NPNs & Kidney Function Tests

A. NON-PROTEIN NITROGEN COMPOUNDS

I. BLOOD UREA NITROGEN
UREA
 NPN of highest concentration in the blood
 major excretory product of protein metabolism
 Formed in the liver from the amino groups and ammonia
 Readily filtered by the glomerulus and mostly excreted BUT SOME ARE REABSORBED BY PASSIVE
DIFFUSION IN THE RENAL TUBULES.
 Concentration of Urea is determined by Renal Function and perfusion, the protein content of the diet, and the
rate of protein catabolism

ANALYTICAL METHODS
I. COUMPLED ENZYMATIC METHOD (MOST COMMON)
 usually starts with conversion of Urea to Ammonium ion by enzyme Urease

a. Glutamate Dehydrogenase Method

 Ammonium ion is reacted with oxoglutarate with the cofactor Reduced Nicotinamide Adenine
Dinucleotide (NADH)
 Measurement is based on the disappearance of NADH at 340nm spectrophotometrically.

b. pH Method
 ammonium from the Urease reaction is reacted with a pH indicator.
 incorporated into instruments using liquid reagents, multilayer film format, reagent strips.

c. Conductimetric Method
 increase in conductivity as ammonium ions are produced
 ammonia contamination is not a problem unlike other methods

II. CHEMICAL METHOD (DIACETYLMONIXIME METHOD)

 based on the Fearon Reaction (diazine is formed and absorbs 540 nm)

III. ISOTOPE DILUTION MASS SPECTROPHOTOMETRY (REFERENCE METHOD)

 detection of characteristic fragments following ionization; quantified by using isotope labelled
compound

SPECIMEN REQUIREMENTS AND INTERFERRING SUBSTANCES

 Plasma, Serum or Urine can be used as a sample
 Ammonium ions and high concentrations of Sodium citrate and fluoride should be avoided (Citrate and
Flouride inhibit Urease).
 fasting is not required non-hemolyzed and freshly collected samples needed.
BUN Reference Interval:
Plasma or Serum: 6-20 mg/dl
24H urine 12-20 g/day
 CLINICAL APPLICATION
 to evaluate renal function, hydration status, nitrogen balance, diagnosis of renal disease and to verify
adequacy of dialysis.
 Urea is usually reported as Urea Nitrogen but can be converted to Urea by multiplying by 2.14.
 Can be converted from Convention units (mg/dl) to SI units (mmol/L) by multiplying by 0.357

PATHOPHYSIOLOGY
 “AZOTEMIA” elevated concentration of NPNs in the body.
 “UREMIA” elevated concentrations of NPNs along with signs and symptoms.

3 KINDS OF AZOTEMIA
A. PRERENAL AZOTEMIA
 caused by conditions leading to reduced blood flow to the kidneys or increase in protein metabolism.
B. RENAL AZOTEMIA
 caused by problems of the kidney itself.
C. POST-RENAL AZOTEMIA
 obstruction of urine flow anywhere in the urinary tract.

 usual cause of decrease in BUN are low protein intake and severe liver disease.
 Normal BUN:Creatinine Ratio 10-20:1

II. BLOOD URIC ACID

 Product of Purine Catabolism (ADENOSINE AND GUANINE) in the liver.

 70% is excreted by Kidneys the rest in the Gastrointestinal Tract.
 Most are reabsorbed by proximal convoluted tubules and reused.
 Insoluble to plasma and can be deposited to the joints.
 Monosodium urate is the dominant form in the plasma (pH of 7).
 Concentrations of > 6.8 mg/dl, crystals may form in the tissues.
 Analysis can be used to assess inherited disorders of purine metabolism, diagnosis and treatment of gout,
diagnosis of renal stones, to detect kidney dysfunction.
ANALYSIS
Uric acid is the final breakdown product of purine metabolism.
CHEMICAL METHOD
A. Caraway Method (Most Common)
 Uric acid is coupled with Phosphotungstic acid.
 Uric acid is oxidized to allantoin while Phosphotungstic acid is reduced to tungsten blue in an alkali
solution.
 lacks specificity

B. Enzymatic Method
 has a similar 1st step: URICASE METHOD
 Uric acid is reacted with Oxygen and water to produce Allantoin, Carbon Dioxide and Hydrogen Peroxide
a. Spectrophotometric
 measures decrease of absorbance of uric acid at 293 nm.
 sensitivity is reduced by proteins; hemoglobin and xanthine are interferences.
b. Coupled Enzymatic Method
 Measures the Hydrogen Peroxide produced.
 Either uses Catalase or Peroxidase enzyme to produce a colored compound to be
analyzed spectrophotometrically.
 Commercial reagent preparations include potassium ferrocyanide and ascorbate oxidase
to counter Bilirubin and ascorbic acid per se that destroys Hydrogen Peroxide.
 ISOTOPE DILUTION MASS SPECTROPHOTOMETRY (REFERENCE METHOD)

SPECIMEN COLLECTION AND INTERFERING SUBSTANCE

 Can be measured in heparinized plasma, serum, or urine.
 Fasting is unnecessary.
 Hemolysis, Lipemia and Bilirubin can falsely decrease results.
 Salicylates and thiazides can falsely increase results.
 EDTA or Flouride inhibits Uricase thus should not be used.
 If urine is needed to be used, it should be alkalinized.
 Pathophysiology
Lesch-Nyhan Syndrome
 X-linked (Seen only in Males)
 Complete deficiency of Hypoxanthine Guanine Phosphoribosyltransferase (HGPRT) used in biosynthesis
of Purines.
 leads to inability to recycle purine bases thus leads to increase in urine and plasma uric acid.
 Increase in Blood uric acid can also be seen secondary to Glycogen Storage diseases and fructose intolerance by
replacing urates with their excess metabolites (Triglycerides and Lactate) in urinary excretion.

 Elevated Plasma Uric Acid is found in Gout, Increased Catabolism of Nucleic acids and in Renal Disease.
 Patients usually develop gout if plasma levels of Uric acid is > 6mg/dl.
 Tophi – deposits of crystalline uric acids and urates in tissues.
 Patients in chemotherapy can lead to increase of uric acid due to destruction of cells. Monitoring is needed to
avoid nephrotoxicity.
 Chronic Renal Disease may lead to increase of Blood Uric Acid due to impaired Filtration and secretion. (Blood
Uric Acid is not useful in assessing Renal Function)
 Hypouricemia is less common than Hyperuricemia. Usually due to liver diseases, defective tubular reabsorption
and chemotherapy of 6-mercaptopurine and Azathioprine (Inhibits purine synthesis)

III. Creatinine
 Creatine is synthesized primarily in the liver from arginine, glycine and methionine.
 It is transported to the muscles and converted to creatine phosphate
 Creatinine is created from creatine and creatine phosphate in muscles and excreted to the plasma at a constant
rate related to muscle mass.
 Plasma concentration is inversely related to glomerular filtration rate.
 Commonly used to assess renal filtration function.
 Creatinine Clearance is also used to assess renal function.

GLOMERULAR FILTRATION RATE (GFR)

 Volume of plasma filtered by the glomerulus per unit of time
GFR= Vp/t
 If a substance is can be measured and freely filtered by the glomerulus and neither secreted nor reabsorbed by
the tubules, Volume of plasma filtered is equivalent to its mass (M) of the substance filtered divided by its
Plasma concentration (Pc).
V=M/Pc
 Mass is equivalent to the product of Urine concentration (Uc) and Urine volume (Vu).
M= Uc x Vu
 If all parameters were collected, GFR may be computed:
 GFR = (Uc x Vu) / (Pc x t)

CLEARANCE
 Volume of plasma from which a substance is removed per unit of time.
 Creatinine Clearance (CrCl):
CrCl = (Ucr x Vu) / (Pcr x t)
 reported in mL/minute
 Creatinine Clearance provides a reasonable estimate of GFR

Methods
JAFFE REACTION (1886)
 creatinine reacts with picric acid in an alkali solution to produce a red-orange chromogen.
 reaction is non-specific and subject to positive interference (acetoacetate, acetone, ascorbate, glucose &
pyruvate)
 was partially resolved by using any of the ff:
a. Fuller’s Earth (Aluminum Magnesium Silicate)
b. Lloyd’s Reagent (Sodium Aluminum Silicate)

A. KINETIC JAFFE
 Serum is mixed with alkaline picrate and the RATE OF CHANGE IN ABSORBANCE IS MEASURED.
 subject to interference by alpha-keto acids and cephalosporins.
 negative bias can be caused by hemoglobin and bilirubin.
 rapid, inexpensive and easy to perform.

COUPLED ENZYMATIC METHODS

 uses creatinase, creatininase, sarcosine oxidase
 for dry slide analyzer (CREATININASE-HYDROGEN PEROXIDE).

ISOTOPE DILUTION MASS SPECTROPHOTOMETRY (REFERENCE METHOD)

SPECIMEN REQUIREMENTS, INTERFERING SUBSTANCES & SOURCES OF ERROR

 Can be measured in plasma, serum or urine.
 Hemolyzed or icteric specimen should be avoided (Particularly if Jaffe method is to be used)
 Lipemic sample can lead to erroneous results in some methods
 Fasting is not required.
 Ascorbate, glucose, alpha-ketoacids, and uric acid may increase creatinine concentration in Jaffe Reaction
specially in temperatures above 30C.
 Bilirubin cause negative bias in both Jaffe and enzymatic methods
 Ascorbate will interfere with peroxidase using enzymatic reaction.
 Cephalosporin taking patients has a false increase creatinine in Jaffe Rxn.
 Lidocaine increases creatinine in enzymatic and Jaffe reaction.
 Lidocaine increases creatinine in enzymatic reaction.
IV. AMMONIA
 Formed form the deamination of amino acids
 Removed from the circulation and converted to urea in the liver.
 Free ammonia is toxic but present in plasma in low concentrations.
 At normal pH, it exists in the blood and is excreted by the kidneys as an ammonium ion.
 Can be used to assess hepatic failure, Reye’s syndrome and inherited deficiencies of Urea cycle.

ANALYTICAL METHODS
CONWAY METHOD
 ammonia gas from the sample diffuses to a separate compartment of a microdiffusion chamber, and is
absorbed in a solution containing pH indicator.
 the amount of ammonia is determined by titration.
BERTHELOT METHOD
 berthelot reagent is composed of phenol and hypochlorite. Sample is reacted to the reagent and the colored
end product (Indolpehol blue) is measured via sphectrophotometry.
GLUTAMATE DEHYDROGENASE METHOD (ENZYMATIC METHOD)
 currently the most common technique
 decrease in absorbance at 340 nm of NADPH in the reaction is proportional to the ammonia concentration of
the specimen.
DRY SLIDE SPECTROPHOTOMETRY
 Ammonia is reacted with Bromophenol blue producing a colored compound that can be measured
spectrophotometrically.

SPECIMEN REQUIREMENTS AND INTERFERING SUBSTANCES

 Whole blood not used because ammonia increases rapidly due to in vitro amino acid deamination.
 Venous blood should be obtained without trauma and placed in ice immediately.
 Heparin or EDTA are the preferred anticoagulants.
 Samples should be centrifuged at 0-4C within 20 mins of collection and the plasma and serum removed.
 Hemolysis should be avoided because rbcs have 2-3 times ammonia.
 Patients should not smoke before being extracted because of possible contamination
Can Increase Ammonia concentration:
 ammonium salts, asparaginase, barbiturates, diuretics, ethanol, hyperalimentation, narcotic analgesics
Can Decrease Ammonia concentration:
 Lactobacillus acidophilus, Diphenhydramine, Lactulose, Levidopa and several antibiotics

Elevated blood sugar more than 600mg/dl can interfere with the slide method.

 Usually there is increase concentrations of Ammonia in severe Liver Disease, leading to neurotoxicity that is ften
associated in encephalopathy.
 Hyperammonemia may be due to inherited deficiency of enzymes

B. Kidney Function Tests

THREE BASIC RENAL PROCESSES:
1. GLOMERULAR FILTRATION
2. TUBULAR REABSORPTION
3. TUBULAR SECRETION
FACTORS THAT AFFECT FILTRATION
 High blood pressure in the glomerular capillaries
 Semi-permeable glomerular basement membrane
 size and charge of molecules
PROXIMAL CONVOLUTED TUBULE
 Reabsorbs almost all essential molecules that have passed through the glomerulus. (Tubular Reabsorption)
 uric acid is secreted on the distal end of the proximal tubule.
RENAL THRESHOLD – Plasma concentration above which a substance appears in the urine.
 does not exist for water because it is always transported passively through diffusion.

GLOMERULAR FILTRATION RATE (GFR)

Volume of plasma filtered by the glomerulus per unit of time
GFR= Vp/t
If a substance is can be measured and freely filtered by the glomerulus and neither secreted nor reabsorbed by the
tubules, Volume of plasma filtered is equivalent to its mass (M) of the substance filtered divided by its Plasma
concentration (Pc).
V=M/Pc
Mass is equivalent to the product of Urine concentration (Uc) and Urine volume (Vu).
M= Uc x Vu
If all parameters were collected, GFR may be computed:
GFR = (Uc x Vu) / (Pc x t)

CLEARANCE
Volume of plasma from which a substance is removed per unit of time.
Creatinine Clearance (CrCl):
CrCl = (Ucr x Vu) / (Pcr x t)
 reported in mL/minute
 Creatinine Clearance provides a reasonable estimate of GFR

-END-

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