an autodidact meets a dilettante…

‘Rise above yourself and grasp the world’ Archimedes – attribution

how statins work 4: blocking the mevalonate pathway and the question of side-effects

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I may be going into this topic in too much detail, but I’m enjoying what I’m learning along the way, and our biochemistry is enormously complex and fascinating and worth knowing a lot about for its own sake as well as a guard against false claims and over-simplification. I haven’t yet ordered Ben Goldacre’s new book Do statins work?: the battle for perfect evidence-based medicine, which might render all this (superficial) research superfluous, but I doubt it…. [I’ve since discovered that the book won’t be available for quite a while – I wonder what’s holding it back, as clearly it has already been written…]

So I left off with the production of citrate. Thiol (HS) groups, like alcohol (HO) groups can be linked to carboxylic acid groups. The alcohol group can be taken from the carboxylic acid group and the hydrogen from the thiol group, and these can be combined to create water molecules. The sulphur atom is bound to the carbon of the carboxylic acid group, and this is called a thioester link (similar to an ester link but with a thiol rather than an alcohol group). The resulting molecule is Acetyl-CoA. Acetyl is essentially acetic acid with the alcohol group removed.

Oxaloacetate combines with Acetyl-CoA and water to create citrate. The water molecule is involved in breaking the thioester link, via hydrolysis, between the carbon atom (of the carboxylic acid group of the acetic acid molecule), and the sulphur atom of the coenzyme A. These are connected by a covalent bond, in which two electrons are shared, one from the carbon and one from the sulphur. This hydrolytic process results in an alcohol group combining with the carbon atom, with the other hydrogen atom combining with the sulphur to recreate a coenzyme A (CoA-SH).

A break in the covalent bond between oxygen and carbon in the ketone group of the oxaloacetate molecule, as well as a break in the covalent bond in the methyl group of the acetic acid component of the Acetyl-CoA molecule, leads to a new molecule with an extra deprotonated carboxylic acid group, the conjugate base of citric acid, i.e. citrate. The conversion is catalyzed by the enzyme citrate synthase.

Next is the transportation of citrate into the inter-membrane space of the mitochondrion. A protein (SLC25A1) magically transports the citrate into the inter-membrane space from where it diffuses through the outer membrane into the cell cytoplasm. The citrate then undergoes a reverse process, thanks to an enzyme called ATP citrate lyase, I won’t go into all the details here, but oxaloacetate is recreated, a condensation reaction occurs, and ADP and inorganic phosphate is formed. The oxaloacetate is returned to the mitochondrial matrix in the form of pyruvate, via malate. We now have Acetyl-CoA in the cytoplasm, and the pyruvate is transported by another protein (SLC16A1) back into the matrix, completing the cycle, as the pyruvate is reconverted to oxaloacetate. As I’ve pointed out, there’s a lot more to this cycle, in terms of enzymes, hydrolysis, ATP and ADP, but we’re now at the point where we can look at cholesterol synthesis in the cytoplasm.

The first stage of this is the formation of mevalonate. Three Acetyl-CoA molecules are required for this. So, according to the videos I’m relying on, the first reaction joins together two Acetyl-CoA’s. This involves breaking a bond between the carbon and the sulphur atom in one (which sends one electron back to the carbon, the other back to the sulphur), and in the other, breaking a bond between carbon and hydrogen. Then the free-electron carbon is bound to the other molecule’s free-electron carbon. This results in a molecule with a methyl group (CH3), a carbonyl group (C=O), a methylene group (CH2<) and a carboxyl group (COOH) bound to the coenzyme A molecule by a thioester link. In the same reaction the hydrogen atom is bound to the sulphur atom to recreate an intact molecule of coenzyme A, with its thiol group. All of this is a reversible reaction catalysed by an enzyme in the cytoplasm called Acetyl-CoA Acetyl Transferase, and the resulting molecular product is called Acetoacetyl-CoA. This is the result of binding two Acetyl-CoA’s together, and the next step is to bind a third Acetyl-CoA to this molecule. This involves breaking one of the carbon-oxygen bonds in the Acetoacetyl-CoA molecule, breaking a carbon-hydrogen bond in the new third molecule, and introducing a water molecule to hydrolyse the thioester link. The free-electron carbons from the two molecules are bound together, and the hydrogen from the new molecule is bound to the oxygen. The product is catalysed by the enzyme HMG-CoA synthase, and the name of the product is HMG-CoA, aka beta-hydroxy-beta-methylglutaryl-CoA. The ‘beta’ refers to the middle carbon in the 5-carbon chain, to which the methyl and alcohol groups are attached.

So, we’re now at the stage of having produced HMG-CoA. The next reaction is vitally important for a couple of reasons. It’s the rate-limiting step for the entire biosynthesis of cholesterol, and it’s the site of action of statins, which block the synthesis here by blocking the enzyme that catalyses this reaction. That enzyme is called HMG-CoA reductase, and the various statins that can block this enzyme have names such as simvastatin, atorvastatin and rosuvastatin.

So I’ll try to make sense of this step, then I’ll go back to how statins interfere with it. HMG-CoA reductase catalyses the reaction which transforms HMG-CoA into mevalonate. This reaction requires two molecules of reduced NADP (2NADPH) which carry hydride anions, and also two protons (2H+). This effectively brings in four neutral hydrogen atoms, resulting in the creation of two oxidised NADP molecules (2NADP+). A thioester link in the HMG-CoA is broken, ultimately recreating a coenzyme A with an intact thiol group and and an aldehyde group. Also one of the bonds between the carbon and the oxygen can be broken, allowing for the binding of other hydrogens, in such a way that two hydrogen atoms are bound to the end carbon of the molecule, as well as an alcohol group. This is the mevanolate molecule, to which phosphate groups are added, converting it to ‘activated isoprenes’.

So the question of whether inhibiting of HMG-CoA reductase by statins has effects beyond the inhibition of cholesterol production de novo, is obviously essential, so I won’t be focusing so much on cholesterol biosynthesis from here, I’ll be looking, in my amateur way, at claims that the inhibition of HMG-CoA reductase by statins can sometimes have somatic side-effects, including SAMS (statin-associated muscle symptoms) ‘which are the most well-documented side effect of statins, although there appears to be no unifying mechanism’, according to this research article published in Circulation Research. Note the vagueness of this acronym, which might be seen as a red flag. Circulation Research is a journal of the American Heart Association, and looks pretty kosher to me, so I will rely on it heavily in this and possibly other posts. The article is also pretty up-to-date having been published earlier this year (2019).

The article summarises the mechanism of action of statins in a single paragraph which I’ll reproduce here, because it also reports on possible positive pleiotropic effects:

Statins work by competitively blocking the active site of the first and key rate-limiting enzyme in the mevalonate pathway, HMG-CoA reductase. Inhibition of this site prevents substrate access, thereby blocking the conversion of HMG-CoA to mevalonic acid. Within the liver, this reduces hepatic cholesterol synthesis, leading to increased production of microsomal HMG-CoA reductase and increased cell surface LDL receptor expression. This facilitates increased clearance of LDL-c from the bloodstream and a subsequent reduction in circulating LDL-c levels by 20% to 55%. In addition to reducing LDL-c and cardiovascular morbidity and mortality, statins may have additional non–lipid-related pleiotropic effects. These include improvements in endothelial function, stabilization of atherosclerotic plaques, anti-inflammatory, immunomodulatory and antithrombotic effects, effects on bone metabolism, and reduced risk of dementia. These additional benefits are primarily thought to arise because of inhibition of the synthesis of isoprenoid intermediates of the mevalonate pathway.

Pleiotropy is a term I’m just learning about. A pleiotropic gene is one that ‘exhibits multiple phenotypic expression’. For our purposes, the word ‘multiple’ is key. The point here is that the blocking of HMG-CoA reductase may have multiple benefits, not all related to high LDL-c levels, which raises the obvious question about the positive purposes of HMG-CoA reductase production. As to the harms or side-effects, the article has this to say:

Currently, no universally accepted definition of statin toxicity/intolerance exists, with several groups attempting to define the condition. The prevalence of statin intolerance is also widely debated, in part because of difficulties in identification and diagnosis, particularly with respect to muscle symptoms. Observational studies suggest it occurs in 10% to 15% of patients, with clinic data putting it as high as 30%. In randomized controlled trials, the incidence is thought to be 1.5% to 5% of patients, although this is believed to be an underestimation as most studies exclude patients with a history of statin intolerance either before randomization or during the run-in period.

As can be seen, it’s very difficult to find a reliable rate of statin intolerance or toxicity, or even a reliable definition of same – the article lists four separate definitions from different associations monitoring lipid problems and atherosclerosis. It concludes the section with this statement:

Despite the difficulties in identifying and diagnosing statin toxicity, however, several international organizations have identified statin intolerance to be of major clinical importance that warrants further research and investigation.

Clearly the statin wars – if I can be so controversial as to use such a term – are far from over. I’ll continue to educate myself about the issue and promise to write more in the future.

it’s complicated

References

Cholesterol biosynthesis part 3, by Ben1994 (video)

Cholesterol biosynthesis part 4, by Ben1994 (video)

https://en.wikipedia.org/wiki/Pleiotropy

https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.118.312782

Written by stewart henderson

October 19, 2019 at 6:50 pm

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