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

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

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,

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

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

NH4+

dye
+ 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 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
Its

creatinine excretion is fairly stable.

a very good test to evaluate renal

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

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
Fanconis 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

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

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
Reyes

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

dl

Interval: Adult Plasma 19 60 g /