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Creatine Supplementation  

Introduction

Creatine (methylguanidine-acetic acid) was discovered in 1832 by Michel Eugene Cheverul.  Later on, in 1834 Justus von Lieburg “confirmed” that creatine was a normal part of meat. It was also found that there was more creatine in wild animals which underwent more exercise than animals that were living in captivity which exercised less. During the early part of the 1900s by using creatine as a supplement allowed for a boost in creatine in animals. Later on, phosphocreatine (creatine phosphate or phosphorylated creatine) was discovered in the year of 1927.  Then in 1934, the creatine kinase (the enzyme that “catalyzes” phosphocreatine was found). Finally, in 1968, phosphocreatine was found in the process of recuperating from exercise.

  In foods, creatine is found primarily in red meat and fish.  Eaten creatine is then eventually sent to the bloodstream. Creatine is also synthesized within the body by the liver, kidney and pancreas, although this primarily takes place in the liver.  This is done in two steps: the first step is when an amidine group from arginine goes to glycine to make guanidinoacetic acid.  Then in step two, a methyl group goes to a guanidinoacetic acid from S-adenoslymthionine forming creatine.  In the synthesis of creatine, there are some controls on it so that when there is less creatine in one’s diet, there will be more synthesis of creatine in the body.  In opposition, if there is a lot of creatine present in one’s diet, then there will be less creatine synthesis in the body.

The storage of creatine in the body occurs in two forms; in the form of phosphocreatine or simply creatine. In the average adult male weighing 70kg, there is 120g of creatine of which 95% is found in the skeletal muscle.  Some of the creatine goes to other various parts of the body such as the heart and brain.  Of all the creatine in the skeletal muscles, 60-70% of that creatine is phosphocreatine.  And because it is phosphocreatine, it cannot leave the membranes.

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Lab : Impact of Creatine Monohydate and Phosphocreatine on Lactic Acid Buildup

The purpose of this lab is essentially to assess the buffering capacity of creatine monohydrate and phosphocreatine.  As well, these two supplements will be contrasted to determine which of the two acts as a better buffer for the H⁺ ions (if at all). 

Among many claims of the benefits of creatine, it has been advertised to “buffer” the build-up of lactic acid in muscles, thus delaying the process of burn in the muscle.  A biological buffer is essentially a mechanism within the body which neutralizes H⁺ ions, which are ultimately excreted.  As discussed before, lactic acid is essentially formed during anaerobic gycolysis, which occurs in the body when there is a lack of oxygen.  After various steps of gycolysis (refer to section VI), fermentation will take place.  There are two types of fermentation, but for the purpose of this lab, lactic acid fermentation will be focused on.  What occurs is the pyruate molecules from gycolosis accept the hydrogen and electrons from NADH yielding NAD⁺.  The pyruvic acid is made into lactic acid, a compound consisting of three carbons.  In the body, lactic acid is formed not long after the acid dissociates, and will form a salt with chemicals such as Na⁺ and K⁺.  Note the term lacate refers to any salt that is made from lactic acid.  As well, when lactic acid builds up it can then cause acute muscle cramps and if enough of the acid is built up, it can cause a heart attack.  Lactic acid is also considered to be responsible for muscle fatigue, as mentioned above. 

It has been shown in various studies that phosphocreatine acts as a buffer in muscles against the hydrogen ions of acids, and in this particular case, lactic acid.  Adenosine triphosphate (ATP) is the body’s form of energy and is made through several metabolic pathways.  The actual structure of this molecule consists of an adenosine, a ribose, and three phosphate groups.  A phosphate group is made of a phosphorus atom, and three oxygen atoms bonded to it.  When one of the phosphate groups bonds to the molecule, it is broken by and enzyme called ATPase, and energy is released.  There is a remaining adenosine diphosphate (ADP) and an “inorganic” phosphate group.  Creatine begins to play a role in this phosphagen system and is phosphorylated by the creatine kinase enzyme, consequently yielding phosphocreatine.  During intense exercise, the phosphate group that was originally added onto the creatine is being removed and placed onto the ADP making it ATP for ready use. (please note for more information on this entire subject please refer to section VI).

Phosphocreatine is responsible for approximately thirty percent of the muscle’s capacity to buffer the H⁺.  As well, phosphocreatine uses up the H⁺ ions during the phosphagen energy system when the ATP is re-synthesized by phosphocreatine and ADP. (for more information to these various subjects refer to section VI).

There are three types of buffers within the human body; protein buffers, carbonic acid-bicarbonate buffers, and phosphate ion buffers.  Although claims have been made that creatine is responsible for the buffering of lactic acid, the buffering is likely due to the phosphate in the phosphorylated creatine.  The role of phosphate buffers within the body is to buffer primarily the intracellular environment and urine, although it plays a small role in buffering extracellular fluid.

(Note that due to the lack of availability of relatively pure phosphocreatine, it was not possible to obtain it.  Thus, the lab will be performed using a creatine supplement containing a small amount of phosphate in it.  However, there are many other ingredients in the mix which may affect the outcome of the lab.  While there appears to be no protein within the supplement, it is not certain, which could significantly alter the results.  For example, in some supplements there is hydrolysed vegetable protein, which would definitely have an unwanted impact on the results.  Nevertheless, for the purpose of this lab, the assumption that any buffering is due to the phosphate will be made.)

To determine the pH of the titration, a universal indicator will be used.  This indicator is unique in the sense that it consists of a series of indicators combined and thus has a higher range or reading level.  And each one of the mixture of indicators shall change its color at a different pH.  A red color indicates a pH of one, while green indicates seven and purple indicating thirteen.

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Materials

·        beakers x 3

·        lactic acid

·        Erlenmeyer flasks x 3

·        white paper towels

·        distilled water in squeeze bottles x 3

·        pipettes and bulbs x 3

·        burette with stopcocks x 3

·        retorque stand and clamp  x 3

·        pH meter

·        universal indicator

·        electric balance (to measure mass)

·        volumetric flask with stopper

·        funnel x 2

·        stringing rod (with police guard)

·        electric pH meter

·        litmus paper (red and blue)

·        creatine monohydrate powder

·        phosphocreatine powder

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Procedure

1. Prepare 100.0mL of a creatine monohydrate solution in a volumetric flask (see procedure for solution preparation­)

2. Rinse the burette and the stopcock with the titrant solution. 
3. Collect approximately 70.0mL of the sample (lactic acid) in a beaker
4. Drain any previous solution from the pipette
5. Cap 5.00mL of lactic acid in the pipette and transfer it to the Erlenmeyer flask
6. Add 2 to 3 drops of the universal indicator to the Erlenmeyer flask and swirl the solution
7. Place the Erlenmeyer flask just under the stopcock of the burette
8. Pour the titrant solution in to the burette and record the initial volume of the burette
9. Turn the stopcock so approximately 25.00mL of the titrant falls in to the flask
10. If the solution in the flask becomes a permanent green (the color does not fade away when swirled), record the final volume in the burette and continue the procedure.  If the color does fade from the solution, keep releasing approximately 25mL at a time in to the flask until it becomes a permanent green.  This step is to determine the approximate amount of titrant needed to render the sample green, so a more precise titration can be performed for the other trials.
11. Pour the contents of the Erlenmeyer flask in to a waste beaker and clean the flask with distilled water
12. Cap 5.00mL of lactic acid in the pipette and transfer it to the Erlenmeyer flask
13. Add 2 to 3 drops of the universal indicator to the Erlenmeyer flask and swirl the solution
14. Place the Erlenmeyer flask just under the stopcock of the burette
15. Pour the titrant solution in to the burette and record the initial volume of the burette
16. Release the stopcock until the volume of the solution in the Erlenmeyer flask is a  little less than the volume recorded previously
17. Let the solution in the burette drip once, then swirl to see if the solution in the flask has become green.  Repeat until it becomes green, then record the final volume of the burette
18. Repeat steps 11 to 17 for as many trials as desired
19. Repeat steps 1 to 18, preparing a solution of mixed creatine (phosphocreatine) and using it as the titrant
20. Repeat steps 2 to 18, using distilled water as the titrant

Table 6.1 –Burette readings and color of solution in the Erlenmeyer flask as creatine monohydrate solution is being titrated in to 5.00mL of lactic acid (first trial)

Table 6.2 –Burette readings and color of solution in the Erlenmeyer flask as creatine monohydrate solution is being titrated in to 5.00mL of lactic acid (second trial)

Table 6.3 –Burette readings and color of solution in the Erlenmeyer flask as creatine phosphate solution is being titrated in to 5.00mL of lactic acid (first trial)

Table 6.4 –Burette readings and color of solution in the Erlenmeyer flask as creatine phosphate solution is being titrated in to 5.00mL of lactic acid (second trial)

Table 6.5 – Burette readings and color of solution in the Erlenmeyer flask as water is being titrated in to 5.00mL of lactic acid

Discussion  

There were several factors which significantly impacted accounted for the outcome of the lab.  Firstly, only two trials were performed (due to time constraint) for each type of creatine, which limited the accuracy of the results.  Secondly, the phosphocreatine was not pure, and contained many other ingredients which may have influenced the acidity of the solution in the Erlenmeyer flask.