Gout and hyperuricaemia - and anti-cancer drugs

Lactic acid competes with uric acid for active secretion into the renal tubule, so he will excreted less and his plasma concentration will be very high.

Nichols and coworkers (1973) determined the plasma concentration and urine excretion of urate in a group of trans-sexual men before and after undergoing oestrogen therapy. The results are shown on the right.

[From data reported by Nicholls A, Snaith ML & Scott JT. Effect of oestrogen therapy on plasma and urinary levels of uric acid. British Medical Journal (i) 449-451 1973.]

These results suggest that, directly or indirectly, oestrogens increase the urinary excretion of uric acid. This means that premenopausally, women will have greater excretion of uric acid and be less at risk of developing gout than will men. However, post-menopausally, when oestrogen secretion falls, they will be at the same risk as men.

The pathway for uric acid synthesis from AMP and GMP is shown on the right.

Click here to download a printable copy of this pathway.

Note that there is continual catabolism of AMP and GMP, and continual salvage of the resultant hypoxanthine (back to AMP) and guanine (back to GMP). This is important for the regulation of intracellular concentrations of purine nucleotides and to maintain the appropriate balance between adenine and guanine nucleotides.

The problem of gout is mainly the very low solubility of uric acid and its salts (urates). The table below shows the solubility of purines and their metabolites

Regardless of the underlying biochemical cause of gout, the same method of treatment is effective: inhibition of the enzyme xanthine oxidase, most commonly with allopurinol.

Allopurinol is a substrate for xanthine oxidase, and competes with hypoxanthine and xanthine; its product, oxypurinol, is also an inhibitor of xanthine oxidase

Hypoxanthine and xanthine are 4 - 5 times more soluble than sodium urate in plasma, meaning that they are very much less likely to form crystals. Since they will be filtered in the glomerulus, and since unlike uric acid they are not actively reabsorbed, they will be excreted in the urine.

In most mammals, uric acid is not the end-product of purine metabolism, but is metabolised onwards to allantoin and allantoic acid, both of which are very much more soluble than uric acid. It is only in animals that have lost uric acid oxidase (human beings and other primates) that gout is a problem.

The diagram on the left shows that uric acid can be converted to allantoin non-enzymically by reaction with superoxide and other oxygen radicals. Indeed, uric acid is one of the major radical trapping antioxidants in the bloodstream.

This suggests that maintaining a high blood concentration of uric acid provides valuable protection against oxygen radical damage to tissues and plasma lipoproteins, which can lead to cancer, atherosclerosis and auto-immune diseases.

The de novo synthesis of purines

The pathway for the de novo synthesis of purines is shown below. THF is tetrahydrofolic acid, the folic acid derivative that is important for transfer of one-carbon units in a variety of metabolic pathways.

Click here to download a printable copy of this pathway.

The onward metabolism on IMP to AMP and GMP is shown in the right

Click here to download a printable copy of this pathway.

The first committed step of the pathway is the reaction of phosphoribosyl pyrophosphate (PRPP) with glutamine to form phosphoribosylamine, catalysed by PRPP amidotransferase. As you would expect, this enzyme is subject to feedback inhibition by purine nucleotides (AMP, GMP and their analogues).

The onward reactions from inosine monophosphate to AMP and GMP are also subject to feedback inhibition by their end-products. Adenylosuccinate synthase is inhibited by AMP, and IMP dehydrogenase is inhibited by GMP.

Mercaptopurine is a synthetic purine analogue that is a substrate for hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the enzyme that is involved in purine salvage (click here to see the pathway of purine catabolism and salvage). The product is a purine nucleotide analogue.

Rapidly dividing cells, such as cancer cells, have a very high requirement for purine (and pyrimidine) nucleotides for DNA synthesis.

The mercaptopurine nucleotide will inhibit all three steps in the de novo synthesis pathway that are subject to end-product inhibition by purine nucleotides: PRPP amidotransferase, adenylosuccinate synthase and IMP dehydrogenase. This will starve the tumour cells of purine nucleotides and so slow growth of the tumour.

Click here to see these steps highlighted in the purine synthesis pathway (there are two diagrams in this file - scroll down to see the second half)

There are two steps in the pathway for de novo purine synthesis that require folic acid derivatives: GAR formyltransferase uses formyl tetrahydrofolate, and AICAR formyltransferase uses formyl dihydrofolate. Methotrexate leads to deficiency of active metabolites of folic acid. This will starve the tumour cells of purine nucleotides and so slow growth of the tumour.

The methylation of cytidine monophosphate to thymidine monophosphate in pyrimidine synthesis also requires a folic acid derivative, so the folic acid deficiency caused by methotrexate will also starve the tumour cells of TTP for DNA synthesis.

Methotrexate also depletes folic acid in normal tissues. However, these generally have a higher capacity to accumulate and store folic acid than tumour cells, and the usual practice is to alternate periods of treatment with methotrexate (hopefully to damage tumour cells more than normal cells) with periods of treatment with folic acid in the form of leucovorin (so-called leucovorin rescue) with the aim of benefiting normal cells more than the tumour cells.

Click here to see these two folate-dependent steps highlighted in the purine synthesis pathway

There are two reactions in purine synthesis in which glutamine is the donor of a nitrogen atom: the first committed reaction, catalysed by PRPP amidotransferase, and the reaction catalysed by formylglycinamide ribonucleotide amidotransferase. Azaserine and diazanorleucine inhibit these glutamine-requiring reactions. Again, this will starve the tumour cells of purine nucleotides and so slow growth of the tumour.

Click here to see these two glutamine-utilising steps highlighted in the purine synthesis pathway

Nitrogen is introduced from glycine in the reaction catalysed by glycinamide ribonucleotide synthetase (GAR synthetase).

There are two reactions in which nitrogen is introduced from aspartate: SAICAR synthase and adenylosuccinate synthetase. In each case the reaction is the same as is seen in the synthesis of argininosuccinate, then arginine, in the urea synthesis pathway. Initially aspartate is condensed with the substrate, then fumarate is released, leaving just the amino group from aspartate in the product.

Click here to see these steps highlighted in the purine synthesis pathway (there are two diagrams in this file - scroll down to see the second half)

Control of purine synthesis by PRPP amidotransferase

The first committed step in de novo synthesis of purines is catalysed by PRPP amidotransferase, which is a multi-subunit enzyme with the following kinetic properties:

[From data reported by: Holmes EW. Kinetic, physical and regulatory properties of amidophosphoribosyltransferase. Advances in Enzyme Regulation 19: 215-231 1981; Kelley WN & Wyngaarden JB. Enzymology of gout. Advances in Enzymology 41: 1-33 1974; Wood AW & Seegmiller JE. Properties of 5-phosphoribosyl-1-pyrophosphate amidotransferase from human lymphoblasts. Journal of Biological Chemistry 248: 138-143 1973; Wyngaarden JB. Regulation of purine biosynthesis and turnover. Advances in Enzyme Regulation 14: 25-42 1976]

The Hill coefficient is a measure the the cooperativity of substrate binding.

A value of 1.0 indicates that there is no cooperativity, and there is a hyperbolic substrate / velocity curve. As the Hill coefficient increases, so there is increasing cooperativity and the substrate / velocity curve becomes increasingly hyperbolic.

Even in the control incubations, the activity of PRPP amidotransferase is very dependent on the availability of PRPP - the Km (140 µmol /L) is very much higher than the normal range of intracellular PRPP, so that even a small increase in the concentration of PRPP will lead to a large increase in the rate of reaction .

In the presence of AMP and GMP the enzyme is significantly inhibited - the substrate / velocity curve is now significantly sigmoid and the apparent Km is now almost four-fold higher, so that the enzyme has little activity at normal intracellular concentrations of PRPP. This suggests that when AMP and GMP are bound the enzyme undergoes a conformational change that hinders the binding of PRPP, and increases cooperativity between the subunits. This is classical allosteric inhibition.

By contrast, the Km for glutamine is significantly lower that the normal range of intracellular glutamine concentrations, and the activity of the enzyme will not be significantly affected by changes in the availability of glutamine, whether or not AMP and GMP are present.

In about 75% of patients with primary gout there is an abnormal form of PRPP amidotransferase that is almost completely insensitive to inhibition by AMP + GMP.

If PRPP amidotransferase is insensitive to feedback inhibition there will be increased synthesis of AMP and GMP, in excess of requirements. These nucleotides will then be catabolised to form uric acid.

Click here or scroll up to see the pathway of purine catabolism

Lesch-Nyhan disease

Lesch-Nyhan disease is a rare X-linked inborn error of metabolism.

Affected boys show extreme hyperuricaemia and gout from an early age, together with a variety of developmental and neurological problems, including compulsive self-mutilation. (The children will chew their lips through, and even bite off their fingers if not restrained, despite screaming with pain as they do so).

This is an X-linked disease - the gene for the defective enzyme is on the X chromosome. Males have only one X chromosome, while females have two copies. This means that a female carrier of the disease has sufficient active enzyme from the normal allele, while a male child who inherits an abnormal X chromosome form his mother will have little or no activity of the enzyme, and will show the disease. Female carriers of Lesch-Nyhan disease may have hyperuricaemia and may develop gout at a relatively early age.

About one-third of cases of Lesch-Nyhan disease are new mutations, with no family history.

Lesch-Nyhan disease is due to more or less complete absence of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT,see the diagram above), which is carried on the X chromosome. The condition is invariably fatal, and to date there is no effective treatment, other than allopurinol or other inhibitors of xanthine oxidase to lower blood and urine uric acid and relieve the gout. The neurological and behavioural problems are associated with developmental defects in dopaminergic neurons, but it is not clear how lack of HGPRT leads to these defects.

Phosphoribosyltransferase is the substrate for only a small number of enzymes, shown in the table below:

We have already seen that PRPP amidotransferase, the first and rate-controlling step in de novo purine synthesis is exquisitely sensitive to the intracellular concentration of PRPP. If one of the small number of enzymes that utilise PRPP is inactive this will lead to an increase in the intracellular concentration of PRPP, and hence increased activity of PRPP amidotransferase, leading to synthesis of more AMP and GMP than is required. The excess will be catabolised to uric acid, leading to an increased blood and urine concentration of urate, and hence the development of gout.

In addition, since hypoxanthine and guanine are not being salvaged, but metabolised onwards to uric acid, there will be less feedback inhibition of PRPP amidotransferase.

Kelley et al. (1970) investigated the effect of added orotic acid on the intracellular concentration of PRPP and the synthesis of purines in fibroblasts from normal subjects and two patients with Lesch-Nyhan disease (lacking HGPRT activity). They measured the rate of purine formation by the incorporation of radioactivity from [14C]formate into formylglycinamide ribonucleotide (FGAR) in the presence of the glutamine analogue azaserine as an inhibitor of onward metabolism of FGAR ( as discussed above, it inhibits FGAR amidotransferase). The results are expressed /mg protein in the incubation:

[From data reported by Kelley WN, Fox IH & Wyngaarden. Regulation of purine biosynthesis in cultured human fibroblasts: (i) effects of orotic acid. Biochimica et Biophysica Acta 215: 512 -516. 1970]

In similar studies, Boyle et al. (1972) investigated the effect of added nicotinic acid on purine synthesis (the results are expressed /million cells):

[From data reported by Boyle JA, Raivo KO, Becker MA & Seegmiller JE. Effects of nicotinic acid on human fibroblast purine synthesis. Biochimica et Biophysica Acta 269: 179-183 1972]

In both cases there is a higher concentration of PRPP in the cells from patients with Lesch-Nyhan syndrome, reflecting the importance of HGPRT as one of the main enzymes using PRPP. As would be expected from the kinetics of PRPP amidotransferase, this increased concentration of PRPP leads to increased synthesis of purines.

Addition of either orotic acid or nicotinic acid as a substrate for a phosphoribosyltransferase that uses PRPP led to a reduction in intracellular PRPP and a reduction in the rate of purine formation in both cells for control subjects and patients with Lesch-Nyhan disease.

These results confirm the reliance of the rate of PRPP amidotransferase activity on the availability of PRPP.

Lesch-Nyhan disease is associated with more or less complete absence of HGPRT immunologically active protein. In a small number of patients with gout and hyperuricaemia various authors have reported the following findings from studies of HGPRT activity in cultured fibroblasts. The amount of enzyme protein for calculation of Vmax was determined by titration against antibodies raised against purified HGPRT):

[From data reported by: Benke PJ, Herrick N & Hebert A. Hypoxanthine-guanine phosphoribosyltransferase variant associated with accelerated purine synthesis. Journal of Clinical Investigation 52: 2234-2240 1973; Fox IH, Dwosh IL, Marchant PJ, Lacroix S, Moore MR, Omura S & Wyhofsky V. Hypoxanthine-guanine phosphoribosyltransferase: characterization of a mutant in a patient with gout. Journal of Clinical Investigation 56: 1239-1249 1975; Sweetman L, Hoch MA, Bakay B, Borden M, Lesh P & Nyhan WL. A distinct human variant of hypoxanthine-guanine phosphoribosyltransferase. Journal of Pediatrics 92: 385-389 1978; Wilson JB, Baugher BW, Mattes PM, Daddona PE & Kelley WN. Human hypoxanthine-guanine phosphoribosyltransferase: demonstration of structural variants in lymphoblastoid cells derived from patients with a deficiency of the enzyme. Wilson JB, Young AB & Kelley WN. Hypoxanthine-guanine phosphoribosyltransferase deficiency: the molecular basis of the clinical syndromes. New England Journal of Medicine 309: 900-910 1983]

Partial lack of HGPRT can lead to the development of gout, but without the severe neurological problems associated with more or less complete lack of the enzyme, as seen in Lesch-Nyhan disease.

In patients A and C the Vmax of the enzyme is significantly lower than normal, suggesting that the mutation has affected the catalytic activity.

In patient A this has not affected the binding of either substrate, since the Km values for hypoxanthine and PRPP are normal. In patient C the mutation has also affected the binding of hypoxanthine (the Km is ~100-fold higher than normal), but not that of PRPP.

In patient B the Vmax of the enzyme is significantly higher than normal, but the Km for hypoxanthine is five times higher than normal. This suggests that the mutation has adversely affected the binding of hypoxanthine, but paradoxically has improved the catalytic efficiency of the enzyme.

For patients D and E we have no information about the Vmax of the enzyme, but the affinity of the enzyme from patient D for PRPP is 6 - 10-fold lower than normal, while the enzyme form patient E has lower affinity (higher Km) for both substrates.

There are thus mutations of HGPRT involving reduced Vmax or increased Km for one or both substrates that result in impaired purine salvage, increased intracellular PRPP and hence increased synthesis of purines in excess of requirements that are therefore metabolised onwards to uric acid, leading to the development of gout.

Knowing which amino acid changes are associated with reduction in Vmax or increase in Km for one or other of the substrates would allow us to elucidate which amino acid side-chains are likely to be involved in catalysis of the reaction and formation of the binding sites for PRPP and hypoxanthine, and which are important for folding of the protein into its active configuration.

For more on Lesch-Nyhan disease, click here or here

Key points from this exercise:

• Uric acid is the end-product of purine catabolism.
• Gout is the result of crystallisation of uric acid in joints and as nodules (tophi) under the skin when the plasma concent5ratrion exceeds the solubility limit of uric acid and its salts. Either excessive synthesis of uric acid or defects in its excretion may be the cause.
• Plasma uric acid is more or less completely filtered by the glomerulus, but 98% of the uric acid in the glomerular filtrate is reabsorbed in the renal tubules. More distally, there is active secretion of uric acid into the lumen of the tubules. Much of the actively secreted urate is also reabsorbed. A number of compounds inhibit the active secretion of uric acid into the renal tubule.
• .Premenopausally, women are less at risk of gout than are men; oestrogens enhance the excretion of uric acid.
• Precursors of uric acid, and especially xanthine and hypoxanthine, are considerably more soluble that uric acid and its salts. Inhibition of xanthine oxidase therefore provides effective treatment of gout.
• The first committed step of de novo purine synthesis is the reaction of phosphoribosyl pyrophosphate (PRPP) with glutamine to form phosphoribosylamine, catalysed by PRPP amidotransferase. This enzyme is subject to allosteric feedback inhibition by AMP and GMP acting synergistically. The Km of PRPP amidotransferase for PRPP is significantly higher than the intracellular concentration of PRPP, so that the activity of the enzyme increases considerably when the intracellular concentration of PRPP rises. This results in increased synthesis of purine nucleotides.
• Impaired sensitivity of PRPP amidotransferase to feedback inhibition is a cause of gout in some cases.
• Onward metabolism of IMP is inhibited by the end-products of the two branches of the pathway (AMP and GMP)
• Two steps in de novo purine synthesis require folic acid derivatives, and folate antimetabolites are effective anti-cancer agents, depriving the tumour cells of purine nucleotides.
• Two steps in de novo purine synthesis use glutamine as a nitrogen donor, and glutamine analogues are effective anti-cancer agents, depriving the tumour cells of purine nucleotides.
• There is continual catabolism of AMP and GMP, with salvage catalysed by hypoxanthine-guanine phosphoribosyltransferase, in order to maintain appropriate intracellular concentrations of the purine nucleotides.
• Mercaptopurine is a synthetic purine analogue that is a substrate for hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the enzyme that is involved in purine salvage The product, a purine nucleotide analogue, inhibits all three steps in de novo purine synthesis that are regulated by feedback inhibition.
• More or less complete lack of hypoxanthine-guanine phosphoribosyltransferase leads to a considerable increase in the intracellular concentration of PRPP, resulting in increased synthesis of purine nucleotides and hence increased synthesis of uric acid. This is Lesch-Nyhan disease, which leads to gout at an early age as well as severe neurological problems that are the result of developmental defects in dopaminergic neurons.
• Partial lack of hypoxanthine-guanine phosphoribosyltransferase is a cause of gout in some people.

  For more on Lesch-Nyhan disease, click here or here