Download Non-protein Nitrogen Compounds

Non-Protein
Nitrogen(NPN)
Compounds
Non-protein Nitrogen Compounds
The determination of nonprotein
nitrogenous substances in the blood has
traditionally been used to monitor renal
function.
 Nitrogen containing compounds that are
not proteins or polypeptides
 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
 These processes release
nitrogen, which is converted to
ammonia
 Synthesized in the liver from
CO2 and Ammonia that arises
from deamination of amino acids
Urea Nitrogen (Blood) BUN
Assays for urea were based on measurement
of nitrogen, the term blood urea nitrogen
(BUN) has been used to refer to urea
determination.
 Excreted by the kidneys – 40% reabsorbed
 <10% of the total are excreted through the
gastrointestinal tract and skin.
 Concentration is determined by:

◦ Renal function
◦ Dietary intake
◦ Protein catabolism rate
Clinical Application
 Measurement of urea is used to:
◦ evaluate renal function,
◦ to assess hydration status,
◦ to determine nitrogen balance,
◦ to aid in the diagnosis of renal disease,
◦ and to verify adequacy of dialysis.
Disease Correlations
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:

◦ Prerenal
◦ Renal
◦ and postrenal
Pre-Renal Azotemia

Reduced renal blood flow
Less blood is
delivered to the kidney
less urea filtered
◦ Anything that produces a decrease in functional
blood volume, include:





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
of 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
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

Analytical methods

Urease → hydrolysis of urea to ammonium ion ,
then detect ammonium ion (NH4+)
 Enzymatic
◦ The most common method couples the urease
reaction with glutamate dehydrogenase
Analytical methods

Indicator dye
NH4+ + pH indicator → color change

Conductimetric
◦ Conversion of unionized urea to NH4+ and
CO32- results in increased conductivity
Reference range of Urea N:
Serum or plasma: 6-20 mg/dl
24 hours Urine: 12-20 g/day
Creatinine/ Creatine
Creatine is 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

Daily creatinine excretion is fairly stable.

It’s a very good test to evaluate renal function
Disease Correlations

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.
Disease Correlations

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
 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
Analytic Methods
creatininase
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

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 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 PCT
 Some secreted by DCT
 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

◦ 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

◦
causes elevated levels of uric acid because
filtration and secretion are hindered.
Disease Correlations
 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
 Free ammonia is toxic;

◦ however, ammonia is present in the plasma in
low concentrations
Disease Correlations

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.
Disease Correlations

Reye’s Syndrome
◦ Most commonly seen in children
◦ Often preceded by viral infection treated with
aspirin
◦ Severe fatty infiltration of liver
◦ May be fatal if ammonia levels remain high
◦ 100% survival if ammonia stays below 5x normal
Disease Correlations
Ammonia is of use in the diagnosis of
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