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

Clinical diagnostic biochemistry - 12

Dr. Maha Al-Sedik2015

CLS 334

Renal function -2

LABORATORY TESTS AIDING

IN THE EVALUATION OF

KIDNEY FUNCTION

Glomerular functionGlomerular filtration rate

( clearance tests )

Non protein nitrogen ( NPN )

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

Definition of glomerular filtration rate:

The amount of plasma pass through glomeruli

per unit time.

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).

VIP

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

volume / 24 hoursHow to measure V per minute ? : 24 X 60

Definition of glomerular filtration rate:

The amount of plasma pass through glomeruli

per unit time.

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%

greater 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

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.

• Urine creatinine: 100 mg / dl

• Serum creatinine: 1.4 mg / dl

• Urine volume: 1000 ml

• Urine collection time : 24 hours

Calculate Creatinine clearance:


• Serum creatinine: 2 mg / dl





• Serum creatinine: 1 mg / dl





• Serum creatinine: 1.5 mg / dl



Creatinine Clearance = [(140 - age) x BW ] / (Serum cr x 72)


Non protein nitrogen

( NPN )

Non protein nitrogen

Creatinine

Urea

Uric acid

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

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.

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.

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,

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.

Creatininase

Creatinine + H2O ------------------ Creatine

Creatinase

Creatine + H2O ------------------- Sarcosine + urea

Sarcosine oxidase

Sarcosine +O 2 + H2O --------------------- Glycine + formaldehyde + H2O2

= 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)

Normal BUN range is 6 - 20 mg/dL.

Normal urea : 10 – 50 mg / dl

Protein

Proteolysis, principally enzymatic

Amino acids

Transamination and oxidative deamination

Ammonia

Enzymatic synthesis in the “urea cycle”

Urea

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.

Plasma concentrations also tend to be slightly

higher in males than females.

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.


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.

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.

Urease

Urea + H2O ---------------------2 NH3 + CO2

2 NH4 + 2- ketoglutarate + 2 NADH --------L- Glutamate + 2 NAD + 2H2O

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

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.

• 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.


Common techniques for measuring uric acid in body fluids include:

(I) phosphotungstic acid (PTA).

(2) uricase.

(3) HPLC-based methods.

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.

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.

Uric acid + O2 + 2 H2O --- Allantoin + H2O2

H2O2

4- AA + DCPS ----------- Quinoneimine + 4 H2O POD

AA : aminoantipyrine

DCPS: dichlorophenol sulphonate

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.

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

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

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