1. Clinical diagnostic biochemistry - 12 Dr. Maha Al-Sedik 2015 CLS 334

2. Renal function -2

4. Glomerular function Glomerular filtration rate ( clearance tests ) Non protein nitrogen ( NPN )

5. U X V C = P Clearance is defined as the quantity of blood or plasma completely cleared of a substance per unit of time. Clearance tests

6. Definition of glomerular filtration rate: The amount of plasma pass through glomeruli per unit time.

7.  Some substances are filtered, enter tubules, but are completely reabsorbed, so they did not reach the final urine, so its clearance is zero (e.g. Glucose).  Some substances when filtered enter the tubules are not reabsorbed and so 100% in urine, So its clearance = GFR (inulin clearance = gold standard for GFR).

8.  Creatinine clearance also used for determination of GFR.(but small amount of creatinine is reabsorbed, so its cclearance <GFR.  Some substances are filtered, enter tubules, and more of the substance is secreted enters the tubules by excretion. Clearance>GFR

10. volume / 24 hours How to measure V per minute ? : 24 X 60

11. Definition of glomerular filtration rate: The amount of plasma pass through glomeruli per unit time.

12.  The most frequently used clearance test is based on the measurement of creatinine.  Small quantity of creatinine is reabsorbed by the tubules, So creatinine clearance is approximately 7% less than inulin clearance.  An estimate of the GFR can be calculated from the creatinine content of a 24-hour urine collection, and the plasma concentration within this period. Creatinine clearance and clinical utility

13.  The volume of urine is measured, urine flow rate is calculated (ml/min) and the assay for creatinine is performed on plasma and urine to obtain the concentration in mg per dl or per ml.  Creatinine clearance in adults is normally about of 120 ml/min,  The accurate measurement of creatinine clearance is difficult, especially in outpatients, since it is necessary to obtain a complete and accurately timed sample of urine.

14. Urine creatinine: 100 mg / dl Serum creatinine: 1.4 mg / dl Urine volume: 1000 ml Urine collection time : 24 hours Calculate Creatinine clearance:

15. Urine creatinine: 110 mg / dl Serum creatinine: 2 mg / dl Urine volume: 1500 ml Urine collection time : 24 hours  Calculate Creatinine clearance:

16. Urine creatinine: 110 mg / dl Serum creatinine: 1 mg / dl Urine volume: 1500 ml Urine collection time : 24 hours  Calculate Creatinine clearance:

17. Urine creatinine: 120 mg / dl Serum creatinine: 1.5 mg / dl Urine volume: 2000 ml Urine collection time : 24 hours Creatinine Clearance = [(140 - age) x BW ] / (Serum cr x 72)  Calculate Creatinine clearance:

18. Non protein nitrogen ( NPN )

19. Non protein nitrogen Creatinine Urea Uric acid

20.  Creatine is formed in the liver from arginine, glycine and methionine.  It is transported to the muscles and converted to phosphocreatine to be used as source of energy.  Creatine phosphate loses phosphoric acid to form creatine.  Creatine loses H2O and form creatinine.  Normal range: o Male 0.6-1.2 mg/dL. o Female 0.5-1.0 mg/dL. Creatinine

21. 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. ☺Consequently, creatinine renal clearance ("creatinine clearance") have been used as markers of the glomerular filtration rate (GFR). ☺Creatinine level can be affected by: Muscle mass. Renal functions.

22. Analytical Methodology: Plasma creatinine is commonly measured using either chemical or enzymatic methods. I. Chemical methodst: Jaffe Reaction Most chemical methods for measuring creatinine are based on its reaction with alkaline picrate. As first described by Jaffe in 1886, creatinine reacts with picrate ion in an alkaline medium to yield an orange-red complex.

23. Disadvantage of Jaffe Reaction:  Lack of specificity of the test for creatinine.  Many compounds produce Jaffee like reaction such as : (1)ascorbic acid, (2)pyruvate., (3) cephalosporins, (4) ketone bodies (5) guanidine,

24. II. Enzymatic Methods: Creatininase and Creatinase: An alternative approach has been the use of creatininase that yields sarcosine and urea, the former being measured with further enzyme mediated steps using sarcosine oxidase. This produces (1) glycine, (2) formaldehyde, and (3) hydrogen peroxide with the latter being detected and measured with a variety of methods.

25. Creatininase Creatinine + H 2 O ------------------  Creatine Creatinase Creatine + H 2 O -------------------  Sarcosine + urea Sarcosine oxidase Sarcosine +O 2 + H 2 O ---------------------  Glycine + formaldehyde + H 2 O 2

26.  = BUN (blood urea nitrogen)  Catabolism of proteins and amino acids results in the formation of ammonia then in the liver urea is formed from ammonia, which is predominantly cleared from the body by the kidneys.  The real urea concentration is BUN x 2.14 because 60 g of urea contains 28 g of nitrogen.  BUN is a sensitive indicator of renal disease.  Urea level depends upon : a. diet b. liver function c. renal function. Plasma urea (BUN)

27.  Normal BUN range is 6 - 20 mg/dL.  Normal urea : 10 – 50 mg / dl

28. Protein Proteolysis, principally enzymatic Amino acids Transamination and oxidative deamination Ammonia Enzymatic synthesis in the “urea cycle” Urea

29.  Urea is the major nitrogen containing metabolic product of protein catabolism in humans.  Its elimination in the urine represents the major route for nitrogen excretion.  More than 90% of urea is excreted through the kidneys, with losses through the GIT and skin.  Urea is filtered freely by the glomeruli then part of it is reabsorped through tubule.  Its clearance is less than GFR.

30.  Plasma concentrations also tend to be slightly higher in males than females.

31. Clinical Significance: Measurement of blood and plasma urea has been used for many years as an indicator of kidney function. However, it is now generally accepted that creatinine measurement provides better information in this respect. Causes of elevated urea ( related to kidney ): Prerenal: renal hypo perfusion. Renal: acute tubular necrosis. Postrenal: obstruction of urinary flow.

32. Analytical Methodology: Both chemical and enzymatic methods are used to quantify urea in body fluids. I. Chemical Methods: Most chemical methods for urea are based on the Fearon reaction in which diacetyl condenses with urea to form the chromogen diazine, which absorbs strongly at 540 nm.

33. II. Enzymatic Methods: Enzymatic methods for the measurement of urea are based on hydrolysis of urea with urease to generate ammonia, which is then quantified. The most clinically used kinetic method couples the urease reaction with L-glutamate dehydrogenase and measure the rate of disappearance of reduced NADH at 340 nm.

34. Urease Urea + H 2 O ---------------------  2 NH 3 + CO 2 2 NH 4 + 2- ketoglutarate + 2 NADH --------  L- Glutamate + 2 NAD + 2H 2 O

35.  In human, uric acid is the major product of the catabolism of the purine nucleosides, adenosine and guanosine.  Purines are derived from catabolism of dietary nucleic acid (nucleated cells, like meat) and from degradation of endogenous nucleic acids. Uric acid

36.  Renal handling of uric acid is complex and involves four sequential steps:  Glomerular filtration of virtually all the uric acid in capillary plasma entering the glomerulus.  Reabsorption in the proximal convoluted tubule of about 98 to 100% of filtered uric acid.  Subsequent secretion of uric acid into the lumen of the distal tubule.  Further reabsorption in the collecting tubule.

37. Hyperuricemia is defined by serum or plasma uric acid concentrations higher than 7.0 mg/dl (0.42mmol/L) in men or greater than 6.0 mg/dl (0.36mmol/L) in women.

38. Analytical Methodology: Common techniques for measuring uric acid in body fluids include: (I) phosphotungstic acid (PTA). (2) uricase. (3) HPLC-based methods.

39. Phosphotungstic Acid Methods:  These methods are based on the development of a blue reaction chromogen (tungsten blue) as PTA is reduced by urate in an alkaline medium.  The absorbance of the chromogen in the reaction mixture is measured at wavelengths of 650 to 700 nm.  PTA methods are subject to many interferences, and efforts to modify them have had little success in improving their specificity.

40. Uricase Methods:  Uricase methods are more specific than PTA approaches.  Uricase is used either as a single step or as the initial step to oxidize uric acid.  Uricase acts on uric acid to produce allantoin, hydrogen peroxide, and carbon dioxide.  The decrease in absorbance as urate is converted and is measured with a spectrophotometaetr 293 nm.

41. Uric acid + O 2 + 2 H 2 O ---  Allantoin + H 2 O 2 H2O2 4- AA + DCPS -----------  Quinoneimine + 4 H 2 O POD AA : aminoantipyrine DCPS: dichlorophenol sulphonate

42. HPLC Methods:  HPLC methods using ion-exchange or reversed-phase columns have been used to separate and quantify uric acid.  HPLC methods are specific and fast; mobile phases are simple; and the retention time for uric acid is less than 6 minutes.  Because of these multiple attributes, HPLC has been used to develop reference methods for measuring uric acid.

43. Reference: Burtis and Ashwood Saunders, Teitz fundamentals of Clinical Chemistry, 4th edition, 2000.