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Monday, 23 February 2015

Why the High-Fat Hep C Diet? Rationale and n=1 results.



I originally started this blog to publicise the hypothesis that a diet low in carbohydrate and linoleic acid, but high in saturated fat and long-chain PUFA, will inhibit HCV replication.

The blog header with the pig above is the abstract for this hypothesis.

I first worked this out in 2010 after reading Dr Atkins New Diet Revolution while studying HCV replication. The lipid patterns in low-carb dieters - low TG and VLDL, high HDL, normal or high LDL - are those associated with lower viral load and improved response to treatment in HCV cases.
The mechanics of HCV replication and infection support this link.


HCV inhibits PPAR-a, a ketogenic diet reverses this inhibition

I wrote a fairly comprehensive version of the hypothesis in 2012:
http://hopefulgeranium.blogspot.co.nz/2012/02/do-high-carbohydrate-diets-and-pufa.html

Recently I was sent a link to an article that cited this paper:
http://www.journal-of-hepatology.eu/article/S0168-8278(11)00492-2/pdfHCV and the hepatic lipid pathway as a potential treatment target. Bassendine MF, Sheridan DA , Felmlee DJ, et al. Journal of Hepatology 2011 vol. 55 j 1428–1440

This review compiles a great deal of supporting evidence regarding the interaction between HCV and lipids, and between lipids and HCV. The only thing missing is the role of carbohydrate. It mentions multiple lipid synthetic pathways as targets for indirect-acting antiviral drugs (IDAA), pathways which are also well documented as targets of low carbohydrate ketogenic diets, or of saturated fat in the diet (in the case of the LDL-receptor complex).

From 2012:
A little n=1 experimental data; 4 years ago (2008) my viral load was 400,000 units, now after 2 years of low carb dieting and intermittent mild ketosis (2012) it is 26,000.

Later in 2012:
Total Cholesterol:  6.7  H     
Triglyceride:          0.8         
HDL:                     1.63              (63.57)
LDL (calc.)            4.7   H    
Chol/HDL ratio:     4.1          

HCV viral load on this day (21st May 2012): 60,690 IU/mL (4.78 log)



Lipid panel from 07 Feb 2012, during ketogenic diet phase (non-fasting)

Total Cholesterol: 8.9   HH  (347.1)
Triglyceride:         1.3          (115.7)
HDL:                    1.65         (64.35)
LDL (calc):           6.7    H    (261.3)
Chol/HDL ratio:     5.4   H

HCV viral load on this day: 25,704 IU/mL (4.41 log)

From 2014:
On a personal note, I have started an 8-week trial of Sofosbuvir and GS-5816 (Vulcan). It is day 11 and it seems tolerable so far.
A pre-trial blood test on 22nd October was normal except for these counts:
AST 74
ALT 174

and viral load was 600,419 (log 5.78), counts consistent with the tests I've had done this last year.

But the day the trial started, 18th November, before my first dose, things were different:
AST 21

ALT 32
Viral load 27,167 (log 4.43)

The low viral load is easy to explain; I get a consistent 1 log drop (to 14,000-60,000*) when I try to eat very low carb (50g/day or lower) and an elevation to 400-600,000 when my carbohydrate intake is over 50g/day. When I ate very high carb (but took antioxidant supps) it was as high as it was on 22nd October. So for me the tipping point seems to be where ketosis begins, and other variations don't have much effect; it's an on/off switch, not a dial (and the name of that switch is PPAR-alpha).
[edit: though the very low scores are at ketogenic, or nearly so, carb intakes, the exact increase in carbohydrate needed to cause a significant increase in viral load seemed to vary]
(I do however, according to CAPSCAN elastography, have zero excess fat in my liver, which is an effect of low carb in general, as well as avoiding vegetable seed oils).

My belief is that my viral load was much higher than any of these counts previous to 2003. This was the year I started taking antioxidant supplements, eating a bit better (in a normal, confused "healthy eating" pattern), and using herbal antivirals like silybin. Prior to that I was seriously ill, and I believe that my viral load would have reflected my extra autoimmune symptoms, signs of liver failure, and elevated enzymes. Unfortunately in those days one didn't get a PCR unless one was considering donating one's body to interferon, which I was not.

* I don't seem to have a record of the date of the 14,000 VL reading, but will include it when I find it.

Summary:
A very low carbohydrate ketogenic diet, without enough PUFA to lower LDL artificially, had a significant inhibitory effect on HCV viraemia in my case.
Effective DAA drugs for HCV infection are now available. There is a ~98% SVR rate at present. These drugs are expensive, they sometimes have side effects (though much less so than interferon + ribavirin), and interferon + ribavirin is still being used.
If my results are more generally applicable, VLCKD diet offers an adjunct therapy for patients with a high viral load, steatosis that relates to diet and lifestyle as well as HCV infection, or a need to postpone treatment. In people who oppose or cannot complete or afford treatment, it offers a way to manage the disease, and in particular to reverse the autoimmune syndromes caused by immune complexes when viraemia is excessive.


Friday, 20 February 2015

Gluconeogenesis Drives Ketogenesis - role of the Nutritional Prometheus.

In trying to explain the findings of Newburgh and Marsh*, and of Karl Petren, from 1923 that switching to a high fat, restricted protein, and very low carbohydrate diet - a ketogenic diet - suppresses diabetic ketoacidosis (DKA) in diabetics without access to insulin, I can't help noticing that gluconeogenesis is a driver of ketogenesis. DKA is a dehydrating syndrome characterized by hyperglycaemia, due in large part to runaway gluconeogenesis, plus levels of ketone bodies, much higher than those seen in starvation or nutritional ketosis, which result in a lethal acidosis. And excess glucose and excess ketones are linked metabolically.

Remember the old saw, that fat burns in a carbohydrate flame? Laugh all you like, but this is true. And it is even more true when the flame is taken away - when carbohydrate (glucose) is being stolen from mitochondrial metabolism. Gluconeogenesis involves a direct loss of oxaloacetate from the citric acid (Krebs, TCA) cycle. Without this oxaloacetate, the fat-burning flame sputters; the smoke that escapes from incomplete combustion is the ketone bodies. I'm not a chemist, but this seems to me a pretty certain way of interrupting the TCA cycle. And a very convenient one in evolutionary terms; at times when you need endogenous glucose, you can use a few extra ketones as well.




Pyruvate from glucose can supply acetyl-CoA or oxaloacetate, fatty acids can only supply acetyl-CoA, if there's no oxaloacetate acetyl-CoA can't be converted to citrate and is converted to ketone bodies instead (not shown).


[In Starvation] degradation of fatty acids in the liver proceeds more rapidly than usual, with augmented production of of acetoacetyl-CoA and acetyl-CoA and their products.
In addition there is a deficit of oxaloacetate and thus a decrease in formation of citrate.
The low level of oxaloacetate is further accentuated because it is being utilized for gluconeogenesis.
This further impairs operation of the citric acid cycle.

Ketosis incident to starvation is most frequently encountered clinically in gastrointestinal disturbances in infancy or pregnancy. Other circumstances in normal individuals in which excessive lipid and diminished carbohydrate are being metabolised may also lead to ketosis, e.g. renal glycosuria and abrupt replacement of a normal diet by one low in carbohydrate and very rich in lipid.

Clinically, the most important cause of ketosis is diabetes mellitus. In the diabetic individual, in contrast to the above situations, glucose is present in excessive amounts in the fluids of the body; however, the metabolic defect, viz., insulin deficiency, prevents glucose utilization from operating at a normal rate. From the point of view of the effect upon lipid metabolism, diabetes and starvation resemble one another.

In diabetic individuals with severe ketosis, urinary excretion of ketone bodies may be as high as 5,000mg/24 h and the blood concentration may reach 90mg/100ml, in contrast to normal values of less than 125mg and less than 3mg respectively.


Ketogenesis, from Principles of Biochemistry, 5th Edn, White A, Handler P, Smith EL. McGraw Hill, 1973, p577-578.


[NB: acetyl-CoA is also a precursor for cholesterol;
"The data suggest that, although acetyl-CoA is channeled towards ketone body formation in both diabetes and fasting, augmented cholesterol synthesis is evident only in diabetes." This suggests that the closer the diabetic diet gets to a ketogenic diet, the less cholesterol synthesis will be augmented - as does seem to be the case in practice.]




So what happens when a diabetic without insulin eats carbohydrate or excess protein?
As we saw in earlier posts, glucagon is released from pancreatic alpha cells in response to carbohydrate and protein. This elevates gluconeogenesis in the liver. Blood glucose is elevated by the meal and by GNG, and hyperglycaemia itself increases hepatic GNG further. Lipolysis is increased by the glucagon, so the liver has additional fatty acids to metabolize. Perfect conditions for ketogenesis to be enhanced above normal levels, because oxaloacetate is being extracted from the TCA in record amounts as this fat is being burned.

What happened when diabetics, in acidosis and without insulin, were switched to the Newburgh and Marsh ketogenic diet in 1923?
With no glucose and minimal protein to trigger glucagon, hepatic GNG is lower. With no glucose to add its load to hyperglycaemia, there is less portal hyperglycaemia to additionally drive GNG.
Less GNG = less ketogenesis.
And, as a bonus, it is likely that dietary fat has an inhibitory effect on lipolysis that is independent of hormonal controls. As long as it's saturated, or not polyunsaturated - in 1923, endocrinologists favoured butter as a source of fat.


Beef tallow diet decreases beta-adrenergic receptor binding and lipolytic activities in different adipose tissues of rat.
Matsuo T, Sumida H, Suzuki M. Metabolism. 1995 Oct;44(10):1271-7.

Abstract
The effects of dietary fats consisting of different fatty acids on lipolytic activity and body fat accumulation were studied in rats. Sprague-Dawley male rats were meal-fed an isoenergetic diet based on either beef tallow or safflower oil for 8 weeks. Lipolytic activities in epididymal and subcutaneous adipose tissues were lower in the beef tallow diet group than in the safflower oil diet group. Body fat accumulation was greater in rats fed the beef tallow diet versus the safflower oil diet. Norepinephrine (NE) turnover rates used as an index of sympathetic activities in adipose tissues were lower in the beef tallow diet group. beta-Adrenergic receptor binding was determined with [3H]dihydroalprenolol. Binding affinities of beta-receptors in adipose tissues were significantly lower in the beef tallow diet group. Membrane fluidities of adipose tissues were also lower in the beef tallow diet group. Membrane fluidities were correlated with the affinities of the beta-receptor. We believe from these correlations that the decreases in beta-receptor binding affinities are due to the changes in membrane fluidities. The results of the present study suggest that intake of the beef tallow diet promotes body fat accumulation by reducing lipolytic activities resulting from lower beta-receptor binding and sympathetic activity in adipose tissues.

Dr Bernstein describes the mechanism of DKA differently; he doesn't consider that the liver is the main source of ketones, or that gluconeogenesis drives ketogenesis. His description addresses the pathology of DKA well, but not I think the early links in the chain of causality. Perhaps the difference is that he is describing the failure of insulin to work, and I am describing the long-term absence of insulin. But we are both agreed; dietary carbohydrate is the cause of DKA in diabetics.

"Furthermore, the higher your blood sugars go, the more insulin resistance you will experience. The more insulin-resistant you are, the higher your blood sugars are going to be.

A vicious circle. To make the circle even more vicious, when you have high blood sugars, you urinate—and of course what happens then is that you get even more dehydrated and more insulin-resistant and your blood sugar goes even higher. Now your peripheral cells have a choice—either die from lack of glucose and insulin or metabolize fat. They’ll choose the latter. But ketones are created by fat metabolism, causing you to urinate even more to rid yourself of the ketones, taking you to a whole new level of dehydration."
See also http://www.diabetes-book.com/ketoacidosis-hyperosmolar-coma/
and http://www.diabetes-book.com/diabetes-dehydration/

Edit: here's a bit more on starvation, from this book
After about 3 days of starvation, the liver forms large amounts of acetoacetate and d-3-hydroxybutyrate (ketone bodies; Figure 30.17). Their synthesis from acetyl CoA increases markedly because the citric acid cycle is unable to oxidize all the acetyl units generated by the degradation of fatty acids. Gluconeogenesis depletes the supply of oxaloacetate, which is essential for the entry of acetyl CoA into the citric acid cycle. Consequently, the liver produces large quantities of ketone bodies, which are released into the blood. At this time, the brain begins to consume appreciable amounts of acetoacetate in place of glucose. After 3 days of starvation, about a third of the energy needs of the brain are met by ketone bodies (Table 30.2). The heart also uses ketone bodies as fuel.

And diabetes:
Because carbohydrate utilization is impaired, a lack of insulin leads to the uncontrolled breakdown of lipids and proteins. Large amounts of acetyl CoA are then produced by β-oxidation. However, much of the acetyl CoA cannot enter the citric acid cycle, because there is insufficient oxaloacetate for the condensation step. Recall that mammals can synthesize oxaloacetate from pyruvate, a product of glycolysis, but not from acetyl CoA; instead, they generate ketone bodies. A striking feature of diabetes is the shift in fuel usage from carbohydrates to fats; glucose, more abundant than ever, is spurned. In high concentrations, ketone bodies overwhelm the kidney's capacity to maintain acid-base balance. The untreated diabetic can go into a coma because of a lowered blood pH level and dehydration.

Note for future research: Mammals can synthesise oxaloacetate from pyruvate, but what if this step depends on insulin (which suppresses ketogenesis) and the conversion of pyruvate to acetyl-CoA doesn't?
The diabetic hepatocyte is swamped with glucose, it can't resist metabolising it, and 65-85% of the carbon from this glucose is recycled as GNG glucose.
What if this glucose, without the guiding hand of insulin, is, like fatty acids, a poor source of oxaloacetate and a good source of acetyl-CoA? After all, its metabolism is not suppressing ketogenesis - the opposite seems to be true.
Ketone bodies for use by heart muscle in normal hepatic metabolism are produced from glycogen, according to the first text I quoted.
So - is glucose itself a ketogenic substrate under certain conditions?
The quest continues...

Sunday, 1 February 2015

The Guinea Pig Model of Atherosclerosis


This is the kind of research that doesn't get as much attention as it used to.
Animal models are vital ways of testing theories that it would be unethical to test on humans, and when theories or novel chemicals are tested on unsuspecting humans, we refer to those humans as "guinea pigs". Not mice, rabbits, rats or any other rodent, but cavia porcellus. The guinea pig was used by Lavosier to demonstrate, by melting snow around his calorimeter, that respiratory gas exchange is a combustion, and by Pasteur, Roux, and Koch in their germ experiments, but fell into neglect at the end of the 20th century: only 2% of laboratory animals in the USA are currently guinea pigs.

In the early history of the lipid hypothesis, the rabbit model of atherosclerosis was developed by Anitschkow in 1911:

"When fed fat and cholesterol, rabbits develop high TC levels and subsequent fatty deposits in their blood vessels. When cholesterol is taken out of their diet, TC levels generally reduce and the fatty deposits may regress. If not used as conclusive evidence as to the process in humans, such experiments are said to be supportive of the theory that under conditions of high TC, cholesterol is more likely to be deposited in human arteries."[1]

The amount of cholesterol fed in these experiments - 0.2% or 0.25% of dry matter - probably exceeds what a human could consume, and of course rodents in nature have a minimal exposure to dietary cholesterol. The lesions seen do not correspond exactly to human atherosclerosis, and the role of saturated and unsaturated fats, or of foods like butter, with regard to progression in rabbits does not always match the lipid hypothesis predictions.

In 2006 Maria Luz Fernandez and Jeff Volek published a paper which should have stirred things up:
"Carbohydrate restricted diets have been shown to reduce plasma triglycerides, increase HDL cholesterol and promote the formation of larger, less atherogenic LDL. However, the mechanisms behind these responses and the relation to atherosclerotic events in the aorta have not been explored in detail due to the lack of an appropriate animal model. Guinea pigs carry the majority of the cholesterol in LDL and possess cholesterol ester transfer protein and lipoprotein lipase activities, which results in reverse cholesterol transport and delipidation cascades equivalent to the human situation. Further, carbohydrate restriction has been shown to alter the distribution of LDL subfractions, to decrease cholesterol accumulation in aortas and to decrease aortic cytokine expression. It is the purpose of this review to discuss the use of guinea pigs as useful models to evaluate diet effects on lipoprotein metabolism, atherosclerosis and inflammation with an emphasis on carbohydrate restricted diets."[2]

Rats and rabbits, on the other hand, don't resemble humans in the way they disburse lipids. The LDL fraction is tiny, they lack CETP and lipoprotein lipase, and generally diverge from human measurements in ways guinea pigs, it seems, don't. Even though rabbits and guinea pigs have pretty much the same natural diet - grasses, and their own poop. Guinea pigs and humans, unlike rats and rabbits, also can't synthesise vitamin C. Is there an orthomolecular connection here?
OMG put that pig on statins stat!
Long story short, guinea pig lipids react to lipid lowering drugs, PUFA and fibre in the same way and via the same mechanisms that produce effects in humans. If you feed a guinea pig cholesterol, fat, and carbohydrate it gets atherosclerosis. If you feed the guinea pig a very low carbohydrate diet (20%E SFA in these experiments - 10%CHO,65%FAT,25%PRO ) it's protected from cholesterol-induced atherosclerosis and inflammation. If you feed it a low-fat diet (55:20:25) the atherosclerotic syndrome progresses [3].

Most recently, the low carb diet reverses the metabolic alterations induced in guinea pigs by high-cholesterol feeding: the high-carb diet doesn't.

"Higher concentrations of total (P < 0.005) and free (P < 0.05) cholesterol were observed in both adipose tissue and aortas of guinea pigs fed the HC compared to those in the LC group. In addition, higher concentrations of pro-inflammatory cytokines in the adipose tissue (P < 0.005) and lower concentrations of anti-inflammatory interleukin (IL)-10 were observed in the HC group (P < 0.05) compared to the LC group. Of particular interest, adipocytes in the HC group were smaller in size (P < 0.05) and showed increased macrophage infiltration compared to the LC group. When compared to the H-CHO group, lower concentrations of cholesterol in both adipose and aortas as well as lower concentrations of inflammatory cytokines in adipose tissue were observed in the L-CHO group (P < 0.05). In addition, guinea pigs fed the L-CHO exhibited larger adipose cells and lower macrophage infiltration compared to the H-CHO group."[4]
Why the guinea pig is such a good model is explained by Maria Luz Fernandez in this 2001 paper, which predates her collaboration with Jeff Volek.[5]
Researchers at our laboratory and other investigators have found that guinea pig cholesterol metabolism does indeed have some analogies to human cholesterol metabolism that merit discussion.
Some of these similarities include the following:
  1. Guinea pigs have high LDL-to-HDL ratios (Fernandez et al. 1990a).
  2. They have higher concentrations of free than of esterified cholesterol in the liver (Angelin et al. 1992).
  3. They have plasma cholesteryl ester transfer protein (CETP)2 (Ha et al. 1982), lecithin-cholesterol acyltransferase (LCAT) (Douglas and Pownell 1991) and lipoprotein lipase (LPL) (Olivecrona and Bengsston-Olivecrona 1993) activities for intravascular processing of plasma lipoproteins.
  4. They exhibit comparable moderate rates of hepatic cholesterol synthesis (Reihner et al. 1990) and catabolism (Reihner et al. 1991).
  5. Similar to humans, the binding domain for the LDL receptor differentiates between normal and familial binding defective apolipoprotein (apo)B-100 (Corsini et al. 1992).
  6. Apo B mRNA editing in the liver is negligible (Greeve et al. 1993)
        7. They require dietary vitamin C (Sauberlich 1978).       
         8. Females have higher HDL concentrations than males (
Roy et al. 2000).
         9. Ovariectomized guinea pigs have a plasma lipid profile similar to that of postmenopausal women (
Roy et al. 2000).         
         10. During exercise in guinea pigs, plasma triacylglycerol (TAG) decreases and plasma HDL cholesterol (HDL-C) increases (
McNamara et al. 1993).
           11. Guinea pigs respond to dietary interventions (
Fernandez and McNamara 1992b1992a and 1995aHe and Fernandez 1998a) and drug treatment (Berglund et al.1989Hikada et al. 1992) by lowering plasma LDL cholesterol (LDL-C)

What we have here is a story of good science that should be better known. Maria Luz Fernandez develops the modern guinea pig lipoprotein model, Jeff Volek recognises its value for testing the carbohydrate hypothesis of atherosclerosis and the safety of LCHF diets, and Fernandez sees the value of such testing in adding to our knowledge of cardiovascular disease and lipid and carbohydrate contributions to inflammation.






[1] Flaws, Fallacies and Facts: Reviewing the Early History of the Lipid and Diet/Heart Hypotheses, Elliot J 2014. Food and Nutrition Sciences. Vol.5 No.19, October 2014 link

[2] Guinea Pigs: a suitable animal model to study lipoprotein metabolism, atherosclerosis and inflammation. Fernandez ML and Volek J. 2006  Nutrition and Metabolism 3:17 doi:10:1186/1743-7075-3-17 link
[3] Low-carbohydrate diets reduce lipid accumulation and arterial inflammation in guinea pigs fed a high-cholesterol diet. Leite JO et al. 2009 Atherosclerosis. 2010 Apr;209(2):442-8. doi: 10.1016/j.atherosclerosis.2009.10.005 link

[4] 
Cholesterol-induced inflammation and macrophage accumulation in adipose tissue is reduced by a low carbohydrate diet in guinea pigs. Aguilar D. et al. 2014 Nutr Res Pract. 2014 Dec;8(6):625-631   http://dx.doi.org/10.4162/nrp.2014.8.6.625  link
[5]  
Guinea Pigs as Models for Cholesterol and Lipoprotein Metabolism. Fernandez, ML 2001 J Nutr. Jan 1 2001 Vol 131 no.1 10-20 link