L-Valine, Zinc supplementation, the Fructose Lipid Hypothesis, and the n=1 Updated

There's a paper casing a big buzz in the Hep C forums, with good cause.

Valine, a Branched-Chain Amino Acid, Reduced HCV Viral Load and Led to Eradication of HCV by Interferon Therapy in a Decompensated Cirrhotic Patient

The HCV viral load plummeted, from 5 log to less than 1.2, by the twelfth week of treatment (and only then was the patient given Interferon-alpha, one of the usual antiviral drugs, not very effective at all used on its own). This required fairly high intake of l-Valine, peaking at 12g/day. Which probably isn't something you'd want to do for more than 12 weeks. Alpha-fetoprotein also dropped back to normal, which is all to the good as elevated AFP is considered a risk marker for hepatocellular cancer. 

OK, so this is just one patient. An n=1 case study, so it's properly documented and peer-reviewed (not to be mistaken for any kind of an anecdote). Case studies tell us what CAN and DOES actually happen, but not how often it might be expected to happen (RCT study) or why it might happen (animal testing, usually).

How might it work? The authors suggest various mechanisms, but there is no detail as to how, for example, l-Valine might activate dendritic cells or improve liver function. I would like to suggest one. This is not a theory, it is not even a hypothesis, just something it might be rewarding to look at.

When amino acids, carbohydrates and (to a lesser extent) fats are metabolized by cells, the energy blocks (called substrates) are fed into the TCA (tricarboxylic acid), or Krebs (after its discoverer), or citric acid cycle. (at the bottom of the above diagram). This cycle generates energy (ATP and GTP), a variety of substrate building blocks for making sugars, amino acids and lipids etc, and reducing equivalents (NADH+ and FADH2) that are used to keep the cycle working and to reduce oxidised ubiquinone (co-enzyme Q10) to ubiquinol.

The TCA cycle generates NADH+ and ATP at a few points but only generates GTP (in hepatocytes) and significant amounts of FADH2 in two consecutive reactions. These are the conversion of succinyl-CoA to succinate, and the conversion of succinate to fumarate, respectively (have a look at table 13 in this paper, where FADH2 is called FPH2, to see what I mean. 484 of 562 mitochondrial FPH2 are generated at this step). Substrates can be removed from the cycle or added to it at many points (and it will presumably run unevenly or at a reduced rate if this intake/exhaust system is out of balance). If the succinyl-CoA substrate was drastically undersupplied, for example by removal of a previous substrate or by inhibition of a previous enzyme, there would be a deficiency of FADH2 (which might lead to inhibition elsewhere in the cycle). 

(The purpose of GTP in hepatocytes is something I know nothing about, but it must have a function distinct from ATP).

Now, how is extra succinyl-CoA made available to the TCA normally? (it's called an anaplerotic reaction, by the way). From propionate supplied by the breakdown of l-Valine mostly. Some also comes from metabolism of odd-chain fatty acids and phytanic acid, which are mainly present in the diet from dairy foods and ruminant fat (note that the l-valine, as far as I know, could be metabolized elsewhere, e.g. muscle, but.the propionate finds its way to the hepatic mitochondria). 

Feeding succinyl-CoA into the TCA cycle might be expected to increase the ratio of FADH2 to NADH+ in the mitochondrial respiratory complex. Peter at Hyperlipid has speculated at length on the implications of the FADH2:NADH+ ratio in his recent Protons series.

Disclosure:
There was a corroborating paper but I lost it. Something about succinate inhibiting viral replication in vitro (as did DHA, AA, and EPA in that order). Or, maybe it was suppressing excess gluconeogenesis in infected hepatocytes (another function of long-chain EFAs) - as l-Valine is a gluconeogenic animo acid this isn't as likely, but I can't rule it out. The first time I've lost a paper and, to my shame, not been able to quickly search it up again. I found it a few weeks before I read the l-Valine paper, while researching fish oil and krill oil. If anyone knows what paper I'm referring to... Anyway, there is definitely a succinate-counters-HCV connection on record out there somewhere.

So is "fixing" the TCA cycle undoing something that HCV has manipulated for its own benefit? Does HCV virion assembly require lots of alpha-oxoglutarate, depleting succinyl-CoA? Does the extra supply of succinyl-CoA mean an equal amount of another substrate has to come out of the cycle (cataplerosis), and if so, what? And does this also apply to dendritic cells? Is l-valine synergistic with alpha-lipoic acid in this scenario (which would tend to confirm the succinyl-CoA mechanism)? I could go on (about HCV core protein inhibiting mitochondrial respiratory complex I and FADH2 feeding complex II, for example), but I've given you enough homework for the Xmas break.

Zinc Supplementation Improves the Outcome of Chronic Hepatitis C and Liver Cirrhosis (2009)

Zinc supplementation therapy improves the outcome of patients with chronic hepatitis C (2012)

This makes sense because HCV sequesters zinc in one of its proteins, and a zinc metalloprotein is required to break down collagen (fibrosis is a repair process and the extra collagen scaffolding - scarring - is meant to be removed). You won't recover from fibrosis if you don't get enough zinc (you need more than just zinc, but these studies show that zinc alone can give significant protection). They used polaprezinc, which is a carnosine-zinc chelate supplying 33mg/day.

A recent review from New Zealand summarizes the evidence that sugar (and high-GI starches), not fat or salt, is the main dietary cause of cardiovascular disease (PDF). John Yudkin's ghost is smiling.

An update on the hygeine hypothesis n=1. I have had one day of hay fever, moderate by previous standards but there nonetheless. I modified my protocol by restricting cheese (the obvious hole in the first n=1 post) and by restoring probiotics (either lactobacillus/bifidus or Del Immune V), which hadn't prevented hay fever without "raw water" therapy, but which seem to be synergistic in combination with it. And everything has been fine since. it's high pollen weather, midsummer, everything's flowering, other people have hay fever but mine is minimal. (updated: another baddish day today. Water gave some relief eventually - I didn't expect it to work like an inefficient drug. Can disease states tend back to homeostasis - is that why drugs often stop working eventually? Next time I write about this I hope to be able to assess it properly)

P.S. Just a little Christmas bonus: 

Consumption of n-3 fatty acids and fish reduces risk of hepatocellular carcinoma

We investigated the association between fish and n-3 PUFA consumption and HCC incidence (n = 398) in a population-based prospective cohort study of 90,296 Japanese subjects (aged, 45–74 y). Hazard ratios and 95% confidence intervals (CIs) for the highest vs the lowest quintile were estimated from multivariable adjusted Cox proportional hazards regression models. We also conducted subanalyses of subjects with known hepatitis B virus (HBV) or hepatitis C virus (HCV) status, and of subjects who were anti-HCV and/or hepatitis B surface antigen positive. All tests of statistical significance were 2-sided.

Results

Among all subjects, consumption of n-3 PUFA-rich fish and individual n-3 PUFAs was associated inversely with HCC, in a dose-dependent manner. Hazard ratios for the highest vs lowest quintiles were 0.64 (95% CI, 0.42–0.96) for n-3 PUFA-rich fish, 0.56 (95% CI, 0.36–0.85) for EPA, 0.64 (95% CI, 0.41–0.98) for DPA, and 0.56 (95% CI, 0.35–0.87) for DHA. These inverse associations were similar irrespective of HCV or HBV status.

What would Bluebelle Eat? Feeding the Paleo Dog.

The other day a new dog owner asked for my advice about what to feed a dog. Bluebelle has been eating mainly paleo after her first year, when she seemed to be getting health problems from a diet of dried food and mince. These included trouble pooing and stiff hips or sore legs. She no longer has any of these problems, but we did have an issue with iodine deficiency on the new diet - see below.

A mainly carnivorous animal like Bluebelle (to wit, a dog) needs to eat muscle, connective tissue (gristle and skin), bones, organ meats, and fat to be healthy, from a range of animals including some fish. It is an omnivore and can tolerate some carbohydrate from starches and fruit, but should not have grain-based dry food often (OK for emergencies but not as regular diet). 

In terms of calories there needs to be [edit] 2-3 parts fat to 1 part protein. Fat is much denser than protein and all meat has some fat in it, so this mostly means using fatty meats or adding a little fat to the dish.

Kelp powder should also be added to the food occasionally to prevent iodine deficiency and hypothyroidism (we learned this the hard way), and salt added to cooked meals.
Symptoms of low iodine include “spooky” behaviour and depression, hair loss, discoloured patches on skin, and in Bluebelle’s case susceptibility to mastitis and allergies and hunched, stiff “aged” posture. This was quickly and completely reversed by kelp, which had a rejuvenating effect like watering a wilting plant. This was only diagnosed by my habit of reading the excellent Hyperlipid blog of vet Peter Dobromylskyj, who mentioned the symptoms in passing, for which I'm eternally grateful; our vet had drugs for the symptoms but didn’t recognise the cause.

Onions and garlic can be toxic to dogs (and cats, causing Heinz body anaemia) but most other veges are safe (and they will generally avoid onions anyway) when feeding leftover meat dishes. The inability to detoxify onions tells you that a dog is not as completely omnivorous as say a pig or human and its ancestors probably never survived by eating roots, shoots and leaves. Though, come to think of it, my goat wouldn't eat onions either. Gophers like them, apparently, and pigs aren't harmed by them (though some warn against feeding onions to pigs, research seems to show benefits). 

We feed Blubelle:

Minced beef and lamb

Beef (ox) heart, lamb heart, cut into cubes, raw

Offal: Liver or kidney (lambs or calves or ox or chicken) COOKED (lightly fried in dripping with salt, once or twice a week). If we don’t include some salt, Bluebelle drinks more sea water, and not all dogs have access to salt water.

Note: pork hearts tend to be flabby, pork kidneys I’ve bought have been deformed, coming from caged animals, so I don’t generally buy these or commercial lard. A bit of bacon scrap accounts for most of the pork in her diet.

Bones - beef bones probably best, also chicken necks, raw.

Kelp powder - a small teaspoon mixed in or sprinkled on food every few days.

Fats - we add a couple of tablespoons dripping (beef and lamb) or if available, free-range lard, to the less fatty meals. If the dog eats dripping (tallow in the U.S.), which is fairly flavourless, then it definitely needs fat. Bacon rinds or bacon grease and offcuts of the meat you prepare for yourself are also good.

Fish - a tin or sardines or half a tin of mackerel once a week keeps the coat shiny.

Chicken - we feed Bluebelle our roast chicken leftovers, including the bones. This may not be for every dog but it hasn't done her any harm.

Cheese - Bluebelle loves cheese, and enjoys milk and cream and yoghurt every so often. When the milk's a bit off, offer it to the dog. Probably not an everyday food though.

Fruit - Bluebelle likes a few raisins (edit - grapes and raisins are toxic to dogs, don't feed these) or diced prunes occasionally. Other dogs like apples and pear cores, but she doesn't seem to. She won't eat potatoes or rice or cooked greens unless these are stirred into meat, so we don't offer these unless there's a very meaty leftover stew.

Dried food - Bluebelle eats toast crusts occasionally and will eat dog biscuits if there's nothing else, so it's a good emergency food occasionally, but it wasn't good for her digestion when she ate it more often. If you get a dog roll, I recommend looking out for the rice-based (gluten-free) versions, or for dog rolls higher in fat and lower in carbohydrate.

Water - Bluebelle doesn't always drink water, but when she does, she needs a lot, so always have some available.

Timing - Bluebelle is fed once a day, in the evening. If she's not interested in food, she's not fed until she asks for it; so every now and then she goes without. She's fed about a pound or more a day; if she asks for more, she'll get it. She might turn up if someone is eating cheese, in which case she'll always be given a bit, and any uneaten or old cheese (cheddar) is given to her.

Illness - it's probably worth giving a little cod liver oil in winter for vitamin D, Bluebelle loves this; just a teaspoon a day.
Parvovirus and distemper are lethal diseases of dogs which are similar to cholera in humans. The dog dies of dehydration, loss of fluids and electrolytes. Dogs can be kept alive till the disease passes (about a week) by force-feeding, twice a day, water and Gastrolyte, or other human "oral rehydration" formula (made up strictly as advised on the packet). I have saved two dogs suffering from parvovirus by this protocol; the dog that received the formula as soon as it became ill suffered no lasting effects, the other dog, which I treated after 3 days, was always a bit skinny afterwards. Another dog that was accidentally taken to the vet and received antibiotics, not rehydration (which a vet could give by IV drip), died there.
I don't know why this isn't widely known.

I'm sure this is a case where many readers will have dogs with unusual dietary needs, or have had experience of deficiencies, diseases and toxins that we can all learn from, so feel free to use the Comments facility. I think my own ideas are working well, but I don't consider them the last word yet.

Rich Van Konynenburg R.I.P. - Methylation and Glutathione.

I was saddened to learn of the death of independent researcher Rich Van Konynenburg PhD. last month.

Rich was a researcher into the causes and treatment of chronic fatigue syndrome/ myalgic encephalomyelitis (CFS/ME) and his outstanding contribution was to grasp the importance of glutathione and the methylation cycle to this disease, then adapt the autism research of Dr Amy Yasko to suggest the concept of methylation cycle blocks and deficient antioxidant protection from glutathione as its characteristics.

There is a vicious cycle mechanism; glutathione protects B12 from the denaturing effects of, for example, mercury or peroxynitrite, and B12 is the co-enzyme that transfers methyl groups to homocysteine, creating SAMe (AdoMet).

This hypothesis struck me as obviously relevant to hepatitis C. Liver disease is produced experimentally by dietary deficiencies, i.e. a diet deficient in choline (a methyl donor) and/or methionine (the source of both SAMe, a methyl donor, and the cysteine moiety of glutathione). Deficiency of selenium will cause hepatic necrosis, and selenium is the co-factor for those enzymes that use glutathione to remove free radicals.

I contacted Rich Van Konynenburg by email and corresponded with him for some time. Re-reading my emails they are breathless and prolix, and there were too many of them, and Rich's replies are detailed and not at all dismissive. He corrects my mistakes gently and encourages my interest.

Rich was like the hedgehog in Isaiah Berlin's parable who knew one thing but knew it very well. He knew the methylation cycle and transsulfuration pathways (the parts of biochemistry concerned with the non-protein functions and metabolism of the essential amino acid methonine) intimately, the proteins and genes involved, the polymorphisms that influenced metabolism one way or another, and how this related to various diseases, especially CFS/ME, autism, and Lyme disease.
The methylation cycle and transsulfuration pathways are a good place to start if you are interested in biochemistry, because their simplicity belies their overall importance to health. There are only 3 vitamins required (folate, B12 and B6) and 3 minerals (magnesium, molybdenum, and selenium), yet the products of the system include many of the chemicals popular as energy supplements; carnitine, creatine, phospholipids, taurine; and of the neurotransmitters, besides the role of glutathione and taurine in bile production and of both methylation and glutathione in neutralizing toxins.

This site gives a link to Rich's many papers; and here is the wiki page on which the hypothesis was expounded and referenced in all its detail. Anyone familiar with the work of Chris Masterjohn at Weston A. Price will know about the importance of glutathione and choline in the diet - here is the repository of the technical and clinical data that underlines that importance.

Both Rich's help, and the example provided by his very existence as a respected independent health researcher, were of the utmost encouragement to me as I strove to educate myself. His scrupulous accuracy and honesty - exemplified by his case reports of adverse reactions to his suggested treatment protocol - set a high standard for others to aspire to. My regret is not being able to catch up with him again before his sudden death.