Non-Protein Nitrogenous

Compounds
 NPN
- Provides an index for renal function
- 45% is urea

1. Urea/ BUN
2. Uric Acid
3. Creatinine
4. Ammonia
 Kidneys
Retroperitoneal
paired organ located
between T12 L3
Located superiorly
are the suprarenal
glands/adrenal
glands
Right is lower than
the left
 Kidneys
Function:
1. Glomerular Filtration
2. Tubular Reabsorption
3. Endocrine
4. Excretion
5. Blood volume and blood pressure
regulation
6. Acid-Base balance
 Kidneys
Three General Processes:
1. Glomerular Filtration
2. Tubular Reabsorption
3. Renal Secretion
 UREA
Major NPN (45%)
Urea Cycle/ Ornithine Cycle/ Krebs
Henseleit Cycle
Liver (from NH2 groups and free ammonia)
End product of protein metabolism
Readily filtered from plasma
 UREA
Pathophysiology
Azotemia increased NPN (urea)
Uremia Increased NPN with organ
involvement
3 Phases of Azotemia:
1. pre-renal
2. Renal
3. Post-renal
 SPECIMEN
Plasma (Heparin)/ Serum
Refrigerate sample (if cannot be processed
immediately)
Do not use hemolyzed sample

NV: 7 18 mg/dl
CF: 0.357
BUN Urea: 2.14
Urea BUN: 0.46
 ASSAYS
METHOD
Colorimetric: Inexpensive; lack
diacetyl specificity
Enzymatic: NH3 High specificity;
formation expensive
Isotope dilution Reference method
mass
spectrophotometry
 DIRECT MEASUREMENT
I. Fearon/ Diacetyl Monoxime
DAM in acid diacetyl + Hydroxylamine
Diacetyl + Urea Diazine (yellow)

440 460 nm
Additives:
Ferric ions, thiosemicabazide
 DIRECT MEASUREMENT
II. O-phthaldehyde
Urea + o phthaldehyde isoindoline
Isoindoline N-I-napthylethylene diamine
(colored compound)
Interfering Substance: Sulfa Drugs
(Sulfamethoxazone)

III. Mass spectrophotometry (isotope dilution)

 INDIRECT MEASUREMENT
I. Enzymatic
Urea + Urease NH4+ + Carbonate
NH4+ + GLDH + oxoglutarate +NADH
glutamate + H2O
 INDIRECT MEASUREMENT
Nesslerization
NH4+ + mercuric iodide + potassium iodide
Ammonium mercuric iodide (yellow orange)

Berthelot
NH4+ + Na hypochlorite + phenol +
nitroprusside indophenol
Other methods:
Folin-Svedberg
Ureastat
Eluted NH3

BUN/CREA ratio 10:1/20:1

Increased = prerenal, postrenal
Decreased = renal
 URIC ACID
End product of purine nucleotide
degradation
Weak acid
Monosodium urate crystals
precipitate in low pH deposit in
joints
 Disease Correlation
Hyperuricemia
1. Gout
2. Tophi
3. Lesch-Nyhan Syndrome HGPRT
deficiency
 GOUT
Defect in uric acid metabolism
Results in deposition of urate crystals
around joints attracting neutrophils
and macrophages inflammatory
reaction
Inflammation of joints (gouty arthritis)
and deposition of urates in tendons and
subcutaneous tissues (tophi)
 LESCH-NYHAN SYNDROME
Defect in HGPRT/ Hypoxanthine
Guanine Phosphoribosyltransferase
Overproduction hyperuricemia
Uric acid lithiasis, neurologic problems
and self-mutilation
 Disease Correlation
Hypouricemia
Liver disease
Fanconis syndrome defect in tubules;
overexcretion
 ASSAYS
I. Caraway/ Phosphotungstic Acid
Uric Acid + Phosphotungstate allantoin +
Tungsten + CO2
Additive: Na Carbonate
II. Enzymatic
U.A. + uricase allantoin + H2O2 + CO2 @
293nm
CF: 0.0595
NV: (M)3.5 7.2 mg/dl; (F)2.6 - 6.0 mg/dl
 CREATININE
Creatine creatinine
End-product of muscle wasting; product of
phosphocreatine
100% filtered by kidneys
100% present in urine
Most specific kidney function test
Increased in renal disease and renal failure
 ASSAYS
1. Jaffe reaction
Creatinine + alkaline picrate solution
janovsky complex/ creatinine picrate (red-
orange color)
Modified Jaffe
2. Lloyds reagent Na aluminum silicate
3. Fullers Earth reagent Magnesium
aluminum silicate
 ASSAYS
2. Enzymatic
Enzyme: creatine kinase
Substrate: Creatinine aminohydrolase
Product: NAD

CF: 88.4
NV (M) 0.6 1.2 mg/dl; (F) 0.5 1.1 mg/dl
 Assays
Enzyme: Creatinase
Coloring agent: benzoquinone amine

Creatinine + H2O + creatinase creatine

Creatine H2O + creatinase sarcosine + urea
Sarcosine + O2 + H2O glycine + formaldehyde +
H2O2
H2O2 + color indicator indicator (oxidized) +
2 H2O
 Creatinine Clearance
Assess GFR
 Ammonia
Deamination of amino acids
Hepatocytes convert ammonia urea
Neurotoxic
NV 11 -32 mol/L
Specimen: plasma (heparin) on ice; not
hemolysed; centrifuged within 20 minutes
 Ammonia
Pathophysiology

Increased in: advanced liver disease

(Reyes syndrome, cirrhosis, viral
hepatitis) and impaired renal function
 Test methodology
ketoglutarate + NH4+ + NADPH
Glutamate + NADP+ + H2O