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Non-protein Nitrogen (NPN) Compounds
Principles of Medical Laboratory Sciences (Universidad de Zamboanga)
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NON-PROTEIN NITROGEN (NPN) COMPOUNDS

 Nitrogen containing compounds that
are not proteins or polypeptides
 The determination of nonprotein
nitrogenous substances in the blood has
traditionally been used to monitor renal
function.
 There are many different unrelated
NPNs, but we are only interested in 4 of
them:
 Blood Urea Nitrogen (BUN) –
45%
 Uric Acid – 20%
 Creatinine – 5%
 Ammonia - .2%

Urea Nitrogen (Blood) BUN
 Highest concentration of NPN in blood
 Major excretory product of protein
metabolism
 These processes release nitrogen, which
is converted to ammonia
 Synthesized in the liver from CO2 and
Ammonia that arises from deamination
of amino acids
 Organisms synthesize urea from
ammonia because ammonia (a common
metabolic waste product) raises pH in
cells to toxic levels. Therefore, urea
synthesis is necessary even though it
costs energy to produce.
 Urea is neither acidic nor basic, so it is a
perfect vehicle for getting rid of
nitrogen waste
Azotemia: elevated conc. of urea in blood
Very high plasma urea concentration
accompanied by renal failure is called uremia, or
the uremic syndrome
 Causes of urea plasma elevations are:
Pre-Renal Azotemia
o Reduced renal blood
flow Less blood is
delivered to the kidney
less urea filtered
o Anything that produces
a decrease in functional
blood volume, include:
 Congestive
heart
failure,shock,
hemorrhage,
dehydration
o High protein diet or
increased catabolism
(Fever, major illness,
stress)
Renal Azotemia
o Decreased renal
function causes
increased blood urea
due to poor excretion
o Acute & Chronic renal
failure
o Glomerular nephritis
o Tubular necrosis &
other Intrinsic renal
disease
Glomerulonephritis, also known as glomerular
nephritis, abbreviated GN, is a renal disease
characterized by inflammation of the glomeruli,
or small blood vessels in the kidneys

Post-Renal Azotemia
Obstruction of urine flow
o Renal calculi
o Tumors of bladder or
prostate
o Severe infections
 Decreased Urea Nitrogen
 Low protein dietary intake
 Liver disease (lack of synthesis)
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

Severe vomiting and/or
diarrhea (loss)
Increase protein synthesis
Analytical methods
 Assays for urea were based on
measuring the amount of nitrogen in
the sample (BUN)
 Current analytic methods have retained
this custom and urea often is reported
in terms of nitrogen concentration
rather than urea concentration (urea
nitrogen).
 Urea nitrogen concentration can be
converted to urea concentration by
multiplying by 2.14
DIRECT METHOD:
 Creatinine is produced as a waste
product of creatine and creatine
phosphate.
 Creatine Phosphate –
phosphoric acid = Creatinine
 Creatine – water = Creatinine
 Creatinine is released into circulation at
stable rate proportional to muscle mass
 Filtered by glomerulus
 Excreted in urine
 Plasma creatinine concentration is a
function of:
 relative muscle mass,
 rate of creatine turnover
 and renal function
 Daily creatinine excretion is fairly stable.
 It’s a very good test to evaluate
renal function
Disease Correlations
 Fearson reaction – uses Diacetyl
monoxime and strong acid to produce
yellow diazine derivative
INDIRECT METHOD
 Urea is first hydrolyzed to yield
ammonium and bicarbonate ions by
enzyme urease
 Berthelot reaction – ammonium is
converted to indophenol with the
addition of nitroprusside.
 Samples specimen include serum,
plasma, and urine
 If not analyzed within a few hours,
specimen should be refrigerated to
prevent bacterial decomposition,
thymol is also added
CREATININE
 Creatine is synthesized in liver from
arginine, glycine & methionine
 Converted to Creatine Phosphate = high
energy source for muscle tissue
 Elevated Creatinine is found with
abnormal renal function (i.e., GFR)
 Measurement of creatinine
concentration is used to determine:
 sufficiency of kidney function
 and the severity of kidney
damage
 and to monitor the progression
of kidney disease.
 GFR is the volume of plasma filtered (V)
by the glomerulus per unit of time
 GFR is used to estimate renal function
 Creatinine Clearance
 A measure of the amount of
creatinine eliminated from the
blood by the kidneys per unit
time
 Plasma concentration of
creatinine is inversely
proportional to clearance
 Therefore, increased plasma levels
mean decreased GFR
Analytic Methods
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 Jaffe reaction Kinetic Jaffe Reaction –
most frequently used, was first
described in 1886
 Creatinine reacts with picric acid in
alkaline solution → red-orange
chromogen
 Kinetic Jaffe Reaction
 Rate of change in absorbance is
measured
ENZYMATIC METHOD
 Using creatininase, creatine kinase,
pyruvate kinase and lactate
dehydrogenase
Uric acid is a final breakdown product of purine
metabolism (adenosine/guanine) in liver
Most other mammals degrade it further to
allantoin
Uric acid is transported to kidney and filtered
(70%)
98% reabsorbed in proximal convoluted tubule
Some secreted by distal convoluted tubule
Net amount 6-12% of filtered amount
Remaining 30% by GIT
 Uric Acid Present in plasma as
Monosodium Urate
 At plasma pH → relatively
insoluble Conc. > 6.8 mg/dl →
plasma saturated → urate
crystals may form & precipitate
in tissue
Uric acid is measured to:
 Assess inherited disorders of purine
metabolism
 To confirm diagnosis and monitor
treatment of gout,
 To assist in the diagnosis of renal calculi,
 To prevent uric acid nephropathy during
chemotherapeutic treatment, and to
detect kidney dysfunction
Disease Correlations
 Gout Primarily in men Onset 30-50
years
 UA greater than 6.0 mg/dL
 Pain & inflammation of joints by
precipitation of sodium urates in tissues
 Increased risk of renal calculi
 Hyperuricemia due to overproduction
of uric acid in 25-30%
DISEASE CORRELATIONS
 Increased catabolism Chronic renal
disease:
 causes elevated levels of uric
acid because filtration and
secretion are hindered.
 occurs in patients on
chemotherapy for diseases such
as leukemia & multiple
myeloma.
 Allopurinol inhibits xanthine
oxidase, an enzyme in the uric
acid synthesis pathway, is used
to treat these patients.
DISEASE CORRELATIONS
 Hypouricemia Secondary to severe liver
disease
 Defective renal tubular
reabsorption
 Chemotherapy with 6mercaptopurine or azathioprine
– inhibit purine synthesis
 Over treatment with allopurinol
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
Fanconi Syndrome (also known
as Fanconi's syndrome) is a
disorder in which the proximal
tubular function of the kidney is
impaired,[1] resulting in
decreased reabsorption of
electrolytes and nutrients back
into the bloodstream
 Most common cause of abnormal
ammonia levels
 Ammonia is not removed from
circulation & not converted to urea
 Elevated ammonia levels are neurotoxic
and are often associated with
encephalopathy.
Analytic Methods
 Primary method uses enzyme uricase
(urate oxidase) to convert uric acid to
allantoin
 Differential absorption at 293 nm
 Uric acid has a UV absorbance peak at
293 nm. Whereas allantoin does not
 Proteins also absorb near this
wavelength
 Newer methods couple uricase with
catalase or peroxidase action on
hydrogen peroxide product from
allantoin production
 Some interferences from reducing
agents Reference range:
 Males 0.5-7.2,
 Females: 2.6-6.0 mg/dl
Ammonia - comes from deamination of amino
acids
 Digestive & bacterial enzymes in
intestine
 Also released from muscle
during exercise
 Consumed by parenchymal cells of liver
and converted to urea
 Free ammonia is toxic;
 However, ammonia is present in
the plasma in low
concentrations
 Disease Correlations - Severe liver
disease
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