that some patients coming off insulin all together. I wonder what these starvation subjects would think if you told them that they could have had equal reductions in insulin usage just by restricting the carbohydrate content of their diets to that 100g/d, while still allowing fat and protein to satiety... I suspect  that a) no one has told them this and b) they might not be best pleased to find out retrospectively.

For a second three months a little food was added to their diet, but not much. For the final six months patients were kept a little hungry but not so much as in the first six months of the study.

Here is what the abstract says:

"Results: Mean weight loss at 12 months was 9.8 kg (SD 4.9) in the intervention and 5.6 kg (SD 6.1) in the control group (adjusted mean difference −4.3 kg, 95% CI −6.3 to 2.3, p less than 0.001)".

Here is what the results show for the intervention group:

Here is the same graph but simplified in to three red lines representing the three phases of the study:

You can argue the exact slopes of the lines but overall the pattern is correct. Something like this:

Now it is time to look into the future. Usually this is difficult but I think that in this case the general shape of the graph lets us predict the shape of things to come when related to weight gain. Plus, because it becomes obvious in the later months of the study (from HbA1c values) that insulin is going to have to be added back in, at this time the rate of weight gain might actually increase (dramatically), but we can't know that.

Using a simple maintenance of the status quo (best case scenario) we get this, looking forwards to around about the 24 month mark:

Weight gain, in the aftermath of a year of hunger, might not stop at baseline mass either.

I think it is also possible to look in to the future of glycaemia too, by extending the plot of HbA1c with time, working from the published graph in the results. Taken forwards to 16 months or so, it looks something like this:

Maybe I'm being pessimistic. Maybe sudden tolerance of chronic hunger might kick in and reverse the adverse trends in weight and glycaemia clearly present at the end of the study. Maybe subjects might suddenly become slim and euglycaemic.

Maybe not.

Peter

Rory Robertson and Protein Restricted Longevity

I looked at this paper when it did the rounds a fair while ago, saw that the only fat source used was soybean oil and decided that living on soybean oil, sucrose, maltodextrin and wheat starch was not a good idea and so I binned it as the junk it is:

The Ratio of Macronutrients, Not Caloric Intake, Dictates Cardiometabolic Health, Aging, and Longevity in Ad Libitum-Fed Mice

I missed the embedded problems which have since been brought to light by Rory Robertson, whose slightly over-the-top concerns are voiced here. I would perhaps disagree slightly with some of his opinions but, overall, he makes a rather good case. The first thing to note is that you have to go to the supplementary data to realise that a significant number of groups of mice were lost (and excluded) due to mortality problems. Table S1 describes all thirty of the diets which the study started out with. Five of these diets had to be discontinued because too many mice either died outright or (I suspect) were ordered to be euthanased on the authority of the supervising veterinary surgeon due to concerns about animal welfare. I'm assuming Oz has a Home Office much as the UK does which requires Named Veterinary Surgeons to be employed to supervise animal welfare in all laboratories.

We know this from the legend to supplementary table S1. Here is the last section of that legend (sorry that the small letter superscripting is lost, that's blogger for you):

"a Diets 2 low energy and 6 medium energy were discontinued within 23 weeks. b Diets 3 low energy, 3 medium energy and 6 low energy were discontinued within 10 weeks of treatment. These diets were discontinued due to weight loss (≥ 20%), rectal prolapse or failure to thrive".

Here is table S1 with the discontinued (and removed) groups outlined in red:

So, they started with 30 diets groups but five of them had such high early death rates that they were excluded from the study. This left 25 groups. Other than the legend to supplementary table S1 I am unable to find any reference to the loss of five diet groups anywhere in the main paper, which gives the distinct impression that 25 groups were all that were included from the start. So 17% of the mice died at under 23 weeks in to the study, many of those within less than 10 weeks, and you have to read the supplementary data to find out.

All of the high mortality groups were eating 5% of calories as protein.

Did you pick that up in the abstract? No, you didn't.

Is there any excuse for failing to discuss this crucial finding in the results and discussion sections of the paper? You can decide that. It's not exactly rocket science.

My feeling is that the authors could argue, if they were convinced that protein restriction was key to longevity (amino acids, cysteine, mTOR etc don'tchano), that studying early life mortality has no relevance to late life longevity. Why not leave early mortality to the paediatricians? That is a potentially arguable position and should, as it involved a huge chunk of the study mice, have been reported and been justified (if possible) in the results, discussion and especially in the abstract.

The other slight hiccup is this line from the main paper:

"Median lifespan was greatest for animals whose intakes were low in protein and high in carbohydrate... (Figure 2A)"

Figure 2A is not in a format which lends itself to simple interpretation and, obviously, excludes all of the mice which died or were euthanased at less than 23 weeks of age, all of which were in low protein groups. Anyway, you might want to see a simple table of median lifespan for each of the surviving groups. Like supplementary table S2. I've high-lighted the group which had the longest median lifespan in red:

Looks to me like the longest median lifespan group might have been eating 42% of it's calories as protein... Hmmmm. Worth repeating:

"Median lifespan was greatest for animals whose intakes were low in protein and high in carbohydrate... (Figure 2A)".

vs Table S2 giving 42% protein for longest median lifespan.

Hmmmmmmmmm...



Let's make this crystal clear: The data demonstrating the actual outcomes are, absolutely, present in the supplementary data of the paper. It is also absolutely crystal clear that the paper itself, excluding supplementary information, does not accurately represent the the actual findings in the study.

If you had to summarise the paper in human terms you could say that applying severe protein restriction to your kids while topping up their calories with sugar and soyabean oil would hopefully result in them being taken away from you and placed in to care before they died.

Please don't try this at home.

Peter

My thanks to Rory Robertson for his attempts to have this paper retracted and more accurately rewritten and to Grant Schofield for tweeting about his efforts.

Coronary Artery Calcium Score and Scleroderma

Dr Malcolm Kendrik has a very interesting post over on his blog relating to coronary artery calcium scoring. I think it is fair to say that he is not in favour of the test.

My ears pricked up (metaphorically) when he mentioned myositis ossificans, about which he comments "This does not end well".

I have spent some time in the past thinking about pathological arterial calcification, as applied to the aorta of of patients with familial hypercholesterolaemia. Bear in mind that the dietary advice for patients with FH is about the worst you could possibly imagine and, of course, has no evidence base. My thoughts and assorted links are in an old blog post here. At the time I had never heard of Sci-hub so was unable to access this rather neat diagram of the mechanism of action of insulin, Pi and pyrophosphate:

Back to pathological soft tissue calcification. Clearly the obvious question about myositis ossificans has to be to ask whether it is in part driven by hyperinsulinaemia/hyperglycaemia or both.

As far as I am aware this is not a question which had been asked. It is simply genetic and that's it.

However, a similar question has already been answered in relationship to a serious generalised soft tissue mineralisation condition described as "calcinosis and scleroderma", back in a publication from 1932 (apologies to the person who tweeted the link, I didn't note their name to acknowledge. And twitter is ephemeral). That is too long ago to be listed on Pubmed so if you would like to read it you can go and ask Elsevier how much they would like to charge you for a peek in to the past or you can go to that awful place that none of use ever use to download any paper for free.

CALCINOSIS AND SCLERODERMA: TREATMENT OF A CASE BY USE OF THE KETOGENIC DIET

"Calcinosis and scleroderm" looks to be one of a family of soft tissue calcification diseases. The case report from 1932 describes the complete remission of this extremely unpleasant condition in a child following a period of time on ketogenic diet of the type used at the start of the last century, before dieticians were invented/summoned from Hades.

Did the ketogenic diet resolve this child's pathological calcification by suppressing insulin levels, glucose levels or both? Does it work by lowering alkaline phosphatase production by cells in/around inflammatory lesions? Or by some other mechanism?

Would it do the same for pathological arterial calcification? Given a tool like the ketogenic diet, perhaps there is some logic to CAC testing?

Unless you feel that tissue calcification is an appropriate part of healing until it gets to scleroderma levels...

Peter

Barja, an aside

I quite enjoyed Barja's review

The Cell Ageing Regulatory System (CARS)

but found this section a little uncomfortable:

Hmmmmmm. Plant based, healthy fruit and vegetables, bad animal fats. Not my sort of outlook really.

In another of his publications here

Highly resistant macromolecular components and low rate of
generation of endogenous damage: Two key traits of longevity

there is this comment

"It was also found that 6–7 weeks of dietary restriction are enough to decrease MitROS production and 8-oxodG in mtDNA and nDNA in rat liver (Gredilla et al., 2001a )".

Gredialla et al (incl Barja) 2001a is

Effect of short-term caloric restriction on H2O2 production and oxidative DNA damage in rat liver mitochondria and location of the free radical source

Here they found, by eyeball, an approximately 50% reduction of in 8-oxodG in mitochondrial DNA after those six weeks of quite severe caloric restriction:

Now let's compare this with the degree of damage reduction (this time using the term 8-OHdG as the marker rather than oxo-8dG, which appears to be the same thing).

Here's the change in mtDNA damage marker in brain mitochondria using F3666, one of the worst ketogenic diets around:

Just by eyeball I make the drop in mtDNA damage out to be greater than 50% by two days and something like 75% by three weeks. On ad-lib food consumption. No hunger.

Considering that F3666 does not extend longevity in mice (it doesn't shorten lifespan either, despite causing liver damage and it does actually improve health during ageing in rodents) this does, for me, slightly knock some of Barjas core ideas.

Sad but true.

Peter

Stearic acid again

Better post this one while I have a few minutes. I picked it up while looking for refs for Gustavo Barja's epic The Cell Ageing Regulatory System (CARS) in which longevity is tied to the Double Bond Index of the mitochondrial inner membrane (Thanks Bob!). BTW it is possible to modify the DBI but, with current data, it looks like you cannot alter the saturated or MUFA percentages, it is replacing omega 3s with omega 6s which mimics the mitochondria of long lived mammals!

Anyway, here is the cocoa butter paper:

Differential effects of saturated versus unsaturated dietary fatty acids on weight gain and myocellular lipid profiles in mice

Here are the diet compositions:

The line in red is the total percent of calories from linoleic acid in each diet. Here are the body weight changes:

The bottom two lines are the low fat high carbohydrate diet which happens to come in at just 1% linoleic acid and the cocoa butter diet which comes in at 1.4% of calories as linoleic acid. The high palmitic acid gives the most weight gain as it delivers 4.5% of calories as PUFA. Olive oil is a close second, also with 4.5% linoleic acid. The oddity is the safflower oil diet which is very high in PUFA but only gives intermediate obesity. Quite what is going on here is difficult to say but you have to wonder at what level of omega 6 PUFA that "next level up" signalling (lipid peroxide based) kicks in. No data on that, just a guess/excuse from the Protons perspective. There are a number of other studies showing this phenomenon of limited weight gain with safflower oil.

Still, stearic acid as cocoa butter is still looking pretty good. All of the high fat diets were based around different fat sources placed in to the D1245 background so are equally high in sucrose and starch too, comparable amounts across all of the higher fat diets.

Peter

Protons (53) a formula

A couple of things came up in emails recently. First is that I never mention that I had a chat with Ally Houston on the Paleocanteen podcast. It was fun. I think I sound like me. It's here

https://paleocanteen.co.uk/peter-dobromylskyj-hyperlipid/

Second is that karl asked if there was a general formula for working out the F:N ratio for assorted fatty acids.

Edit: cavenewt pointed out that for people unfamiliar with the FADH2:NADH ratio concept there is a reasonable introduction at Protons: FADH2:NADH ratios and MUFA. PubMed-ing Dave Speijer and CoQ makes good reading too. End edit.

There wasn't but given a few minutes and some algebra it works out like this for even-numbered, fully saturated fatty acids of carbon skeleton length n:

F/N   =   (n-1)/(2n-1)

So stearate (C18) is 0.486

Palmitate (C16) is 0.484

Caprylate (C8) is 0.467



For MUFA/PUFA you just subtract one FADH2 per double bond (db). This doesn't affect the NADH term.

F/N  =  (n-1-db)/(2n-1)

Oleate (db = 1) is 0.457

Oleate is the MUFA of stearate. Saturated fats allow us to resist insulin, MUFA allow insulin to act.

Linoleic acid, also C18 but with two double bonds, gives 0.429

This is lower than stearate or oleate. The switch for ROS generation occurs between roughly 0.486 (high physiological ROS) and 0.457 (low physiological ROS). LA is lower than oleic acid.

Glucose has an F/N ratio, from memory, of 0.2 so LA is the "glucose-like" of the common fatty acids, in Mike Eades' terminology, and so will fail to generate fatty acid appropriate ROS. Which will allow continued insulin action when it should be resisted. That will make you fat, and the loss of calories in to adipocytes will make you hungry. The exact opposite of stearic acid...

Happy New Year all.

Peter