Monday, September 21, 2020

Protons (64) The miracle of fish oil (6)

Preamble: I started this current series of post about the ability of fatty acids with multiple double bonds to limit weight gain. To me, this is a paradox. Paradoxes are, without a doubt, the most productive sources for the development of an idea. Even as I started this current post I had no idea where it was going to end up and was bit surprised at where the metabolism took me. So be it. Let's begin.


Beta oxidation in peroxisomes consumes half the amount of oxygen as in mitochondria. The first step of oxidation of saturated fats runs like this:

R-CH2-CH2-COOH + FAD -> R-CH=CH-COOH + FADH2

In peroxisomes this is followed by

FADH2 + O2 -> FAD + H2O2

then

2xH2O2 -> Signalling -> Catalase -> 2xH2O + O2

The energy from FADH2 is released as heat and half the oxygen is regenerated.

The NADH from beta oxidation is of no immediate use in a peroxisome and has to be transferred to mitochondria before it can be utilised. I suppose it could be phosphorylated to NADPH for anabolism but I have no data on that. It's not clear how reducing equivalents might be transferred from peroxisomes to mitochondria. There is speculation about something along the lines of the malate-aspartate shuttle used to import cytoplasmic NADH in to mitochondria.

It's also something of a truism that peroxisomes cease beta oxidation at C8 and then export this (by uncertain mechanism) to mitochondria for completion of oxidation. Digging back through the reference trail leads to the origin of this as the finding that isolated peroxisome preparations happily oxidise lauric acid but won't oxidise caprylic acid (much). Clearly oxidising DHA will never produce caprylic acid directly because there are double bonds within the residual eight carbon atoms. What exactly happens to truncated DHA at the C8 length appears to be an unasked question.

So beta oxidation in peroxisomes produces heat, NADH, acetyl-CoA and signalling H2O2. And perhaps some caprylic acid from any saturated fatty acids being oxidised. It requires markedly reduced oxygen consumption and appears to result in proportionately lower CO2 production, which gives an unchanged respiratory exchange ratio (RER).

Going back to











The round symbols are the fish oil fed groups. Average VO2 through 24h is reduced by fish oil from about 3500ml/kg/h to about 3000ml/kg/h, ie that's a just under 15% reduction.

Here are the RER figures, still fish oil as circles. As expected high fat diets show a low RER, low fat diets show the converse. The reduced O2 consumption is exactly balanced by a reduced CO2 production and the RER is still largely set by the dietary carbohydrate-fat ratio.











Clearly, under fish oil, approximately 15% of calories are being used to generate heat and anabolic substrate without consuming oxygen or being transferred to the ETC.  Provided there is enough fish oil to stimulate peroxisomal proliferation the changes are quantitively independent of the absolute amount of fish oil. 

So with fish oil at as low as 10% of calories, not all of which are PUFA, VO2 is dropped by 15% suggesting that the peroxisomes are activated and are oxidising more fatty acids than just the PUFA from the diet. Presumably on the low fat fish oil diet the peroxisomes are also metabolising palmitate and oleate derived from carbohydrate by de novo lipogenesis too.

If we go to this paper:

Peroxisomal and Mitochondrial Oxidation of Fatty Acids in the Heart, Assessed from the 13C Labeling of Malonyl-CoA and the Acetyl Moiety of Citrate

we can see, by clever carbon 13 labelling, that peroxisomal derived acetyl-CoA in cardiac muscle (and I would guess most other extra-hepatic sites) does not enter mitochondria, it all stays in the cytoplasm as malonyl-CoA.














These data are from perfusing hearts with docosanoate, a C24, fully saturated, fully peroxisome targeted fatty acid. We get lots of labelled malonyl-CoA in the cytoplasm, minimal labelled citrate in the mitochondria.

The next fascinating paper (HT to Peter Schmitt for the link) used erucic acid, another peroxisome targeted fatty acid.

Peroxisomal oxidation of erucic acid suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation in the rat liver

In the liver peroxisomal oxidation of fatty acids generates acetate but this is still converted to acetyl-CoA and then malonyl-CoA without entering mitochondria. We know from the Randle cycle that malonyl-CoA is an inhibitor of fatty acid oxidation so it should come as no surprise that erucic acid feeding to peroxisomes inhibits fatty acid oxidation in mitochondria. So we end up with lipid accumulation within the liver, progressing to fatty liver and NASH. I have mention before that in rodent models of alcoholic fatty liver disease fish oil is one of the most effective generators of alcohol induced liver damage...

But perhaps the best line from this last paper is:

"Peroxisomal metabolism of erucic acid also remarkably increased the cytosolic NADH/NAD+ ratio..."

It seems very, very unlikely that fish oil will be any different.

We find ourselves in a situation where peroxisomal oxidation of fatty acids generates benign heat combined with large amounts of anabolic substrate and a high NADH:NAD+ ratio while requiring reduced oxygen consumption and simultaneously inhibiting mitochondrial fatty acid oxidation and shifting metabolism to glucose.

Does that look like a recipe for cancer?

It does to me.

I had no idea that there is a large literature looking at the role of peroxisomes in all sorts of cancer types. Woohoo, they are a drug target! Perhaps avoiding peroxisome activating fatty acids and their derivatives might be a better approach. Apart from accepted Bad Things like drinking erucic acid or 4-HNE (a superb peroxisome activator) we might ask serious questions about drinking bulk fish oil.

Perhaps don't.

Peter

Addendum: I recall this study (observational but not a food frequency questionnaire in sight), which I was fairly uncertain about back in 2013

Plasma phospholipid fatty acids and prostate cancer risk in the SELECT trial

Now I'm more convinced...

23 comments:

raphi said...

interesting to see you bring it back to (intracellular) NADH/NAD+ and cancer. i have to wonder what role peroxisomes play in cell's who's mitochondria broken...

Peter said...

raphi,

Not my idea, the data push me there!

I'm wondering at the moment if it is the peroxisomes which break the mitochondria... It's an interesting possibility. Obviously both are physiological under evolutionary conditions. But under modern conditions? And clearly a low bodyweight and a low insulin exposure, while good, may be obtained at significant cost if they are secondary to managing lots of double bonds/lipid peroxides via peroxisomes. Stuff is so interesting.

Peter

ctviggen said...

Okay, you people are killing me. ;-) As I sit here realizing that I did not bring enough to eat for my first meal (at 11:00) of the day, and turn toward my canned fish, which is my emergency food supply at work... And this fish is in water of course, since I'm afraid even "olive oil" has been adulterated with PUFAs.

You keep upending everything I thought was sacrosanct, like fish and Omega 3s. (Gave up on the oil pills a while ago, but still thought fish was "good".)

Does anyone make canned beef? ;-)

Peter said...

Yep, it's called corned beef (no corn). Just beef, beef fat and salt. A true health food apart from probably zero ascorbate.

BTW it's the data that are the problem. I have absolutely no choice in where they lead me. They're a bugger.

Peter

mroush731 said...

Hi, Peter, I’m somewhat new to your fascinating blog. I have for some years taken a teaspoon of Cod Liver Oil each day, amounting to about 50 calories. I guesstimate that’s about 2% of my daily total.
I credit the addition of CLO to improving my dental health and resolving some mental health issues like anxiety and seasonal affective disorder. I’m not asking for medical advice, but would you feel the same level of concern with someone who is consuming CLO that amounts to 2% of calories as you do with some of the data you’ve presented, which often include subjects who consumed greater than 10% of calories from fish oil? Other than the CLO, I’m not a heavy consumer of seafood. Perhaps 2-3 servings per month, typically oysters or salmon.
Thank you for the thoughtful information you provide.

Puddleg said...

This seems as good a place as any to drop this idea -

as far as I can see, the level of l-carnitine in the lean tissues of an animal closely correlates with the proportion of SFA in the fat of that animal.
Is l-carnitine synthesis driven specifically by the amount of SFA requiring oxidation? (perhaps because other fats can avoid the carnitine shuttle in various way?)

Also, some really cool theories about hydrogen selenide as an antiviral oxidant and as the fourth gasotransmitter, and a note on the synthesis of selenocysteine from selenide in humans,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246001/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915170/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065758/

The sweet spot seems to be 100-200mcg/day with a risk of insulin resistance at 300 mcg-day

https://pubmed.ncbi.nlm.nih.gov/24774067/

Peter said...

Hi mroush731, ah, the trade off between repleting DHA/vitA etc and activating peroxisomes.I doubt a few grams a day could do any harm. Personally I wouldn't go for 10% of my calories! There are early studies from supplementing fish/fish oil which reliably induced an elevated fasting glucose in humans but as researchers have gotten better at supporting the prevailing paradigm these no longer occur and any sort of review can now conclude fish oil is magic while being glucose neutral. It amazes me how easy it seems to be to get the result you need with fish oil! The 4HNE study with fat rodents on fish oil is convincing to me because they were not looking at fish oil, so got the biological effect, not the paradigm supporting result!

George, thanks! I finally got to follow your links, it's been on my to-do list since you posted them previously. I have to say the gaseous transmitter aspect id fascinating, having come up through nitric oxide/ICU and ventilation perfusion mismatch in horses... I am also wondering about how the balance of Se intake/excretion is controlled and whether this fails in metabolic syndrome, much as it does for magnesium...

Peter

Peter said...

George,

Do you have a link for the carnitine comment?

Ta, Peter

Puddleg said...

Hi Peter,

there's a table here where you can see that ruminant meat has much higher carnitine content than pork, which has much more than fish or chicken.
https://lpi.oregonstate.edu/mic/dietary-factors/L-carnitine

And another more detailed one here
https://lib.dr.iastate.edu/cgi/viewcontent.cgi?article=2865&context=etd

This generally follows the SFA/PUFA ratio of these foods, and I think it needs some explaining!

"Furthermore, increasing muscle total carnitine content in resting healthy humans (via insulin-mediated stimulation of muscle carnitine transport) reduces muscle glycolysis, increases glycogen storage and is accompanied by an apparent increase in fat oxidation. By increasing muscle pyruvate dehydrogenase complex (PDC) activity and acetylcarnitine content at rest, it has also been established that PDC flux and acetyl group availability limits aerobic ATP re-synthesis at the onset of exercise (the acetyl group deficit). Thus, carnitine plays a vital role in the regulation of muscle fuel metabolism."
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2075186/


Puddleg said...

The question of selenium causing insulin resistance and increasing mortality in high-dose supplements, not mirrored as far as I can see in natural high-dose populations, may have a simple explanation - supplements allow us to consume micronutrients without protein.
If you have no cysteine or methionine coming in when you take Se (either because you're not eating protein, or perhaps it can happen naturally if the Se level is high in a low-protein food and diet) then the selenocysteine formed will be incorporated into all proteins, not just the ones that require it. Including the insulin receptors, which will suffer a relative loss of function.
(similarly, though for different reasons, pyridoxine toxicity can be triggered by supplementing on a low-protein diet)

If we think that insulin resistance causes CVD, then the increased risk from (mostly) natural high Se levels is not great, see fig 5 here, but the intervention studies have more alarming results, and I think the competition of selenium- vs sulphur-amino acids in protein fed vs unfed states can explain this. There is next to no evidence of Se toxicity from Brazil nuts, which are high in both Se and protein.
https://www.tandfonline.com/doi/full/10.1080/10408398.2020.1803200

Passthecream said...

George, that's an interesting idea about the supplements vs appropriate protein intake. Do you think it's more widely relevant ie to other micronutrients?

Peter, what's the situation with magnesium vs ms?

Martin Sip said...

If I read stochiometry well, oxygen is consumed although somewhat less because half of it is regenerated, is that right or I am missing something?:
2xFADH2 + 2xO2 -> 2xFAD + 2xH2O2 -> (Signalling -> Catalase) -> 2xFAD + 2xH2O + 1xO2

Peter said...

Sipak, yes, you're absolutely correct. I'm now also wondering how much carbon from peroxisomal beta oxidation does actually end up as CO2... Too late at night to correct the post now, a job for tomorrow. And how much peroxisomal beta oxidation is actually happening to get that 15% drop in O2 consumption.

Thanks

Peter

Passthecream said...

George, or Peter or whoever might know, do peroxisomes in human cells increase or decrease in number to keep up with substrate changes as they do in yeast?

Will.that affect the regulation of carnitine?

Peter said...

Hi sipak,

Corrected now, many thanks.

Pass, the impression I get ie that they increase in number, enzyme content and enzyme activity but it's not been something I've felt the need to pin down. I'd not seen the carnitine connection. I spent some time last night looking at clofibrate and peroxisomes, activity of which it induces potently. There has long been interest in it as a wonder drug and also long term niggling concern that it might be a carcinogen (hence I was looking, bias again). There is now a raft of retrospective observational papers showing it is preventative but the challenges of correcting for other variables make this so challenging that you can probably show what you like with observational studies...

Peter

Tucker Goodrich said...

"Fat and Weight Gain (a Note to Peter) and the Essentiality of Linoleic Acid"

https://yelling-stop.blogspot.com/2020/09/fat-and-weight-gain-note-to-peter-and.html

Passthecream said...

(If this is double posted I will delete.)

Tucker:

This 2001 paper

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC64677/

Is one often quoted source of this idea:

" Vertebrates lack the Δ12 and Δ15 fatty acid desaturases responsible for converting oleic acid (18:1n-9) into linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) and thus are unable to biosynthesize polyunsaturated fatty acids (PUFA) de novo (1, 2). "

Followed immediately by this conclusion derived from the same sources:

" PUFAs therefore are essential dietary nutrients for vertebrates (1, 2). "

1. Tinoco J. Prog Lipid Res. 1982;21:1–45.

2. Holman R T. J Am Coll Nutr. 1986;5:183–211.

I haven't yet tracked it back beyond that but I suspect I'll find that same early work you refer to as the Ultimate Oracle of it.


The fact that countless generations of vertebrates have never regained the ability to produce LA and ALA from OA which itself can be produced by native desaturases from saturated fats present, seems like a great big pointer to me, a sign, a billboard: "Look here, smoking gun, can't make 'em, don't need to, stop eating these things, put the marg down and step away from the cabinet!". Etc.

Isn't that the simplest and most basic conclusion you could draw???

Frunobulax said...

Peter, sorry for responding to an older post. This doesn't relate strictly to this post, more to n-3 fatty acids in general. I'm trying to wrap my head around the fact that trial results with n-3 PUFAs are all over the place, and not always for the usual reasons (bad study design or wrong assumptions/axioms, like cholesterol=spawn_of_hell). Reading your recent PUFA posts seems to confirm something that has been a gut feeling of mine.

To sum it up in two sentences for someone who doesn't want to dig too deep into research, I'd say "Both n-3 and n-6 FAs are bad for us if we consume more than we should (compared to a hunter-and-gatherer baseline). But if you were on a diet high in n-6 PUFAs, you might want to consume some n-3 PUFAs like flaxseed oil to offset the effects of high n-6 PUFAs." Would you agree?

It seems clear to me that n-6 FAs are inflammatory and prevent us from manufacturing DHA and EPA from n-3 alpha-linoleic acid (https://scialert.net/abstract/?doi=ijbc.2016.1.6). n-3 PUFAs might keep n-6 LA in check though (https://iubmb.onlinelibrary.wiley.com/doi/full/10.1002/iub.1428, https://scialert.net/abstract/?doi=ijbc.2016.1.6).

As n-6 LA is stored in significant amounts in our lipid cells (Guyenets study), it seems to me that this could create some havoc when we lose weight and this fat is burned off/released into the bloodstream. So it might be beneficial to supplement some n-3 PUFAs there.

If this was true, it could explain the wildly varying study results involving n-3 PUFAs. They could be highly beneficial in some trials where people on western diet lose weight, and they could be inefficient or obesogenic in trials that are not designed to lose weight. (And of course n-3 PUFAs would appear beneficial if n-6 PUFAs were used as controls, as you remarked in an earlier post. But I'd put this under "bad study design".)

Peter said...

Hi Frunobulax,

The omega 3 idea would be fine except that in the rodent study ALA was as good an inhibitor of DHA synthesis as LA was. So unless there is some core function of ALA per se it’s not going to help through DHA synthesis. I have never looked in to whether lipids derived from ALA are useful or harmful. There are suggestions that those from DHA may be helpful but I agree this may simply reflect the usual high LA background to most studies. I have to accept that consuming DHA in amounts which might meet overall needs could possibly be a good idea, there are suggestions that the brain does need a few tens of milligrams a day and there might be other functions I’ve never thought about…

I would also agree that losing weight when you have adipocytes loaded with LA is putting you between a rock and a hard place.

I recall this study from Volek’s group https://pubmed.ncbi.nlm.nih.gov/18046594/ where VLCKD showed a greater increase in both LA and all omega 6 PUFA combined cf the low fat group. Whether you could off-set this with omega 3s, of any length, seems like a pretty open question. Quite an important question as a huge percentage off the world population is in this bind.

Peter

Frunobulax said...

Peter, I probably have plenty of LA in my fat reserves (only moderate overweight remains, lost 40 pounds with another 30 to go). I can't fast for more than 2 days apiece, due to massive fatigue and horrendous restless legs. Now I do have ME/CFS so there may be special circumstances, and I suspect that my lipid cells have stored plenty of BPA, pesticides and whatever these buggers store that is released when fasting (or when losing weight quickly, for whatever reason -- so I figure it's not a lack of electrolytes). So I do occasionally fast a day or two, but no longer.

With all limitations of n=1 experiments, a few months ago I went on a longer fast, where I fasted for a week and fatigue set on only after 5 days. The difference was that I consumed maybe 30-40ml of fresh flaxseed oil during the first days, along with a cup of bone broth. (I didn't take notes, unfortunately. But I do remember that at some point I decided "let's go water fast for the last couple of days".) I will repeat this experiment as soon as it is feasible (maybe not next week when I'm on vacation with my little daughter) :)

Peter said...

I think Tucker Goorich suggests crashing the PUFA out as fast as possible, preferably before there is a tumour for 13-HODE to act one. I have to say I think he is probably correct. It's interesting that the Paleomedicina group have success without strict limiting of PUFA in the diet. So maybe enough saturated fat can offset the PUFA. Lots to find out!

Peter

cavenewt said...

This showed up on the JAMA Most Read This Week list today: Effect of High-Dose Omega-3 Fatty Acids vs Corn Oil on Major Adverse Cardiovascular Events in Patients at High Cardiovascular Risk—The STRENGTH Randomized Clinical Trial.

"Interventions Participants were randomized to receive 4 g/d of omega-3 CA (n = 6539) or corn oil, which was intended to serve as an inert comparator (n = 6539), in addition to usual background therapies, including statins." >snort< "inert comparator".

"Conclusions and Relevance Among statin-treated patients at high cardiovascular risk, the addition of omega-3 CA, compared with corn oil, to usual background therapies resulted in no significant difference in a composite outcome of major adverse cardiovascular events. These findings do not support use of this omega-3 fatty acid formulation to reduce major adverse cardiovascular events in high-risk patients."

https://jamanetwork.com/journals/jama/fullarticle/10.1001/jama.2020.22258

Unknown said...

How do our mammal brethren the whales get fat? Via insulinogenic action from eating carbs that are from algae eating plankton? Any insight or opinion?