[Histonet] RE: Phosphorylase method

Wed Mar 28 17:17:48 CDT 2012

This is the method we use:

Phosphorylase
Use
McArdle's disease (myophosphorylase deficiency) is the most common glycogenosis affecting skeletal muscle. The biochemical hallmark of the disease is impaired muscle glycogenolysis due to lack of muscle glycogen phosphorylase (M-GP) activity. M-GP is the muscle-specific isoform of the enzyme, with two others being the liver- and the brain-specific forms.

Muscle biopsies of McArdle's patients are characterized by subsarcolemmal storage of normal glycogen and by absent stain with the histochemical phosphorylase reaction. In some patients, however, it is possible to find scattered fibers with positive phosphorylase staining.
Underlying Principle
Phosphorylase catalyses the breakdown of Glucose-1-phosphate to glucose and phosphate ions. The Glucose is subsequently stained with iodine to give a purple localisation.

Fixation and Sectioning

Air dried unfixed 8µM cryostat sections
Reagents

1.	0.2M Sodium Acetate
Sodium acetate 		 3.6g
Distilled water 		500ml

2.	0.2M Acetic Acid
Glacial acetic acid( 17N) 	5.9ml
Distilled water  		make up to 500ml total volume

3.	0.1M Sodium hydroxide
Sodium hydroxide pellets 	 2.0g
Distilled water			500ml

4.	Incubating medium

0.2M sodium acetate 				15ml
0.2M acetic acid				5ml
Absolute alcohol 				5ml
Glucose 1 phosphate 				0.015 g
Adenosine monophophate (AMP)		0.015 g
EDTA						0.025 g
Sodium fluoride 				0.020 g
Dextran 				           1.000 g
Adjust pH to 5.9 using 0.1M NaOH

5.	Grams iodine
Iodine 				10.0g
Potassium iodide 	             2.0g
Distilled water 		300ml

Dissolve the Potassium Iodide in a small amount of water, dissolve the iodine in this, make up to 300ml.

Method

1.	Allow frozen sections to dry approx. 45min.
2.	Incubate in above medium for 60min at 37°C 
3.	Wash slides in distilled water 
4.	Stain in Grams iodine 1 min, 
5.	Wash well in distilled water
6.	Air dry 
7.	Clear in xylene
8.	Mount in DPX

Results
Phosphorylase positive muscle fibres		purple
Deficient muscle fibres and background 	yellow-brown
Smooth muscle in artery walls		purple
References
Martinuzzi et al (1999) "McArdle's Disease - The Unsolved Mystery of the Reappearing Enzyme" American Journal of Pathology.;154:1893-1897

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Sharon Allen
Sent: Thursday, 29 March 2012 12:47 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Phosphorylase method

Hi,

Does anyone have a good enzyme method for Phosphorylase done on muscle bx's. We have been doing the method from Dubowitz, 2nd edition for years but would like to get away from the messy PVP & coverslipping.  We are trying cytoseal, which seems to work fine for coverslipping, but would like to compare other methods without PVP.  Also, does anyone know the purpose of PVP, glyceron or dextrin in this test?

Thanks for any help,

Sharon Allen

Senior Medical Technologist

Neuropathology Lab-MS435U

Health Sciences Centre

820 Sherbrook Street

Winnipeg,MB, CA 

R3A 1R9

e-mail: sallen <@t> dsmanitoba.ca

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