Biochemistry I

Lesson 5Lesson 5

Gout and Routine Urinalysis

Uric Acid

Is the final breakdown product of purine metabolism

Purines (e.g. adenosine and guanine) result from breakdown of nucleic acidsfrom breakdown of nucleic acids

They are either ingested or come from the destruction of tissue cells - converted into uric acid by liver

From liver, uric acid are transported to kidney where it is filtered by glomeruli

Uric Acid

Nearly all filtered uric acid is reabsorbed in the proximal tubules, small amounts are then secreted by distal tubules and finally ending up in urine

Uric acid in plasma is normally in the form of monosodium urate

Urates in this form are relatively insoluble

At high levels (>6.4 mg/dL), plasma is saturated, urate crystals may form and precipitate in the tissues

Purine Metabolism and Uric Acid

Purines are simple cyclic organic molecules

containing nitrogen - are essential

components of nucleic acids (involved in

energy transformation and phosphorylation energy transformation and phosphorylation

reaction and acts as intracellular messengers)

3 sources of purines in human : diet,

degradation of endogenous nucleotide and de

novo (new) synthesis

Purine Metabolism and Uric Acid

Since purines are metabolised to uric acid, the body urate pool (and hence plasma concentration) depends on the rate of urate formation and urate excretion

Urate is excreted by kidney ( of the total) and alimentary canal

Urate secreted into alimentary canal is metabolised to CO2 and ammonia by bacterial action (uricolysis)

Urate Handling by the Kidney

It is filtered at the glomeruli and almost totally reabsorbed in the proximal convoluted tubules

Distally, both secretion and reabsorption Distally, both secretion and reabsorption occur

Normal urate clearance is about 10% of the filtered load

Urate excretion increases if the filtered load is increased

Urate Handling by the Kidney

In chronic renal failure, the plasma

concentration rises only when the glomerular

filtration rate falls below about 20 ml/min

Dietary purines makes up about 30% of Dietary purines makes up about 30% of

excreted urate

The introduction of a purine-free diet typically

reduces plasma urate concentration by only

10 - 20%

Metabolic Pathways of Uric Acid Synthesis

De novo synthesis leads to the formation of

inosine monophosphate (IMP), which can be

converted to the nucleotides adenosine

monophosphate (AMP) and guanosine monophosphate (AMP) and guanosine

monophosphate

Nucleotide degradation involves formation of

respective nucleosides (inosine, adenosine

and guanosine), these are then metabolised to

purines

Metabolic Pathways of Uric Acid Synthesis

Purine derived from IMP is hypoxanthine,

which is then converted by the enzyme

xanthine oxidase first to xanthine and then to

uric aciduric acid

Guanine can be metabolised to xanthine (and

then to uric acid) directly but adenine cannot

Metabolic Pathways of Uric Acid Synthesis

However, AMP can be converted to IMP by the

enzyme AMP deaminase, and at the

nucleoside level, adenosine can be converted

to inosineto inosine

Thus, surplus GMP and AMP can be converted

to uric acid and excreted

Plasma Urate Concentration

Plasma urate concentration are generally

higher in men than in women

Marked increase occur at puberty in males

(there is lesser increase in females at this (there is lesser increase in females at this

time) and peri-menopausal (before

menopause) women

Gout

Acute gout = severe joint

pain of rapid onset

associated with swelling and

rednessredness

Risk of gout increases with

increasing plasma urate

concentration

At any particular urate

concentration, the risk is

similar in males and females

Gout

Gout can be precipitated by a sudden change

(either increase or decrease) in urate

concentration

When urate concentration has fallen rapidly in When urate concentration has fallen rapidly in

a hyperuricaemic individual, the plasma urate

concentration may not be elevated when the

patient presents with gout

Classification of Gout

Gout customarily defined as:

1. Primary (idiopathic, of unknown cause)

2. Secondary (when a condition known to cause

hyperuricaemia is present)hyperuricaemia is present)

1. Primary Gout

Characterised by recurrent attacks of

monoarticular arthritis

It is likely that supersaturation of urate (>0.42

mmol/L) occur in patients with mmol/L) occur in patients with

hyperuricaemia

This cause crystals to accumulate in

connective tissues over a long period prior to

the development of symptoms

1. Primary Gout

Most patients have impaired renal fractional

excretion of urate (an inappropriately low

urinary urate output if raised plasma urate is

taken into consideration)taken into consideration)

Patients with primary gout often show

deposition of urate as tophi (deposit of urates

in the skin and tissue around a joint) in soft

tissue

Some develop renal stones (from uric acid)

1. Primary Gout

A genetic component to primary gout appears

to be present but the precise genetic basis in

unclear

Diagnosis of Primary Gout

Diagnosis is often made clinically on the basis of:

Distribution of the joint involvement

A past history of similar episodes

Presence of a raised plasma urate Presence of a raised plasma urate

Note that not all cases are typical clinically and

must remember that:

A high plasma urate makes the diagnosis probable

but not certain

A small minority of patients with gout have a normal

plasma urate at the time of attack

Diagnosis of Primary Gout

For definitive diagnosis of gout, it may be

necessary to aspirate joint fluid during an

acute attack

The finding of needle-shaped urate crystals The finding of needle-shaped urate crystals

when examined microscopically establishes

the diagnosis

Pathogenesis of Gout

Acute symptoms of gout - due to trauma or

local metabolic changes

This cause crystals of monosodium urate to

shed into the joints cavityshed into the joints cavity

The crystals are phagocytosed by leukocytes

and macrophages - cause damage to

membranes within the leukocytes

Pathogenesis of Gout

Lysosomal contents and other mediators of the

acute inflammatory response (cytokines,

prostaglandins, free radicals, etc.) are released,

causing both systemic and local acute formation

of goutof gout

The solubility of monosodium urate declines

rapidly with decreasing temperature and this

may explain the tendency of more peripheral

joints (hands, feet and knees) to be affected

Reason: those joints have lower intra-articular

temperatures

Pathogenesis of Gout

The progress of gout may be over many years

Patients may have asymptomatic

hyperuricaemia for many years before the first

acute attackacute attack

This may be followed by periods which are

symptom-free

Then it will lead to frequent acute attacks

If unchecked, will progress to chronic

tophaceous gout

Treatment of Primary Gout

Anti-inflammatory drugs are normally

prescribed (e.g. indomethacin)

Colchicine is also effective - has prophylactic

(preventive and protective) effect(preventive and protective) effect

Long-term treatment = reduce plasma urate

Avoid factors which increases plasma urate

(e.g. high-protein diet, alcohol and certain

drugs)

Treatment of Primary Gout

Weight reduction, uricosuric (promoting the

excretion of uric acid in the urine) drugs and

inhibitors of urate synthesis may be required

Because acute changes in plasma urate Because acute changes in plasma urate

(increase or decrease) can increase attacks of

gout, drugs mentioned above should be

avoided within several weeks of an acute

attack

2. Secondary Gout

Hyperuricaemia may occur as a complication

of several disorders, all of which affect either

urate production or excretion, or both

These conditions, though commonly cause These conditions, though commonly cause

hyperuricaemia, are only uncommonly

associated with formation of gout in joints

They include:

Overproduction

Defective elimination of urate

Overproduction of Urate

Myeloproliferative

Polycythaemia rubra vera is the most common of

these disorders, may be associated with signs of

goutgout

This is due to increased turnover of red cell

precursors causing hyperuricaemia

Overproduction of Urate

Cytotoxic Drug Therapy

Increased rates of cell turnover cause

hyperuricaemia

Renal failure may occur due to deposition of urate Renal failure may occur due to deposition of urate

crystals in the collecting ducts and ureters

Maintenance of a high fluid intake and prophylaxis

with inhibitors of urate synthesis can prevent this

Overproduction of Urate

Psoriasis

Hyperuricaemia is thought to be due to an

increased rate of cell turnover in the skin

Hypercatabolic states and starvation Hypercatabolic states and starvation

There may be both an increased rate of cell

destruction and impaired urate excretion due to

an associated acidosis (a blood condition in which

the bicarbonate concentration is below normal)

Defective Elimination of Urate

Chronic Renal Disease

Plasma urate rises in uraemia due to reduced GFR,

but clinical gout is very unusual

Diuretic Therapy Diuretic Therapy

Most effective diuretics (increasing the volume of

the urine excreted) cause hyperuricaemia by

reducing distal tubular secretion of urate

Defective Elimination of Urate

Inherited metabolic disorders

Those that are associated with lactic acidosis,

often cause hyperuricaemia

Hypertension and ischaemic (local deficiency Hypertension and ischaemic (local deficiency

of blood supply produced by vasoconstriction

or local obstacles to the arterial flow) heart

disease

Associated with hyperuricaemia for a variety of

reasons, e.g. obesity and drug treatment

Assay Method Uric Acid -

Colourimetric Method

Uric acid is converted by uricase to allantoin

and hydrogen peroxide

Under the catalytic influence of peroxidase, Under the catalytic influence of peroxidase,

hydrogen peroxide oxidizes 3,5-dichloro-2-

hydroxybenzenesulfonic acid and 4-

aminophenazone to form a red-violet

quinoneimine compound

Principle

Uric acid + O2 + 2H2O uricase Allantoin + CO2 + H2O2

2H O + 3,5-dichloro-2-hydroxybenzenesulfonic acid 2H2O2+ 3,5-dichloro-2-hydroxybenzenesulfonic acid

+ 4-aminophenazone peroxidase N-(4-antipyryl)-3-

chloro-5-sulfonate-p-benzo-quinoneimine

Samples for Uric Acid Test

It is best to use human serum for this test

Besides, other samples that can be used

includes:

Heparinised plasma Heparinised plasma

EDTA-plasma

Urine

When urine is used, it needs to be diluted

1+10 with distilled water

Calculation of Uric Acid Concentration

Serum or plasma

Uric acid conc. = x conc. of standardA sampleAstandard

Urine

Uric acid conc. = x conc. of standard x 11

Astandard

A sampleAstandard

Interference

Haemoglobin up to 100 mg/dL and bilirubin

up to 20 mg/dL DO NOT affect the

determination by using this method

Normal Values

For serum:

mol/L mg/mL

Men 202 - 416 3.4 - 7.0

Women 142 - 339 2.4 - 5.7

Normal Values

FOR URINE:

24-hour urine sample must be used

Reference ranges - same for both gender Reference ranges - same for both gender

1.5 - 4.5 mmol / 24-hr

250 - 750 mg / 24-hr