Ketosis

Q- What are ketone bodies ? Discuss their biological significance ?

Answer- Ketone bodies can be regarded as  water-soluble, transportable form of acetyl units. Fatty acids are released by adipose tissue and converted into acetyl units by the liver, which then exports them as ketone bodies.

Acetoacetate, D(-3) -hydroxybutyrate (Beta hydroxy butyrate), and acetone are often referred to as ketone bodies (Figure-1).

Figure-1- showing the structure of ketone bodies.

The term “ketones”  is a misnomer because 3-hydroxybutyrate is not a ketone and there are ketones in blood that are not ketone bodies, eg, pyruvate, fructose.

Biological Significance

Ketone bodies serve as a fuel for extra hepatic tissues

The  brain  is an  important  organ.  It  is  metabolically  active  and  metabolically privileged. The brain generally uses 60-70% of total body glucose requirements, and always  requires  some  glucose for  normal  functioning.  Under  most  conditions, glucose is essentially the sole energy source of the brain. The brain cannot use fatty acids, which  cannot cross  the  blood-brain  barrier. Because animals  cannot synthesize significant amounts of glucose from fatty acids, as glucose availability decreases, the brain is forced to use either amino acids or ketone bodies for fuel.

Individuals eating diets extremely high in fat and low in carbohydrates,or starving, or suffering  from  a severe lack of insulin  (Type I diabetes  mellitus) therefore increase the synthesis and utilization of ketone bodies

During high rates of fatty acid oxidation, primarily in the liver, large amounts of acetyl-Co A are generated. These exceed the capacity of the TCA cycle, and one result is the synthesis of ketone bodies. The synthesis of the ketone bodies (ketogenesis) occurs in the liver mitochondria allowing this process to be intimately coupled to rate of hepatic fatty acid oxidation. Conversely, the utilization of the ketones (ketolysis) occurs in the peripheral cells, in the cytosol.

The acetyl CoA formed in fatty acid oxidation enters the citric acid cycle only if fat and carbohydrate degradation are appropriately balanced. The reason is that the entry of acetyl CoA into the citric acid cycle depends on the availability of oxaloacetate for the formation of citrate, but the concentration of Oxaloacetate is lowered if carbohydrate is unavailable or improperly utilized. Oxaloacetate is normally formed from pyruvate, the product of glycolysis, by pyruvate carboxylase (Figure-2).This is the molecular basis of the adage that fats burn in the flame of carbohydrates.

Figure-2-showing the pathway of ketogenesis in conditions of non availability of Oxaloacetate

In fasting or diabetes, oxaloacetate is consumed to form glucose by the gluconeogenic pathway (figure-2) and hence is unavailable for condensation with acetyl CoA. Under these conditions, acetyl CoA is diverted to the formation of acetoacetate and β-hydroxybutyrate.

These substances diffuse from the liver mitochondria into the blood and are transported to peripheral tissues. These ketone bodies were initially regarded as  degradation products of little physiological value. However, the results of studies revealed that these derivatives of acetyl CoA are important molecules in energy metabolism. Acetoacetate and β-hydroxybutyrateare normal fuels of respiration and are quantitatively important as sources of energy. Indeed, heart muscle and the renal cortex use acetoacetate in preference to glucose. In contrast, the brain adapts to the utilization of acetoacetate during starvation and diabetes. In prolonged starvation,75% of the fuel needs of the brain are met by ketone bodies.

Q.- Describe the pathway for the synthesis of ketone bodies by naming substrates, the first ketone body made in the pathway, the next two ketone bodies made in the pathway, the intermediates in the pathway that can be used either for ketone body synthesis or cholesterol synthesis, and the enzyme that actually produces the first ketone body as a product.

Answer-Ketogenesis takes place in liver using Acetyl co A as a substrate or a precursor molecule. Enzymes responsible for ketone body formation are associated mainly with the mitochondria.

Steps of synthesis-Acetoacetate (First ketone body) is formed from acetyl CoA in three steps (Figure-3 ).

1)Two molecules of acetyl CoA condense to form acetoacetyl CoA. This reaction, which is catalyzed by thiolase, is the reverse of the thiolysis step in the oxidation of fatty acids.

2) Acetoacetyl CoA then reacts with acetyl CoA and water to give 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) and CoA. The reaction is catalyzed by HMG co A synthase. This enzyme is exclusively present in liver mitochondria. There are two isoforms of this enzyme-cytosolic and mitochondrial. The mitochondrial enzyme is needed for ketogenesis while the cytosolic form is associated with cholesterol biosynthesis.

This condensation resembles the one catalyzed by citrate synthase. This reaction, which has a  favorable equilibrium owing to the hydrolysis of a thioester linkage, compensates for the unfavorable equilibrium in the formation of acetoacetyl CoA.

3) 3-Hydroxy-3-methylglutaryl CoA is then cleaved to acetyl CoA and acetoacetate in the presence of HMG Co A  lyase (Figure-3)

The carbon atoms split off in the acetyl-CoA molecule are derived from the original acetoacetyl-CoA molecule. Both enzymes must be present in mitochondria for ketogenesis to take place. This occurs solely in liver and rumen epithelium,

The sum of these reactions is

The other two ketone bodies-Acetone and D(-)- 3-Hydroxybutyrate are formed from Acetoacetate, the primary ketone body.

4) Acetone is formed by decarboxylation in the presence of decarboxylase enzyme and, because it is a beta-keto acid, acetoacetate also undergoes a slow, spontaneous decarboxylation to acetone. The odor of acetone may be detected in the breath of a person who has a high level of acetoacetate in the blood.  “Acetone-breath” has been used as  a  crude  method  of  diagnosing  individuals  with  untreated Type I diabetes mellitus.

 5) D (-)-3-Hydroxybutyrate is formed by the reduction of acetoacetate in the mitochondrial matrix by D(-)3-hydroxybutyrate dehydrogenase. D(-)-3-Hydroxybutyrate is quantitatively the predominant ketone body present in the blood and urine in ketosis.

The β-hydroxybutyrate dehydrogenase reaction has two functions: 1) it stores energy equivalent to an NADH in the ketone body for export to the tissues, and 

2) it  produces  a  more  stable molecule.

Acetoacetate and β-hydroxybutyrate, in particular, also serve as major substrates for the biosynthesis of neonatal cerebral lipids.

The ratio of β hydroxybutyrate to acetoacetate depends on the NADH/NAD+ ratio inside mitochondria. if NADH concentration is high, the liver releases a higher proportion of β-hydroxybutyrate.

In vivo, the liver appears to be the only organ in nonruminants to add significant quantities of ketone bodies to the blood. Extrahepatic tissues utilize them as respiratory substrates. The net flow of ketone bodies from the liver to the extrahepatic tissues results from active hepatic synthesis coupled with very low utilization. The reverse situation occurs in extra hepatic tissues.

While an active enzymatic mechanism produces acetoacetate from acetoacetyl-CoA in the liver, acetoacetate once formed cannot be reactivated directly except in the cytosol, where it is used in a much less active pathway as a precursor in cholesterol synthesis. This accounts for the net production of ketone bodies by the liver.

Why are three enzymes required to synthesize acetoacetate?

An enzyme that cleaves the thioester  bond of the thiolase  product  acetoacetyl-CoA  would  also produce acetoacetate, but such a thioesterase does not seem to exist. The reason for the multienzyme pathway is not really understood. However, the pathway that does exist is not especially wasteful; the third acetyl-CoA used merely acts catalytically.

Because the cell needs to have HMG-CoA synthase for other purposes, the choice is in having HMG-CoA lyase. It is possible that having two mitochondrial enzymes (HMG-CoA synthase and HMG-CoA lyase) reuired for ketone body synthesis assists in controlling the pathway.

Figure-3- Showing the steps of ketogenesis

Q.- Name the tissues that oxidize ketone bodies. Why not the liver? What happens to blood ketone bodies? Name the intermediates in the pathway from β-Hydroxybutyrate to acetyl CoA.

Answer- The ketone bodies are water soluble and are transported across the inner mitochondrial membrane as well as across the blood-brain barrier and cell membranes. Thus they can be used as a fuel source by a variety of tissues including the CNS. They are preferred substrates for aerobic muscle and heart, thus sparing glucose when they are available.

Tissues that can use fatty acids can generally use ketone bodies in addition to other energy sources. The exceptions are the liver and the brain. The liver synthesizes ketone bodies, but has little β-ketoacyl-CoA transferase, and therefore little ability to convert acetoacetate into acetyl-CoA. The brain does not normally use fatty acids, which do not cross the blood-brain barrier; under ordinary circumstances, the brain uses glucose as its sole energy source.

The metabolic rate of the brain is essentially constant. While other tissues reduce their metabolic requirements during starvation, the brain is unable to do so. After a few  days  of  fasting,  the  brain  undergoes  metabolic  changes  to  adapt  to  the decreased availability of glucose. One major change is increased amounts  of the enzymes necessary to metabolize ketone bodies.

Ketone bodies are utilized by extrahepatic tissues via a series of cytosolic reactions (Figure-4) that are essentially a reversal of ketone body synthesis, the ketones must be reconverted to acetyl CoA in the mitochondria:

Figure-4- Showing the steps of utilization of ketone bodies.

Steps- (Figure-4)

1) Beta-hydroxybutyrate, is first oxidized to acetoacetate with the production of one NADH (1). It is important to appreciate that under conditions where tissues are utilizing ketones for energy production their NAD⁺/NADH ratios are going to be relatively high, thus driving the β-hydroxybutyrate dehydrogeanse catalyzed reaction in the direction of acetoacetate synthesis. 

2) Coenzyme A must be added to the acetoacetate. The thioester bond is a high energy bond, so ATP equivalents must be used. In this case the energy comes from a trans esterification of the CoAS from succinyl CoA to acetoacetate by Coenzyme A transferase (2), also called Succinyl co A : Acetoacetate co A transferase, also known as Thiophorase.

The succinyl CoA comes from the TCA cycle. This reaction bypasses the succinyl-CoA synthetase step of the TCA cycle, hence there is no GTP formation at this steps although it does not alter the amount of carbon in the cycle.

The liver has acetoacetate available to supply to other organs because it lacks this particular CoA transferase and that is the reason that “Ketone bodies are synthesized in the liver but utilized in the peripheral tissues”. The latter enzyme is present at high levels in most tissues except the liver. Importantly, very low level of enzyme expression in the liver allows the liver to produce ketone bodies but not to utilize them. This ensures that extrahepatic tissues have access to ketone bodies as a fuel source during prolonged fasting and starvation, and also,lack of this enzyme in the liver prevents the futile cycle of synthesis and breakdown of acetoacetate.

3) The acetoacetyl CoA is now cleaved to two acetyl CoA’s with Thiolase (3).

This implies that the TCA cycle must be running to allow ketone body utilization; a fact  which is necessarily true,  because  the TCA cycle is necessary to allow generation of energy from acetyl-CoA.

D(-)-3-Hydroxybutyrate is oxidized to produce acetoacetate as well as NADH for use in oxidative phosphorylation.

If the blood level is raised, oxidation of ketone bodies increases until, at a concentration of approximately 12 mmol/L, they saturate the oxidative machinery. When this occurs, a large proportion of the oxygen consumption may be accounted for by the oxidation of ketone bodies.

In most cases, ketonemia is due to increased production of ketone bodies by the liver rather than to a deficiency in their utilization by extrahepatic tissues. While acetoacetate and D(-)-3-hydroxybutyrate are readily oxidized by extrahepatic tissues, acetone is difficult to oxidize in vivo and to a large extent is volatilized in the lungs.

Both β-hydroxybutyrate and acetoacetate are organic acids. These compounds are released in the protonated form, which means that their release tends to lower the pH of the blood. In  normal  individuals, other mechanisms compensate  for  the increased proton release. Individuals with untreated Type I diabetes mellitus often release  ketone  bodies  in  such  large  quantities  that the normal  pH-buffering mechanisms are overloaded; the reduced pH, in combination with a number of other metabolic  abnormalities  associated  with lack  of  insulin results  in  diabetic ketoacidosis, a life-threatening acute disorder of Type I diabetes. In most cases, the increase in ketone body concentration in blood is due to increased synthesis in liver; in severe ketoacidosis, cells begin to lose ability to use ketone bodies also.

------------------------------------------ Best Wishes: Dr.Ehab Aboueladab, Tel:01007834123 Email:ehab10f@gmail.com,ehababoueladab@yahoo.com ------------------------------------------