L-16 Purine Biochemistry and Uric Acid Metabolism.xml

Lecture 16. Purine Biochemistry and Uric Acid Metabolism


Lecture 16. Purine Biochemistry and Uric AcidMetabolismMcGill Department of MedicineDivision of RheumatologyMontreal General Hospital937-6011 loc. 4178

M. [email protected]

Objectives

The objectives of these lectures and this handout are for you to learn:1. Basic clinical concepts of urate crystal deposition disease.2. Basic biochemistry of purine compounds: the substrates and major enzymes involved in

pathways of purine metabolism and uric acid formation.3. Basic physicochemical properties of urate and uric acid.4. The mechanisms of urate synthesis and elimination.5. To understand the mechanisms that lead to hyperuricemia and how dietary modification

or drugs could be used to restore normouricemia.

I. Gout

HISTORY

Excellent descriptions of gout can be found in the earliest medical writings. The aphorisms ofHippocrates documented the fact that gout is predominantly a disease of adult men and onlyappears in women after the menopause. Sydenham was himself a gout sufferer and his vividdescriptions of the disease, written in 1683 can still make the big toe itch! Gout was extremelycommon in Georgian England, and many of the best known figures in English political andcultural life suffered from the disease; the extreme pain of acute attacks of gout has beenresponsible for anger, frustration, and misjudged decisions in the highest political circles (e.g. Pittand the Boston Tea Party).

DEFINITION

Gout a term first used in the 13th century A.D. is derived from the Latin 'gutta' (a drop) whichreflects the notion that gout resulted from a local instillation of a malevolent humour. We havesince recognized that gout is a crystal deposition disease; its clinical and pathologicalmanifestations are due to the presence of crystalline salts of urate within the affected tissues.



CLINICAL FEATURES• Hyperuricemia: a serum or plasma urate concentration greater than 7.0 mg/dl (0.42

mmol/l) in males and 6.0 mg/dl (0.36 mmol/l) in females.• Acute gouty arthritis: sudden, intense inflammation in and ``around the joints (most

typically, but not limited to, the big toe) and other areas of soft tissues such as theAchilles tendon (heel), olecranon bursa (elbow), helix of the ear.

• Urolithiasis: uric acid stones in the kidney and urinary collecting system.• Chronic interstitial nephropathy: disease related to the deposition of monosodium

urate monohydrate crystals in the substance of the kidney itself.

EPIDEMIOLOGY OF GOUTAND HYPERURICEMIA

Gout is predominantly a disease of adult males, with a peak incidence in the fifth decade. Theprevalence in males varies from about 10-20/1,000 and in females 1-6/1,000. It is the second mostcommon cause of inflammatory arthritis in males over the age of 30 in the U.S.A. It causessignificant short-term disability, occupational limitation, and utilization of medical services.

Hyperuricemia is demonstrable in at least 5% of asymptomatic American (and probablyCanadian) men on at least one occasion during adulthood. The risk of developing clinicallyapparent urate crystal deposition disease increases as the level of serum uric acid rises.

II. Purine Metabolism

INTRODUCTION

Recognition of the role of purines in human disease began with the observation that uric acid, apurine base, was a component of some renal calculi and in the form of monosodium urate, was amajor constituent of tophi (deposits of urate in soft tissues), was elevated in the serum of goutypatients, and was present, in crystalline form, in synovial (joint) fluid during the acute attack ofgouty arthritis.

In humans, uric acid is derived both from the ingestion of foods containing purines and from theendogenous synthesis of purine nucleotides, which are building blocks in the synthesis of nucleicacids.

FIG. 1. PURINE BIOCHEMISTRY

The parent compound, the purine base, is composed of a six-membered pyrimidine ring fused tothe five- membered imidazole ring.

Fig. 1

The most important purines are:

The purines all show lactam-lactim isomerism and may be written in either form (Fig. 2).



• adenine• guanine• hypoxanthine• xanthine• uric acid

Fig. 2

Purine nucleosides are composed of a purinebase plus a pentose joined to the base by a -N-glycosyl bond between carbon atom 1 of the pentose and nitrogen atom 9 of the purine base.

There are two series of nucleosides:



• ribonucleosides, which contain D-ribose as the sugar component• deoxyribonucleosides, which contain 2-deoxyribose

(Fig 3)

Fig. 3

Purine nucleotides and deoxynucleotides consist of a nucleoside or a deoxynucleoside with aphosphate group in ester linkage with carbon 5 of the pentose (Fig. 4).





Fig. 4

The nucleosides and deoxynucleosides may exist as:

The phosphoric acid residues of these compounds are designated by the symbols a, ß, γ(Fig. 5).

• 5'-monophosphates• 5'-diphosphates• 5'-triphosphates



Fig. 5

These compounds serve:

PURINE METABOLISM (Fig. 6)

The synthesis of purine nucleotides involves alternative biochemical pathways that are closelyregulated. In the pathway of purine synthesis de novo, a purine ring is synthesized from smallmolecule precursors of uric acid, sequentially added to a ribose-phosphate backbone donated by 5-phophoribosyl 1-pyrophosphate (PRPP).

The first reaction committed to the pathway, catalyzed by the enzyme amidophosphoribosyltransferase (reaction 1 in Fig.), is the major site of the regulation of this pathwayby means of an antagonistic interaction between inhibition by purine nucleotide products andactivation by PRPP, a substrate usually present in limited concentrations in the cell. Additional

1. as building blocks for RNA and DNA2. as precursors of the cyclic nucleotides, adenosine 3',5'-cyclic phosphate, and guanosine

3',5'-cyclic phosphate3. as a source of chemical energy4. as precursors of various purine cofactors and coenzymes such as nicotinamide adenine

dinucleotide (NAD)



sites of control of purine nucleotide production have been identified at the level of the PRPPsynthesis (reaction 3) and at the distal branch point governing distribution of newly formednucleotides into adelylate and guanylate derivatives.

The alternative pathway of purine nucleotide synthesis involves two enzymes, adeninephosphoribosyltransferase (reaction 4) and hypoxanthine-guanine phosphoribosyltransferase(HGPRT, reaction 2), which catalyze reactions between PRPP and the respective purine basesubstrates in the single-step synthesis of purine nucleotides.

Among the factors governing the relationship between rates of purine base salvage and purinesynthesis de novo are the availability of PRPP and the concentrations of the nucleotide productscommon to both pathways.

The catabolicsteps that generate uric acid from nucleic acids and free purine nucleotides involvedegradation through purine nucleoside intermediates to hypoxanthine and xanthine. The latterare ultimately oxidized to uric acid in sequential reactions catalyzed by the enzyme xanthineoxidase (reaction 8).

Fig. 6



III. Physicochemical Properties of Uric Acid and Urate

Uric acid is a weak acid which is ionized at normal body pH and thus occurs in the blood ortissues in the form of the urate ion. When ionized, uric acid can form salts with various cations but98% of extracellular uric acid is in the form of the monosodium urate. However the proportion inthe form of uric acid or urate is pH dependent so that the ratio between these two forms may varyconsiderably in urine.

Effect of pH on Uric Acid

Fig. 7

At pH 5.7 equal amounts of uric acid and urate are present in solution.

The solubility of urate and uric acid is critical to the development of crystals. Urate isconsiderably more soluble in plasma, synovial fluid and urine than in aqueous solutions. Even so,above a concentration of about 7.0 mg/dl (0.42 mmol/l), plasma is supersaturated with urate.Perhaps because this is often a stable situation and because of the other constituents of plasma,there may be no tendency to crystal formation. The solubility of uric acid in urine rises



exponentially as the pH increases above 4. However, there is little change in the solubility of uratewithin the pH range that may exist in the plasma, synovial fluid and other tissues. Both urate anduric acid solubility fall with decreasing temperatures (Fig. 7).



IV. Urate Synthesis and Elimination

Urate is derived from purines that may be ingested or synthesized from ingested foods as well asbeing reutilized following cell breakdown. Urate is then eliminated via the kidneys and alimentarytract. (Fig. 8).

Fig. 8

URATE PRODUCTION

The pathways of production of uric acid were outlined in Fig. 6 and in a simpler form in Fig. 13.Urate production is therefore dependent upon purine nucleotide metabolism and the function ofxanthine oxidase. Whereas there are several mechanisms for the reutilization of purine bases tonucleosides and nucleotides, degradation of these purine bases by xanthine oxidase is irreversible.



Purine availability can be altered by several factors as illustrated by Fig. 8. Purines can be deriveddirectly from the diet or by synthesis from small molecular precursors. Purine bases derived fromtissue nucleic acids may also be reutilized. These arise from normal cell turnover but the amountscan be greatly increased in proliferative disorders of the bone marrow, particularly hematologicmalignancies and by cytotoxic therapy.

URATE ELIMINATION

In order to maintain homeostasis, urate must be eliminated from the body as there is no uricaseand thus no metabolism of urate in human tissues.

Approximately 2/3 of produced urate is eliminated via the kidneys with the remainder beingexcreted into the alimentary tract.

It is probable that the elimination of urate via the alimentary tract is a passive process and varieswith plasma urate concentrations. Under normal conditions, negligible amounts of urate are foundin the feces because it is degraded by colonic bacteria. In a sterilized bowel, on the other hand,urate excreted into the lumen does not undergo uricolysis and may be found in the feces.

Almost all of the urate in the plasma is filtered at the glomerulus. Although there is some bindingof urate to plasma proteins, this is of low affinity in vivo, readily reversible and not of physiologicsignificance in man. Following filtration, there is almost complete reabsorption of urate in theproximal tubule such that only a small amount of the filtered urate passes to the loop of Henle.Thus, in proximal tubular disorders such as the Fanconi syndrome or acquired nonselectiveproximal tubular transport defects, a failure of urate reabsorption resulting in hyperuricosuria andhypouricemia may occur.



Fig. 9

Perhaps the most important mechanism for maintaining normal renal urate excretion is the activetubular secretion of urate. This may account for as much as 50% of the urate which is filtered.Neither the site nor mechanism for this urate secretion has been precisely identified in man.Lactate, -hydroxybutyrate and, acetoacetate are thought to decrease urate excretion by inhibitingtubular secretion of urate.

The variation in the amount of urate that is finally excreted by the kidney is most likely due toalterations in the reabsorption of secreted urate. The precise site of postsecretory reabsorption inman is uncertain, as is whether it occurs distally to or in the same segment as urate secretion. Inall, approximately 10% of filtered urate is finally excreted in the urine.

Clearly, abnormalities at any of the four stages of renal urate excretion may have a profound effecton urate homeostasis. (Fig 9).



V. Etiology of Hyperuricemia

Hyperuricemia may result from overproduction or underexcretion of urate. In overproductionof urate, the purine precursors may be of endogenous or exogenous (i.e. dietary) origin. Withrespect to underexcretion of urate, this depends principally upon renal handling of urate. Overall,the great majority of patients with gout and hyperuricemia demonstrate urate underexcretion asjudged by a urate clearance of less than 6 ml/min or a urate to creatinine clearance ratio of lessthan 6%. However, in practical terms, the combination of a relative excess of dietary purineconsumption together with urate underexcretion is likely to be the basis of hyperuricemia in manypatients with gout.

Primary hyperuricemia refers to those circumstances in which elevated serum urate levels ormanifestations of urate deposition are due to inherently disordered uric acid metabolism notassociated with another acquired disorder and in which gout is a prominent feature of the clinicalpicture.

Secondary hyperuricemia refers to circumstances in which gout is a minor clinical fearuresecondary to any of a number of genetic or acquired processes.

URATE OVERPRODUCTION

About 10% of patients with hyperuricemia are overproducers of uric acid of which no more than10% (i.e. 1% of all hyperuricemic people) have primary identifiable inherited derangements inmechanisms regulating purine nucleotide synthesis de novo.

In both partial deficiency of HGPRT and milder forms of superactivity of PRPP synthetase,early adult- onset gout and a high incidence of uric acid urinary tract stones constitute the usualclinical phenotype.

Severe HGPRT deficiency is associated with spasticity, choreoathetosis, mental retardation, andcompulsive self-mutilation (Lesch-Nyhan Syndrome). Lesser neurologic lesions may be found ina minority of patients with partial HGPRT deficiency.

Intracellular accumulation of PRPP is a result of diminished utilization of this regulatory substratein purine base salvage. This drives purine synthesis de novo at an increased rate in HGPRTdeficiency. In addition, in the absence of HGPRT, hypoxanthine, once formed, cannot be reutilizedand can only be degraded. In the case of variant forms of PRPP synthetase with excessive activity,increased PRPP availability for purine synthesis results from increased rates of PRPP generation.

Both of these enzymes are produced from X-linked genes, and thus heterozygous men areaffected. Hyperuricemia detected in prepubertal boys suggests that one of these enzymatic defectscould be the cause.

An increase in the net intracellular degradation of the adenine nucleotide, ATP, has been proposedas a common thread linking hyperuricemia in several conditions which are listed in Fig. 11,below. In particular, the accelerated degradation of ATP to AMP via the conversion of acetate toacetyl CoA in the metabolism of ethanol appears to be a critical mechanism in causingoverproduction of uric acid and hyperuricemia associated with alcohol ingestion (Fig. 10).



Fig. 10

Click here for the Flash Animation

Fig. 11. Classification of hyperuricemia

Uric acid overproduction Uric acid underexcretion Primary hyperuricemia Primary hyperuricemia

1. Idiopathic2. HGPRT deficiency (partial and

complete)3. PRPP synthetase superactivity

• Idiopathic



Fig. 12

Secondary hyperuricemia Secondary hyperuricemia

1. Excessive dietary purine intake2. Increased nucleotide turnover (e.g.

my e l o p r o l i f e r a t i v e a n dlymphoproliferative disorders,hemolytic disorders, psoriasis)

3. Accelerated ATP degradation glycogenstorage diseases (Types I, III, V, VII)fructose ingestion, hereditary fructoseintolerance hypoxemia and tissueunderperfusion severe muscle exertionalcohol abuse

1. Diminished renal function2. Inhibition of tubular urate secretion

competitive anions (e.g. keto- andlactic acidosis)

3. Enhanced tubu la r u ra t ereabsorption dehydration, diuretics

4. Mechanism incompletely definedhypertension hyperparathyroidismcertain drugs (e.g. low dose aspirin,many diuretics, lead nephropathy



Fig. 13

Source of figures and tables:

Figures 76.6, 76.7, 76.8, 76.9, 76.10, 76.11, 76.17 and Table 76.10 are from William N. Kelly andH. Ralph Schumacher, Jr. "Crystal-Associated Synovitis" from Textbook of Rheumatology. W.N.Kelley, E.D. Harris, S. Ruddy, C.B. Sledge editors, W.B. Saunders, Philadelphia, U.S.A., 1993,pp1291-1336.

Figures 18.1, 18.2, 18.3 are from "Gout" from Atlas of Clinical Rheumatology. P.A. Dieppe, P.A.Bacon, A.N. Bamji, I. Watt editors, Gower Medical Publishing Ltd, London, England, 1986, p18.2.

Figure 31A-1 and Table 31A-1 are from Robert Terkeltaub "Gout" from Primer on the RheumaticDiseases, H.R. Schumacher, Jr., J.H. Klippel, W.J. Koopman editors, The Arthritis Foundation,Atlanta, U.S.A. 1993, pp209-213.



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