this is regarding the gut hormone,peptide YY,(3-36).the PYY will rise within 15 min after taking food and the peak level reached 90 min?.,,and gives the signal to the brain about the appetite regulation and also produce the normal post prandial level and reduce the food intake.so,how can we analyse and diagnose this effective gut hormone PYY(3-36) ? especially for diabetes patients ?
Dear Andrew, I think you can determine PYY (3-36) in blood sample of the mentioned diabetic patients. There are several valid methods for PYY determintion including ELISA (EIA & IEMA), RIA, IRMA, HPLC,...
My suggestion is considering at least 2 points. PYY is not very stable molecule, so it is better blood collection in test tube containing cocktail of antiprotases and EDTA too.
In this way, you will prevent false negative results obtaining.
For correct sampling and exact timing, it is better you have angiocath in the antecubital vein of the patients.
In this way you can sampling at different time with on needling.
best
mehdi
Andrew,
I concur with Mehdi. Inclusion of protease inhibitor and EDTA mixture is essential for all labile peptide hormones. You can purchase a 10x concentrated mixture of EDTA+Trasylol (Aprotinin) from various suppliers, such as Sigma Chemicals.
All plasma or serum samples can be kept chilled until the last sample is collected before beginning measurements by (ELISA or other methods of your choice). If measurements are to be made on a different day than sample collection, you can freeze samples for storage but avoid repeated freeze-thaw cycles. This means freeze samples in aliquots sufficient for desired measurements.
Best...
dear anrdew
you can detect this peptide by blood sampling in a scheduled time before and after feeding. then you can apply several method such as ELIZA or HPLC. but you must act promptly because it is very un-stable.
good luck
Thank you so much for Drs.Mehdi,Tausif and Habib..and one more doubt is,if i am collecting the blood sample,until the collection of last subject( few months),can i freeze the sample at -20 or else,what temp.is suitable for storage and how many days i can freeze the samples.
and one more thing,when i use the refrigerated centrifuge,how much of temp. and spin needed.
Hi Andrew,
I would collect into pre-chilled tubes containing an inhibitor such as DPP-IV, aprotinin and/or other chemicals to stabilize the sample. I would spin at ~1,500g or ~3,500rpm depending upon your centrifuge and at 4 degrees Celsius for 10 mins. Storage at -20 Celsius should suffice but -40 better and -80 ideal.
Hope this helps,
Jamie
Andrew, I didn't realize that your duration of sample collection was over a period of several months. As Jamie pointed out, you could collect blood sample in tubes containing an anticoagulant (EDTA, Citrate or Heparin), isolate plasma at 4ºC by spinning at 2,000xg for 15-20min (at this speed, you remove platelets), and store the plasma in polypropylene tubes containing a cocktail of protease inhibitors in aliquots that are suitable for individual use in your measurements. Repeated freeze thaw is not good for survival of many sensitive hormones and peptides.
Alternatively, you can also measure the peptide on the same day as you go along. With appropriate controls and care, day-to-day variations in measurements are minimal.
dear jamie and Tausif, really appreciated and thank you so much for your answers
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