Non-Protein Nitrogen (NPN) Compounds

Non-protein
Nitrogen(NPN)
Compounds
NPN
• Nitrogen containing compounds that are not

proteins or polypeptides
• Tested by making a protein-free filtrate
• Total NPN Nessler’s Reaction
– HgI2 / KI
– Yellow color
• Useful clinical information is obtained from
individual components of NPN fraction
Clinically Significant NPN
• The NPN fraction comprises about 15 compounds
• Majority of these compounds arise from catabolism of proteins
and nucleic acids
Urea Nitrogen (Blood) BUN
• Highest concentration of NPN in blood
• Major excretory product of protein
metabolism
• Synthesized in the liver from CO2 and
Ammonia that arises from deamination of
amino acids
• Excreted by the kidneys – 40%
reabsorbed
• Concentration is determined by:
– Renal function
– Dietary intake
– Protein catabolism rate
Disease Correlations
• Azotemia: elevated conc. of urea in blood

• Uremia or uremic syndrome: very high
conc. accompanied by renal failure
• Causes of urea plasma elevations:
– Prerenal
– Renal
– and postrenal
Pre-Renal Azotemia
• Reduced renal blood flow = reduced urea to

the kidneys and less filtered
– Anything that produces a decrease in functional
blood volume
– E.g. Congestive heart failure, shock, hemorrhage,
dehydration
• High protein diet or increased catabolism
– Fever, major illness, stress
Renal Azotemia
• Decreased renal function causes increased

blood urea due to poor excretion
– Acute & Chronic renal failure
– Glomerular nephritis
– Tubular necrosis
– & other Intrinsic renal disease
Post-Renal Azotemia
• Obstruction of urine flow

– Renal calculi
• Tumors bladder or prostate
• Severe infections
Decreased Urea Nitrogen
• Low protein dietary intake

• Liver disease (lack of synthesis)
• Severe vomiting and/or diarrhea (loss)
• Increase protein synthesis
Analytical methods
• Based on measurement of amount of

nitrogen
• Urea is often reported in terms of nitrogen
conc. (urea nitrogen or BUN)
• Urea nitrogen conc. can be converted to
urea conc. by multiplying by 2.14 – mg/dl
Analytical methods
• Urease → hydrolysis of urea to ammonia, then detect
ammonia (NH4+)
• Colormetric Methods
– Coupled to Nessler’s Reagent
• HgI2 / KI – yellow color
– Berthelot reaction with nitroprusside (blue color)
Analytical methods
• Now kinetic with glutamate dehydrogenase (GLDH)
– Consumption of NADH
Reference range: Serum or plasma 6-20 mg/dl

Urine 12-29 g/day
Creatinine/ Creatine
• Creatine synthesized in Liver from arginine, glycine &
methionine
• Converted to Creatine Phosphate = high energy source
for muscle tissue
• Creatinine is produced as a waste product of creatine
and creatine phosphate.
Creatine Phosphate – phosphoric acid = Creatinine
Creatine – water = Creatinine

Creatinine production
Creatinine/Creatine
• 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
• The blood creatinine conc. is reasonably stable
• It’s a very good test to evaluate renal function
• Elevated Creatinine is found with abnormal renal
function (i.e. GFR)
• Creatinine Clearance
– Clearance of a substance is the volume of plasma
from which the substance is removed per unit time
(ml/min)
• Plasma concentration of creatinine inversely

proportional to clearance
• Therefore increased plasma levels mean decreased GFR
• Renal damage
Creatine
• Elevated in plasma and urine in

– Muscular dystrophy, hyperthyroidism, trauma,
• Plasma creatinine levels usually normal, but
urinary is elevated
• Specialized testing – not part of routine lab
Analytic Methods
• Jaffe Reaction 1886
– Creatinine reacts with picric acid in alkaline
solution → red-orange chromogen
• Kinetic Jaffe Reaction

– Rate of change is measured
• Enzymatic Method
– Using creatininase, creatine kinase, pyruvate
kinase and lactate dehydrogenase
Analytic Methods
Assay of creatine
• Analyzing the sample for creatinine

before and after heating in acid
solution using an endpoint Jaffe
method.
• Heating converts creatine to creatinine
and the difference between the two
samples is the creatine concentration.
Uric Acid
• Uric acid is final breakdown product of purine
metabolism (adenosine/guanine) in liver
• Most other mammals degrade further to
allantoin
• Uric acid is transported to kidney and filtered
(70%)
• 98% reabsorbed in PCT
• Some secreted by DCT
• Remaining 30% by GIT
Uric Acid
• Present in plasma as monosodium urate
• At plasma pH → relatively insoluble
• Conc. > 6.4 mg/dl → plasma saturated → urate
crystals may form & precipitate in tissue
• Disease states with increased plasma uric acid

– Gout
– Increased catabolism of nucleic acids
– And renal disease
• Gout
– Primarily men
– Onset 30-50 years
– Pain & inflammation of joints by precipitation
of sodium urates in tissues
– Increased risk of renal calculi
– Overproduction of uric acid in 25-30%
• Increased intake, alcohol, drugs
• Increased catabolism
– 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.
• Chronic renal disease also causes elevated

levels of uric acid because filtration and
secretion are hindered.
• Hypouricemia
– Secondary to severe liver disease
– Defective renal tubular reabsorption
• Fanconi’s Syndrome
– Chemotherapy with 6-mercaptopurine or
azathioprine – inhibit purine synthesis
– Over treatment with allopurinol
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 absorpance peak at 293 nm.
Whereas allantoin does not
– Proteins also absorb near this wavelength
Analytic Methods
• 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
• Not dependent on renal function
• Severe liver disease

– 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.
• Reye’s Syndrome
– Most commonly seen in children
– Often preceded by viral infection treated with
aspirin
– Acute metabolic disorder of the liver
– Severe fatty infiltration of liver
– May be fatal if ammonia levels remain high
– 100% survival if ammonia stays below 5x normal
• Inherited deficiencies of urea cycle

enzymes
• Measurement of ammonia used to diagnose
and monitor treatment
Analytic Methods
• Low concentration, volatile nature,

instability, easy contamination – testing
difficult
• Historical Methods
– Conway 1935 – volatilize, absorbed then
titrated
– Dowex 50 cation-exchange column + Berthelot
reaction
Analytic Methods
• Glutamate dehydrogenase
– Decrease in absorbance at 340 as NADPH is
consumed (oxidized)
• Direct ISE
– Change in pH of solution as ammonia diffuses
through semi-permeable membrane
• Reference Interval: Adult Plasma 19 – 60 μg / dl

Non-Protein Nitrogen (NPN) Compounds

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Non-protein

• Nitrogen containing compounds that are not

• Azotemia: elevated conc. of urea in blood

• Reduced renal blood flow = reduced urea to

• Decreased renal function causes increased

• Obstruction of urine flow

• Low protein dietary intake

• Based on measurement of amount of

Reference range: Serum or plasma 6-20 mg/dl

Creatine Phosphate – phosphoric acid = Creatinine

Creatine – water = Creatinine

• Plasma concentration of creatinine inversely

• Elevated in plasma and urine in

• Kinetic Jaffe Reaction

• Analyzing the sample for creatinine

• Disease states with increased plasma uric acid

• Chronic renal disease also causes elevated

• Primary method uses enzyme uricase (urate

Reference range: Males 0.5-7.2, Females: 2.6-6.0 mg/dl

• Comes from deamination of amino acids

• Severe liver disease

• Inherited deficiencies of urea cycle

• Low concentration, volatile nature,

• Reference Interval: Adult Plasma 19 – 60 μg / dl

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