“Extraordinary Measures” – A Movie about Pompe’s Disease


 

Extraordinary Measures is a 2010 film about parents trying to save their children affected by Pompe Disease, A Glycogen Storage Disease produced by mutations on a gen that makes the enzyme acid alpha Glycosidase (GAA), a lysosomal hydrolase.

 Pompe disease is a rare (estimated at 1 in every 40,000 births), inherited and often fatal disorder that disables the heart and muscles.

The movie is based on the true story of John and Aileen Crowley, whose two youngest children were affected with Pompe Disease.

The real John Crowley

As you know Glycogen storage diseases are genetic enzyme deficiencies that result in excessive glycogen accumulation within cells. Additional symptoms depend on the particular enzyme that is deficient.

There are different forms of Glycogen Storage Diseases (aka Glycogenoses), including the Type Ia GSD or Von Gierke’s disease, caused by hepatic deficiency of Glucose 6 Phosphatase, the Type IV or Andersen’s Disease, caused by deficit of branching enzyme in various organs, including the liver, and the GSD Type V or McArdle’s Disease (caused by muscle deficiency of Glycogen Phosphorylase), among others.

GSD Type II or Pompe’s Disease was described by Pompe in 1932, when he studied a girl who suffered from a cardiopathy caused by glycogen accumulation.

The National Institute of Neurological Disorders and Stroke (NINDS), an Institute of the National Institutes of Health System, describe the disease in these terms:

 

“Early onset (or infantile Pompe disease is the result of complete or near complete deficiency of GAA.  Symptoms begin in the first months of life, with feeding problems, poor weight gain, muscle weakness, floppiness, and head lag. Respiratory difficulties are often complicated by lung infections.  The heart is grossly enlarged. More than half of all infants with Pompe disease also have enlarged tongues.  Most babies with Pompe disease die from cardiac or respiratory complications before their first birthday. 

 

Late onset (or juvenile/adult) Pompe disease is the result of a partial deficiency of GAA.  The onset can be as early as the first decade of childhood or as late as the sixth decade of adulthood.  The primary symptom is muscle weakness progressing to respiratory weakness and death from respiratory failure after a course lasting several years.  The heart may be involved but it will not be grossly enlarged.  A diagnosis of Pompe disease can be confirmed by screening for the common genetic mutations or measuring the level of GAA enzyme activity in a blood sample — a test that has 100 percent accuracy.  Once Pompe disease is diagnosed, testing of all family members and consultation with a professional geneticist is recommended.  Carriers are most reliably identified via genetic mutation analysis.

A diagnosis of Pompe disease can be confirmed by screening for the common genetic mutations or measuring the level of GAA enzyme activity in a blood sample — a test that has 100 percent accuracy.  Once Pompe disease is diagnosed, testing of all family members and consultation with a professional geneticist is recommended.  Carriers are most reliably identified via genetic mutation analysis.”

“…Individuals with Pompe disease are best treated by a team of specialists (such as cardiologist, neurologist, and respiratory therapist) knowledgeable about the disease, who can offer supportive and symptomatic care.  The discovery of the GAA gene has led to rapid progress in understanding the biological mechanisms and properties of the GAA enzyme.  As a result, an enzyme replacement therapy has been developed that has shown, in clinical trials with infantile-onset patients, to decrease heart size, maintain normal heart function, improve muscle function, tone, and strength, and reduce glycogen accumulation.  A drug called alglucosidase alfa (Myozyme©), has received FDA approval for the treatment of infants and children with Pompe disease.  Another alglucosidase alfa drug, Lumizyme©, has been approved for late-onset (non-infantile) Pompe disease. ..”

“…Without enzyme replacement therapy, the hearts of babies with infantile onset Pompe disease progressively thicken and enlarge.  These babies die before the age of one year from either cardiorespiratory failure or respiratory infection.  For individuals with late onset Pompe disease, the prognosis is dependent upon the age of onset.  In general, the later the age of onset, the slower the progression of the disease.  Ultimately, the prognosis is dependent upon the extent of respiratory muscle involvement. …”

 It is interesting that even when the Acid Alpha-glycosidase is only involved in the degradation of about 3 % of the Glycogen, its deficit provokes such important damages. Since this enzyme is not related to the main pathways of degradation of glycogen, its deficit does not produce hypoglycemia or a direct lack of metabolic energy. Cellular damage is caused mainly by accumulation of glycogen in the cytoplasm and the lysosomes.

As describe above, nowadays the treatment is based on the use of a recombinant human acid Glycosidase as a replacement of the normal enzyme.  “Extraordinary Measures” describes, in fact, the events that triggered the development of the enzyme for the treatment of this disease.

My favorite quotes of this movie:

John Crowley (Looking at the college-aged kids hired to work under Dr. Stonehill):

-These guys make me feel old.
Dr. Robert Stonehill:

– Scientists get all sensible & careful when they get old. Young ones like risk, not afraid of new ideas… and you can pay ’em less.

 

John Crowley (talking with Dr. Stonehill after an argument):

–  “Fine, spend the rest of your life dreaming up great ideas that don’t get funded. Draw your diagrams on the wall that cure diseases in theory but never help a single human being in reality.”

 

John Crowley (arguing with a corporate executive about drug research):

–  “This is not about a return on an investment, it’s about kids. Kids with names, dreams, families that love them.”

 

Recommended articles and links:

NINDS Pompe Disease Information Page

Ibrahim, J.; McGovern, M. M.

Glycogen Storage Disease Type II

Some pictures of the Crowley family

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Cataracts and Biochemistry of the Lens


 

Original questions:

 

https://biochemistryquestions.wordpress.com/2008/11/03/q-about-cataracts-in-diabetes/

 

https://biochemistryquestions.wordpress.com/2008/10/20/about-a-baby-with-cataracts/

 

The light should pass through the cornea, aqueous humor, lens and vitreous humor before reaching the retina for triggering the process of vision. These structures should be transparent in order to allow the path of light.

 

 

The lens (m in the figure) is bathed in its anterior side by the aqueous humor (i) and in its posterior side by the vitreous humor (o). It has no blood capillaries (that would interfere with light path), so the aqueous humor is responsible of the nutrition of the lens and the disposal of metabolic products.

 

The energy necessary for the lens is provided mainly through anaerobic glicolysis; the Krebs cycle, located in peripheral cells, only provide about 5 % of the necessary energy. Pentose phosphate cycle is also another important metabolic pathway in lens since it provides NADPH necessary for the maintenance of the redox status of the lens proteins.

 

The majority of the proteins in the lens are alpha, beta and gamma crystallines. They should maintain a transparent environment, so they should be in a native, non aggregate state. Some disturbances, as changes in the redox states of these proteins or changes in osmolarity in the lens can produce lost of the native state and aggregation of these proteins.

 

Cataracts results from changes in solubility and aggregation of the crystallin proteins.

The most frequent kinds of cataracts are those that appear as result of aging (senile cataracts) or as a result of Diabetes Mellitus (diabetic cataracts). Other conditions can also result in cataracts: cataracts that appear in galactosemia are very similar in the way of production to the cataracts that appear in Diabetes.

 

Aldose reductase is an enzyme that usually reduce aldehyde group of aldoses to a primary alcohol, so the aldose becomes a polyalcohol. The enzyme uses NADPH as hydrogen donor.

 

Typical reactions catalyzed by aldose reductase are the formation of sorbitol (glucitol) and the formation of dulcitol (galactitol):

 

Glucose +NADPH —à Glucitol (Sorbitol) + NADP+

 

Galactose +NADPH —à Galactitol (Dulcitol) + NADP+

Sorbitol (Glucitol)

Sorbitol (Glucitol)

 

 

Aldehyde reductase function is mainly in the conversion of glucose to fructose.

Dulcitol (Galactitol)

Dulcitol (Galactitol)

 

 

 

 

The sequence of reactions is:

 

1. – Reaction of Aldehyde reductase:

Consist in the reduction of the aldehyde group of glucose to a primary alcohol group, with the conversion of the aldohexose glucose to a polyalcohol.

 

Glucose + NADPH.H+ —à Sorbitol + NADP+

 

2. – Reaction of Sorbitol dehydrogenase (SORD):

      Consist in the oxidation of the secondary alcohol group of Carbon 2 of Sorbitol to

       a ketone group. It results in the conversion of Sorbitol in Fructose, a ketohexose.

  

      Sorbitol + NAD+ –à Fructose + NADH.H+

 

This sequence of reactions is particularly important in the formation of fructose in the seminal vesicles and the liver, and it has the advantage over the use of the sequence in glycolysis for obtaining fructose – Glucose 6 (P) to Fructose 6 (P) – that this polyalcohol pathway does not require the expending of ATP.

 

Lens contains aldehyde reductase and also a very low activity of sorbitol dehydrogenase, so some of the glucose that enter in the lens is converted in fructose. This quantity is usually very low since the enzyme aldehyde reductase has a very high Km for glucose.

 

In conditions of hyperglycemia, since the concentration of aldehyde reductase substrate (glucose) is high, this enzyme becomes very active, and a high quantity of sorbitol is formed. Unfortunately for diabetic patients, the activity of sorbitol dehydrogenase in lens is very low (enough for normal conditions, but not for this abnormal situation) and for complicating more the problem, sorbitol formed in the lens diffuse with difficulty out of it.

 

As a result, Sorbitol accumulates and increases the osmotic effects producing cell swelling and structural damage (this effect would explain also the neuropathy and vascular problems present in Diabetic patients).

 

In the lens, these changes in osmolarity will affect the native conformation of the crystalline proteins, in such a way that they aggregate and form structures that scatter the light: Cataracts are being formed.

 

In patients with galactosemia, a congenital disease in which the patient can not metabolize galactose and this sugar accumulates, the physiopathology of cataracts formation and nerve damage apparently is similar to the mechanism described for diabetes:

 

Through the reaction of Aldehyde reductase occurs the reduction of the aldehyde group of galactose to a primary alcohol group, with the conversion of the aldohexose galactose to its corresponding polyalcohol, galactitol:

 

Galactose + NADPH.H+ —à Galactitol + NADP+

 

Galactitol accumulates increasing the osmotic pressure with similar results to those found in sorbitol accumulation in Diabetes Mellitus.

 

 

For more information, please visit the following links:

 

About Cataracts

 

Sorbitol: A hazard to diabetes

 

About side effects of Sorbitol

 

About Polyol pathway and arterioral dysfunction in hyperglicemia

 

About galactitol and cataracts formation in galactosemia

 

 

Energetic Balance of the oxidation of 1 mol of Pyruvate up to CO2 and water


Answers to Questions about Carbohydrate Metabolism CM-07 and CM-08

 

Answer to (CM-07 )

 

Which is the energetic balance (expressed in moles of ATP) of the total oxidation of 1 mol of Glucose up to CO2 and water, assuming that the Glycolytic Pathway and the Glycerol Phosphate Shuttle have been used.

 

Answer: 36 moles of ATP

 

Note: We are assuming also, as described in the question, that each mol of NADH.H+ oxidized in the respiratory chain yields 3 moles of ATP and each “mol” of FADH2 oxidized in the respiratory chain yields 2 moles of ATP)

 

Details here.

 

 

Answer to CM-08

 

Which is the energetic balance of the oxidation of 1 mol of pyruvate up to CO2 and water?

 

Answer: 15 moles of ATP

 

Note: We are assuming also, as described in the question, that each mol of NADH.H+ oxidized in the respiratory chain yields 3 moles of ATP and each “mol” of FADH2 oxidized in the respiratory chain yields 2 moles of ATP)

 

Details:

 

Oxidative Decarboxylation of Pyruvate occurs in mitochondria, in a reaction catalyzed by the Pyruvate Dehydrogenase Complex.

 

The global reaction is:

Pyruvate +  NAD+ + CoA –à  Acetyl CoA +  CO2 +  NADH.H+ 

 

The resultant Acetyl Co A is oxidized  in the Krebs Cycle yielding:

 

Isocitrate to  alfaketoglutarate  (+CO2)                              1 NADH.H+

alfaketoglutarate to  Succinyl CoA (+CO2)                         1 NADH.H+

Succinyl CoA to Succinate (SLP)                                           1 GTP

Succinate to Fumarate                                                              1 FADH2

Malate to Oxalacetate                                                                1 NADH.H+

 

Observe that at the end of the Kreb Cycle the 3 carbons of pyruvate have been oxidized to 3 CO2

 

At the same time, from the energetic point of view:

1 ATP have been obtained in the Krebs Cycle as GTP through Substrate level Phosphorylation (1 GTP is equivalent, from the energetic point of view,  to 1 ATP).

1 FADH2 have been obtained from the Krebs Cycle

4 NADH.H+ have been obtained inside the mitochondria (1 from Oxidative decarboxylation of Pyruvate and 3 from the Krebs cycle)

 

If we use the convention that each NADHH+ produce approximately 3 ATP in the Respiratory chain, and each FADH2 produce 2 ATP, the total ATP production is:

 

1 Substrate Level Phosphorylation (SLP)                           01 ATP

4 NADH.H+  x 3 ATP/NADH.H                                                12 ATP

1 FADH2  x  2 ATP/FADH2                                                        02 ATP

 Total                                                                                               15 ATP

 

 

 

 

 

 

 

Energetic Balance of the total oxidation of one mol of glucose up to CO2 and H2O: Understanding the contradictions.


 

For understanding the process of total oxidation of glucose, it is necessary to consider the different steps and metabolic pathways involved as well as the cellular location of these processes:

 

   

STEPS

M. PATHWAY

CELLULAR LOCATION

Glucose to  Pyruvate

Aerobic Glycolysis

Cytosol

Pyruvate to Acetyl CoA

Oxidative Decarboxylation of Pyruvate

Mitochondria

Acetyl Co A to CO2

Krebs Cycle

Mitochondria

  

 

Aerobic Glycolysis describes the oxidation of one mol of Glucose (6 carbons)  up to the formation of two moles of pyruvate (3 carbons each).

This conversion involves different reactions where ATP is produced or consumed:

Glucose to Glucose-6-(P)                                                                          – 1 ATP

 

Fructose-6-(P) to Fructose 1,6- bisphosphate                                 – 1 ATP

 

Since Fructose 1,6 bisphosphate becomes two trioses phosphate, the reactions after the aldolase reaction occurs twice (one for each triose that continues the glycolysis). In one reaction, NADH.H+  is produced; in other two reactions, a Substrate Level Phosphorylation (SLP) occur

 (2) Gliceraldehide 3 (P) to (2) 1,3 bisphosphoglycerate           +(2) NADH.H+

 

(2) 1,3 bisphosphoglycerate to (2) 3 phosphoglycerate (SLP*)     + 2 ATP

 

(2) Phosphoenol pyruvate to Pyruvate (SLP*)                                    + 2 ATP

 

(*Maybe it is necessary to recall now that there are two different ways of synthesizing ATP:  (a)SLP, in which ATP is synthesized using energy from some reactions of metabolism, like these reactions, and (b) Oxidative Phosphorylation, synthesis of ATP using the energy released in the Respiratory chain, from the oxidation of reduced cofactors)

 

Total energetic Balance from Glucose to (2) Pyruvate

(Aerobic Glycolysis): 

   _ 2 ATP  +  2 NADH.H+  + 4 ATP  = 2 ATP + 2 NADH.H+ in cytosol

 

 

An excellent animation of this process up to pyruvate can be found here.

 

Pyruvate will enter the mitochondria and will experiment Oxidative decarboxylation, in a reaction catalyzed by the pyruvate dehydrogenase complex.

The global reaction is:

2 Pyruvate + 2NAD+  +2 CoA —à 2 Acetyl CoA + 2 CO2 + 2 NADH.H+

This reaction occurs twice since each glucose (6 carbons) produce 2 pyruvates (3 carbons each), consequently these process produce

2 NADH.H+ in the mitochondria

 

Each Acetyl Co A is oxidized  in the Krebs Cycle yielding:

 

2 Isocitrate to 2 alfaketoglutarate            (+2CO2)                     2 NADH.H+

2 alfaketoglutarate to 2 Succinyl CoA (+2CO2)                          2 NADH.H+

2 Succinyl CoA to 2 Succinate (SLP)                                               2 GTP

2 Succinate to 2 Fumarate                                                                 2 FADH2

2 malate to 2 oxalacetate                                                                   2 NADH.H+

 

An animation with these reactions can be found in this link. 

Observe that at the end of the Kreb Cycle the 6 carbons of glucose have been oxidized to 6 CO2

 

At the same time, from the energetic point of view:

4 ATP have been obtained through Substrate level Phosphorylation (ATP synthesis without intervention of the energy of respiratory chain): 2 were obtained during the Aerobic Glycolysis and 2 were obtained in the Krebs Cycle as GTP (1 GTP is equivalent, from the energetic point of view,  to 1 ATP).

2 FADH2 have been obtained from the Krebs Cycle

8 NADH.H+ have been obtained inside the mitochondria (2 from Oxidative decarboxylation of Pyruvate and 6 from the Krebs cycle)

2 NADH.H+have been obtained in the cytosol through the Aerobic Glycolysis.

 

It is important to do these distinctions about the cellular location of the NADH.H+. since those produced in the cytosol should enter the mitochondria to be oxidized. Since the internal mitochondria membrane is impermeable to these nucleotides, (the mitochondria has their own pool of NAD) the NADH.H+ produced in the cytosol should enter using one of the shuttles already described for transporting the reduction equivalents of cytosolic NADH.H+  through the internal membrane of the mitochondria:

 

a)     the malate aspartate shuttle.

b)     The glycerophosphate shuttle

 

As described in other post, the malate-aspartate shuttle regenerates NADH.H+  inside the mitochondria, the energy yielding of the cytoplasmatic NADH.H+  is the same as if it was generated directly in the mitochondria

 

With the glycerophosphate shuttle, the reduction equivalents of the cytosolic NADH.H+  are transferred to FAD in the inner membrane. It means that the cofactor that will be oxidized in the respiratory chain when this shuttle is used, is FADH2 (see the explanation in this related post)

 

In conclusion, when the malate aspartate shuttle is used for transporting the reduction equivalents from the cytosolic NAD+ inside the mitochondria, we can consider that the t oxidation of glucose has produced:

4 ATP

10 NADH.H+  to be oxidized in the Respiratory chain

2 FADH2 to be oxidized in the Respiratory Chain.

 

If we use the convention that each NADHH+ produce approximately 3 ATP in the Respiratory chain, and each FADH2 produce 2 ATP, the total ATP production is:

 

Substrate Level Phosphorylation (SLP)                           04 ATP

10 NADH.H+  x 3 ATP/NADH.H                                          30 ATP

02 FADH2  x  2 ATP/FADH2                                                04 ATP

 Total                                                                                           38 ATP

 

Using the same convention (each NADHH+ produce approximately 3 ATP in the Respiratory chain, and each FADH2 produce 2 ATP,), but now assuming that the shuttle use is the glycerophosphate shuttle:

 

Substrate Level Phosphorylation (SLP)                         04 ATP

08 NADH.H+  x 3 ATP/NADH.H                                        24 ATP

04* FADH2  x  2 ATP/FADH2                                            04 ATP

 Total                                                                                         36 ATP

(*02 from the use of the glycerophosphate shuttle and 02 from the Krebs Cycle)

 

It explains that some textbooks say that the energetic Balance of the Total Oxidation a a mol of Glucose is 36-38 moles of ATP (since it depends on the shuttle that is used for entering the reduction equivalents of the NADH.H+ produced in the cytosol through glycolysis).

 

Other possible results:

 

As described in other post, some books use the convention that each mol of NADH.H+, when oxidized in the respiratory chain, produce approximately 2.5 moles of ATP, while each mol of FADH2 produce 1.5 moles of ATP. Using this convention:

When the malate-aspartate shuttle is used:

Substrate Level Phosphorylation (SLP)                         04 ATP

10 NADH.H+  x 2.5 ATP/NADH.H                                     25 ATP

02 FADH2  x  1.5 ATP/FADH2                                           03 ATP

 Total                                                                                         32 ATP

 

When the glycerophosphate shuttle is used:

Substrate Level Phosphorylation (SLP)                         04 ATP

08 NADH.H+  x 2.5 ATP/NADH.H                                    20 ATP

04* FADH2  x  1.5 ATP/FADH2                                        06 ATP

 Total                                                                                        30 ATP

(*02 from the use of the glycerophosphate shuttle and 02 from the Krebs Cycle)

 

It explains that some textbooks say that the energetic Balance of the Total Oxidation a a mol of Glucose is 30-32 moles of ATP

 

An advice: When solving a problem of this kind is absolutely necessary to know the conventions used for the yielding of the reduced cofactors (2.5 or 3 ATP/ NADH.H+ ? 1.5 or 2 ATP/ FADH2?) and the kind of shuttle that has been used for entering the reduction equivalents from the cytosol to the mitochondria.  A fair question will have both information. If not information is provided in the question, use the conventions followed by your professor during the lectures.

For students preparing for USMLE exams, the most used review books, like Harvey and Champe, in the “Lippincott Illustrated Reviews” of Biochemistry, Dawn B. Marks in “Biochemistry, Board Review Series”, Kaplan Biochemistry Lecture Notes for USMLE and “First Aid for the USMLE Step I”, agree in using the equivalence of  ‘approximately” 3 ATP for each NADH.H+  that is oxidized and ‘approximately” 2 ATP per FADH2. 

 

Related post:

To understand the mechanism of the shuttles and their difference in yielding ATP, I strongly recommend to read this post.

ATP yield in Aerobic Glycolysis: 5, 6, 7 or 8 ATP/glucose?


Sometimes students get very confused when they found that the energetic balance in his Biochemistry book does not agree with the energetic balance studied in classroom. The student can get even more confused when he/she uses some consultation books and found that values also can differ from one book to another.

The intention of this post is to clear the apparent contradictions in energetic balance of the oxidation of glucose, in some of the books more used by biomedical students.

Aerobic Glycolysis describes the oxidation of one mol of Glucose (6 carbons)  up to the formation of two moles of pyruvate (3 carbons each).

This conversion involves different reactions where ATP is produced or consumed:

Glucose to Glucose-6-P                                                                              – 1 ATP

 

Fructose-6-P to Fructose 1,6- bisphosphate                                     – 1 ATP

 

Since Fructose 1,6 bisphosphate becomes two trioses phosphate, the reactions after the aldolase reaction occurs twice (one for each triose that continues the glycolysis). In one reaction, NADH.H+  is produced; in other two reactions, a Substrate Level Phosphorylation (SLP) occur.

(2) Gliceraldehide 3 (P) to (2) 1,3 bisphosphoglycerate              (2) NADH.H+

 

(2) 1,3 bisphosphoglycerate to (2) 3 phosphoglycerate (SLP)            + 2 ATP

 

(2) Phosphoenol pyruvate to Pyruvate (SLP)                                           + 2 ATP

 

 

Total energetic Balance from Glucose to (2) Pyruvate

(Aerobic Glycolysis): 

·        _ 2 ATP  +  2 NADH.H+  + 4 ATP  = 2 ATP + 2NADH.H+

 

 

An excellent animation of glycolysis can be found here. 

 

The NADH.H+ will be oxidized in the Respiratory Chain and during the process, will release energy enough for the synthesis of additional ATP.

Maybe it is necessary to recall now that there are two different ways of synthesizing ATP:

a)               Substrate Level Phosphorylation (SLP) in which a ATP is synthesized using energy from some reactions of metabolism, (like the two reactions indicated above)

b)               Oxidative Phosphorylation: Synthesis of ATP using the energy released in the Electron Transport Chain, from the oxidation in the Respiratory Chain of reduced cofactors.                                                          

 

So, up to this moment it has been obtained in the aerobic glycolysis 2 ATP (through SLP) and 1 NADH.H+. This NADH.H+ will release energy for the synthesis of ATP in the Respiratory Chain…. but exactly how much ATP can be synthesized from the oxidation of this NADH.H+ ?

It is a question kind of complicated, since there are different factors affecting the answer:

1.- Which “energetic yield” criteria is used?

Traditionally Biochemistry textbooks have used the criteria that with the energy released by each mol of NADH.H+ that is oxidized in the Respiratory Chain, 3 moles of ATP can be produced. More accurate calculations indicates that in fact, the energy released when 1 mol of NADH.H+  is oxidized, is just enough for the synthesis of 2.5 moles of  ATP and not 3 ATP as considered before. Those calculations indicate also that the equivalent of the oxidation of 1 “mol” of FADH2 is enough for the synthesis of 1.5 moles of ATP and not 2 moles of ATP as was considered before. Anyway,  different textbooks continue using the equivalents of 3 and 2 ATP for each NADH.H+  and FADH2 oxidized.

“Lehninger’s principles of Biochemistry”, Devlin’s “Textbook of Biochemistry”,” and Marks’Essential of Medical Biochemistry” base the calculations in an energetic yield of 2.5 ATP per NADH.H+  that is oxidized and 1.5 ATP for each FACH2 oxidized in the Respiratory Chain.

Harvey and Champe, in the “Lippincott Illustrated Reviews” of Biochemistry, Dawn B. Marks in “Biochemistry, Board Review Series”, Kaplan Biochemistry Lecture Notes for USMLE and “First Aid for the USMLE Step I”, use the equivalence of  3 ATP for each NADH.H+  that is oxidized and 2 ATP per FADH2. (some of them have introduced the word “approximately”, so they say that these cofactors yield approximately 3 ATP and 2 ATP respectively)

To increase the confusion, some authors, like Wilcox ( “High Yield Biochemistry”, 2nd. Edition) use sometimes an equivalence and sometimes other in the same book.

(In my opinion, the continue use of a yield of 3 ATP and 2 ATP for NADH.H+ and FADH2, is based on tradition and also because these are more intuitive values and easier to understand when talking in terms of molecules and not in term of moles. It is obvious that it is quite difficult to understand that the oxidation of one molecule of NADH.H+ releases energy for the synthesis of 2.5 molecules of ATP, since expressions that are valid when talking about moles, become absurd when are applied to molecules.)

Besides this factor that affects all the calculations that involve the production of ATP from the oxidation of NADH.H+  or reduced flavoproteins, there is other factor that is specifically related to the oxidation of NADH.H+  produced in the cytosol; the kind of shuttle that is used for transporting the cytosolic NADH.H+  to the mitochondria, where this reduced cofactor will be oxidized in the Respiratory Chain.

2.- Which shuttle is used for transporting NADH.H+  produced in the cytosol to the mitochondria?

The internal mitochondria membrane is not permeable to NAD+ or NADH.H+  (the mitochondria has their own pool of these nucleotides).

There are two different mechanisms for transporting the reduction equivalents of cytosolic NADH.H+  through the internal membrane of the mitochondria:

a)      the malate aspartate shuttle.

b)      The glicerolphosphate shuttle.

a)          Malate-Aspartate Shuttle:

 

The reduction equivalents of the cytosolic NADH.H+  are transferred to oxalacetate to form malate, in a reaction catalyzed by a cytosolic malate dehydrogenase:

Cytosol: Oxalacetate + NADH.H+  —-à Malate + NAD+

Malate can pass through the mitochondria membranes and enter the matrix. Once in the matrix the malate is dehydrogenated by a mitochondrial malate dehydrogenase:

Mitochondria: Malate + NAD+ ——à oxalacetate + NADH.H+ 

The oxalacetate is transaminated to aspartate to go out of the mitochondria and once in the cytosol, the aspartate is transaminated to oxalacetate beginning a new cycle.

Since this malate-aspartate shuttle regenerates NADH.H+  inside the mitochondria, the energy yielding of the cytoplasmatic NADH.H+  is the same as if it was generated directly in the mitochondria (2.5 or 3 ATP, depending on the equivalence followed)

b)       The glycerol phosphate shuttle

 

With this shuttle, the reduction equivalents of the cytosolic NADH.H+  are transferred to  dihydroxyacetone phosphate to form glycerol 3-phosphate, in a reaction catalyzed by a cytosolic glycerol 3-phosphate dehydrogenase, that oxidizes the cytosolic NADH.H+  :

 

                                    

Cytosol: dihydroxyacetone (P)+ NADH.H+  —-à glycerol 3 (P) + NAD+

 

The glycerol 3 (P) is dehydrogenated by a mitochondrial glycerol 3 (P) dehydrogenase  located on the outer surface of the inner membrane of the mitochondria, that transfer the reduction equivalents to FAD in the inner membrane:

Inner membrane: glycerol 3 (P) + FAD —à dihydroxiacetone (P) + FADH2

Observe that the reduction equivalents have been transferred to FAD and not to NAD. It means that FADH2 is the cofactor that will be oxidized in the Respiratory Chain, and so, the use of this shuttle will yield less ATP: 1.5 or 2 ATP, depending on the equivalence that is followed.

So, the answer to the original question, “ATP yield in Aerobic Glycolysis: 5, 6, 7 or 8 ATP/glucose?”, can be:

a)      2 ATP from SLP + 3 ATP if it is considered that the reduction equivalents of the cytoplasmatic NADH.H+  are transported  through the glycerol phosphate shuttle, and that for each FADH2 oxidized in the respiratory chain, the energy released can yield 1.5 ATP

b)      2 ATP from SLP + 4 ATP if it is considered  that the reduction equivalents of the cytoplasmatic NADH.H+  are transported through the glycerol phosphate shuttle, and that for each FADH2 oxidized in the respiratory chain, the energy released can yield 2 ATP

c)      2 ATP from SLP + 5 ATP if it is considered that the reduction equivalents of the cytoplasmatic NADH.H+  are transported through the malate aspartate shuttle and that for each NADH.H+ oxidized in the respiratory chain, the energy released can yield 2 .5 ATP

d)      2 ATP from SLP + 6 ATP if it is considered that the reduction equivalents of the cytoplasmatic NADH.H+  are transported  through the malate aspartate shuttle and that for each NADH.H+ oxidized in the respiratory chain, the energy released can yield 6 ATP

 

In summary, the answer to the original question…“ATP yield in Aerobic Glycolysis: 5, 6, 7 or 8 ATP/glucose?”, can be:

“ATP yield in Aerobic Glycolysis: 5, 6, 7 or 8 ATP/glucose!”.

 

So, the bottom line is that for answering a question involving energetic balance of a metabolic pathway, it is important to know:

         which equivalence is used (which one use your professor? Which one is used in the kind of exam you are taking? Most of the Biochemistry review books for USMLE use the equivalence of 3 ATP per NADH.H+ and 2 ATP per FADH2).

         In the case of NADH.H+  generated in the cytosol (NADH.H+  from glycolysis is not the only NADH.H+  generated in cytosol!), which shuttle is used to move the reduction equivalents from the cytosol to the mitochondria.

 

 Related Post:

Energetic Balance of the Total Oxidation of one mol of glucose up to CO2 and H2O: Understanding the Contradictions.

 

About Muscle Glycogen Phosphorylase Deficiency (GSD V)


Answer to CM-02

 

Original Question

Answer: (c)

                                    https://i2.wp.com/www.gfmer.ch/International_activities_En/Images/Leonardo/Muscles1.jpg

An important energy source for exercising muscle is anaerobic glycolysis, using glucose from the blood, that is converted to glucose 6 (P) inside the muscle cells, and glucose 6 (P) formed from glycogen stored in the muscle. The final product of anaerobic glycolysis is lactate.

The lactate diffuses from the muscle to the blood and then is transported to the liver where it is converted in pyruvate.

This patient is not able to use the glycogen stored in his muscle to obtain energy, since he lacks muscle glycogen phosphorylase, which degrades glycogen to glucose 1 (P). Normally this glucose 1 (P) becomes glucose 6 (P) in a reaction catalyzed by phosphoglucomutase and then glucose 6 (P) enters glycolysis. Due to this patient inability to degrade glycogen, it accumulates in the muscle fiber (it is called Glycogen Storage Disease Type V or McArdle Disease), the patient present fatigue very early in the test and the concentration of lactate in blood drawn from the forearm is lower than in a normal person.

Glucose in blood is not affected since muscle glycogen does not buffer blood glucose.

 

For more information about McArdle Disease, click on these links

Cupler, E.J.: Glycogen Storage Disease Type V

Stojanov, L.:Glycogen Storage Diseases Type I-VII

Association for Glycogen Storage Disease

The association for Glycogen Storage Disease UK

                        Leonardo anatomy muscle drawing

A: About a baby with Fructose Intolerance (C-02)


Original question 

 

Short answer: (f)

 

Sucrose or table sugar is a disaccharide formed by glucose and fructose. When digested in the small intestine by the action of sucrose, glucose and fructose are released. For the patient, the effect is the same as consuming fructose.

(Usually the baby is asymptomatic during the first weeks of life, until he/she consumes fructose (in fruit juices) or milk sweetened with sucrose)

 

Extended answer:

 

Sucrose is formed by glucose linked by an alpha-1, beta-2 O-glycoside linkage to fructose.  Sucrose is hydrolyzed by the action of Sucrase, a disaccharidase of the epithelial brush border, to free glucose and fructose. These monosaccharides are absorbed in the small intestine and travel to the portal system up to the liver.

 

In the liver, glucose is phosphorylated to Glucose 6(P) by Glucokinase (present in the liver) or the Hexokinase (present in the liver and in most of the tissues). (See Kinases enzymes).

 

Fructose also needs to be phosphorylated to be metabolized. This phosphorylation can happens in a reaction catalyzed by Fructokinase, (yielding fructose 1 phosphate), or in a reaction catalyzed by Hexokinase (the specificity over substrate of Hexokinase is relative, so it can catalyzes the phosphorylation of different hexoses).

 

Fructose 6 (P) + ADP <—————-Fructose  + ATP ————-> Fructose 1 (P) + ADP

                                            Hexokinase                                   Fructokinase

 

The Fructose 6 (P) formed by the action of Hexokinase, can be degraded through the glycolysis pathway

 

Fructose 6 (P) + ATP ———————->fructose 1,6 bis (P) + ADP

                                      Phosphofructokinase

 

 

Fructose 1,6 bis (P) <—- ——–> 3 (P) glyceraldehyde + (P) dihydroxyacetone)

                                          Aldolases

 

…until completion.

 

 

The Fructose 1 (P) formed by the action of Fructokinase, will be split by the Aldolase B, aka fructose 1 (P) aldolase, to two trioses,  (just one of them is a phosphorylated triose):

 

Fructose 1 (P) ——————> Glyceraldehyde + (P) dihydroxiacetone

         

 

Errors in Fructose Metabolism:

 

These errors appears usually as a result of the deficit of the enzymes Fructokinase or Aldolase B

 

Fructokinase: Fructose +ATP  ———–> Fructose 1 (P) + ADP

 

Aldolase B: Fructose 1 (P)——> Glyceraldehyde + (P) dihydroxyacetone

 

If the deficitary enzyme is Fructokinase, the patient will present Benign Fructosuria, a disease that usually does not have consequences for the patient and sometimes is discovered by accident when fructose is detected in urine 

 

If the deficitary enzyme is Aldolase B, the disease that appears is Hereditary Fructose Intolerance, an autosomal  recessive disease with a frequency of 1:20 000 newborns.

The lack of this enzyme can produce deleterious consequences, apparently as a result of the trapping of (P) in form of fructose 1 (P) inside the cell, since fructose 1 (P) can not be hydrolyzed to fructose, or isomerized to fructose 6 (P) or glucose 1 (P), or be phosphorylated to Fructose 1,6 bisphosphate.

 

The increase of Fructose 1 (P) in the cell inhibits the action of fructokinase, causing fructosemia.  More dangerous, the decrease of availability of  phosphate inside the cell – since it is trapped as Fructose 1 (P) – causes a decrease in the formation of ATP.  It produces a decline in gluconeogenesis and consequently the patient presents hypoglycemia. It has been described that an important factor in decreasing gluconeogenesis is that aldolase B has also a similar effect to other Aldolases in the catalysis of the interconversion between phosphotrioses and fructose 1, 6 bisphosphate.

The lack of energy in the hepatocytes affects the synthesis of plasma proteins including coagulation factors and the patient can present hemorrhages.

 

Since the production of ATP is impaired, Adenine accumulates and its conversion to Uric acid increases (recall that in the catabolism of purines adenine and Guanine become Uric Acid), and the patient presents Hyperuricemia.

 

So, this patient can present sever hypoglycemia, vomiting, failure to thrive, jaundice, coagulopathy, hepatomegaly, sever metabolic acidosis and the ultimate consequence in a non treated patient is hepatic failure and death.

 

The long term treatment consists basically in the elimination of Fructose and Sucrose from the diet. Since many fruits are rich in fructose, and sucrose and High Fructose Corn Syrup (HFCS) are used for sweeter in the commercial production of many foods, the patient should follow the indications of an experienced nutritionist.

 

For more information about Hereditary Fructose Intolerance:

 

Roths, K.S.: Fructose 1 (P) Aldolase Deficiency (Fructose Intolerance)

  

  

 A good summary about Fructose metabolism can be found at:

The Medical Biochemistry page:  Metabolism of Major Non-glucose Sugars