NON-PROTEIN
NITROGENOUS
COMPOUNDS (NPN)
Non-Protein Nitrogenous
Compounds (NPNs)
• Compounds containing nitrogen other than proteins
urea 45-50%
amino acids 25%
Renal Profile or Kidney
uric acid 10% Function Tests
creatinine 5%
creatine 1-2%
ammonia 0.2%
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UREA
• major excretory product of protein metabolism
• synthesized in the liver from carbon dioxide and
ammonia from deamination of amino acids
40% reabsorbed
50% excreted in urine
<10% excreted through GI and skin
**Concentration of urea is dependent on renal
function and perfusion, protein content of the diet, and
the amount of protein catabolism.
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Disease Correlations
• Azotemia:
• Elevated concentration of urea or other NPNs in the
blood
• Uremia/Uremic Syndrome:
• Increased urea in the blood accompanied by renal
failure
• Fatal if not treated by dialysis or transplantation
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Causes/Types of Azotemia
1. Pre-renal Azotemia:
• due to reduced renal blood
flow
• causes:
• Congestive heart failure
• Shock
• Hemorrhage
• Dehydration
• Other: high protein intake and
high protein catabolism
• Stress, fever, major illness,
corticosteroid therapy, GI
hemorrhage
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Causes/Types of Azotemia
2. Renal Azotemia:
• due to decreased renal
function
• causes:
• Acute or chronic renal
failure
• Glomerular nephritis
• Tubular necrosis
• Intrinsic renal disease
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Causes/Types of Azotemia
3. Post-Renal Azotemia:
• due to obstruction of the
urine flow anywhere in the
urinary tract
• causes:
• Renal calculi
• Tumors of the
bladder/prostate
• Severe infection
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Causes/Types of Azotemia
• Pre-renal High urea: normal creatinine
• Renal High urea: high creatinine
• Post-Renal Normal urea: high creatinine
Urea Nitrogen: Creatinine
Normal= 10:1 to 20:1
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Disease Correlations
• Decreased levels of urea in the blood
• causes:
• decreased protein intake
• increased protein synthesis (late pregnancy and infancy)
• severe liver damage
• severe vomiting and diarrhea
Decreased urea nitrogen:creatinine ratio
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Urea Determination
1. Direct method: measures urea as a
whole
2. Indirect Method: measures the nitrogen
content of urea (BUN)
Uses:
BUN to urea : 2.14
• Evaluate renal function
Urea to BUN: 0.467
• Assess hydration status
• Determine nitrogen balance
BUN in mg/dL to mmol/L: 0.357
• Verify adequacy of dialysis
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Urea Determination
1. Condensation with Diacetyl Monoxime Method:
• Fearon’s Reaction
• Also known as Friedman’s Method or Xanthydrol Method
• Reagents: strong acid, oxidizing agent, ferric ions,
thiosemicarbazide
• Product: yellow diazine derivative
Ammonia does not interfere
DAM + water ===(H+) diacetyl Used in autoanalyzers
Diacetyl + urea ==== (Fe+3) diazine non-specific
Uses toxic substances
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Urea Determination
2. Reaction with o-phthalaldehyde and
naphthylethylenediamine
• Product: chromogen or colored product
• Urea + o-phthalaldehyde (H+) isoindoline +
naphthylethylene diamine (H+) colored product
No ammonia interference
Used in automation
Sulfa containing drugs interfere
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Urea Determination
3. Micro-Kjeldahl Nessler Method
• digestion: urea (H2SO4 + H3PO4) NH4+
• NH4+ (alkalinized) NH3 + K2HgI4 NH2Hg2I3
• Product: yellow compound
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Urea Determination
4. Enzymatic Methods: urease
4.1. UREASE-NESSLER’s Method
• Urea (urease) HCO3- + NH4+ + Gum Ghatti (alkaline)+
Nessler’s reagent NH2Hg2I3 (yellow)
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Urea Determination
4. Enzymatic Methods: urease
4.2. UREASE-BERTHELOT’s Method
• Urea (urease) HCO3- + NH4+ + sodium nitroprusside
(alkaline)+ phenol hypochlorite indophenol blue +
NaCl + H2O
Not specific
Very sensitive to interference from endogenous ammonia
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Urea Determination
4. Enzymatic Methods: urease
4.3. L-Glutamate Dehydrogenase Method (GLDH method)
• Urea (urease) 2NH4+ + HCO3-
• NH4+ + 2-oxoglutarate + NADH (GLDH) glutamate + NAD+ + H+
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Urea Determination
4. Enzymatic Methods: urease
4.4. UREASE-Conductimetric Method
• Urea (urease) HCO3- + NH4+
• Conductivity of ammonium is measured
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Specimen Considerations
• Urine
• Serum
• Plasma: do not use citrate or fluoride as anticoagulant
Protein content of diet influences urea but minimal:
no fasting requirement
Urea is susceptible to bacterial decomposition:
refrigerate!!!
Reference Interval:
Serum/plasma: 7-18 mg/dL (2.5-6.4 mmol/L)
Urine, 24 hr: 12-20 g/day (0.43-0.71 mol/day)
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Creatinine
• CREATINE (C4H9N3O2): synthesized in the liver from arg,
gly, and met
• CREATININE (C4H7N3O): anhydride of creatine
• product of muscle catabolism
• 99% excreted in the urine and <1% reabsorbed
Creatine muscles phosphocreatine
Phosphocreatine – phosphate creatine
Creatine – water creatinine
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Clinical Applications
• Creatinine measurement:
used to determine the sufficiency of kidney
function
To determine the severity of kidney damage
To monitor the progression of kidney
disease
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Disease Correlations
• Abnormal renal function (glomerular function)
Creatinine Clearance (CrCl) = (urine creatinine)(urine volume)
(plasma creatinine)(time)
Reference interval:
Male: 97 – 137 mL/min
Female: 88 – 128 mL/min
• creatine and urinary creatinine (normal plasma creatinine):
muscle diseases like muscular dystrophy, poliomyelitis,
hyperthyroidism, trauma
Plasma creatinine: insensitive to mild renal dysfunction
Not affected by diet
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Creatinine Determination
1. Jaffe’s Method
• Reagents: picric acid, 10% sodium hydroxide
Creatinine + alkaline picrate creatinine picrate (JANOVSKI complex)
ascorbic acid, glucose, glutathione, ketoacids, UA, cephalosporins
1.1. Kinetic Jaffe:
ketoacids, cephalosporins
bilirubin, hemoglobin
1.2. Jaffe with adsorbent:
• Fuller’s earth (aluminum magnesium silicate)
• Lloyd’s reagent (sodium aluminum silicate)
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Creatinine Determination
2. Enzymatic Method
2.1. Creatininase-CK
Creatininase, CK, PK, LD- NAD is produced
creatinine aminohydrolase
Creatinine + H2O creatine
CK
Craetine + ATP creatine phosphate + ADP
PK
ADP + phosphoenolpyruvate ATP + pyruvate
LD
Pyruvate + NADH lactate + NAD+
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Creatinine Determination
2. Enzymatic Method
2.2. Creatininase-H2 O2 -
- Adapted for use as dry slide method to replace Jaffe
Positive bias due to lidocaine
No interference from acetoacetate and cehalosporins
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Creatinine Determination
3. 3,5-Dinitrobenzoic acid Method (DNBA)
• used in reagent strips
• Creatinine + DNBA (OH-)
• Product: purple colored compound
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Specimen Considerations
• Urine
• non-lipemic, non-hemolyzed, non-icteric serum or
plasma
• No fasting requirement
Reference Interval Jaffe Enzymatic
Adult Male 0.9-1.3 mg/dL (80-115 μmol/L) 0.6-1.1 mg/dL (53-97 μmol/L)
Adult Female 0.6-1.1 mg/dL (53-97 μmol/L) 0.5-0.8 mg/dL (44-71 μmol/L)
Child 0.3-0.7 mg/dL (27-62 μmol/L) 0-0.6 mg/dL (0.53 μmol/L)
Urine Male 800-2000 mg/d (7.1-17.7 mmol/d)
Urine Female 600-1800 mg/d (5.3 15.9 mmol/d)
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Uric Acid
• Final breakdown product of purine metabolism
• 98-100%:reabsorbed in the glomerular filtrate
• <1%: secreted in the proximal tubules
• Renal excretion: 70% of UA
• GI excretion: 30%
• In plasma:
• monosodium urate (pH 7 >6.8 mg/dL)
• uric acid crystals (pH <5.75)
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CLINICAL APPLICATIONS
Uric Acid is measured:
To confirm diagnosis and treatment of
gout
To prevent uric acid nephropathy during
chemotherapeutic treatment
To detect kidney dysfunction
To assist in the diagnosis of renal calculi
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Disease Correlation
Hyperuricemia: >6.0 mg/dL
1. Gout: due to precipitaion of sodium urates
• susceptible to development of renal calculi
• tophi: deposition of urates in tissues
• male 30-50 y.o.;menopausal women
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Disease Correlation
2. Increased metabolism of
cell nuclei
• seen in patients on
chemotherapy
• UA monitoring to avoid
nephrotoxicity
• Treatment: allopurinol
(inhibit xanthine oxidase)
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Disease Correlation
3. Kidney Diseases
• impaired filtration and secretion
• not a good indicator of renal function
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Disease Correlation
• Lesch Nyhan Syndrome
• X-linked genetic disorder
• Caused by complete deficiency of hypoxanthine
guanine phosphoribosyl transferase (HGPRT)
• HGPRT: important for the biosynthesis of purines
• Symptoms: mental retardation and self-mutilation
Increasing purine synthesis,
increases the degradation product
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Disease Correlation
• Other conditions with
increased UA:
• Mutations on
phosphoribosylpyrophosphate
synthetase
• Toxemia of pregnancy
• Lactic acidosis (competition
for binding sites in renal
tubules)
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Causes of Hyperuricemia
• Increased dietary intake of purine rich food
• Increased urate production
• Decreased excretion
• Catabolic pathways enzyme defects
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Disease Correlation
Hypouricemia
• Causes:
• Secondary to severe liver disease
• Defective tubular reabsorption (Fanconi’s syndrome)
• Chemotherapy with 6-mercaptopurine or azathiopurine
(inhibits de novo purine synthesis)
• Overtreatment with allopurinol
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Uric Acid Determination
1. Direct REDOX Method: Caraway Method/ Henry’s
Method
• Based on the oxidation of UA in PFF and reduction of PTA
UA + PTA + sodium carbonate allantoin + CO2 + tungsten blue
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Uric Acid Determination
2. Iron Reduction Method
ligand
Uric acid + ferric ion Ferrous ion + chromophore
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Uric Acid Determination
3. Uricase Method (Blauch and Koch)
• measurement of the differential absorption of UA and
allantoin at 290-293 nm
• more specific
• proteins cause high background absorbance
• negative interference due to Hb and xanthine
UA + O2 + 2 H2O allantoin + CO2 + H2O2
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Uric Acid Determination
4. Coupled Enzymatic Method
UA + O2 + 2 H2O allantoin + CO2 + H2O2
H2O2 + indicator dye colored compound + 2 or 3 H2O
Bilirubin and ascorbic acid may destroy peroxide
Remedy: addition of potassium ferricyanide and ascorbate oxidase
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Uric Acid Determination
5. HPLC
6. Isotope dilution mass spectrometry (IDMS)
• Detection of characteristic fragments after ionization
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Specimen Considerations
• Serum, urine, heparinized plasma
• EDTA and fluoride inactivate uricase
• No fasting requirement
• Bilirubin: false decreases UA
• salicylates and thiazides: false increase UA
Reference Interval
Male 3.5-7.2 mg/dL 0.21-0.43 mmol/L
Female 2.6-6.0 mg/dL 0.16-0.36 mmol/L
Urine 250-750 md/d 1.48-4.43 mmol/d
child 2.0-5.5 mg/dL 0.12-0.33 mmol/L
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Ammonia
• Produced in the deamination of amino acids during
protein metabolism - liver
• Absorbed from the intestinal tract where it is
formed by bacterial degradation of dietary protein
and the urea present in the GI secretions.
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Disease Correlation
1. Hepatic Failure
2. Reye’s Syndrome
3. Inherited
deficiency of all
urea cycle
enzymes
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Ammonia Determination
1. Conway Method (1935):
• Volatility of ammonia
• NH3 diffuses into a separate compartment
• Absorbed in a solution with pH indicator
• Amount of ammonia is determined by titration
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Ammonia Determination
2. Cation Exchange Resin
• Ammonia is isolated
• Eluted with NaCl
• Quantified by Berthelot’s reaction
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Ammonia Determination
3. Glutamate Dehydrogenase Method
• decrease in A at 340 nm as NADPH is consumed
Ammonium + 2-oxoglutarate + NADPH + H+
glutamate +NADP+ + H2O
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Ammonia Determination
4. ISE (Direct)
• Electrode measures the change in pH of a solution of
NH4Cl as NH3 diffuses across a semi-permeable
membrane
5. Thin Film Colorimetry
• NH3 reacts with an indicator (BPB) to produce a colored
(blue) compound
• spectrophotometry
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Specimen Considerations
• Whole blood (heparin or EDTA)
• Blood collection without trauma
• Place on ice ASAP
• Centrifuge at 0-4°C within 20 minutes and separate
plasma from red cells
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Specimen Considerations
hemolysis (false increase), urine contamination
cigarette smoking, detergent contamination
False elevations: ammonium salts, asparaginase,
barbiturates, diuretics, alcohol, hyperalimentation,
narcotic analgesics
False decrease: diphenhydramine, L. acidophilus,
lactulose, L-dopa, antibiotics
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Reference Interval
• Plasma 19-60 μg/dL (11-35 μmol/L)
• Urine 140-500 mg N/d (10-107 mmol N/d)
• Child 68-136 μg/dL (40-80 μmol/L)
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Quiz Next Meeting!
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