CLINICAL CHEMISTRY

LECTURE / FIRST SEMESTER

UNIT 7: NONPROTEIN NITROGEN COMPOUNDS
Nonprotein Nitrogen Compounds  Chemical Method (Direct Method)
 have been used to monitor renal function, to assess the A. Diacetyl monoxime method
functioning capabilities of the kidneys a) Urea + DAM → Yellow Diazine Derivative
 the term NPN originated when analytic methodology
required removal of protein from sample before analysis  Enzymatic Methods (Indirect Methods)
 Deproteinization - PFF or Protein-free Filtrate A. Urease (Urea aminohydrolase)
 concentration of nitrogen-containing compounds was B. Coupled Urease/ Glutamate Dehydrogenase method
quantified spectrophotometrically by converting nitrogen to
ammonia
 Subsequent reaction with Nessler’s reagent to
produce a yellow color
 majority of these compounds arise from catabolism of
proteins & nucleic acids

 Indicator Dye - reagent strip

 Ammonium ion + pH indicator → Color change
 Conductometric method – uses electrode, measures the
rate of conductivity as ammonium ions are produced from
hydrolysis of urea
Urea  Isotope dilution mass spectrometry (IDMS) - proposed
 Highest concentration in the reference method, the sample will be needing a of a known
blood amount of stable isotope and then the concentration of urea
 Major excretory product of will be measured by mass spectrometer
protein metabolism
 Formed in the liver
 Ammonia + carbon
dioxide → carbamoyl phosphate → enters the urea
cycle → urea
 Blood urea nitrogen (BUN) - has been used to refer to
urea determination
 Urea nitrogen (Urea N) - a more appropriate term

Biochemistry *Specimen is volatilized and ionized to form fragments. Mass

 After its synthesis in the liver, urea is carried in the blood to analyzer separates fragments according to mass:charge ratio. It
the kidney is measured in the Ionization detection. Data is displayed in the
 Most of the urea in the glomerular filtrate is excreted in the read-out device.
urine.
 Small quantities of urea (<10% of the total) are Specimen Requirements
excreted through the GIT and skin.  Plasma, serum, or urine
 Factors that affect the plasma urea concentration -  Citrate and fluoride inhibit urease
protein content of the diet, the rate of protein catabolism,  Fasting sample is not required
and renal function and perfusion  Urea is susceptible to bacterial decomposition
 Timed urine samples should be refrigerated
Analytical Methods
 Traditional method – Protein-free filtrate of whole Clinical Significance
blood; based on the amount of nitrogen  Azotemia: elevated urea in the blood
 Current method – urea is reported in terms of nitrogen  Prerenal
concentration rather than urea concentration  Renal
 urea N can be converted to urea concentration by  Postrenal
multiplying by 2.14  Uremia (uremic syndrome): elevated urea in the blood
 Urea N X 2.14 = urea concentration accompanied by renal failure
 Unit of expression - mmol/L
 CLINICAL CHEMISTRY
LECTURE / FIRST SEMESTER
 Decrease Plasma Urea
 Low protein intake - urea is produced from protein
metabolism
 Severe liver disease - liver can no longer produce
urea
Azotemia
 Prerenal: a result of reduced renal blood flow
 congestive heart failure, shock, hemorrhage,
dehydration, high protein diet or high protein
catabolism
 Renal: a result of diminished glomerular filtration which
occurs in wide variety of kidney diseases. Intrinsic renal
damage
 acute and chronic renal failure, glomerular nephritis,
tubular necrosis, and other intrinsic renal disease
 Postrenal: happens due to obstruction of the urine flow
and anywhere in the urinary tract by renal calculi, tumors of
the bladder or prostate or severe infection. Urinary Tract
obstruction
Reference Intervals
Conversion Factor:
mg/dL → mmol/L
0.357
Uric Acid
 the product of catabolism of the purine nucleic acids
 most uric acid is reabsorbed in the proximal tubules and
reused
 insoluble in plasma and, at high concentrations, can be
deposited in the joints and tissue, causing painful
inflammation
 Gout - deposited in joints, susceptible to formation of
kidney stones
 Tophi (in severe cases) - deposited in tissues
Biochemistry
 purines are converted into uric acid, primarily in the liver
 98% to 100% of uric acid is reabsorbed in the proximal
convoluted tubule
 nearly all of the uric acid in plasma is present as
monosodium urate
Analytical Methods
 Chemical method: Caraway method (lacks specificity)
 Enzymatic methods
 Uricase (urate oxidase): uric acid → allantoin
 Enzymatic methods
 Coupled enzymatic method (Peroxidase): hydrogen
peroxide + indicator dye → colored compound
 Bilirubin and ascorbic acid destroys peroxide
(potassium ferricyanide and ascorbate oxidase to
minimize these interferences)
 IDMS: proposed reference method
Specimen Requirements
 Heparinized plasma, serum, or urine
 High bilirubin concentration may falsely decrease results
(peroxidase method)
 Significant hemolysis (with glutathione release) may result
in low values
 Drugs such as salicylates and thiazides increase uric acid
values
 Serum samples may be refrigerated for 3-5 days
 Urine collections must be alkaline (pH 8)
Clinical Significance
 Hyperuricemia: increased plasma uric acid concentration
 Gout / Tophi (severe cases)
 Increased metabolism of cell nuclei (chemotherapy -
when cancer cells die, they release uric acid)
 Allopurinol – inhibits xanthine oxidase, enzyme
needed in uric acid synthesis
 Hemolytic or megaloblastic anemia, increased tissue
catabolism. Can be a risk factor for gout development
 Inherited disorders of purine metabolism
 Lesch-Nyhan syndrome: occurs in men only,
caused by complete deficiency of hypoxanthine-
guanine phosphoribosyltransferase, enzyme
needed in recycling purines
 Deficiency of phosphoribosylpyrophosphate
synthetase
 Secondary to glycogen storage disease (deficiency
of glucose-6- phosphatase, Glycogen Storage
Disease type 1 or Von Gierke Disease) and fructose
intolerance (deficiency of fructose-1- phosphate
aldolase, GSD Type 1a)
 Toxemia of pregnancy (preeclampsia, high blood
measure meaning reduced glomerular filtration rate)
 CLINICAL CHEMISTRY
LECTURE / FIRST SEMESTER
and lactic acidosis (high ethanol consumption which
increases production of uric acid)
 Hypouricemia: decreased plasma uric acid concentration
 Secondary to severe liver disease (damaged to
hepatocytes will impair uric acid production)
 Defective tubular reabsorption (Fanconi syndrome -
proximal convoluted tubule). Uric acid is excreted and
not reabsorbed
 Overtreatment with allopurinol. It reduces plasma uric
acid concentration
 Treatment with 6-mercaptopurine or azathioprine,
potent inhibitors of uric acid synthesis
Reference Intervals
Conversion Factor:
mg/dL → mmol/L
0.0595
Creatinine / Creatine
 Creatinine is formed from creatine and creatine phosphate
in muscle and is excreted into the plasma at a constant rate
related to muscle mass.
 Plasma creatinine is inversely related to glomerular filtration
rate (GFR).
Biochemistry
 Creatine is synthesized primarily in the liver from arginine,
glycine, and methionine.
 Creatine (to other tissues such as muscle) → creatine
phosphate (high energy source)
 Creatine phosphate loses phosphoric acid and
creatine loses water → creatinine
 Creatinine is removed from the circulation by glomerular
filtration and excreted in the urine.
 Small amounts of creatinine are secreted by the
proximal tubule and reabsorbed by the renal tubules.
 Creatinine clearance (CrCl) and Glomerular filtration
rate (GFR): to gauge renal function
 CrCl: a measure of the amount of creatinine
eliminated from the blood by the kidneys
 Glomerular Filtration Rate
 Creatinine clearance (CrCl) and Glomerular filtration
rate(GFR): to gauge renal function
 CrCl (mL/min)
Analytical Methods: Creatinine
Chemical Methods
 Jaffe Reaction
 creatinine + alkaline picrate → red-orange chromogen
 Jaffe reagent (alkaline picrate): saturated picric acid
and 10% NaOH
 Kinetic Jaffe
 serum is mixed with alkaline picrate and the rate of
change in absorbance is measured between 2 points
 Jaffe with adsorbent
 Creatinine is adsorbed onto Fuller’s earth (aluminum
magnesium silicate) or Lloyd’s reagent (sodium
aluminum silicate), then reacted with alkaline picrate
 Other methods – IDMS as the accepted reference method
Enzymatic Methods
- Creatininase (Creatinine Aminohydrolase)-CK
method
 Creatininase : Creatinine + H20 → Creatine
 CK: Creatine + ATP → Creatine PO4 + ADP
 PK: ADP +Phosphoenolpyruvate → ATP + Pyruvate
 LDH: Pyruvate + NADH → Lactate + NAD
- Creatininase-Hydrogen Peroxide method
 Creatininase : Creatinine + H2O → Creatine
 Creatinase: Creatine + H20 → Sarcosine + Urea
 Sarcosine oxidase: Sarcosine + H2O + O2 → Glycine +
HCHO + H2O2
 Peroxidase: H2O2 + colorless substrate → colored product
+ H2O
Analytic Methods: Creatine
 Endpoint Jaffe method for creatinine before and after it is
heated in acid solution.
 Enzymatic methods: creatininase (creatine kinase, pyruvate
kinase, lactate dehydrogenase)
 HPLC
Specimen Requirements: Creatinine
 Plasma, serum, or urine (refrigerated after collection or
frozen if stored longer than 4 days)
 CLINICAL CHEMISTRY
LECTURE / FIRST SEMESTER
Sources of Error
 Ascorbate, glucose, α-keto acids, and uric acid may
increase creatinine concentration (Jaffe reaction; above
30°C)
 Bilirubin causes a negative bias (Jaffe and enzymatic
methods)
 Ascorbate will interfere in enzymatic methods (peroxidase)
 Drugs that increase creatinine concentration –
cephalosporin, dopamine, lidocaine
Reference Intervals
Conversion Factor:
mg/dL → µmol/L
88.4
Clinical Significance: Creatinine
 Increased plasma creatinine: abnormal renal function
Clinical Significance: Creatine
 Increased plasma creatine: muscle disease (measurement
of creatine kinase is typically used)
Ammonia
 is produced in the deamination of amino acids during
protein metabolism and by bacterial metabolism in the
lumen of the intestine
 is removed from the circulation and converted to urea in the
liver
 free ammonia is toxic
Biochemistry
 most ammonia in the blood exists as ammonium ion
 ammonia is excreted as ammonium ion by the kidney and
acts to buffer urine
Analytical Methods
 Enzymatic method: Glutamate Dehydrogenase
 most commonly used method; 340 nm
 NH4 + 2-oxoglutarate + NADPH → glutamate + NADP
 Thin film colorimetric assay
 ammonia + indicator → colored compound
 Direct measurement using an Ion Selective Electrode
 measures the change in pH of an ammonium chloride
solution as ammonia diffuses across a membrane
Specimen Requirements
 Venous blood should be placed on ice immediately
 Heparin and EDTA are suitable anticoagulants
 Samples should be centrifuged at 0 to 4°C within 20
minutes of collection and the plasma or serum should be
removed immediately
 Frozen plasma is stable for several days at –20°C
 Hemolysis should be avoided
 Cigarette smoking is a source of ammonia contamination
 Increases plasma ammonia
 Ammonium salts, asparaginase, barbiturates,
diuretics, ethanol, hyperalimentation (total parenteral
nutrition), narcotic analgesics
 Decreases plasma ammonia
 Diphenhydramine, Lactobacillus acidophilus,
lactulose, levodopa, and several antibiotics
 Glucose at >600 mg/dL (33 mmol/L) interferes in dry slide
methods
Reference Intervals
Conversion Factor:
µg/dL → µmol/L
0.587
Sources of Error
 Sources of contamination: tobacco smoke, urine, and
ammonia in detergents, glassware, reagents, and water
 Ammonia content of serum-based control material is
unstable
 Frozen aliquots of human serum albumin containing
known amounts of ammonium chloride or ammonium
sulfate may be used
Clinical Significance
Hyperammonemia
 Severe liver disease - ammonia cannot be converted to
urea and cannot be removed in the circulation
 Reye’s syndrome - acute metabolic disorder of the liver,
preceded by viral infection and administration of aspirin.
Ammonia cannot be remove from the circulation and cannot
be converted to urea
 CLINICAL CHEMISTRY
LECTURE / FIRST SEMESTER
 Inherited deficiency of enzymes of the urea cycle