DiMauro on fresh vs. frozen

[Guest guest]

Some of you might be interested in this info from DiMauro on the MDA site:

QUESTION: Can you tell if a mitochondrial myopathy is or is not maternally

inherited by a frozen muscle biopsy alone? What information does a fresh

biopsy give as opposed to a frozen biopsy?

REPLY from MDA: Salvatore DiMauro, M.D., Prof. of Neurology, New York

A frozen muscle biopsy can provide clues suggesting a mitochondrial DNA

(mtDNA) mutation and, therefore, maternal inheritance of the related

disorder. The clues are of two types: (i) if the biopsy is adequate for

histochemistry, evidence of scattered cytochrome c oxidase-deficient

(COX-negative) fibers, having a " mosaic " distribution among COX-positive

(normal) fibers, is a good indication of a mtDNA mutation, probably

affecting mtDNA genes involved in protein synthesis (such as tRNA genes);

(ii) biochemical analysis of respiratory chain enzymes may reveal decreased

activities of those complexes that contain mtDNA-encoded subunits, i.e.

complexes I, III, and IV (complex V is not assayed routinely in frozen

tissue). This is additional circumstantial evidence of a mtDNA mutation

affecting mitochondrial protein synthesis.

A fresh muscle biopsy allows careful isolation of a mitochondrial fraction;

the isolated mitochondria can be used to assess respiration and

phosphorylation using different substrates and an oxygen electrode

( " polarographic analyses " ), or to study ATP synthesis from ADP and

radio-labeled inorganic phosphate (Pi). These studies can provide

information on the site of a metabolic block in the electron transport chain

(complexes I to IV) and on the function of ATP synthetase (complex V).

------------

There is also an interesting 2003 abstract on the subject:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_ui

ds=12542905&dopt=Abstract

As I understand it, this says something similar to DiMauro but may be a

little easier to understand. They both say that complexes I-IV can be

assayed in frozen tissue, but to look at overall OXPHOS capacity one needs

fresh tissue. According to this abstract of a study in the Netherlands,

about 25% of their mito cases do not manifest as specific deficiencies in

one of the complexes, but only show up as reduced overall OXPHOS

capacity--which can only be measured in fresh tissue. If correct, this would

mean that 25% of mito patients could only get a diagnosis from fresh muscle

tissue. I don't know if all experts agree with these authors (or their

percentages) that a person can have normal activity in complexes I-V and

still have reduced overall OXPHOS capacity that causes disease. But this

distinction may explain why Dr. Korson and others feel some of their

patients with typical mito symptoms do have mito in spite of normal complex

I-V activity in muscle.

B

[Guest guest]

---Thank you so much for posting this valuable information!

Dawn A

In , " Barbara Seaman "

wrote:

> Some of you might be interested in this info from DiMauro on the

MDA site:

>

> QUESTION: Can you tell if a mitochondrial myopathy is or is not

maternally

> inherited by a frozen muscle biopsy alone? What information does a

fresh

> biopsy give as opposed to a frozen biopsy?

>

> REPLY from MDA: Salvatore DiMauro, M.D., Prof. of Neurology, New

York

>

> A frozen muscle biopsy can provide clues suggesting a

mitochondrial DNA

> (mtDNA) mutation and, therefore, maternal inheritance of the

related

> disorder. The clues are of two types: (i) if the biopsy is

adequate for

> histochemistry, evidence of scattered cytochrome c oxidase-

deficient

> (COX-negative) fibers, having a " mosaic " distribution among COX-

positive

> (normal) fibers, is a good indication of a mtDNA mutation, probably

> affecting mtDNA genes involved in protein synthesis (such as tRNA

genes);

> (ii) biochemical analysis of respiratory chain enzymes may reveal

decreased

> activities of those complexes that contain mtDNA-encoded subunits,

i.e.

> complexes I, III, and IV (complex V is not assayed routinely in

frozen

> tissue). This is additional circumstantial evidence of a mtDNA

mutation

> affecting mitochondrial protein synthesis.

>

> A fresh muscle biopsy allows careful isolation of a mitochondrial

fraction;

> the isolated mitochondria can be used to assess respiration and

> phosphorylation using different substrates and an oxygen electrode

> ( " polarographic analyses " ), or to study ATP synthesis from ADP and

> radio-labeled inorganic phosphate (Pi). These studies can provide

> information on the site of a metabolic block in the electron

transport chain

> (complexes I to IV) and on the function of ATP synthetase (complex

V).

>

> ------------

> There is also an interesting 2003 abstract on the subject:

> http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?

cmd=Retrieve&db=PubMed&list_ui

> ds=12542905&dopt=Abstract

>

> As I understand it, this says something similar to DiMauro but may

be a

> little easier to understand. They both say that complexes I-IV can

be

> assayed in frozen tissue, but to look at overall OXPHOS capacity

one needs

> fresh tissue. According to this abstract of a study in the

Netherlands,

> about 25% of their mito cases do not manifest as specific

deficiencies in

> one of the complexes, but only show up as reduced overall OXPHOS

> capacity--which can only be measured in fresh tissue. If correct,

this would

> mean that 25% of mito patients could only get a diagnosis from

fresh muscle

> tissue. I don't know if all experts agree with these authors (or

their

> percentages) that a person can have normal activity in complexes I-

V and

> still have reduced overall OXPHOS capacity that causes disease.

But this

> distinction may explain why Dr. Korson and others feel some of

their

> patients with typical mito symptoms do have mito in spite of

normal complex

> I-V activity in muscle.

>

> B

[Guest guest]

---Thank you so much for posting this valuable information!

Dawn A

In , " Barbara Seaman "

wrote:

> Some of you might be interested in this info from DiMauro on the

MDA site:

>

> QUESTION: Can you tell if a mitochondrial myopathy is or is not

maternally

> inherited by a frozen muscle biopsy alone? What information does a

fresh

> biopsy give as opposed to a frozen biopsy?

>

> REPLY from MDA: Salvatore DiMauro, M.D., Prof. of Neurology, New

York

>

> A frozen muscle biopsy can provide clues suggesting a

mitochondrial DNA

> (mtDNA) mutation and, therefore, maternal inheritance of the

related

> disorder. The clues are of two types: (i) if the biopsy is

adequate for

> histochemistry, evidence of scattered cytochrome c oxidase-

deficient

> (COX-negative) fibers, having a " mosaic " distribution among COX-

positive

> (normal) fibers, is a good indication of a mtDNA mutation, probably

> affecting mtDNA genes involved in protein synthesis (such as tRNA

genes);

> (ii) biochemical analysis of respiratory chain enzymes may reveal

decreased

> activities of those complexes that contain mtDNA-encoded subunits,

i.e.

> complexes I, III, and IV (complex V is not assayed routinely in

frozen

> tissue). This is additional circumstantial evidence of a mtDNA

mutation

> affecting mitochondrial protein synthesis.

>

> A fresh muscle biopsy allows careful isolation of a mitochondrial

fraction;

> the isolated mitochondria can be used to assess respiration and

> phosphorylation using different substrates and an oxygen electrode

> ( " polarographic analyses " ), or to study ATP synthesis from ADP and

> radio-labeled inorganic phosphate (Pi). These studies can provide

> information on the site of a metabolic block in the electron

transport chain

> (complexes I to IV) and on the function of ATP synthetase (complex

V).

>

> ------------

> There is also an interesting 2003 abstract on the subject:

> http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?

cmd=Retrieve&db=PubMed&list_ui

> ds=12542905&dopt=Abstract

>

> As I understand it, this says something similar to DiMauro but may

be a

> little easier to understand. They both say that complexes I-IV can

be

> assayed in frozen tissue, but to look at overall OXPHOS capacity

one needs

> fresh tissue. According to this abstract of a study in the

Netherlands,

> about 25% of their mito cases do not manifest as specific

deficiencies in

> one of the complexes, but only show up as reduced overall OXPHOS

> capacity--which can only be measured in fresh tissue. If correct,

this would

> mean that 25% of mito patients could only get a diagnosis from

fresh muscle

> tissue. I don't know if all experts agree with these authors (or

their

> percentages) that a person can have normal activity in complexes I-

V and

> still have reduced overall OXPHOS capacity that causes disease.

But this

> distinction may explain why Dr. Korson and others feel some of

their

> patients with typical mito symptoms do have mito in spite of

normal complex

> I-V activity in muscle.

>

> B