2. CREATININE UREA AND URIC ACID 2_2.pptx

Creatine measurements and understanding is one on the best way in estimating renal function

1. CREATININE, UREA AND URIC ACID t [email protected]

2. INTRODUCTION Creatinine is the cyclic anhydride of creatine that is produced as the final product of decomposition of phosphocreatine It is excreted in the urine, measurements of plasma creatinine and its renal clearance are used as diagnostic indicators of kidney function

3. BIOCHEMISTRY AND PHYSIOLOGY Creatine is synthesized in the kidneys, liver and pancreas by two enzymatically mediated reactions In the 1 st transamidation of arginine and glycine forms guanidinoacetic acid In the second reaction, methylation of guanidinoacetic acid occurs with S – adenosylmethionine as the methyl donor. Creatinine is then transported in the blood to other organ, such as muscle and brain, where it is phosphrylated to phosphocreatine, a high – energy compound

4. Cont... Interconversion of phosphocreatinine and creatine is a particular feature of the metabolic processes of muscle contraction A proportion of the free creatine in muscle (thought to be between 1% and 2% day ) spontaneously and irreversibly converts to its anhydride waste product - creatinine The amount of creatinine produced each day is relatively constant and is related to the muscle mass

5. Cont... In the health person, the concentration of creatinine in the bloodstream is relatively constant. Also diet intake especially meat influence the value in the bloodstream Creatinine is present in all body fluids and secretions, and is freely filtered by the glomerulus It is not reabsorbed to any great extent by the renal tubules, there is a small but significant tubular secretion

6. Cont... Creatinine production also decreased as the circulating level of creatinine increases, and several mechanism for this have been proposed including, feedback inhibition of production of creatinine, reconversion of creatinine to creatine and conversion to other metabolites Each day 1 – 2% of muscle creatine is converted to creatinine Men tend to have higher levels of creatinine than women because, in general they have a greater mass of skeletal muscle

7. CLINICAL SIGNIFICANCE Creatinine is produced endogenously and released into body fluids at a constant rate and its plasma concentration is maintained within narrow limits predominantly by glomerular filtration Both plasma creatinine concentration and its renal clearance (creatinine clearance) have been used as markers of the glomerular filtration rate (GFR )

8. Cont.... Measuring serum creatinine is a simple test, and it is the most commonly used indicator of renal function A rise in blood creatinine level is observed only with marked damage to functioning nephrons Therefore testing creatinine level is unsuitable for detecting early stage of kidney disease A better estimation of kidney function is given by calculating the estimated glomerular filtration rate

9. Cont... Estimated GFR can be calculated accurately using serum creatinine concentration and some other variables like sex, age, weight, race etc without a 24 – hrs urine collection The typical human reference ranges for serum creatinine are 45- 90micromol/l (0.5 – 1.0 mg/dl) for women and 60 – 90micromol/l (0.7 – 1.2 mg/dl ) for men A patient with a greater muscle mass will have a higher creatinine level

10. Specimen type Serum is the specimen of choice for measurement of creatinine level Store samples for no longer than 8 hours at room temperature (25-35 C) and 7 days at 2-8 C and above 7 days it should be freezed For a longer duration, store in the freezer

11. Jaffe reaction Jaffe reaction is the common analytic technique employed for measurement of creatinine levels Buffered kinetic jaffe reaction without deproteinization. Compensated for serum/plasma in alkaline solution creatinine reacts with picric acid to form a yellow – red adduct Creatinine + picric acid forms yellow – red complex under the aid of enzyme hexokinase

12. UREA Catabolism of protein and amino acids results in the formation of urea, which is predominantly cleared from the body by the kidney Protein is cleavage into amino acids by proteolysis principally enzymatic Then amino acids are converted into ammonia under transamination and oxidative deamination Finally ammonia is converted to urea under enzymatic synthesis in the urea cycle

13. Cont... Urea (CO[NH2]2) is the major nitrogen – containing metabolic product of protein catabolism in humans, accounting for more than 75% of the non protein nitrogen eventually excreted The biosynthesis of urea from amino acids – nitrogen derived ammonia is carried out exclusively by hepatic enzymes of the urea cycle During the process of protein catabolism, amino acid nitrogen is converted to urea in the liver by the action of the so called urea cycle enzymes

14. Cont... More than 90% of the urea is excreted through the kidneys , with losses through the gastrointestinal tract and skin accounting for the most of the remaining minor fraction Kidney diseases is associated with accumulation of urea in the blood An increase in the plasma urea concentration characterizes the uremic (azotemic) state Urea is neither actively reabsorbed nor secreted by the tubules but is filtered freely by the glomeruli

15. Cont... In the normal kidney, 40% to 70% of the highly diffusible urea moves passively out of the renal tubule and into the interstitium, ultimately to reenter plasma The back diffusion of the urea is also dependent on the urine flow rate , with less entering the interstitium in highly – flow states (e.g pregnancy) and vice versa

16. CLINICAL SIGNIFICANCE Measurement of blood and plasma urea has been used for many years as an indicator of kidney function But currently creatinine measurement is accepted to provides better information than urea Plasma and urinary urea measurement, however, may still provide useful clinical information in particular circumstances, and the measurement of urea in dialysis fluids is widely used in assessing the adequacy of the renal replacement therapy

17. Cont... A number of extrarenal factors influenced the circulating urea concentration, limiting its value as a test of kidney function for example, plasma urea concentration is increased by High protein diet Increased protein catabolism Reabsorption of blood proteins after gastrointestinal haemorrhage Treatment with cortisol or its synthetic analogues

18. Cont... Dehydration Decreased perfusion of the kidneys e.g heart failure Urea clearance is an inferior indicator of GFR, as its production is dependent on several nonrenal factors, including diet and the activity of the urea cycle enzymes A high – protein diet causes significant increases in urinary urea excretion Normal ranges of urea in plasma is 0.0 – 8.0 mmol/l

19. Specimen types, collection and storage Serum is the specimen of choice . Store samples for no longer than 8 hours at room temperature (25-35 C) and 7 days at 2-8 C. For a longer duration, store in the freezer. If the samples show evidence of bacterial contamination, do not use these for urea estimation. Plasma could also be used for urea estimation

20. PRINCIPLE FOR UREA MEASUREMENTS Enzymatic methods are used mostly and frequently in clinical laboratories Urease Urea + 2 H 2 O 2NH4 + + CO3 2- GLDH 2NH4 + + 2 – Oxoglutarate Glutamate + H 2 O The reaction takes places under the oxidation reaction of NADH to NAD. GLDH means Glutamate dehydrogenase

21. URIC ACID Uric acid is nitrogenous compound (C5H4N4O3)2,8 – trihydroxypurine) present as the principal nitrogenous component of the excrement of reptiles and birds It found in small amounts in mammalian urine and its salts occur in the joints It forms ions and salts known as urates and acids water such as ammonia acid waste Uric acid is a product of the metabolic breakdown of purine nucleotides

22. Cont... Uric acid is produced by the breakdown of purines, which are essential components of nucleic acids (DNA and RNA) as well as other important biomolecules such as ATP, cyclic AMP and NADH.

23. Cont... High concentration of uric acid can lead to gout The chemical is associated with other medical conditions including diabetes mellitus and the formation of ammonium acid urate kidney stones In general, water solubility of uric acid and its alkali metal and alkaline metal and earth salts is rather low All of these salts exhibit greater solubility in hot water and cold allowing for easy recrystallization This low solubility is significant for the solubility of Gout

24. Cont... The normal value of uric acid in human plasma: In males: 3.5 - 7 mg/dl. In females: 3.0 - 6 mg/dl. The enzyme xanthine oxidase makes uric acid from xanthine and hypoxanthine which in turn are produced from other purines

25. BIOCHEMISTRY AND PHYSIOLOGY In humans, uric acid is the major product of the catabolism of purine nucleosides adenosine and guanosines Purines from catabolism of dietary nucleic acid are converted to uric acid directly The bulk of purines excreted as uric acid arise from degradation of endogenous nucleic acids The daily synthesis of uric acid is approximately 400mg Dietary sources contribute another 300mg

26. Cont.... Patients with gouty arthritis and tissue deposition of urate may have urate pools as large of 18,000 to 30,000mg Overproduction of uric acid may result from increased synthesis of purine precursors Renal handling of uric acid is complex and involves four sequential steps which are Glomerular filtration of virtually all the uric acid in capillary plasma entering the glomerulus

27. Cont... Reabsorption in the proximal convoluted tubule of about 98% to 100% of filtered uric acid Subsequent secretion of uric acid into lumen in the distal portion of the proximal tubule Further reabsorption in the distal tubule The net urinary excretion of uric acid is 6% to 12% of the amount filtered

28. CLINICAL SIGNIFICANCE More than 20 inherited disorders of purine metabolism giving rise to both hyperuricemias and hypouricemias Most are very rare and the diagnosis requires support from a specialist purine laboratory Symptoms that should raise suspicion include Kidney failure or stones in a child or young adult Gravel in an infants diaper Unexplained neurological problems in an infant, child, or adolescent

29. Cont... Gout presenting in a man or women less than 30 years old Disease states with increased plasma uric acid Gout Increased catabolism of nucleic acids And renal disease In Gout increased serum levels of uric acid lead to formation of monosodium urate crystals around the joints. Uric acid test is useful to assess for gout and to monitor patients with renal failure

30. Cont... To monitor if uric acid levels are too high after chemotherapy or radiation. Hypouricemia is seldom observed and associated with rare hereditary metabolic disorders.(e.g. Wilson’s disease , Fanconi’s Syndrome)

31. HYPERURICEMIA Hyperuricemia is most commonly defined by plasma uric acid concentrations greater than 7mg/dl (0.4mmo/l) in men or greater than 6mg/dl (0.36mmol/l) in women High level of uric acid can be caused by High intake of purine rich diet Impaired excretion of uric acid in the kidney Chemotherapy- related side effects(due to breakdown of tumour cells and release of purines

32. Cont... Stress or excessive exercise Toxemia of pregnancy The association of high serum uric acid with insulin resistance . Study showed that high serum uric acid is associated with higher risk of type 2 diabetes

33. Hyperuricemia may lead to Formation of kidney stones: Most of uric acid is removed by the kidneys and disposed of in the urine. In Hyperuricemia uric acid crystals precipitate in the kidney and may block filtering tubules leading to renal failure. Uric acid crystals in a urine sample Sodium urate/ kidney stones

34. specimen Serum or plasma may be used Stability in serum / plasma: 6 months at -20°C 7 days at 4-8°C 3 days at 20-25°C

35. ANALYTIC METHODS—URIC ACID Chemical Method (old) : Phosphotungstic Acid, read the absorbance( Ab ) at 700nm (UV). blue Colored product Enzymatic Method: is More specific By using Couple reaction of Uricase and Peroxidase. Pink solution (Ab at 500nm spectrophotometric ) Pink color solution.

36. PRINCIPLE OF ESTIMATION Uricase Uric acid + O 2 +H 2 O Allantoin + H 2 O 2 Peroxidase H 2 O 2 + 4AA + ADPS Quinoneimime + 4 H 2 O The method used is a colorimetric enzymatic method . Uric acid is oxidized by Uricase to allantoin with the formation of Hydrogen peroxide. Hydrogen peroxide can oxidize a mixture of ADPS and 4-aminoantipyrine (4AA) to form a quinoneamine that absorbs maximally at 550 nm.

37. GOUT Gout occurs when monosodium urate precipitates from saturated body fluids The deposits of urate are responsible for the clinical signs and symptoms Gouty arthritis may be associated with urate crystals in joint fluid and with deposits of crystals (tophi) in tissue surrounding the joint The deposits may occur in other soft tissue

38. Cont... Wherever they occur they elicit an intense inflammatory response consisting of polymorphonuclear leukocytes and macrophages The big toe (first metatarsophalangeal) joint is the classic site for gout Gout is the condition characterized by the occasional attacks and long period of remission

39. Cont... It is important to appreciate that the plasma uric acid concentration is often normal during an acute attack Kidney disease associated with Hyperuricemia may take one or more of several forms Gouty nephropathy with urate deposition in the renal parenchyma Acute intratubular deposition of urate crystals Urate nephrolithiasis

40. Cont... Gout is classified into two groups Primary gout Secondary gout Primary gout is associated with essential hyperuricemia, which has polygenic basis In greater than 99% of cases, the cause is uncertain but is probably due to a combination of : Metabolic overproduction of purines Decreased renal excretion (80% of the patients show decreased renal tubular secretion of uric acid)

41. Cont... Increased dietary intake Very rarely, primary gout is attributable to inherited defects of enzymes in the pathways of purine metabolism like hypoxanthine – guanine phosphoribosyl transferase (HGPRT).

42. Cont... Secondary gout is a result of hyperuricemia attributable to several identifiable causes Renal retention of uric acid may occur in acute or chronic kidney disease of any type or as a consequences of administration of drugs and some diuretics Increased nucleic acid turnover and a consequent increased in catabolism of purines may be encountered in rapid proliferation of tumor cells and in massive destruction of tumor cells on therapy with certain chemotherapeutic agents

43. Cont... Management of an acute attack of gout generally involves the use of non steroidal ant inflammatory drugs (NSAIDs) Patients should be advised to avoid Foods that have a high purine content (e.g liver, kidney, red meat and sardines) Drugs that affect urate excretion (thiazide diuretics and salicylates)

44. PREECLAMPTIC TOXEMIA This condition is associated with increasing plasma uric acid concentration, probably caused by uretoplacental tissue breakdown and decreased kidney perfusion Plasma urate measurement has been used as an indicator of the severity of preeclampsia Concentration in excess of 6 mg/dl (0.36mmo/l) at 32 weeks of gestation have been noted to be associated with a high perinatal mortality rate

45. HYPOURICEMIA Is the condition where plasma urate concentrations are less than 2.0mg/ dl (0.12 mmol/l) It is much less common then hyperuricemia It may be secondary due to the following Severe hepatocellular diseases with reduced purine synthesis or xanthine oxidase activity Defective renal tubular reabsorption of uric acid. Defective may be due to congenital, as in general fanconi syndrome or acquired

46. Cont... Very rarely, hypouricemia may a result of inherited metabolic defect Xanthine oxidase is an Fe – mo enzymes, so people with fe deficiency ( most common cause of anaemia in young women) may experience hypouricemia Oestrogen increases half life of copper, women with very high oestrogen levels and intense blood loss during menstruation are likely to have a high cu/fe and present with hypouricemia