CLIN CHEM | NON-PROTEIN NITROGENOUS
TRISHA LORAINNE R. VILLOCENO
COMPOUNDS (NPNS)
INTRODUCTION
 NPNs – not proteins but contain nitrogen in
their structure
 Products of protein and nucleic acid
catabolism
 Usually tested in lab to determine renal and
liver functions
 Includes:
 Urea – renal function test
 Creatinine – renal function test
 Uric Acid – test in cases of gout
 Ammonia – assess liver function/
detoxification
NPNs: UREA
 Highest in concentration in blood among all
NPNs (45-50%)
 Most abundant of all
NPNs
 Major excretory product
of protein metabolism
 Formed in the liver from amino groups and
free ammonia generated during protein
catabolism
 Often times referred to as BUN or Blood
Urea Nitrogen – since urea determination is
based on measurement of nitrogen (prev.)
A. BIOCHEMISTRY
o Synthesized in the liver then carried in
the blood to the kidney
o In the kidney, urea is readily filtered from
plasma by glomerulus
o Most of urea (approx. 90%) is excreted in
urine; only less than 10% is reabsorbed via
passive diffusion
o Concentration of urea in the plasma is
determined by:
Protein content of diet (consumption) –
directly proportional w/ urea
Rate of protein catabolism
Renal function and perfusion
B. CLINICAL APPLICATION
o Measurement of urea is used to:
Evaluate renal function
Assess hydration status – if
dehydrated, urea is increased due to
low liquid levels
Determine nitrogen balance
Aid in diagnosis of renal disease
Verify adequacy of dialysis – before
dialysis urea and crea must be high;
after dialysis it must be low
o Reported as Nitrogen Concentration
(BUN)
o Conversion factor:
To convert BUN mg/dL to urea in
mg/dL, BUN must be multipled with
2.14
To convert BUN mg/d L to urea in
mmol/L, BUN must be multiplied with
0.36
Note: mg/d L = conventional unit; mmol/L = SI unit
C. PATHOPHYSIOLOGY
o Normally, urea produced by body should be
excreted in the urine; urea’s level in the
blood must be at low levels
o In cases of impaired renal function, urea
is not cleared and not excreted, it is built
up in blood
o Azotemia – term to describe elevated
concentration of urea in the blood (renal
failure is not yet diagnosed)
o Uremia – term to describe high plasma
urea concentration accompanied by renal
failure (diagnosed by renal failure)
o Increased plasma urea concentration can
be classified into: Prerenal, Renal and
Postrenal
CLIN CHEM | NON-PROTEIN NITROGENOUS
COMPOUNDS (NPNS)
Prerenal
 Due to reduced renal blood flow or
increase in protein diet (note: kidney is not
impaired)
 Causative factors: Congestive Heart
failure, Shock, Hemorrhage, Dehydration
Renal
 Due to decreased renal function or
damaged kidneys
 Causative factors: Acute/Chronic Renal
failure, Glomerula Nephritis
 RBC cast: Hallmark sediment in
glomerular nephritis
Postrenal
 Due to obstruction of urine flow in the
urinary tract
 Causative factors: Renal Calculi, Tumors,
Severe infection
o Major causes of decreased plasma urea
concentration:
Low protein intake
Severe vomitting
Liver disease
Pregnancy
o To differentiate the cause of abnormal
urea concentration, calculate BUN to
creatinine ratio – normally between 10:1 or
20:1 (simply divide BUN/Crea)
High Urea:Creatinine ratio w/ normal
creatinine levels = Prerenal
High Urea:Creatinine ratio w/ elevated
creatinine levels = Postrenal
Low Urea:Creatinine ratio = Conditions
associated w/ Decreased Urea Conc.
D. METHODS
o Specimen: Serum/Plasma (preferrably
note hemolyzed)
o Do not use citrate and fluoride as
anticoagulant – Inhibits urease
TRISHA LORAINNE R. VILLOCENO
o Specimens should be refrigerated if
analysis is not done w/in a few hours
o Reference method: Isotope Dilution Mass
Spectrometry (IDMS)
o Other Methods: Direct, Indirect and
Enzymatic
Direct Method
 Aka Rosenthal Method; directly measures
urea
 Reagent: Diacetyl monoxime
 Positive color: Yellow diazine
Indirect Method
 Aka Microkjeldahl Method
 Measures nitrogen instead of urine
Enzymatic Method
 Most commonly used method
 Urease – hydrolyzes urea to produce
ammonium ion (NH4+)
 Ammonium ions can be measured:
 Directly (Berthelot’s method)
 Reaction w/ glutamate dehydrogenase
(GLDH) w/c requires NADH; rate of
disappearance of NADH is measured at
340 nm
 Color change via pH indicator (alkaline)
 Conductivity
NPNs: CREATININE
 Formed from creatine and creatine phosphate
in the muscles
 Formed as by-product of muscle activity
 Excreted into plasma at constant rate related
to muscle mass
 Inversely related to glomerular filtration rate
(GFR)
 Commonly used to assess renal filtration
function
CLIN CHEM | NON-PROTEIN NITROGENOUS
COMPOUNDS (NPNS)
 Better renal function marker than urea
A. BIOCHEMISTRY
o Conversion of creatine to creatine
phosphate happens in the muscle
o When creatine loses water  Creatinine
o When creatine phosphate loses inorganic
phosphate or phosphoric acid  Creatinine
o When creatinine is formed, it is released
into circulation then travels to kidney. It
is released into circulation at constant
rate that has been shown to be
proportional to individual’s muscle mass
o It is removed from circulation by
glomerular filtration and excreted in the
urine
o Small amounts are secreted by proximal
tubule and reabsorbed by renal tubules
o Daily creatinine excretion is reasonably
stable making it a marker for glomerular
filtration
B. CLINICAL APPLICATION
o Measurement of creatinine conc. is used
to:
TRISHA LORAINNE R. VILLOCENO
Determine sufficiency of kidney
functions
Determine severity of kidney damage
Monitor progression of kidney disease
o Plasma creatinine conc. is dependent on:
Relative muscle mass
Rate of creatinine turnover
Renal function (increased creatinine due to
impaired kidney function)
o Not heavily affected by diet, the reason
why creatinine is preferred over urea
o Endogenously produced by the muscles
o Used as measure of completeness of 24-
hour urine samples
o Creatinine clearance – measure of GFR (if
high creatinine, low GFR and vice versa)
C. PATHOPHYSIOLOGY
o High creatinine concentration in the blood
is associated w/ abnormal renal function
o Plasma creatinine concentration is
inversely proportional to creatinine
clearance or GFR
o Renal damage is suspected = when plasma
creatinine is elevated/ GFR is decreased
o Plasma creatine concentration is not
elevated in renal diseases, it is elevated in:
Muscular dystrophy
Poliomyelitis
Trauma
D. METHODS
o Specimen: Serum/Plasma/Urine
o Hemolyzed, icteric and lipemic samples
must be avoided
o Urine should be refrigerated after
collection or frozen if longer storage is
required (> 4 days)
o Reference method: Isotope Dilution Mass
Spectrometry (IDMS)
o Methods: Chemical and Enzymatic method
CLIN CHEM | NON-PROTEIN NITROGENOUS
TRISHA LORAINNE R. VILLOCENO
COMPOUNDS (NPNS)

o Transported to the kidney where it is

Chemical Method reabsorbed
 Jaffe Reaction o Most of uric acid in plasma is in the form
 Reagent: Picric acid in alkaline solution of monosodium urate
(10% NaOH) or aka Alkaline picrate o At plasma pH, uric acid is insoluble
 Positive color: Red-orange complex o At > 6.8 mg/dL conc., urate crystals may
(Creatinine picrate) form and precipitate in tissues
 Modification: o At acidic urine pH (pH <5.75), uric acid
 Jaffe-kinetic: Used to avoid crystals may form
interferences of non-creatinine
chomogens such as ascorbic acid and B. CLINICAL APPLICATION
glucose (used in laboratory) o Uric acid is measured to:
 Jaffe w/ Adsorbent: Adsorbent Confirm diagnosis and monitor
increase specificity, it adsorb treatment of gout
interferences and remove it. The ff Prevent uric acid nephropathy during
are adsorbent: chemotherapy
 Fuller’s earth reagent (Aluminun Assess inherited disorders of purine
Magnesium Silicate) metabolism
 Lloyd’s reagent (Sodium Aluminum Detect kidney dysfunction
Silicate) Assist in diagnosis of renal calculi
Enzymatic Method
 Uses the ff. enzymes for dry slide C. PATHOPHYSIOLOGY
analyzers o Abnormally increased uric acid
 Creatininase concentration is founc in:
 Creatinase Gout – uric acid deposition in joints
 Sarcosine oxidase Tophi – uric acid deposition in tissues
 Peroxidase Patients on chemotherapy – increased
catabolism of nucleic acids
NPNs: URIC ACID Chronic renal disease
Disorders in purine metabolism
 Major product of purine and nucleic acid
(enzyme deficiencies)
catabolism
Enzymatic Deficiencies
 Not a kidney function test
 Lesch-Nyhan syndrome – Hypoxanthine
 98-100% of uric acid is reabsorbed in the
guanine phosphoribosyl transferase
proximal tubules
deficiency; exhibit in males because it is
 Relatively insoluble in plasma
X-linked (HPRT1 gene mutation)
 Deposited in tissues and joints at high
 Phosphoribosylpyrophosphate synthetase
concentrations (gout arthritis)
deficiency
 Glycogen storage disease type 1 – Glucose
A. BIOCHEMISTRY
6-Phosphatase deficiency
o Purines (adenine and guanine) are
 Fructose intolerance – Fructose-1-
converted to uric acid in the liver
Phosphate aldolase deficiency
CLIN CHEM | NON-PROTEIN NITROGENOUS
TRISHA LORAINNE R. VILLOCENO
COMPOUNDS (NPNS)

o Uric acid nephtolithiasis (kidney stones)

o Hypouriciemia – Less common (decrease NPNs: AMMONIA
uric acid)  Liver function test (detoxification test)
 Produce of amino acid deamination during
D. METHODS protein metabolism
o Specimen: Serum/Heparinized Plasma  Rapidly removed from circulation and
o Can be stored in refrigerator temp. for 3- converted to urea in the liver
5 days  Free amomonia is toxic
o Grossly lipemic samples must be avoided  Not a kidney function test
o EDTA or Fluoride additives must not be
used if specimen will be tested via uricase A. BIOCHEMISTRY
method o Present in plasma in very low concentration
o Methods: Chemical and Enzymatic o Ammonia (NH3) is consumed by
methods parenchymal cells of liver in production of
Chemical Method urea
 Caraway Method or Phosphotungstic Acid o If liver is damaged, ammonia levels in the
Method blood is increased because detoxification
 Reagent: Phosphotungstic acid will not happen
 Principle: Oxidation-Reduction o At normal physiologic pH, ammonia exists
reaction as ammonium ion (NH4+)
 Produce Allantoin and Tungsten blue
complex B. CLINICAL APPLICATION
 Reaction: o Ammonia determination is helpful in
Uric acid + phosphotungstic acid + O2  detecting:
Allantoin + tungsten blue + CO2 Hepatic failure (most common cause of
Enzymatic Method disturbed ammonia metabolism)
 Most commonly used in laboratory Hepatic coma
 Uricase – Catalyze oxidation of uric Reye’s syndrome (usually affects
acid to allantoin children)
Inherited enzyme deficiencies
involved in urea cycle

C. PATHOPHYSIOLOGY
 Reaction: Measured at 293 nm o Normally, liver clears/detoxify ammonia
 Difference in absorbance before from the circulation
and after incubation w/ enzyme is o In severe liver disease, ammonia is not
proportional to uric acid removed  increased in concentration in
concentration the blood
 Coupled enzyme methods measure o High concentration of ammonia leads to
hydrogen peroxide produced in the neurotoxicity and encephalopathy
reaction o Hyperammonemia can also be caused by
deficiencies of enzymes in urea cycle
CLIN CHEM | NON-PROTEIN NITROGENOUS
TRISHA LORAINNE R. VILLOCENO
COMPOUNDS (NPNS)

REFERENCE VALUES
D. METHODS
o Specimen: Venous Whole Blood Urea Nitrogen – Adult
o Must be placed on ice immediately Plasma or 6-20 mg/dL 2.1–7.1 mmol/L
o Heparin and EDTA may be used as serum
anticoagulants – Heparin is commonly used Urine 24 hrs 12-20 g/day 0.43-0.71 mol
o Centrifuged at 0-4o C w/in 20 mins of urea/day
collection
o Plasma must be removed immediately Creatinine (Plasma or Serum)
o Specimens must be tested immediately Jaffe method Enzymatic
o If not, store at -20o C method
o Hemolyzed samples must be avoided as Adult Male 0.9-1.3 mg/dL 0.6-1.1 mg/dL
RBCs contain ammonia (80-115 umol/L) (53-97 umol/L)
Adult Female 0.6-1.1 mg/dL 0.5-0.8 mg/dL
o Methods: Titration, Berthelot and
(53-97 umol/L) (44-71 umol/L)
Enzymatic method
Child 0.3-0.7 mg/dL 0.0-0.6 mg/dL
(27-62 umol/L) (0=53 umol/L)
Titration Method Creatinine (Urine)
 Aka Conway Method Adult Male 800-2000 mg/day (7.1-17.7
 Uses volatility of ammonia to separate mmol/day)
it from sample using a microdiffusion Adult Female 600-1800 mg/day (5.3-15.9
chamber mmol/L)
 Ammonia gas from sample diffuses
into the chamber and absorbed in Uric Acid
solution w/ pH indicator Adult Male 3.5-7.2 mg/dL ; 0.21-0.43 mmol/L
Berthelot Reaction Adult Female 2.6-6.0 mg/dL ; 0.16-0.36 mmol/L
 Chemical method Child 2.0-5.5 mg/dL ; 0.12-0.33 mmol/L
 Reagent: Sodium Nitroprusside
 Positive color: Indophenol blue Ammonia
Adult 19-60 ug/dL ; 11-35 umol/L
 Reaction:
Child 68-136 ug/dL ; 40-80 umol/L
NH3 + phenol + hypochlorite  indophenol
blue
Enzymatic Method CONVERSION FACTORS
 Most commonly used  Urea – 0.357
 Uses glutamate dehydrogenase  Creatinine – 88.4
(GLDH)  Uric acid – 0.059
 Measures decrease in absorbance at  Ammonia – 0.59
340 nm
 NADPH consumed is proportional to
ammonia concentration