Purine Metabolism and Gout

Dr Sunila BHMS,MD(Hom)
Purine Metabolism
The chief purines found in the nucleotides and nucleic acids are adenine and guanine. Uric acid is the final oxidation product (in man) of these purines. Purines combine through their 9-nitrogen position with sugar residues →nucleoside. If the sugar residue is also phosphorylated a nucleotide results. Purines are occasionally found as free bases, more usually as nucleosides and nucleotides, and as nucleic acids.

Synthesis of purine nucleotides
The synthesis of purine nucleotides occurs along two pathways, referred to as the de novo and salvage pathways.
The de novo pathway involves synthesis of purines and then uric acid from non purine precursors. The starting substrate for this pathway is ribose-5- phosphate.

• Formation of 5- Phosphoribosyl- 1- pyrophosphate (PRPP).    Ribose is converted by successive phosphorylations of C1 and C5 to form 5- phosphoribosyl-1- PP. ATP is required as phosphate donor.
• The pyrophosphate in C1 is replaced by an NH2 group from glutamine to form 5- phosphoribosyl amine. This is a “committed step” in purine biosynthesis. It is subject to feed back inhibition by purine nucleotides.
• Glycine combines with 5- phosphoribosyl amine to form glycinamide ribotide.
• A formyl group is added to the N at position (7) to form formyl glynamide ribotide. This step requires tetra hydrofolic acid.
• Glutamine will now add the NH2 of position (3) and closure of ring occurs between C8 and N9 to form aminoimidazole ribosylphosphate.
• CO2 (from CO2 pool of the body) and NH3 from aspartic acid are added as carbamate to C5 to form the C6 and N1 and the compound now formed is 5- amino- 4- imidazole- carboxamide- ribotide. This requires biotin.
• A formyl group is now added to the amino group of N3. This step requires tetrahydrofolic acid. The intermediate now formed is 5- formamido- 4- imidazole- carboxamide- ribotide.
• Ring closure now occurs between N1 and C2 to form inosine monophosphate or inosinic acid.
• Inosine is converted to adenine by taking an amino group at C6 from aspartic acid.
• Inosine can be converted to guamine by oxidation C2 to C=O and later amination from glutamine to form C.NH2. Reactions 9 and 10 occur while still in the nucleotide (as inosinic acid). Hence the product formed in step9 is adenylic acid (AMP) and in step 10 it is guanylic acid (GMP).

• The purine ribonucleotide is converted to deoxyribonucleotide by reduction of the second carbon of ribose. The reaction requires a protein cofactor called reduced thioredoxin’ which is converted to the oxidized form in the reaction. It is converted to the reduced form again by the enzyme ‘thioredoxin reductase’ using NADPH + H+ as coenzyme.

• The erythrocyte, the polymorphonuclear leukocyte and the mammalian brain do not have the ability to synthesize the purine base. They depend on exogenous supply. The liver supplies the purine base to these tissues.

Purine Salvage Pathways
The salvage of these preformed purine compounds can occur by two general mechanisms. The quantitatively more important mechanism is the phosphoribosylation of the free purine bases by specific enzymes requiring PP riboseP as the ribose phosphate donor. The second general mechanism is the phosphorylation of purine nucleosides on their 5- hydroxyl group.

AMP : Phosphoribosylation of adenine catalysed by adenine phosphoribosyl transferase.

Phosphoribosylation of hypoxanthine and guanine to form IMP and GMP, respectively. The reactions are catalysed by the enzyme hypoxanthine- guanine phosphoribosyl transferase.

The salvage of purine ribonucleosides to purine ribonucleotides is carried out in humans by adenosine kinase only.

Phosphorylation of adenosine to AMP by adenosine kinase

In humans, there is a cycle in which IMP and GMP as well as their respective deoxyribonucleotides are converted to their respective nucloesides (inosine, deoxyinosine, guanosine and deoxy guanosine) by purine-5-nucleotidase. These purine ribonucleocides and 2-deoxy nucleosides are converted to hypoxanthine and guanine by purine nucleoside phosphorylase. The hypoxathine and guanine can then again be phosphorylated by PP ribose P to IMP and GMP to complete the cycle. In the human organism, the consumption of pp ribose P by this salvage cycle is greater than the consumption of pp ribose p for the synthesis of purine nucleotides de novo.

There is a lateral pathway of this cycle that involves the conversion of IMP to AMP with subsequent      conversion of AMP to adenosine. The adenosine thus protected is then either salvaged directly back to AMP via adenosine kinase or is converted to inosine by the enzyme adenosine deaminase.

Regulation of purine biosynthesis
The single most important regulator of de novo purine biosynthesis is the intracellular concentration of PP ribose P. The rate of the synthesis of PP ribose P is dependent upon the availability of its substrates, particularly ribose-5- phosphate and the catalytic activity of PP ribose P synthetase. The rate of utilization of PP ribose P is dependent to a large extent on its consumption by the salvage pathway that phosphorylates hypoxanthine and guanine to their respective ribonucleotides.

The first enzyme uniquely commited to de novo purine synthesis, PP ribose P amidotransferase, demonstrates in vitro sensitivity to feedback inhibition by purine nucleotides, particularly adenosine monophosphate and guanosine monophosphate. These feedback inhibitors of the amidotransferase are competitive with the substrate PP ribose P, and thus again, PP ribose P plays a major role in the regulation of de novo purine synthesis.

The conversion of IMP to GMP is regulated by 2 mechanisms. AMP feedback regulates its own synthesis at the level of adenylosuccinate synthetase; GMP regulates its own synthesis by feedback inhibition of IMP dehydrogenase. Furthermore, the conversion of IMP to AMP requires GTP. The conversion of xanthinylate to GMP requires the presence of ATP. Thus there is significant cross regulation between the divergent pathways in the metabolism of IMP. This regulation prevents the synthesis of one purine nucleotide when there is a deficiency of the other.

 Catabolism of Purins
The end product of purine metabolism in primates including Dalmatian dog is uric acid. In the lower animals, birds and reptiles this is further broken down by the enzyme uricase to form allantoin and other products. The oxidation of the purine ring can occur while it is still in nucleotide combination or nucleoside combination. Adenase is absent in men. Instead, adenosine deaminase will convert adenine to hypoxanthine while in nucleoside combination. Similarly adenylic acid deaminase will act while in nucleotide combination.

Disorders of purine metabolism

• Those exhibiting Hyperuricemia.
• Those exhibiting Hypouricemia.
• Immunodeficiency diseases.

Hyperuricemia and Gout 

• Individuals with hyperuricemia can be divided into 2 groups:
• Those with normal urate excretion rate.
• Those excreting excessive quantities of total urates.

Lesch- Nyhan Syndrome and Von Gierke’s disease
There are persons with identifiable enzyme abnormalities of PP ribose P synthetase, the HGPRTase (hypoxanthine- guanine phosphoribosyl transferase) deficiencies (both the complete- Lesch Nyhan syndrome and incomplete deficiencies) and glucose 6- phosphate deficiency. There exists also a group of patients exhibiting idiopathic overproduction hyperuricemia.

The Lesch Nyhan syndrome is an inherited X- linked recessive disorder characterized by cerebral palsy with choreoathetosis and spasticity, a bizarre syndrome of self mutilation and severe overproduction hyperuricemia. The mothers of affected children exhibit hyperuricemia but no neurological manifestations.

Gout
Gout (also called metabolic arthritis) is a disease caused by a disorder of purine metabolism resulting in hyperuricemia. In this condition sodium urate crystals are deposited on the articular cartilage of joints and in the particular tissue like tendons and clinically manifesting as recurrent acute arthritis progressing to chronic deforming arthritis, formation of tophi and development of systemic complications like renal failure.

Normally, the human bloodstream only carries small amounts of uric acid. However, if the blood has an elevated concentration of uric acid, uric acid crystals are deposited in the cartilage and tissue surrounding joints. Plasma levels of uric acid vary from 2-7 mg/ dl in health. The term hyperuricemia denotes values above 7 mg/ dl.

Causes

• Primary or genetic gout (95%): It is either due to primary overproduction or under excretion of uric acid.
• Secondary gout (5%): Hyperuricemia results from a demonstrable disorder, leading either to overproduction or defective excretion of uric acid.

Causes of overproduction of uric acid

1. Increased break down of cellular nuclei occurs in malignant disease, especially when treated by anticancer drugs.
2. Several inborn errors of metabolism lead to overproduction of uric acid:

• 1)      Lesch Nyhan syndrome.
• 2)      Type 1 glycogen storage disease.
• 3)      Phosphoribosyl pyrophosphate synthetase over activity.

Impairment of excretion of uric acid

The excretion of uric acid is impaired

• 1.  in chronic renal failure
• 2. during intake of drugs like thiazides
• 3. in lactic acidosis
• 4. In miscellaneous conditions like hypertension, hyperparathyroidism, myxoedema, Down’s syndrome, toxemia of pregnancy, starvation and exercise. 

Pathogenesis
Although the exact cause of gout is not known, it is thought to be linked to defects in purine metabolism. The essential abnormality in primary gout is increased formation of uric acid without intermediary incorporation into nucleic acids. In secondary gout, there is an increased breakdown of nucleic acids leading to an excess of the end-product, uric acid.

Arthritis is caused by the deposition of monosodium urate crystals in the synovium. Polymorphonuclear leucocytes ingest the crystals. They release lysosymal enzymes which cause inflammation. Crystals are demonstrable in the synoviyum and articular cartilage in the stage of acute arthritis. In the chronic stage, erosion of the articular cartilage, proliferation of synovial membrane, pannus formation, cystic erosions of bone and secondary osteoarthritc changes develop. Tophi are nodular deposits found in and around the joints and in the articular cartilage. Histollogically these consists of monosodium crystals surrounded by mononuclear infiltration and foreign body giant cells. These lead to osteoarthritic changes, ankylosis of joints and tissue destruction.

Urate deposition and inflammatory reaction in the parenchyma of kidneys lead to hyalinization or fibrosis of glomeruli. Multiple urate calculi, chronic pyelonephritis and arteriosclerosis are other changes seen in long standing gout.

Clinical features

Gout has four distinct stages:

1. asymptomatic
2. acute
3. intercritical
4. Chronic.

In the first (asymptomatic) stage, plasma uric acid level increases, but there are no symptoms. The first attack of gout marks the second or acute stage. The classic picture is of excruciating and sudden pain, swelling, redness, warmness and stiffness in the joint. Low-grade fever may also be present. The patient usually suffers from two sources of pain. The crystals cause intense pain especially when the affected area is moved. The inflammation of the tissues around the joint also causes the skin to be swollen, tender and sore if it is even slightly touched. For example, a blanket draping over the affected area would cause extreme pain. Mild attacks usually go away quickly, whereas severe attacks can last days or even weeks.

After the initial attack, the person enters the intercritical stage or symptom-free interval that may last months or even years. Most gout patients have their second attack within 6 months to 2 years from their initial episode.

Gout usually attacks the big toe (approximately 75% of first attacks). The term podagra denotes painful affection of the foot occurring as a result of metatarsophalangeal arthritis. However it can also affect other joints such as the ankle, heel, instep, knee, wrist, elbow, fingers, and spine. In some cases the condition may appear in the joints of the small toes which have become immobile due to impact injury earlier in life, causing poor blood circulation that leads to gout.

In the last or chronic stage, gout attacks become frequent and become polyarticular (affecting multiple joints at one time). Large tophi can also be found in many joints. Most common sites of tophi are around the olecranon, ankles, tendo-achilles, and helix of the ear and over other joints.

In advanced cases of chronic gout, kidney damage, hypertension, ischemic heart disease and kidney stones can also develop.

Diagnosis
The diagnosis is generally made on a clinical basis, although tests are required to confirm the disease. Hyperuricemia is a common feature; however, urate levels are not always raised. Hyperuricemia is defined as a plasma urate (uric acid) level greater than 420 μmol/L (7.0 mg/dL) in males (the level is around 380 μmol/L in females); despite the above, high uric acid level does not necessarily mean a person will develop gout. Additionally, urate falls to within the normal range in up to two-thirds of cases. If gout is suspected, the serum urate should be repeated once the attack has subsided. Other blood tests commonly performed are full blood count, electrolytes, renal function and erythrocyte sedimentation rate (ESR). This serves mainly to exclude other causes of arthritis, most notably septic arthritis.

A definitive diagnosis of gout is from light microscopy of joint fluid aspirated from the joint (this test may be difficult to perform) to demonstrate intracellular monosodium urate crystals in synovial fluid polymorphonuclear leukocytes. The urate crystal is identified by strong negative bi-refringence under polarised microscopy, and their needle-like morphology. A trained observer does better in distinguishing them from other crystals.

Radiological changes: in well developed chronic gout periarticular bone shows small punched out erosions due to urate deposits, with superadded osteoarthritic changes. 

Treatment
All precipitating factors should be avoided. Dietary change can make a contribution to lowering the plasma urate level if a diet low in purines is maintained, because the body metabolizes purines into uric acid. Avoiding alcohol, high-purine foods, such as meat, fish, dry beans (also lentils and peas), mushrooms, spinach, asparagus, and cauliflower, as well as consuming purine-neutralizing foods, such as fresh fruits (especially cherries and strawberries) and most fresh vegetables, diluted celery juice, distilled water, and B-complex and C vitamins can help. Low fat dairy products such as skim milk significantly reduced the chances of gout.

Improved blood circulation in the immediate area of an affected immobile joint can be encouraged with a warm bath. This assists in the relief of swelling and reduction in uric acid crystallization. Ensure area is dry before putting on clothes.

Surgery For extreme cases of gout, surgery may be necessary to remove large tophi and correct joint deformity.

Hypouricemia
Hypouricemia is either due to enhanced excretion or decreased production of urate and uric acid. Deficiency of enzyme xanthine oxidase results in hypouricemia and increased excretion of the oxypurines; hypoxanthine and xanthine. A deficiency of the enzyme purine nucleoside phosphorylase is associated with hypouricemia.

Two immunodeficiency diseases associated with purine metabolizing enzymes; adenosine deaminase deficiency, purine nucleoside phosphorylase deficiencies have been described. Both of these diseases are inherited as autosomal recessive disorders.

References

1. Samson Wright’s Applied Physiology
2. A Textbook of Biochemistry by A. V. S. S. Rama Rao
3. Harper’s Textbook of Biochemistry
4. Text book of medicine- K V Krishna Das
5. Harrison’s principles of internal medicine
6. Pathologic basis of disease- Robbins, Cotran and Kumar