How are Ketone Bodies formed?

In the liver, ketone bodies are synthesized in the mitochondrial matrix from acetyl-CoA generated from fatty oxidation and this process is known as ketogenesis. Acetyl-CoA results from the breakdown of carbohydrates, lipids and certain amino acids. Normally, the acetyl group of acetyl-CoA enters the citric acid cycle to generate energy in the form of ATP, but it can also form ketone bodies; this happens if acetyl-CoA levels are high and the TCA cycle capacity is exceeded (the limiting factor is the availability of oxaloacetate).

Steps in ketogenesis:

1. Catalyzed by enzyme thiolase, 2 molecules of acetyl-CoA condense to form acatoacetyl-CoA.
2. Catalyzed by enzyme HMG-CoA synthase, acetoacetyl-CoA combines with another molecule of acetyl-CoA to produce HMG-CoA (β-hydroxy β-methyl glutaryl-CoA).
3. HMG-CoA lyase cleaves HMG-CoA to produce acetoacetate and acetyl-CoA.
4. Acetoacetate can undergo spontaneous decarboxylation to form acetone.
5. Acetoacetate can be reduced by a dehydrogenase to β-hydroxybutyrate.

The dehydrogenase reaction is readily reversible and interconverts the 2 ketone bodies, which exist in an equilibrium ratio determined by NADH/NAD+ ratio of the mitochondrial matrix. Under normal conditions, the ratio of the 2 ketone bodies in the blood is approxiamtely 1:1.

Regulation:

When fat enters liver it has 2 fates: either

1. Beta oxidation (Occurs during glucose deficiency)
2. Storage as triacylglycerol (Occurs when liver has sufficient supplies of glycerol 3 phosphate from glycolysis)

Hormones:

1. Hunger stimulates Glucagon hormone responsible for stimulating Fatty acid oxidation and Ketogenesis
2. Fed state stimulates Insulin hormone responsible for inhibiting ketogenesis

How are Ketone Bodies utilized?

The ketone bodies being water soluble, are easily transported from the liver to various tissues. Acetoacetate and β-hydroxybutyrate can be oxidized as fuels in most tissues includin skeletal muscle, brain, certain cells of kidney and cells of the intestinal mucosa. The tissues which lack mitochondria (eg. erythrocytes) cannot utilize ketone bodies. During prolonged starvation, ketone bodies are the major sources of fuel for the brain and other parts of central nervous system (CNS).

Steps in oxidation of ketone bodies (ketogenolysis):

1. β-hydroxybutyrate is converted back to acetoacetate.
2. Acetoacetate is activated to Acetoacetyl-CoA by a mitochondrial enzyme thiophorase (succinyl-CoA : acetoacetate-CoA transferase). Thiophorase is absent in liver, hence ketone bodies are not utilized in the liver.
3. Thiolase cleaves acetoacetyl-CoA to 2 moles of acetyl-CoA
4. The principal fate of the acetyl-CoA synthesized is the oxidation in TCA cycle.

Ketosis:

The concentration of ketone bodies in blood is maintained around 1 mg/dl. The excretion in urine is very low and undtectable by routine tests (Rothera’s test).

When the rate of synthesis of ketone bodies exceeds the rate of utilization, their concentration in blood increases, this is known as ketonemia. This is followed by ketonuria – excretion of ketone bodies in urine. The overall picture of ketonemia and ketonuria is commonly referred to as ketosis. Smell of acetone in breathe is a common feature of ketosis. It is most commonly associated with following conditions:

Starvation: Starvation is accompanied by increased degradation of fatty acids to meet energy needs of the body. This causes an overproduction of acetyl-CoA which cannot be entirely handled by TCA or citric acid cycle. Furthermore, TCA cycle is impaired due to deficiency of oxaloacetate, since most of it is diverted for glucose synthesis (gluconeogenesis) to meet the essential requirements (often unsuccessful) for tissues like brain. The result is an accumulation of acetyl-CoA and overproduction of ketone bodies leading to ketosis.

Diabetes Mellitus type I (Insulin Dependent):

1. Hyperglycaemia occurs due to decreased glucose uptake in fat and muscle cells due to insulin deficiency
2. Lipolysis in fat cells now occurs promoted by the insulin deficiency releasing Free fatty acids (FFA) into the blood which provide substrate to the liver
3. A switch in hepatic lipid metabolism occurs due to the insulin deficiency and the glucagon excess, so the excess FFA is metabolised resulting in excess production of acetyl CoA
4. The excess hepatic acetyl CoA (remaining after saturation of TCA cycle) is converted to ketone bodies which are released into the blood
5. Ketoacidosis and hyperglycaemia both occur due to the lack of insulin and the increase in glucagon and most of the clinical effects follow from these two factors

Components of a PCR reaction:

1. A DNA template
2. Heat stable Taq DNA polymerase (derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature of 90c, so is not denatured by the high temperatures)
3. Nucleotides
4. A pair of DNA primers
5. Buffers containing magnesium ions

Comparison of PCR components with photocopier items

The principle of PCR is as follows:

1. Starting with a sample of the DNA (template) to be amplified, add the 4 nucleotides (deoxyribonucleoside triphosphates) and the enzyme heat stable DNA polymerase to the solution.

2. Normally (in vivo) the DNA double helix would be separated by the enzyme helicase, but in PCR (in vitro) the strands are separated by heating to 95 c for 2 minutes. This breaks the hydrogen bonds between the 2 DNA strands.

3. Initiation of DNA polymerisation always requires short lengths of DNA (about 20 bp long) called primers. The primers are 2 synthetic oligonucleiotides: one is complementary to a short sequence in one strand of the DNA to be amplified, and the other is complementary to a sequence in the other DNA strand. In vivo the primers are made during replication by DNA polymerase, but in vitro they must be synthesized separately and added at this stage. This means that a short length of the sequence of the DNA must already be known.

4. The DNA must be cooled to 40 c to allow the primers to anneal to their complementary sequences on the separated DNA strands.

5. The DNA polymerase enzyme can now extend the primers and complete the replication of the rest of the DNA. Its optimum temperature is about 72 c, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible.

5. Each original DNA molecule has now been replicated to form 2 molecules. The cycle is repeated from step 2, each time doubling the number of DNA molecules. This is why it is called a chain reaction, since the number of molecules increases exponentially. Typically PCR is run for 20–30 cycles.

PCR can be completely automated; a minute sample of DNA can be amplified millions of times with little effort.

Description of the schematic diagram for PCR technique: Strand 1 and Strand 2 are original DNA strands. The short dark blue fragments are the primers. After multiple heating and cooling cycles, the original strands remain, but most of the DNA consists of amplified copies of the segment (shown in lighter blue) synthesized by the heat stable DNA polymerase.

Uses of PCR:

1. It is appropriate for forensic testing procedures because only a very small sample of DNA is required as the starting material which can be obtained from a single strand of hair or a single drop of blood or semen.

2. Tissue typing for organ transplantation

3. DNA based phylogeny or functional analysis of genes useful for research

4. Detection of genetic diseases: Prenatal genetic diagnosis, Hereditary diseases

5. Detection of infectious diseases: HIV, TB, etc.

6. Parental testing, where an individual is matched with their close relatives

Disadvantage of PCR:

A potential problem for PCR is obtaining a pure sample of DNA to start with; any contaminant DNA will also be amplified.

Use this animation for better understanding of PCR technique from DNA Learning Center.

Sources:

• Essential Biochemistry for Medicine
• Mark’s Medical Biochemistry

Further Reading:
Isoenzymes and their Diagnostic Importance

• Also called isozymes
• Are multiple forms of an enzyme that catalyzes the same reaction
• Arise through gene duplication
• Differ in their physical and chemical properties, Km and Vmax values, optimum pH, substrate affinity, etc.

Lactate Dehydrogenase (LDH)

Lactate ←LDH→ Pyruvate

LDH is a tetrameric enzyme with 2 types of subunit “H” and “M” :

1. M (for muscle) : basic
2. H (for heart) : acidic

Isoenzymes of LDH:

1. LDH1 (H4) : Heart and RBC
2. LDH2 (H3M) : Heart and RBC
3. LDH3 (H2M2) : Brain and kidney
4. LDH4 (HM3) : Liver and skeletal muscle
5. LDH5 (M4) : Liver and skeletal muscle

LDH1 has high Km (low affinity) and LDH5 has low Km (high affinity) for pyruvate.

Diagnositc importance of LDH:

1. Normal: LDH2 > LDH1
2. Myocardial infarction (within 12-24 hours): LDH1>>LDH2 (flipped LDH pattern)
3. Liver diseases: increased LDH5 in serum
4. Increased LDH suggests other following diseases:
   • Hemolytic anemia
   • Hypotension
   • Infectious mononucleosis
   • Intestinal ischemia and infarction
   • Muscle injury
   • Muscular dystrophy
   • Pancreatitis
   • Lung tissue death
   • Stroke
   • Ischemic cardiomyopathy

Creatine Phosphokinase (CPK)

Phosphocreatine ←CPK→ Creatine

CPK is a dimeric enzyme consisting of 2 subunits:
a. M : for muscle
b. B : for brain

Isoenzymes of CPK:

1. CPK1 (BB) : Brain
2. CPK2 (MB) : Heart
3. CPK3 (MM) : Skeletal muscles

Diagnositic importance of CPK:

1. Normal: low CPK2 (<2%) in serum
2. MI (within 6-18 hrs): increased CPK2 (20%) in serum
3. Increased CPK-1 suggests:
   • Brain cancer
   • Brain injury
   • Pulmonary infarction
   • Seizure
4. Increased CPK-2 suggests other diseases like:
   • Electrical injuries
   • Heart injury
   • Myocarditis
5. Increased CPK-3 suggests:
   • Crush injuries
   • Rhabdomyolysis
   • Muscular dystrophy
   • Myositis
   • Recent seizures

Alkaline Phosphatase (ALP):

The enzyme is a monomer and the isoenzymes are due to the difference in the carbohydrate content:

1. Alpha1-ALP
2. Alpha2-Heat labile ALP
3. Alpha2-Heat stable ALP
4. Pre Beta-ALP
5. Gamma-ALP ,etc.

Diagnostic importance of ALP:

1. Increased Alpha2-Healt labile ALP:
   • Liver diseases : Biliary obstruction, hepatitis
2. Increased pre beta-ALP:
   • Bone diseases: Paget’s disease, Osteoblastic bone tumors, Osteomalacia, Rickets, Skeletal disease
3. Increase in ALP also suggests other diseases like:
   • Pregnancy
   • Healing bone fracture
   • Anemia
   • Leukemia
   • Thyroid gland inflammation
   • Hyperparathyroidism
   • Chronic alcohol ingestion
4. Decreased ALP suggests:
   • Malnutrition

Sources:

• Harper’s Illustrated Biochemistry
• Biochemistry – Satyanarayana
• Medline Plus : Isoenzyme tests