Biochemistry

Diabetes Mellitus: Sugar Pee and A1C

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Diabetes mellitus, or “sugar” diabetes, occurs when the body inappropriately manages its blood sugar levels. The key player in diabetes mellitus is a hormone called insulin. For simplicity we will use the term “glucose” as a relatively generic term for “sugar” going forward, but remember that glucose is actually one type of sugar.

Insulin is a protein secreted by specialized cells in the pancreas known as β-cells. When the body senses a high level of glucose floating around in the blood it causes the pancreas to secrete insulin. Many individual insulin molecules then travel throughout the body where it binds to receptors on many different tissue types. For example, if insulin binds to hepatocytes (liver cells) it instructs those cells to remove glucose from the blood and store it in a polymerized form known as glycogen. Insulin’s goal is to bring blood sugar levels to within normal limits.

The problem in diabetes mellitus is that insulin is either not secreted by the pancreas, or it does not perform its functions appropriately. Either way, glucose is not removed from the blood stream. The result is a higher than normal blood sugar concentration.

There are two distinct forms of diabetes mellitus: type 1 diabetes (sometimes referred to as juvenile onset or insulin dependent diabetes) and type II diabetes (sometimes referred to as adult onset or insulin independent diabetes).

Type 1 diabetes is an auto-immune disorder, which means that the bodies’ immune system attacks itself. In this case, the body attacks the β-cells in the pancreas. Once enough β-cells are destroyed the pancreas can no longer secrete insulin.

The pathology of type 2 diabetes mellitus is more complicated. There is no single "cause" of type 2 diabetes, and as such, it is believed to be the result of a combination of factors. Genetics, lifestyle, and diet all play an important role in the development of this form of the disease. I like to think of type 2 diabetes as a slew of factors that have beaten down the bodies ability to properly manage blood sugar levels.

In type 2 diabetes the pancreas is still able to produce insulin. However, the problem is that insulin does not cause its normal effect on body tissues. This is known as "insulin resistance." In an effort to combat this resistance the pancreas secretes more insulin. Once the insulin resistance becomes too severe the pancreas can not keep up, and blood sugar levels begin to rise. If the levels rise beyond a specific laboratory threshold the diagnosis of diabetes is made.

How Does Diabetes Mellitus Present?

High blood sugar levels lead to the symptoms of diabetes mellitus. One of the most common symptoms of diabetes is a frequent need to urinate. The reason this occurs is because the excess blood sugar exceeds the kidney’s ability to re-absorb it. As a result, glucose leaks into the urine. The body is forced to dilute this excess solute load by secreting more water into the urine. Hence more urine production –> increased trips to the bathroom!

Since diabetics have difficulty using sugar as a fuel they often lose weight (although most type II diabetics are overweight or obese secondary to excessive caloric intake). Diabetes, in metabolic terms, is similar to slowly starving. The body is unable to utilize sugar appropriately, which is the main “fuel” for most people. Normally, excess sugar gets converted to fat (ie: for you “need-to-know it all types” glucose is converted into acetyl-CoA fragments. If these are not “burned” by the Krebs cycle they get polymerized into long chain fatty acids, which get stored in fat tissue). However, in diabetics the sugar is "lost" in the urine and not used for metabolic purposes.

Diabetes, in metabolic terms,
is similar to slowly starving.
If blood sugar levels become ridiculously high severe complications, and potentially death can result. These patients can become very dehydrated and will often have significant electrolyte abnormalities. These factors can lead to coma, heart arrhythmias, and death if left untreated. These severe complications usually occur during periods of "stress." Conditions like infections and certain drugs (both prescription and illicit) can cause the "stress" needed to induce a diabetic crisis.

When a diabetic crisis occurs in type 1 diabetics it is referred to as "diabetic ketoacidosis". It is known as "non-ketotic hyperosmolar syndrome" if it occurs in a patient with type 2 diabetes.

What Else Can Happen to People with Diabetes?

There are many complications of diabetes mellitus. These complications are the result of years of high sugar levels in the blood. Over time the excess sugar undergoes a metabolic transformation known as "glycosylation". Glycosylated sugar is toxic to nerves and blood vessels.

Diabetic Ulcer
Diabetic foot ulcer
If damage occurs to the nerves that innervate the stomach a condition known as "gastric paresis" can occur, which is a fancy term for the stomach not being able to contract as well as it used to.

In addition, nerves that carry sensory information can also be damaged, especially at the finger tips and toes. Patients with this type of nerve damage may not feel cuts and blisters on their toes. These areas can become secondarily infected resulting in gangrenous digits that are often amputated (see image to left). This is part of the reason why all diabetics, especially poorly controlled diabetics, should see a foot specialist regularly.

Perhaps the most frightening complication of diabetes is the havoc it wreaks on the cardiovascular system. Diabetics have a much larger risk of heart attack and stroke.

Patients with diabetes are at risk for retinopathy, or damage to the retina of the eye. This can lead to blindness. Damage to the kidney’s filtering area known as the glomerulus can also occur; this can lead to chronic kidney disease. If severe enough, patients may need permanent dialysis or kidney transplantation.

Diagnosis

The diagnosis of diabetes is made by measuring the amount of sugar in the blood. There are three common ways to diagnose diabetes today. The first is by checking a molecule known as hemoglobin A1C. Diabetes can also be diagnosed with fasting blood sugar levels or by a glucose tolerance test. There are three possible results of these tests: normal, impaired (pre-diabetic), or diabetic.

Hemoglobin A1C levels are drawn from the blood. Two separate results above 6.5% indicate diabetes. Levels between 5.7 and 6.4% indicate pre-diabetes. Levels below 5.7% are normal.

"Fasting" blood glucose levels are usually taken in the morning before breakfast. It is considered "normal" if the blood glucose levels are between 60 and 100 mg/dL. Impaired is between 100 and 126 mg/dL. Diabetes is diagnosed if there are two fasting blood glucose levels greater than 126 mg/dL.

If fasting is not an option, or the patient ate breakfast, the clinician can do a "glucose tolerance test." In this test the patient drinks a liquid that is rich in sugar (75 grams of sugar is used for most adults). The blood sugar levels are then tested two hours later. If the blood sugar level at two hours is less than 140 mg/dL the test is "normal." If the level falls between 140 and 200 mg/dL the patient is impaired or pre-diabetic. And if the levels are greater than 200 mg/dL the diagnosis of diabetes is made.

Diagnosing Diabetes Mellitus

  Hemoglobin A1C Fasting blood glucose levels Glucose tolerance test levels at 2 hours after a 75g sugar load
Normal Less than 5.7% < 100 mg/dL < 140 mg/dL
Impaired 5.7% to 6.4% 100-126 mg/dL 140-200 mg/dL
Diabetes 6.5% or greater > 126 mg/dL > 200 mg/dL

Heal Me Doctor

Treatment of diabetes involves either replacing insulin or improving insulin sensitivity.

Type 1 diabetics require insulin since their bodies no longer produce it. This is why type 1 diabetes was previously referred to as “insulin dependent diabetes”. Insulin as a treatment is discussed in a separate article.

Treatment Options:
(1) Replace insulin
(2) Improve insulin
       sensitivity
A healthy diet and exercise are very important treatments in managing diabetes. However, many patients require medications.

There are numerous medicines used to control blood sugar levels in type two diabetics. One option is a class of medications known as the sulfonylureas. These medications work by increasing pancreatic production of insulin.

A second category of medications, known as the thiazolidinediones, improve the ability of insulin to act on peripheral tissues like fat, muscle, and liver.

The third category of medications, known as the biguanides, perform a similar function to the thiazolidinediones, and also inhibit liver cells from secreting stored sugar into the blood stream.

Regardless of which medications are used, the ultimate goal of medical therapy is to maintain the blood glucose levels within a specified range. Fasting blood glucose levels should ideally be kept between 90 and 130 mg/dL; post-meal glucose levels should ideally be less than 180 mg/dL. Maintaining glucose values within these ranges helps keep glycosylated hemoglobin (aka: hemoglobin A1C) levels below 7%, which decreases the risk of the diabetic complications.

In addition to replacing insulin, or increasing insulin sensitivity, patients with diabetes should also be treated for other co-morbid conditions. For example, statins are often started to control hyperlipidemia. Dialysis might be necessary to control severe kidney disease. Frequent foot and neurological exams are also necessary to prevent complications of diabetic neuropathy (ie: foot ulcers, gastric paresis, etc).

It is currently recommended that patients with diabetes have a blood pressure goal of 130/80 or better. In addition the LDL cholesterol level should be maintained below 100 mg/dL, HDL should be above 40 mg/dL, and triglycerides should be kept below 150 mg/dL.

The Replay…

Diabetes is classified as type 1 or type 2. In type 1 diabetes destruction of the pancreatic cells responsible for insulin secretion occurs through an auto-immune process. In type 2 diabetes, insulin sensitivity is lost by peripheral body tissues causing the pancreas to secrete more and more insulin until it “burns out”. Diagnosis is based off of blood sugar levels, usually measured when the patient is fasting (although other methods exist). Treatment is with insulin or insulin-sensitizing medications.

Just Keep Learning, Just Keep Learning (That’s a Dory Reference)…

References and Resources

  • Swanson A, Watrin K, Wilder L. Clinical Inquiries: How can we keep impaired glucose tolerance and impaired fasting glucose from progressing to diabetes? J Fam Pract. 2010 Sep;59(9):532-3.
  • Judge EP, Phelan D, O’Shea D. Beyond statin therapy: a review of the management of residual risk in diabetes mellitus. J R Soc Med. 2010 Sep;103(9):357-62.
  • Fowler GC, Vasudevan DA. Type 2 diabetes mellitus: managing hemoglobin A(1c) and beyond. South Med J. 2010 Sep;103(9):911-6.
  • Kumar V, Abbas AK, Fausto N. Robbins and Cotran Pathologic Basis of Disease. Seventh Edition. Philadelphia: Elsevier Saunders, 2004.
  • Le T, Bhushan V, Grimm L. First Aid for the USMLE Step 1. New York: McGraw Hill, 2009.
  • Flynn JA. Oxford American Handbook of Clinical Medicine (Oxford American Handbooks of Medicine). First Edition. Oxford University Press, 2007.
  • Champe PC. Lippincott’s Illustrated Reviews: Biochemistry. Second Edition. Lippincott-Ravens Publishers, 1992.
  • American Diabetes Association. Standards of medical care in diabetes. Diabetes Care. 2004 Jan;27 Suppl 1:S15-35.

Vitamin B12: Function and Disease

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Vitamin B12 and its derivatives serve many roles in the body. It functions primarily as a co-factor to help enzymes perform their molecular reactions. It is composed of the metal cobalt, which is coordinated to a large corrin ring structure composed of carbon and nitrogen atoms. Several important biochemical reactions in which vitamin B12 is a crucial component include:

(1) The conversion of odd chain fatty acids (specifically propionate) into succinate.
(2) The conversion of homocysteine into methionine via methyl group donation.

In the first reaction above B12 serves as a cofactor for the enzyme methylmalonyl-CoA mutase, which converts the odd chain fatty acid L-methylmalonyl-CoA into succinyl-CoA. Succinyl-CoA can then enter the citric acid cycle (Krebs cycle).

In the second reaction vitamin B12 serves as a methyl donor (ie: a "-CH3" unit) allowing the conversion of homocysteine into methionine. Interestingly, this reaction also requires a form of vitamin B9 (aka: folate or folic acid) known as N-methyltetrahydrofolate. The reason this reaction is important is because methionine will eventually go on to form S-adenosylmethionine, which is an important contributor of single carbon fragments to various other molecules.

Additionally, vitamin B12 is needed to convert N-methyltetrahydrofolate back into tetrahydrofolate, which serves as a cofactor in a slew of other important biochemical reactions.

Role in Disease

Patients typically become deficient in vitamin B12 in one of three ways: strict vegan diets, an autoimmune condition known as pernicious anemia, or impaired absorption via the gut.

Vitamin B12 is found mainly in animal products and vegans may become deficient if they fail to supplement. Patients with pernicious anemia have antibodies that attack the cells in the stomach that secrete a molecule (intrinsic factor), which helps the intestine absorb B12 from the diet. Any damage to the gut lining (ie: inflammatory bowel disease, celiac disease, etc.) can also cause impair absorption of B12.

Deficiencies of any cause usually take several years to develop because excess vitamin B12 is stored in the liver. These stores can last several years before becoming depleted.

When a person’s vitamin B12 level is low it can cause the accumulation of odd chain fatty acids. These are thought to "gunk-up" cellular membranes. This is most noticeable in the nervous system, and can result in neurological signs and symptoms including numbness and tingling, loss of proprioception (ie: the ability to "feel" where your limbs are in space), and difficulty with coordination. In its most severe form a condition known as subacute combined degeneration of the spinal cord can occur.

Vitamin B12 deficiency can also cause a megaloblastic anemia. This occurs because rapidly dividing cells in the bone marrow require a constant supply of tetrahydrofolate for methylation reactions in order to produce enough DNA. When B12 is deficient, N-methyltetrahydrofolate cannot be converted back to tetrahydrofolate. When this occurs all of the tetrahydrofolate gets trapped in the N-methyltetrahydrofolate form, which is not used as a methyl donor in nucleotide synthesis. The resulting DNA deficiency causes red blood cells to exit the bone marrow deformed, dysfunctional, and larger than normal (hence the term "megalo"-blastic anemia).

There is no evidence that excess vitamin B12 causes any significant health problems. It is well excreted in the urine.

Diagnosis

Deficiencies of vitamin B12 are diagnosed by measuring the amount of B12 in the blood. However, "normal" B12 levels do not necessarily rule out B12 deficiency, making this test less than useful.

Other blood tests that are often sent include methylmalonyl-CoA and homocysteine levels. Both of these will be elevated in B12 deficiency since the vitamin is necessary for the enzymatic conversion of these molecules to succinyl-CoA and methionine, respectively.

Although rarely used today, another test called the Schilling test can be performed. It involves several steps in which radiolabelled B12 is given orally. If the patient is having difficulty absorbing B12 because of either intrinsic factor deficiencies or intestinal problems the Schilling test will be able to differentiate the cause.

Treatment

Treatment of vitamin B12 deficiency is, you guessed it, giving the patient B12. Patients with pernicious anemia or intestinal absorption problems are given the vitamin intramuscularly. Other patients can supplement with oral forms.

Overview

Vitamin B12, also known as cobalamin, is important in several cellular reactions including the conversion of odd chain fatty acids into succinate and the replenishing of tetrahydrofolate (a form of vitamin B9 or folate). Deficiencies may occur in those who are vegan, have pernicious anemia, or have intestinal absorption problems. Diagnosis is made by measuring blood B12, homocysteine, and methylmalonyl-CoA levels. Treatment is with supplementation, either orally or intramuscularly.

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References and Resources

Thiamine (Vitamin B1): Wernicke-Korsakoff and Beriberi

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Vitamin B1 (thiamine) and its derivatives serve many roles in the body. It functions primarily as a co-factor to help enzymes perform their molecular reactions. Several important biochemical reactions in which thiamine plays a crucial role include:

(1) Conversion of pyruvate to acetyl-CoA.
(2) Conversion of α-ketoglutarate to succinyl-CoA.
(3) Conversion of glyceraldehyde-3-phosphate to ribose-5-phosphate.
(4) It is used by the enzyme branched-chain α-keto acid dehydrogenase.

All of these reactions are crucial to human biochemistry. The conversion of pyruvate to acetyl-CoA feeds important molecules into the Krebs cycle, which allows the cell to produce energy.

The conversion of α-ketoglutarate to succinyl-CoA is necessary for the Krebs cycle to continue “spinning”. Without vitamin B1 (thiamine) the cycle would slow resulting in decreased energy production.

Thiamine is also important in the production of NADPH via the pentose phosphate pathway. NADPH plays a crucial role in biochemistry because it donates its electron pairs to numerous anabolic reactions.

The breakdown of branched chain amino acids such as valine, isoleucine, and leucine are also dependent on thiamine. The product that results from the breakdown of these amino acids are α-keto acids. They can also be fed into the Krebs cycle to replete molecules in the pathway that may have been siphoned off for other purposes. Maple syrup urine disease is a disorder of branched chain amino acid catabolism.

Since thiamine is involved in many energy producing pathways it is found abundantly in tissues that produce and use lots of energy. These include, but are certainly not limited to, the brain, muscle, and liver.

Role in Disease

Thiamine is a water soluble vitamin that is not actively stored in the body. It therefore must be obtained in the diet. Deficiencies can cause numerous disorders.

One such disorder is Wernicke-Korsakoff’s syndrome. It is seen in chronic alcoholics who may not be obtaining adequate nutrition. In Wernicke’s encephalopathy patients present with nystagmus (ie: jerky eye movements), ophthalmoplegia (ie: inability to move the eyes), and ataxia (ie: a wobbly unsteadiness).

These patients are often very confused. If Wernicke’s encephalopathy is left untreated it can progress to Wernicke-Korsakoff’s syndrome, in which the patient also develops short term memory problems and confabulations (ie: making up stories that aren’t true).

Another disease of thiamine deficiency is known as beriberi. There are numerous forms of this disease. It can affect infants who are being breast fed by thiamine deficient mothers, or who are receiving formulas with inadequate thiamine. In its worst case, infantile beriberi can manifest as severe heart problems.

Beriberi can also affect adults. The adult form exists in two types: wet and dry. Dry beriberi refers to the development of symmetric neuropathies (ie: damage to the peripheral nerves). Both sensory and motor nerves can be involved, and it tends to affect the parts of the nerve furthest from the spinal cord (ie: near the hands and feet). Wet beriberi is the combination of neuropathy plus heart problems, which can range in severity from cardiomegaly (ie: an enlarged heart) to congestive heart failure.

There is no evidence that excess thiamine (vitamin B1) causes any significant health problems. It is well excreted by the kidneys.

Diagnosis

Deficiencies of vitamin B1 (thiamine) are diagnosed by adding the vitamin to a preparation of the patient’s red blood cells. An increase in the activity of the transketolase enzyme is diagnostic of deficient levels.

Treatment

Treatment of thiamine deficiency is, you guessed it, giving the patient thiamine. If the patient has significant disease such as Wernicke’s encephalopathy thiamine can be given IV. Otherwise, oral supplementation and/or "prescribing" a diet rich in thiamine can be used to replenish any deficiencies.

Overview

Vitamin B1, also known as thiamine, is a vital component of numerous cellular reactions, especially those involving carbohydrate and amino acid catabolism (ie: breakdown). When deficiencies exist, several different diseases can occur. They include, but are not limited to, Wernicke-Korsakoff’s syndrome and beriberi. There are no known problems with excessive levels of thiamine since excess vitamin is efficiently excreted in the urine. Diagnosis of deficiencies are made by adding the vitamin to a preparation of the patient’s red blood cells. An increase in the activity of transketolase, an enzyme that relies on thiamine as a co-factor, is considered diagnostic of deficiency.

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References and Resources

Maroteaux-Lamy Syndrome: GAGs and Dermatan Sulfate

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Maroteaux-Lamy syndrome falls into a family of conditions called the “mucopolysaccharidoses”. This fancy term describes any disorder where patients are unable to metabolize long chains of repeating sugar units.

Another term for mucopolysaccharide is glycosaminoglycan, or “GAG”. GAGs are composed of long, repeating, un-branched sugar chains. Each repeating unit has a six carbon sugar (ie: the general term for a simple six carbon sugar is a “hexose”; glucose, mannose, galactose, etc. are specific examples) attached to another six carbon sugar that has a nitrogen group sticking off of it; the general name for a six carbon sugar with an attached nitrogen group is a “hexosamine”.

These two-sugar units, a hexose plus a hexosamine, connect to one another over and over again to form a very long chain called a glycosaminoglycan.

GAGs and mucopolysaccharides are important in the human body because they make up a significant proportion of connective tissue. Connective tissues provide support and structure to many of the organ systems. As such, the body is constantly forming, breaking down, and re-molding GAGs to provide the structural framework so that the rest of our cells can do their jobs well.

So what do GAGs have to do with Maroteaux-Lamy disease? A specific GAG known as dermatan sulfate is not broken down appropriately in patient’s suffering from this condition.

The reason these individuals are unable to break down dermatan sulfate is because they have a genetic mutation in the enzyme arylsulfatase B. In a normal person, arylsulfatase B degrades excess and unwanted dermatan sulfate; however, when the enzyme is defective it causes dermatan sulfate to build up to abnormally high levels within cells.

This excess dermatan sulfate eventually “gunks” up various organ systems and results in the characteristic signs and symptoms associated with the disease.

Signs and Symptoms

It is important to realize that there are numerous genetic mutations that can cause Maroteaux-Lamy syndrome and not all mutations are created equal! Therefore, there is a wide spectrum of signs and symptoms that may occur quickly, or slowly, depending on which particular mutation the patient has.

Most patients are short and have progressive changes in their facial features. Patients often develop early onset vision problems because their corneas become clouded by excess dermatan sulfate. Joint stiffness and skeletal abnormalities are also very common, as are heart and lung complications.

The lining of the brain and spinal cord can become excessively thick which can cause weakness secondary to spinal cord and nerve compression.

Patient’s also often have enlargement of the spleen – a condition called “splenomegaly”. It is also not uncommon for patients to have various types of hernias.

Unlike patients who suffer from some of the other mucopolysaccharidoses, people with Maroteaux-Lamy are of normal intelligence.

Diagnosis

Overall, Maroteaux-Lamy disease is more appropriately a “syndrome”, or constellation of signs and symptoms. By themselves, each sign or symptom is not diagnostic of the disorder, but when present together can support the diagnosis.

Genetic testing for mutations of the arylsulfatase B gene are done once the syndrome is suspected on clinical grounds. Additionally, urine samples contain elevated amounts of dermatan sulfate.

Treatment

Treatment consists of using a replacement enzyme known as Naglazyme® (aka: galsulfase). This enzyme performs the function of the deficient arylsulfatase B and helps in the degradation of dermatan sulfate.

Stem cell transplants can also provide the missing enzyme, but at significant risk. Unfortunately, few patients are candidates for stem cell transplant given difficulty with donor matching.

A very important component of treatment is monitoring for the development or progression of symptoms. This will allow specialists to step in and perform palliative procedures that can prevent worsening disability. For example, corneal transplants can be done to restore vision loss.

Overview

Maroteaux-Lamy is one of the mucopolysaccharidoses. It is caused by a genetic deficiency in the enzyme arylsulfatase-B, which prevents the degradation of dermatan sulfate. It presents with numerous signs and symptoms, which may be rapidly or slowly progressive depending on the specific genetic mutation. Treatment is with enzyme replacement therapies and palliative management of symptoms.

Related Articles

References and Resources

  • Calleja Gero ML, González Gutiérrez-Solana L, López Marín L, et al. Neuro-imaging findings in patient series with mucopolysaccharidosis. Neurologia. 2011 Dec 15.
  • Mtar A, Charfeddine B, Braham I, et al. Maroteaux-Lamy syndrome: a case report. Ann Biol Clin (Paris). 2011 Dec 1;69(6):693-697.
  • Golda A, Jurecka A, Tylki-Szymanska A. Cardiovascular manifestations of mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome). Int J Cardiol. 2011 Jul 5.
  • Valayannopoulos V, Wijburg FA. Therapy for the mucopolysaccharidoses. Rheumatology (Oxford). 2011 Dec;50 Suppl 5:v49-v59.

Hans Krebs and His Cycle: Oxidation and Energy

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The main role of the citric acid cycle is to continue the oxidation (ie: energy "stripping") of acetyl-CoA. Acetyl-CoA can be obtained from the oxidation of pyruvate (the final molecule of glycolysis) or from the oxidation of long chain fatty acids.

The cycle begins when acetyl-CoA combines with oxaloacetate to form citrate. Several chemical reactions occur and citrate gets converted back to oxaloacetate (see cycle below). At this point, oxaloacetate re-combines with another acetyl-CoA to restart the cycle. Note that pyruvate and acetyl-CoA are NOT technically considered part of the cycle.

The Kreb’s cycle (aka: citric acid cycle) produces both energy and "waste". The waste are two molecules of carbon dioxide, which are formed from the two carbons present in the original acetyl-CoA molecule. The energy comes from removing electron pairs from the molecules in the cycle.

Citric Acid Cycle
These electrons are stored in the energy rich molecules NADH and FADH2. NADH and FADH2 donate the electrons to carrier molecules in the electron transport chain. The final result is the production of adenosine triphosphate (ATP), an energy rich molecule that can be used by cells for various purposes. In addition, the citric acid cycle also produces an ATP equivalent molecule known as guanosine triphosphate (GTP).

The cycle itself operates in the mitochondrial matrix. This is no mistake because the enzymes of the electron transport chain are located on the adjacent inner mitochondrial membrane; the NADH and FADH2 produced by the cycle do not have to travel far in order to donate their electron pairs.

Regulation

Two key enzymes control the citric acid cycle. The first is isocitrate dehydrogenase. This enzyme converts isocitrate to α-ketoglutarate. It is inhibited by ATP and NADH, and stimulated by ADP. If we think about it, this makes sense because if ATP and NADH are abundant (ie: the cell has adequate energy stores), then we want the cycle to slow down. ADP is the breakdown product of ATP and represents an energy depleted state. Thus, ADP stimulates the cycle so that more energy can be produced.

The second regulatory enzyme is α-ketoglutarate dehydrogenase. This enzyme converts α-ketoglutarate to succinyl-CoA. It is inhibited by succinyl-CoA (an example of feedback inhibition), NADH, and ATP. The rationale behind this inhibition is similar to isocitrate dehydrogenase.

These two steps are also regulatory because they are highly exergonic. This means that they release lots of energy, so much so, that they are effectively irreversible reactions.

Overview

The main goal of the citric acid cycle is to produce energy by oxidizing acetyl-CoA. The energy produced comes in the form of NADH and FADH2. These molecules donate their electrons (energy) to the electron transport chain, which ultimately drives oxidative phosphorylation. This allows the formation of energy rich ATP, which can be used by the cell for numerous processes.

References and Resources

  • Nelson DL, Cox MM. Lehninger Principles of Biochemistry. Fifth Edition. New York: Worth Publishers, 2008.
  • Champe PC. Lippincott’s Illustrated Reviews: Biochemistry. Second Edition. Lippencott-Ravens Publishers, 1992.
  • Le T, Bhushan V, Grimm L. First Aid for the USMLE Step 1. New York: McGraw Hill, 2009.

Phenylketonuria: A Musty Aminoacidopathy

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Phenylketonuria is one of the “aminoacidopathies”. An aminoacidopathy is a fancy medical term for a malfunction in the bodies’ ability to handle specific amino acids, in this case, phenylalanine.

But before we get too crazy, first things first… What is an amino acid? Amino acids are the building blocks of proteins. Each amino acid consists of a carbon atom that has four “things” hanging off of it: an amine group, a carboxylic acid group, a hydrogen atom, and finally an “R” group. The “R” group varies depending on the specific amino acid being discussed. For example, the “R” in phenylalanine is a benzyl group.

Amino acids link up together via “peptide bonds”. A peptide bond is simply the carboxylic acid group of one amino acid “hooking” up to the amine group of another amino acid. This linking occurs over and over again resulting in a protein that can be thousands of amino acids in length.

With that background, let’s discuss one specific amino acid, phenylalanine, and what happens when the body has difficulty using it – a condition called phenylketonuria.

An enzyme in the liver called phenylalanine hydroxylase is necessary to transform phenylalanine into another amino acid called tyrosine. In certain individuals, the genes on chromosome 12 that encode the hydroxylase enzyme are deficient. Mutations in both of your hydroxylase genes (ie: the one you got from mom and the one you got from dad) must be deficient for phenylketonuria to develop; in other words, one adequate gene can produce enough normal hydroxylase to handle a normal dietary phenylalanine load. However, when both genes are deficient, phenylalanine can accumulate to abnormally high levels in the blood.

It is important to note that phenylketonuria can occur even if the hydroxylase enzyme is normal! How is this possible? The answer is because the hydroxylase enzyme requires another molecule called tetrahydrobiopterin (try saying that 3 times fast!) to function properly. When the enzyme (dihydrobiopterin reductase) that produces tetrahydrobiopterin is deficient, it gunks up the entire system and slows the ability of phenylalanine hydroxylase to convert phenylalanine to tyrosine.

Phenylketonuria

Either way you slice it, the excess phenylalanine interferes with the ability of other amino acids to enter the brain. This hinders brain development and, if left untreated, can cause mental retardation.

Signs and Symptoms

When excess phenylalanine backs up in the blood stream it blocks amino acid transporters in the brain. As a result, the brain does not get access to other essential amino acids, and patients that are left untreated, develop mental retardation and seizures.

Children affected by the condition also commonly have a musty odor. The odor is caused by phenylacetate, which is a breakdown product of excess phenylalanine.

Interestingly, patient’s who suffer from phenylketonuria are also usually blond haired and blue eyed. The reason for this is that tyrosine is necessary for melanin formation. Melanin is the pigment that gives hair, skin, and eyes their color. In phenylketonuria, phenylalanine is not converted to tyrosine and therefore melanin is not produced. The resultant lack of pigment causes blond hair and blue eyes.

Diagnosis

Many countries screen new babies for phenylketonuria because mental retardation can be prevented with strict dietary control. Diagnosis is based on finding an elevated phenylalanine to tyrosine ratio in the blood. Additionally, phenylpyruvate (a breakdown product of phenylalanine) will be elevated in urine samples of affected individuals.

Treatment

Treatment is, you guessed it, avoiding phenylalanine! This is done by ingesting a low protein diet, and in babies, using formula that is free of phenylalanine.

In phenylketonuria, tyrosine becomes an essential amino acid. Therefore, an important aspect of treatment is ensuring adequate intake of tyrosine via supplementation.

Overview

Phenylketonuria results when the body is unable to convert the amino acid phenylalanine into tyrosine. It can be caused by a primary genetic deficiency in the enzyme phenylalanine hydroxylase. It can also be caused by defects in the production of the hydroxylases’ necessary cofactor, tetrahydrobiopterin. Symptoms, if left untreated, include mental retardation. Patients with phenylketonuria are generally fair skinned and blue eyed because they have difficulty producing the melanin. Treatment is avoidance of phenylalanine and supplementation with tyrosine.

Related Articles

References and Resources

  • Cunningham GC. Phenylketonuria – early detection, diagnosis and treatment. Calif Med. 1966 July; 105(1): 1–7.
  • van Spronsen FJ, Huijbregts SC, Bosch AM, et al. Cognitive, neurophysiological, neurological and psychosocial outcomes in early-treated PKU-patients: A start toward standardized outcome measurement across development. Mol Genet Metab. 2011;104 Suppl:S45-51. Epub 2011 Oct 6.
  • Mitchell JJ, Trakadis YJ, Scriver CR. Phenylalanine hydroxylase deficiency. Genet Med. 2011 Aug;13(8):697-707.
  • Werner ER, Blau N, Thöny B. Tetrahydrobiopterin: biochemistry and pathophysiology. Biochem J. 2011 Sep 15;438(3):397-414. Review.

Sugar Strip Down: Glycolysis and Energy Formation

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Glycolysis is the biochemical pathway that strips glucose of its energy. It starts with glucose and ends with pyruvate. Pyruvate is converted to acetyl-CoA, which can be stored as fat, or further metabolized in the citric acid cycle. The fate of pyruvate depends on the energy needs of the cell.

The net yield of energy from glycolysis is two adenosine triphosphates (ATP) and two nicotinamide adenine dinucleotide (NADH) molecules per glucose "burned" by the pathway. Each NADH molecule from glycolysis eventually generates 1.5 ATPs from the electron transport chain. Therefore, a total of 5 ATP molecules are produced per molecule of glucose consumed by the glycolysis pathway.

The net energy used or created (in the form of ATP or NADH) are shown in the schematic below:

Glycolysis

Want to know what happens to the NADH? Check out the article on the electron transport chain.

Regulation

Glycolysis is regulated at several steps in order to ensure that glucose molecules are used appropriately by the cell. The first point of regulation is at the conversion of glucose to glucose-6-phosphate (G6P). This reaction is catalyzed by an enzyme known as hexokinase. This enzyme is inhibited by its own product – G6P. This is known as "feedback inhibition". Once glucose is converted to G6P it becomes "trapped" inside the cell. Therefore, when there are adequate G6P levels any particular cell can shut off the flow of glucose into glycolysis so that it can be sent to other cells that may need it.

Phosphofructokinase 1
(PFK1) is the key
regulatory enzyme in the
glycolytic pathway.
The second, and key, regulatory point of glycolysis is at the enzyme phosphofructokinase-1 (PFK1). PFK1 converts fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate. This enzyme is inhibited by ATP and citrate, and activated by ADP, AMP, and fructose-2,6-bisphosphate (note that this is a different molecule than fructose-1,6-bisphosphate). In other words, when the body is in an energy depleted state (ie: low levels of ATP and high levels of ADP and AMP) glycolysis is activated; under conditions of high energy (ie: high levels of ATP) glycolysis is inhibited. It is important to regulate the enzyme PFK1 closely because once F6P is converted to fructose-1,6-bisphosphate it MUST continue down the glycolytic pathway.

Side note: fructose-2,6-bisphosphate is produced by the enzyme phosphofructokinase-2 (PFK2) and degraded by the enzyme fructose bisphosphatase-2 (FBPase2). PFK2 becomes active under conditions of satiety (ie: well fed states in which lots of glucose/sugar is being absorbed from the gut). When PFK2 is active the concentration of fructose-2,6-bisphosphate increases. This activates PFK1 and increases the activity of the glycolytic pathway.

The final point of regulation is at the enzyme pyruvate kinase. This enzyme converts phosphoenolpyruvate into pyruvate. It is inhibited by ATP, acetyl-CoA, and long chain fatty acids, which are all markers of high energy levels.

In general, the overall regulation of glycolysis is related to how much energy the cell has. In energy rich states (ie: high ATP and low ADP/AMP) the cell slows glycolysis so that it can store glucose for use at a latter time. In energy depleted states (ie: low ATP and high ADP/AMP) the rate of glycolysis increases so that more energy can be formed by "burning" more glucose molecules per unit time.

Overview

Glycolysis breaks down glucose molecules. In the process the energy rich molecules ATP and NADH are formed. It is regulated at several enzymatic steps, most importantly at the enzyme phosphofructokinase-1. Pyruvate can be further metabolized to acetyl-CoA.

References and Resources

  • Nelson DL, Cox MM. Lehninger Principles of Biochemistry. Fifth Edition. New York: Worth Publishers, 2008.
  • Champe PC. Lippincott’s Illustrated Reviews: Biochemistry. Second Edition. Lippencott-Ravens Publishers, 1992.
  • Le T, Bhushan V, Grimm L. First Aid for the USMLE Step 1. New York: McGraw Hill, 2009.

Electron Transport Chain in All Its Glory

virtualmedstudent

The electron transport chain is a series of interconnected proteins and carrier molecules that are embedded in the inner mitochondrial membrane. In order to understand the function of the electron transport chain in all its glory, we have to first appreciate two important molecules: NADH and FADH2. These two molecules can be thought of as energy dump trucks that strip sugars, fats, and proteins of their energy rich electrons (see the articles on glycolysis and the Kreb’s cycle for more details on how sugars and fats get stripped of their energy).

NADH and FADH2 then transfer their electrons to specific carrier molecules that constitute the “electron transport chain”. From there, the electrons "bounce" from carrier molecule to carrier molecule as if they were falling down an energy waterfall. Each time they bounce between carrier molecules energy is emitted; this energy is used to pump protons out of the mitochondrial matrix.

As protons get pumped out of the matrix they form an electrochemical gradient. As more electrons bounce down the chain, more protons get pumped out of the matrix. Eventually the concentration of protons outside the mitochondrial matrix is much higher than the concentration inside the matrix.

This is where the plot thickens… At this point all of the protons outside the matrix want to diffuse down their concentration gradient to their "home" in the matrix. However, the membrane that lines the matrix is impermeable to protons… Except at one specific point!

That point is a specialized enzyme embedded in the membrane called ATP synthase. The sole function of this enzyme is to rejuvenate a molecule known as adenosine triphosphate (ATP).

As the protons flow down their concentration gradient through the ATP synthase they release energy. The synthase uses this energy, via a wonderfully complicated molecular gear shifter, to form ATP from ADP (adenosine diphosphate) and free phosphate. This process is formally known as oxidative phosphorylation.

ATP is an energy rich molecule that can be thought of as the gasoline used to run a car. In simple terms, if a cell is building a molecule (ie: protein, DNA, RNA, etc.) it is likely using the energy in ATP to do it. When ATP runs out, the cell stops producing molecules and is in jeopardy of dying.

Electron Transport Chain Schematic

The whole point of the electron transport chain is to produce more ATP by using the energy obtained from the electron pillaging of molecules like glucose, long chain fatty acids, and proteins!

So what happens to the poor old electrons? Oxygen absorbs them at the end of the chain and forms water! This is why humans require oxygen to live! Without oxygen, electrons back up in the chain and eventually the whole transport system grinds to a halt.

Regulation

Like every other biochemical pathway in the body, the electron transport chain is under strict regulation. When ATP is abundant in the cell the chain slows down (ie: why drop electrons down the chain and strip sugars, fats, and proteins of their energy when you don’t have to?).

However, when the cell has little ATP around, then the chain springs in to action as the body burns sugars, proteins, and fats to create the electron carriers necessary to run the chain.

There are several points in the chain where things can turn awry. The first is if there is no oxygen. Oxygen is the last point in the electron "waterfall". Once the electrons get to the last carrier in the chain they have no where to go but to reduce oxygen to form water.

Without oxygen the electrons back up like lemmings. Eventually the whole chain becomes saturated with electrons and causes the whole system to stop, which can be disastrous to cellular function.

Overview

So let’s bring it all together… Cells strip sugars, proteins, and fats of their energy rich electrons. These electrons hitch a ride on special molecules like NADH and FADH2. NADH and FADH2 donate their electrons to special carrier proteins embedded in the inner mitochondrial membrane. The electrons bounce from carrier to carrier releasing energy. The released energy is used to pump protons out of the matrix. The resulting energy rich proton gradient is then used to create ATP as the protons flow down their concentration gradient back into the matrix through the ATP synthase unit… Now go drink another cup of coffee!

References and Resources

  • Le T, Bhushan V, Grimm L. First Aid for the USMLE Step 1. New York: McGraw Hill, 2009.
  • Kumar V, Abbas AK, Fausto N. Robbins and Cotran Pathologic Basis of Disease. Seventh Edition. Philadelphia: Elsevier Saunders, 2004.
  • Champe PC. Lippincott’s Illustrated Reviews: Biochemistry. Second Edition. Lippincott-Ravens Publishers, 1992.
  • Nelson DL, Cox MM. Lehninger Principles of Biochemistry. Fifth Edition. New York: Worth Publishers, 2008.