20 12 2013
The Biochemistry of Creatine
Breaking the silence! The first article on MASS written by yours truly, the webmaster (GetXXL): the Biochemistry of Creatine. Creatine never seems to become a dull subject now does it? All the bodybuilding boards on the internet are flooded on a daily base with topics about creatine. In this article I will not discuss its ergogenic effect, nor whatever new study came out concerning the subject (okay maybe a little at the end). I will discuss the biochemistry of creatine into great detail, enabling the reader to accurately reason about creatine and its usage among athletes.
What is creatine and how does it work?
Creatine is, in chemical terms, an organic acid with a nitrogen containing group (NH2) attached to it. Quite impractical definition isn’t it? In more practical terms, it is a molecule which aids cells in their energy requirements through the (re)formation of an important energy carrier: Adenosine TriPhosphate (ATP). ATP is often referred to as the “energy currency” of cells, as it provides the energy for a wide variety of biochemical reactions which take places within them.
Of particular interest for most of you reading this, is its role as an energy supplier for muscle contraction. Muscle contraction can be described by the so called “Sliding Filament Model”, as muscle contraction is in essence the sliding of filaments (thick and thin) over each other. This model is illustrated in the figure below, in which you can observe a sarcomere (the basic contractile unit of a muscle fibre) in two states, the top one being relaxed and the bottom one being contracted.
So basically what you see are the blue and pink/reddish filaments, actin and myosin respectively. Myosin ‘lies’ in the center and actin is tightly attached to each end of the sarcomere onto the Z-disks (but absent in the center). In a relaxed state, the myosin and actin have a low degree of overlap, however, when contracted the two have a high degree of overlap. This is due to the two sliding over each other (hence the ‘Sliding Filament Model’) during contraction as a result of the myosin heads ‘walking’ over the adjecent actin filament, in essence pulling them to the center. This walking consumes energy and the energy is obtained from the hydrolysis of ATP. Each step a myosin head takes is fueled by hydrolyzing one molecule of ATP. And this is exactly why creatine is interesting.
Our muscles have a small pool of ATP, which is generated during rest so it can be utilized during action. However, this pool would rapidly ‘dry out’ if the body had no means of regenarating it quickly. When ATP is hydrolyzed energy is released, and two products remain: Adenosine DiPhosphate (ADP) and inorganic phosphate (Pi). Now, one of the ways, and in particular a quick one too, to regenerate ATP from the ADP, lies in the Creatine Phosphate (PCr) the cell contains. The PCr pool serves as a buffer, as it can donate its Pi to ADP to regenerate ATP again. Another product of the reaction is creatine. So, to wrap this specific reaction up into one formula, the Creatine Kinase (CK) reaction read as follows :
H+ + PCr + ADP ↔ Cr + ATP (of course Creatine is not required to hydrolyze ATP, but this is the reaction as catalyzed by the Creatine Kinase enzyme, maintaining equilibrium)
The bidirectional sign means the reaction is reversible: it can go both ways depending on certain conditions. Without going into much detail, the main determinant concerning the direction in which this reaction goes is the concentration of the substrates and products. Now, when ADP increases, as for example during exercise by hydrolysis of ATP, this reaction is favored into the right direction. When ATP increases again, equilibrium will eventually be reached.
The ATP to ADP ratio is, under normal conditions, very high. The ATP storage itself is quite small and therefore the muscles heavily rely on the quick regeneration of ATP from ADP (for which the catalysation by CK is perfectly adequate in doing so). The result of creatine suppletion is that it increases the total muscle creatine content, including phosphorylated creatine, resulting in a larger buffer to quickly regenerate ATP from ADP when its hydrolyzed during muscle contraction. This is the core ‘function’ on how creatine enhances performance.
In addition, it also exerts its effects by enhancing mitochondrial respiration and facilitating intracellular energy transport in which the PCr functions as an energy transporter from the mitochondria to the cytosol (Cr getting rephosphorylated in the mitochondria utilizing ATP derived from oxidative phosphorylation). Furthermore, it affects the expression of several genes, of which some are involved in skeletal muscle hypertrophy.
Measurement of the protein content of a wide variety of protein kinases in skeletal muscle after short-term Creatine Monohydrate (CrM) supplementation show, among others, upregulation of: p38 MAPK, ERK and Akt. Activation of the MAPK/ERK pathway can lead to phosphorylation of p70S6K (also one of mTORC1’s downstream effectors) which can increase gene transcription. Akt on the other hand works through the canonical PI3K/Akt/mTOR pathway. This could be another mode of action through which creatine positively influences protein synthesis.
Moreover, creatine induces proliferation and differentiation of satellite cells in vitro. Indeed, it has been shown in a clinical trial that creatine supplementation enhances the training-induced increase in satellite cell and myonuclei number in skeletal muscle. However, the exact biochemical mechanism through which creatine does so remains to be elucidated.
Another proposed mechanism is that of creatine being an ‘androgenic aid’, solely based on a single study showing a (testosterone indepedent) increase of dihydrotestosterone (DHT). DHT is the product of testosterone by 5α-reduction and has a higher potency in terms of androgen receptor activation. Now, this proposed mechanism of action is problematic in a multitude of ways. DHT is a 3-keto 5α-reduced steroid, and as most of these steroids, the enzyme 3α-HSD is happy to reduce the steroid on C3 resulting in a useless metabolite. One of the main sites of action of this enzyme is (don’t be shocked): skeletal muscle. So DHT gets metabolized rather quickly in the tissue where it should work according to this proposed mechanism. Now we aren’t done yet: skeletal muscle lacks significant amounts of any of the 5α-reductase isozymes. So there isn’t much conversion going on in the respective tissue to begin with. Surely the DHT diffuses from the serum to the skeletal muscle, yet these concentrations are quite low (around the 1 nmol/l mark). All in all, it is highly unlikely that this is one of the modes of action.
Finally, I would like to discuss its effects on serum myostatin, since creatine appears to decrease serum myostatin and it is currently another hot theory. For those of you who are unaware, myostatin is a protein involved in the regulation of muscle hypertrophy and hyperplasia. Now, I’m willing to go as far as to believe that this entire decrease measured in serum is solely the result of its decrease in skeletal muscle (which isn’t even a rare assumption I must admit, since its main site of synthesis is skeletal muscle). When looking at the big and bulky myostatin null mice in the original study wherein the protein was discovered (then still dubbed ‘GDF-8′) by McPherron et al. in 1997, it seems tempting to believe that any reduction in myostatin translates to gains. However, when closely examining their results and mechanism of action of the protein, I would hold back with drawing that conclusion. For starters, the myostatin null mice had NO myostatin, which makes it difficult to extrapolate the results to various concentrations. Nevertheless, this is a useful way to examine its biological function. As it seems in the myostatin null mice, its main function is the inhibition of myoblast proliferation and differentiation. Indeed the mouse also underwent significant hyperplasia. Myostatin therefore seems to put a brake on hyperplasia, as it does not occur under physiological conditions. Of course it also influences hypertrophy, but be it to a lesser extent. It mildly decreases Akt phosphorylation and some of its downstream substrates. Furthermore it upregulates a ubiquitine ligase (atrogine-1) via FoxO1, thus increasing protein breakdown. Nevertheless, when comparing the cross sectional area (CSA) increases of the mice both due to hyperplasia and hypertrophy, hyperplasia seems to take the crown. Although it would be stupid to say this mechanism of action isn’t involved in creatine’s mode of action, it is far from certain to conclude it actually is a significant mode of action.
When creatine is orally ingested, it gets absorbed from the gastrointestinal tract. The body has creatine transporters located throughout the intestinal wall in order to achieve this (creatine can not diffuse past the cell membranes, nor is there any significant paracellular movement). In order for the body to absorb it however, the creatine must be dissolved. Indeed, when creatine absorption by solution or lozenge is compared, the area under the concentration-time curve of the latter is significantly lower. Therefore, make sure you take in creatine with ample water.
After passing the intestinal wall it hits the bloodstream going through the hepatic portal and into the circulation. From the circulation it gets absorbed by a variety of tissues, e.g. brain, muscle and even your balls. Nevertheless, more than 95% of a body stores of creatine is found in skeletal muscle tissue. The absorption into the cells is also transporter-mediated. This plasma membrane transporter has an estimated Michaelis-Menten constant (Km) of around 15 to 77 µM. So when the creatine plasma concentrations are around 15-77 µM, the transport into the cell is half of its maximal influx. This has some practical implications for ‘creatine loading': maintaining creatine plasma concentrations well above this mark to ensure maximal creatine uptake by the muscle. This is achieved by taking roughly around 15 gr spread over three times a day.
Finally, after absorption from the gastrointestinal tract into the bloodstream and its subsequent absorption by the cells (mainly muscle cells), its clearance remains to be discussed. Since creatine requires a transporter to cross cell membranes due to its polarity, and there is no transporter emitting the substance out of the cells, it is effectively trapped within. Creatine therefore leaves the cells in the form of creatinine. The formation of creatinine from creatine is spontaneous and is NOT catalyzed by any enzym or accelerated by whatever process whatsoever. Nor does its phosphorylated state significantly affect its rate of degradation. Only pH and temperature affect its rate of degradation. Since both these factors are fairly constant within the cells, the degradation is constant. This leads to the conclusion that the amount of creatine degraded into creatinine is linearly proportional to the amount of creatine. Contrary to popular believe, this is not accelarated by lifting weights or any other activity (hence it makes no sense to only supplement it on training days). Additionally, any creatine not absorbed by the cells is cleared by the kidney (a part of it reabsorbed tho), and so is creatinine.
How should creatine be supplemented?
In essence, the ergogenic effect of creatine supplementation is due to its effect on muscle creatine stores. Hence, a supplementation schema should focus on: 1) saturating the muscle tissue with creatine as fast as possible, 2) maintaining the saturated level of creatine in the muscle tissue. As evidenced by the Km of the CRT as presented in the previous section, it probably does not take that much to saturate it. However, a single dose of creatine will not elevate the plasma concentration for the entire day, so multiple doses are required, spread throughout the day. The traditional loading schema of 15-20 gr per day, as also applied in literature, seems adequate in achieving this, spreading it out over 3-4 dosages a day. Saturation is achieved after only a couple of days, after which a maintenance dose can be used. Since literature reports an average breakdown of creatine to creatinine of approximately 2 gr per day, 2 gr per day is supposed to be enough. However, bodybuilders are bigger than the regular folks recruited in literature. Carrying around all that extra muscle mass also implies carrying around more creatine. Since the total amount of creatinine broken down per day is directly proportional to the amount of creatine in the body, bodybuilders will probably breakdown somewhat more. Additionally, of course, we are supplementing creatine and thus increasing our total creatine amount as well. Finally, creatine is dirt-cheap and most distributors supply it with a scoop of 5 gr. This leads me to the recommendation of just maintaining on 5 gr per day, instead of some more ‘difficult’ number such as 3 or 4 gr (which could be perfectly fine as well).
Now, one question remains: does timing matter? Considering there is a saturation limit, and my recommendation of the maintenance dose is on the extremely safe side of being sufficient, the answer is: no. However, there recently has been one study, conducted by Jose Antonio and Victoria Ciccone (published of course in Antonio’s JISSN), which suggests it does. Antonio and Ciccone compared the effect of supplementing 5 gr of creatine pre and post workout on strength and body composition. The outcome was statistically significant between both groups, and the winner was: post workout. Now, I would like to note that there obviously is a difference going on there. However, I wonder if the same result would be observed if both groups applied a loading phase. It might have been that the post workout group reached saturation faster than the pre workout group, and thus reaped the benefits of it earlier. Nevertheless, this makes a case for taking your creatine after your workout (if compliance does not suffer from it!). For me personally, I simply take it in the morning ( or afternoon actually, as I usually don’t wake up in the morning ) as I’m sure I won’t forget it then.
Now you’re aware of the biochemistry of creatine, you can reason about it. Loading phases, maintenance dosage, supplementing it on the days you’re not training, even the particular dosage you’re using: it all makes sense in the context of its biochemistry. Being my first article here on MASS, I hope you all enjoyed reading it as much as I enjoyed writing it. I would like to apologize however for the lack of references: I simply didn’t have the time to reference all of this. I might add references later when I find some time (and motivation) for it.