#5 Creatine & Creatinine

1. Outline biosynthesis of creatine & creatinine 2. Role of phosphocreatine in ATP homeostasis 3. Creatinine excretion in urine

Phosphagens

1. Role in storage high energy phosphate in

MUSCLE

2. [ATP] & [ADP] = small reserve (not stored as fuels) 3. Most cells: ATP use  ATP syn Muscle: ATP use >> ATP syn 4. Phosphagens also provide directionality (control), increase in ATP would affect all cell processes 5. Vertebrates: Phosphocreatine Some invertebrates: phospho Arg

Synthesis of Creatine

( i) 3 AA are involved in syn: GLY, ARG, MET (as SAM) (ii) First RXN: Transamidinase transfers amidine (H 2 N-C=NH 2 + ) group from ARG to GLY  guanidinoacetate + ornithine.

(ii) Second RXN: guanidinoacetate undergoes methylation (CH 3 ) via SAM  creatine This RXN represents the major use of SAM in the body (quantitatively). More SAM is used than in all other methyl rxns (> 50%)

Synthesis of Phosphocreatine

Creatine + ATP = ADP + creatine-P

(phosphagen) enzyme=creatine kinase (i) At rest RXN  (ATP demand low) At work  RXN (ATP demand increases) (ii) Creatine kinase  “functional” availability of ATP and directionality (only phosphorylates ADP) (iii) ATP is immediate donor free energy, rapid turnover & must be rapidly generated

ATP / Phosphocreatine Energetics

(i) ATP is a high energy compound  G ° = -7.3 kcal/mol (ii) Phosphocreatine also high energy  G ° = -10.3 kcal/mol (iii)Phosphocreatine is a compound designed to keep [ATP] constant Where creatine-P + ADP  Creatine + ATP Keq = [products] / [substrates] = 54 where  G ° = -3 CrP  Cr ATP  ADP Resting muscle 20 Working muscle 2 10 28

5.94

4.52

0.055

1.17

mM mM 54 = Keq resting = [10] [5.94] working = [28] [4.52] [20] [.055]  [2] [1.17] 

Stable

Thus ATP remains at 76% original [ ] although

Variable

[creatine - P] decreased

10x

(10%)

ENERGY USAGE

TIME

< 1 SEC ~ 4 SEC (100 m sprint) ~ 10 SEC (1 min run).

ENERGY

ATP Phosphocreatine Glycogen (muscle / liver) anaerobic glycolysis 1000 m Oxidative phosphorylation Marathon (hours) Fatty acid oxidation * Abundance of CrP * High phosphoryl transfer  effective phosphate energy buffer

Formation of Creatinine

(i) Spontaneous cyclization occurs  creatinine Occurs at a relatively constant rate Creatinine excreted in urine  constant and  (ii) Amount creatinine excreted urine  muscle mass time over which sample is taken measurement of total creatinine in 24 h urine collection indicates accuracy of collection (iii) Creatinine increases with elevated catabolism in muscle increase in muscular dystrophy

Creatine Kinase

Creatine Kinase (muscle enzyme) increase with muscle damage. CK exists as isozymes (MM, MB, BB) Only MB is found in the heart (15% of total CK) Measured in blood (after trauma)

Index of: myocardial infarction muscle trauma muscular dystrophy severe muscular exertion

#5. Creatine : Creatine Kinase and Myocardial Infarction Case Discussion

An obese, middle-aged man was brought to the emergency room following an automobile accident. The patient stated that he had been short of breath and very dizzy just before the crash. Examination suggested either a cerebrovascular accident or a myocardial infarction. The patient was admitted for observation, and blood samples for creatine kinase (CK) and other enzyme assays were periodically collected.

Discussion

1. Using a reaction described in this chapter, how might CK be assayed?

2. What is the relation of CK activity in the blood to tissue damage?

1. Assay creatine kinase using artificial substrate (instead of creatine): creatinine + ATP



H+ which can be measured through changes in pH 2. Diagnostic Value: CK is present in all tissues but only SELECTIVE RELEASE (brain, muscle but not liver) therefore different from liver damage Caused by:MI, muscle trauma, muscle dystrophy, severe exertion, IM injections, hypothyroidism, chronic alcoholism (with myopathy)

Discussion

3. What would be the advantage of measuring heart-specific isozyme and how could the isozymes of CK be differentiated?

4. What is the purpose of assaying for CK over a period of time?

3. Isozymes creatine kinase are tissue specific CK is a dimer of MM, MB, BB isozymes, and only MB is present in the myocardium (15% TOTAL CK) Analysis by antibody assay specific for isozymes (which can differentiate between proteins) 4. MB-CK: myocardial specific injury - 100% increase in MB-CK within 4 hr although total blood CK may be N (Why?) - Peaks at 8-24 hr then decreases, with aminotranserase change much slower



peak ~ 60hrs (To maximize treatment, prompt recognition essential) - Usually [CK] cardium & % MB/total CK are constant, [MB-CK



] is proportional to degree injury to myo cardium