Creatine Case Study

Case Study

History

Learning Goals /
Concept Map


Creatine and Related Compounds

Structure

Amino Acids

Creatine in the Body

Equilibrium

Creatine-Creatinine Equilibrium

Creatinine Test for Kidney Function

Detection

• Regulation and Ethics

Amine & Nitrile Chemistry

Laboratory Synthesis

Chemical Analysis

Creatine-Phosphocreatine Equilibrium

Uses & Side Effects


Detection

We have seen that creatinine levels in the blood and urine are used as indicators of renal function. In addition, elevated levels of urinary creatine (in people who do not use creatine supplements) can indicate degenerative muscle disease[1]. Finally, some studies have suggested that short-term creatine supplementation leads to increased urinary creatinine levels that return to normal once creatine intake ends[2], whereas other studies have indicated that short-term creatine supplementation leads to elevated levels of creatine in the urine but no change in creatinine levels[3]. Clearly, it is critically important to have reliable analytical methods for the detection of both creatine and creatinine in order to monitor health and possibly supplementation in sport. Many methods exist for the detection of creatine and creatinine. To give a sense of the variety of methods available, a few of these techniques are described here.

Chemical Methods for the Determination of Creatinine and Creatine
Jaffé Reaction (Creatinine)

Originally discovered in 1886[4], the Jaffé reaction of creatinine with picric acid and base has long been used as a colorimetric test for measuring creatinine levels.  The product of the reaction is an orange-red complex and is postulated to be an enol tautomer of creatinine complexed with picric acid[5], but the mechanism of this reaction is not well understood[6].

The advantage of this test is that it is inexpensive, but the disadvantage is that other metabolites—such as ketones and ketoacids, protein, and bilirubin—interfere with the readings for creatinine, making it challenging to obtain accurate results[3].

Folin Method (Creatine)
The Folin method, which is an indirect method for the determination of creatine, takes advantage of both the creatine-creatinine equilibrium and the Jaffé reaction.  In this method, it is assumed that the sample contains both creatine and creatinine.  This test is carried out by first removing two aliquots from the original sample.  In the first aliquot, the amount of creatinine is measured using the Jaffé reaction.  In the second aliquot, all of the creatine is converted into creatinine.  As we have seen in CREATINE-CREATININE EQUILIBRIUM, creatine and creatinine interconvert in solution (outside of the body), and creatinine is favored under acidic conditions and at high temperatures.  Therefore, in order to convert creatine into creatinine, the aliquot is boiled in acid for one hour.  Alternatively, the aliquot may be autoclaved for 20 minutes at 120 °C[12].

The amount of creatinine in the second aliquot is then measured using the Jaffé reaction.  Because the second aliquot contains both the creatinine present in the original sample and the creatinine that was converted from creatine, the amount of creatinine should be greater in the second sample than in the first.  Therefore, the amount of creatine in the original sample may be determined by difference[12]:

Like the Jaffé method, this method is inexpensive, but it also leads to large errors in the reported concentration of creatine.  Furthermore, heating creatine in acid for one hour also produces sarcosine[12], causing the measured value of creatinine in the second aliquot to appear artificially low.

Enzymatic Methods for the Determination of Creatinine and Creatine
Levels of creatinine in the blood and urine may be determined using a series of enzymatic reactions, as shown below.  Creatinine is first converted into creatine through the action of creatinine amidohydrolase, and then creatine kinase is used to convert creatine into phosphocreatine.  These first two reactions are coupled to the NADH indicator system, in which ADP produced from the conversion of creatine into phosphocreatine reacts with phosphoenolpyruvate (PEP) as it is dephosphorylated to produce pyruvate and ATP.  The subsequent reduction of pyruvate into lactate using the reduced form of nicotinamide adenine dinucleotide (NADH) as an indicator allows the concentration of creatinine (or creatine) to be determined[7,12].

By omitting the first reaction in this sequence, this method may also be adapted to measure creatine levels.  Creatinine and creatine content are directly proportional to the decrease in NADH concentration, which is measured by absorbance[7].

The enzymatic methods have two advantages:  (1) blood and urine samples do not have to be pretreated, and (2) there is no interference from other common endogenous substances, such as pyruvate, bilirubin, and lactate in blood, as well as oxalate in urine[7].  The disadvantage of the enzymatic methods is their higher cost, which has prevented them from completely replacing the Jaffé method[12].

Biosensor Method for the Determination of Creatinine
Biosensors are used to detect analytes of biological interest, such as creatinine, glucose, lactate, and cholesterol.  These analytes can all be detected through the use of amperometric enzyme biosensors, in which an enzymatic reaction is coupled to an electrochemical reaction, and the resulting electron current is measured[8].

While some biosensors for creatinine are commercially available and currently used in clinical settings[12], others (such as the one described here) are still under development.  In the detection of creatinine, a series of enzymatic reactions converts creatinine into glycine, formaldehyde, and hydrogen peroxide[8,9]:

Application of a voltage causes the oxidation (loss of electrons) of hydrogen peroxide.  As a result, a current is produced; this current is proportional to the concentration of creatinine in the original sample[8]:

There are three limitations to this method:  (1) creatine and other substances present in the sample interfere with the accurate measurement of creatinine[8]; (2) three enzymes are required to retain their activity for this system to work, so this system is only good for a few days[8,9]; and (3) silver ions from the Ag/AgCl reference electrode can inactivate the required enzymes[9].  The second limitation has been addressed by the chemical modification, immobilization, and stabilization of the three required enzymes, thereby increasing their half-lives from a few days to a month or more, depending on the enzyme[9,10,11].  The third limitation has been addressed by spotting some of the silver reference electrodes with cellulose acetate solution; after drying, the cellulose acetate acts as a cover that prevents the silver chloride from leaching into solution and deactivating the enzymes.  This modification has extended the stability of the complete biosensor from less than one day to five days[11].

High-Performance Liquid Chromatography (HPLC) for the Determination of Creatine and Creatinine
Unlike the methods described above, chromatography makes it possible to analyze mixtures containing both creatine and creatinine (as well as other analytes) simultaneously.  In high-performance liquid chromatography (HPLC), a high-pressure pump forces a liquid solvent phase (also called the eluent) through a stationary solid phase (the adsorbent) that is tightly packed in a long stainless steel column.  As in thin layer chromatography (TLC), different components are separated on the basis of polarity.  However, because HPLC is often used to separate biomolecules, most of which are water-soluble, aqueous buffer systems are often used as the solvent phase in HPLC.  Aqueous solvents dissolve polar packing materials such as silica, so the analysis of water-soluble biomolecules requires a “reverse-phase” (RP) adsorbent that is extremely nonpolar.  A common packing material used in reverse-phase HPLC columns is an 18-carbon (C18) chain that is attached to silica gel particles[13].

Because RP packing materials are nonpolar, the nonpolar analytes adhere most tightly to the HPLC column and elute last, whereas the polar analytes elute first[[13].  This is the opposite of the trend we observed in the TLC analysis of the creatine reaction mixture in the CHEMICAL ANALYSIS module. The components of the eluted sample are analyzed in a variety of different ways, including mass spectrometry[13] and UV absorbance[13,14], as well as enzymatic detection and fluorescence[1].

Analysis of creatine and creatinine by HPLC is usually done using a phosphate buffer with careful attention paid to the preparation of the sample[1].  The presence of protein in the sample can wreak havoc on RP columns, so protein may be minimized by dilution[1,14] or removed either by centrifugation or use of a pre-column[1]

As we have seen, the advantage of HPLC is that many components of a serum or urine sample may be analyzed at the same time.  In addition to the high cost of the required instrumentation, a disadvantage of HPLC is that some components (including creatine, creatinine, and other substances) elute at similar times, making separation difficult.  A popular method for increasing the hold-up time of some substances that interfere with creatine is to add a quaternary ammonium ion-pairing agent (such as tetra-butyl ammonium sulfate)[12].

Summary
As we have seen, a wide variety of methods—including chemical, enzymatic, and instrumental—have been used to analyze serum and urine samples for creatine and creatinine.  The technique appropriate for each circumstance may vary and may be selected by considering cost, convenience, speed, and required level of precision.

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References

[1]   Smith-Palmer, Truis. “Separation Methods Applicable to Urinary Creatine and Creatinine.” J. Chromatogr. B, 2002, 781, 93-106.

[2]  Hultman, E.; Soderland, K.; Timmons, J. A.; Cederblad, G.; Greenhaff, P. L. “Muscle Creatine Loading in Man.” J. Appl. Physiol., 1996, 81, 232-237.

[3]  Burke, Darren G.; Smith-Palmer, Truis; Holt, Laurence E.; Head, Brian; Chilibeck, Philip D. “The Effect of 7 Days of Creatine Supplementation on 24-Hour Urinary Creatine Excretion.” J. Strength Cond. Res., 2001, 15(1), 59-62.

[4]   Bonsnes, Roy W.; Taussky, Hertha. “On the Colorimetric Determination of Creatinine by the Jaffé Reaction.” J. Biol. Chem., 1945, 158(3), 581-591.

[5]   Greenwald, Isidor. “The Chemistry of Jaffé’s Reaction for Creatinine II. The Effect of Substitution in the Creatinine Molecule and a Possible Formula for the Red Tautomer.” J. Am. Chem. Soc., 1925, 47(5), 1443-1448.

[6]   Wyss, Markus; Kaddurah-Daouk, Rima. “Creatine and Creatinine Metabolism.” Physiol. Rev., 2000, 80(3), 1107-1213.

[7]   Beyer, Cornelis. “Creatine Measurement in Serum and Urine with an Automated Enzymatic Method.” Clin. Chem., 1993, 39(8), 1613-1619.

[8]   D’Orazio, Paul. “Biosensors in Clinical Chemistry.” Clin. Chim. Acta, 2003, 334, 41-69.

[9]   Berberich, Jason A.; Yang, Lee Wei; Madura, Jeff; Bahar, Ivet; Russell, Alan J. “A Stable Three-Enzyme Creatinine Biosensor. 1. Impact of Structure, Function and Environment on PEGylated and immobilized sarcosine oxidase.” Acta Biomaterialia, 2005, 1(2), 173-181.

[10] Berberich, Jason A.; Yang, Lee Wei; Bahar, Ivet; Russell, Alan J. “A Stable Three-Enzyme Creatinine Biosensor. 2. Analysis of the Impact of Silver Ions on Creatine Amidinohydrolase.” Acta Biomaterialia, 2005, 1(2), 183-191.

[11] Berberich, Jason A.; Chan, Andy; Boden, Mark; Russell, Alan J. “A Stable Three-Enzyme Creatinine Biosensor. 3. Immobilizaton of Creatinine Amidohydrolase and Sensor Development.” Acta Biomaterialia, 2005, 1(2), 193-199.

[12] Smith-Palmer, Truis. “Clinical Analysis: Sarcosine, Creatine, and Creatinine.” in Encyclopedia of Analytical Science, 2nd ed.; Worsfold, Paul J.; Townshend, Alan; Poole, Colin F., Eds. Elsevier: Amsterdam, 2005; pp 166-174.

[13] Williamson, Kenneth L.; Minard, Robert D.; Masters, Katherine M. Macroscale and Microscale Organic Experiments, 5th ed. Houghton Mifflin: Boston, 2007; Ch. 9, p 200.

[14] Yang, Yue-dong. “Simultaneous Determination of Creatine, Uric Acid, Creatinine and Hippuric Acid in Urine by High Performance Liquid Chromatography.” Biomed. Chromatogr., 1998, 12, 47-49.