SKM Metabolism practical

Question 1

Why is Gas Chromatography-Combustion-Isotope Ratio Mass Spectometry (GC-C-IRMS) important?

Answer 1

• Allows us to measure very low levels of 13 C labelling in amino acids
• Important as mussccle turns over v slowly, only small amounts of label are incorporated

Question 2

How does GC-C-IRMS work?

Answer 2

• GC sepates the Amino Acid mixture into individual AA peaks
• They are combusted to CO2 and N2 gases at high temperature
• They enter the mass spec for analysis of the isotope ratio of the CO2
• System has 3 detectors which analyse the sample simultaneously for mass 44-46

Question 3

How do we measure both the concentration and labelling/enrichment of amino acids ?

Answer 3

• Make the compounds volatile for gas chromatographic separation
  -amino acids need to be made more volatile and less polar

Question 4

How does GC separate complex mixtures?

Answer 4

• By increasing the temp of the oven so the compund becomes more volatile and is pushed through the column by Helium flow

Question 5

What does mass spec fragmentation allow?

Answer 5

• To identify the compound and measure the amount of tracer in the compound

Question 6

What are the applications of GC-MS ?

Answer 6

• To identify compounds and measure concentration
• relevant to:
• Drug Abuse
• Doping in Sport
• Human Metabolic Research
• Protein, Fat and Carbs Metabolism

Question 7

How are stable isotope tracers used?

Answer 7

• Tracer is infused into the body
• Muscle Biopsy taken

Question 8

What is the concept of “Muscle full” in relation to whey protein intake?

Answer 8

• Despite continued availabilty of amino acids, MPS resources are TIME LIMITED

Question 9

What is anabolic resistance?

Answer 9

MPS responses to protein nutrition are blunted with advancing age even with enhanced DOSES
- Seen with inactivity and numerous disease states

Question 10

What is enrichment?

Answer 10

The amount of heavy/stable isotope present in a compound
- Enrichment can be diluted by adding more unlabelled compound

Question 11

How is mass spec used to measure concentration?

Answer 11

• Amino acid conc curves are prepared by accurately pipetting amounts of aa stadards and internal standards (isotopicallu labelled)
• Samples are dried, derivatised , transferred to autosampler vials and then run on GC-MC overenight
• Data will be collected and then forwarded to demonstrate the accurace and reproducinility og GC-MS
• Plasma AA standard (pAA) mix containing all proteinogenic AA and an internal standard AA mix, containing stable isotopically labelled AA (ISmix) to distinguish them from the unlabelled pAA is given