NON-PROTEIN

NITROGENS (NPNs)
OUTLINE
• Urea
• Uric Acid
• Creatinine/Creatine
• Ammonia
 OUTLINE
• Physiology
• Methods
• Specimen Requirements and Interfering
subs.
• Reference Intervals
• Clinical Application
• Pathophysiology
 INTRODUCTION
• Generally, NPNs are used to monitor renal
function → KIDNEY FUNCTION TESTS.
• Historically, most analytical methods
require removal of proteins before analysis
→ NON-PROTEIN NITROGENS.
 COMPONENTS OF NPNs
Approximate Plasma Concentration
Compound (% of Total NPN)
Urea 45
Amino acids 20
Uric Acid 20
Creatinine 5
Creatine 1-2
Ammonia 0.2
UREA
 UREA: Physiology
• It is synthesized in the liver from Co2 and
ammonia arising from the deamination of
amino acids by means of the ornithine.
• Approximately, 50% is reabsorbed and 50%
is excreted.
UREA: Methods
• The test is called BUN – Blood Urea Nitrogen

3 METHODS:
1. Urease Enzymatic method – oldest method
2. Colorimetric method – uses both enzyme and
a coloring reagent
3. Spectrometric method – to quantify levels
UREA: Enzymatic Method
Principle: Urea is hydrolyzed in presence of
UREASE to produce AMMONIA and CO2. The
ammonia combines with oxoglutarate and
NADH in presence of GLDH to yield glutamate
and NAD.
UREA: Colorimetric Method
Nessler’s Method Berthelot Method
Enz: Urease Enz: Urease
Rgt: Potassium Rgt: Phenol and
tetraiodomercurate - hypochlorite
K2(HgI4)
Principle – The reagent Principle – The reagent
becomes deep yellow in becomes blue in the
the presence of ammonia. presence of ammonia.
At higher levels, a brown
precipitate may form.
 REMEMBER!
• Nessler – YELLOW
• Berthelot - BLUE
UREA: Spectrometric Method
• Isotope-Dilution Mass spectrometry
–Considered as the reference method.
–Can quantify the levels of BUN
UREA: Specimen Requirements
and Interfering Substances

• Fluoride or citrate will inhibit the urease

utilized in the coupled enzymatic methods.
• Fasting sample is usually not required.
• Avoid high protein intake.
UREA: Specimen Requirements
and Interfering Substances

• A non-hemolyzed sample is recommended.

• Hemoglobin is also a protein.
• Urea is quite susceptible to bacterial
decomposition, so samples (especially urine)
that cannot be analyzed within a few hours
should be refrigerated.
UREA: Reference Interval

CONVERSION FACTOR: 0.357

UREA: Clinical Application
INCREASED RESULTS:
• Azotemia – elevated urea level in the blood
• Uremia – Elevated level with signs and
symptoms of renal failure
DECREASED RESULTS:
• Low protein intake
• Severe vomiting and diarrhea
• Liver disease
• Pregnancy
UREA: Pathophysiology
RENAL DISORDERS:
• PRERENAL
• RENAL
• POSTRENAL
 PRERENAL AZOTEMIA
• Events occurring “before“ the kidneys
Causes:
• Reduce blood flow → less urea is excreted →
ELEVATED BUN
• High protein diet → oversaturation at the
kidneys → ELEVATED BUN
• High protein catabolism → oversaturation at
the kidneys → ELEVATED BUN
 PRERENAL AZOTEMIA
• Conditions/Events:
REDUCED HIGH PROTEIN HIGH PROTEIN
BLOOD FLOW DIET CATABOLISM
• Congestive • STEAK!!! • Stress
heart failure (YUM!) • Fever
• Shock • Cancer
• Hemorrhage • Steroid
• Dehydration therapy
 RENAL AZOTEMIA
• Events occurring “inside“ the kidneys
• Glomerular disorders
• Tubular disorders
 POSTRENAL AZOTEMIA
• Events occurring “after“ the kidneys
• Obstruction of urine flow – Stones (Calculi),
tumors, enlarged prostate, severe
inflammation due to infection.
 How to differentiate the TYPES?
• Check for BUN, Creatinine, and
BUN/Creatinine Ratio (BCR)
• BCR: 10:1 or 20: 1

BUN CREATININE BCR

Prerenal High Low/Normal High
Renal High High Normal
Postrenal Low/Normal High Low
URIC ACID
URIC ACID: Physiology
• Final Breakdown product of PURINE
metabolism by the liver.
• In some mammals – ALLANTOIN is the end-
product which is more water-soluble in
nature.
URIC ACID: Physiology
• 98 % to 100% is REABSORBED in the
proximal tubule, small amounts of uric
acid are then SECRETED by the distal
tubules and ultimately appear in the
urine.
URIC ACID: Methods
3 METHODS:
1. Uricase Enzymatic Method
2. Caraway Method – colorimetric method
3. Spectrometric Method – REFERENCE
METHOD
• Isotope-Dilution Mass spectrometry
URIC ACID: Enzymatic Method
Principle: Uricase oxidizes uric acid to form
allantoin and it will be readily measured
spectrophotometrically.
URIC ACID: Caraway Method
Principle: Uricase oxidizes uric acid to form
allantoin. Allantoin is used as reducing
agent, for reduction of phosphotungstic
acid to tungsten blue.
• Rgts:
–Uricase
–Phosphotungstic acid
URIC ACID: Specimen Requirements
and Interfering Substances
• Significant hemolysis
• Releases glutathione, also may give
low values
• Drugs, such as thiazides and salicylates
(ASPIRIN), have been shown to cause
elevated values for uric acid.
• Do not use EDTA or Fluoride additives.
URIC ACID: Reference Interval
C

CONVERSION FACTOR: 0.0595

URIC ACID: Clinical Application
• HYPERURICEMIA – elevated uric acid
• HYPOURICEMIA – low uric acid

• Gout – occurs in elevated levels of uric acid.

• Patients have pain and inflammation of the
joints caused by precipitation of sodium
urates in the joint resulting from the high
levels of uric acid.
URIC ACID: Pathophysiology
 Lesch-Nyhan Syndrome
• It affects the MALE gender only.
• Deficiency of HGPRT – Hypoxanthine
Guanine Phospho-Ribosyl Transferase
• Increased Purine production → increase uric
acid
Lesch-Nyhan Syndrome
L Lips and fingers are bitten
E Elevated uric acid
S Spasticity
C Calculi in kidney
H HGPRT deficiency
Nyhan - Ny-MAN
 Glycogen storage disorder type I
VON GIERKE DISEASE
• Deficiency of Glucose-6-Phosphatase
• Diverts the glucose-6-phosphate metabolism
to uric acid synthesis → increased uric acid
 Gouty Arthritis
• Form of arthritis characterized by
recurrent attacks of red, tender, hot and
swollen joint.
• Most affected joint – base of the big toe –
commonly called as the PODAGRA.
• Tophi – pathognomonic for gout. These are
stones of uric acid crystals
 Hypouricemia
• Fanconi syndrome – disorder of the tubules
in the kidneys responsible for the
reabsorption of substances.

• Allopurinol intake
• Xanthine Oxidase inhibitor; used to
treat gouty arthritis.
CREATININE
CREATININE: Physiology
• Creatine is synthesized mainly in the liver
from glycine, arginine, and methionine.
• It is then transported to other tissues, such
as muscle, where it is converted to:
phosphocreatine, which serves as a high
energy source.
CREATININE: Physiology
CREATINE
↓
Phosphocreatine (Muscle)
↓
Loss of water or phosphoric acid
↓
CREATININE (Plasma)
CREATININE: Physiology
It is a function of:
• Relative muscle mass
• Creatine turnover
• Renal function
CREATININE: Methods
3 Methods:
1. Enzymatic method
2. Jaffe reaction – colorimetric method
3. Spectrometric method
–Isotope-Dilution Mass spectrometry –
considered as the reference method.
CREATININE: Enzymatic Methods
Enzymes:
a. Creatininase
b. Creatinase
c. Sarcosine oxidase
d. Peroxidase
CREATININE: Jaffe Reaction
• Principle – Creatinine reacts with picric
acid in alkaline solution to form a red-
orange chromogen.
CREATININE: Specimen Requirements
and Interfering Substances
• Hemolysis should be avoided, since there
are a significant number of nonspecific
chromogens in the red cells.
• Fasting is not required.
• A heavy protein ingestion may elevate levels
of creatinine.
CREATININE: Reference Interval

• CONVERSION FACTOR = 88.4

CREATININE: Clinical Application
• Creatinine clearance – measure of the amount
of creatinine eliminated from the blood by the
kidneys.
• Glomerular Filtration Rate:
Ucr – Urine creatinine UCrVu
PCr – Plasma creatinine GFR =
PCrT
Vu – Volume of urine
CREATININE: Clinical Application
• MDRD – Modification of Diet in Renal Disease
equation
• Dependent on plasma creatinine
concentration, age, gender and ethnicity.
• No need to memorize this formula ☺
CREATININE: Pathophysiology
• Recall the Prenal, Renal and Postrenal
disorders. ☺
AMMONIA
AMMONIA: Physiology
• It arises from the deamination of amino acids,
which occurs mainly through the action of
digestive and bacterial enzymes on proteins
in the intestinal tract.
• Most ammonia (NH3) in the blood exists as
AMMONIUM (NH4).
• It is excreted by the kidney and acts to buffer
urine.
AMMONIA: Methods
1. Conway Method – one of the first methods.
2. Ion-selective Electrode Method –
modification of Conway Method
3. Enzymatic Method
4. Colorimetric Method
AMMONIA: Conway Method
• aka Microdiffusion method
• PRINCIPLE: The ammonia diffuses into a
separate compartment and is absorbed in
a solution containing a pH indicator. The
amount of ammonia is determined by
titration.
AMMONIA: ISE Method
• PRINCIPLE: The ammonia diffuses through
selective membrane into NH4Cl causing pH
change, which is measure by
potentiometry.
• Good accuracy but unstable.
AMMONIA: Enzymatic Method
• MOST COMMONLY USED
• Principle: The ammonia combines with
oxoglutarate and NADH in presence of GLDH
to yield glutamate and NAD.
AMMONIA: Colorimetric Method
• PRINCIPLE: The ammonia reacts with an
indicator called bromophenol blue to produce
a BLUE colored compound that is detected
spectrophotometrically.
AMMONIA: Specimen Requirements
and Interfering Substances
• Ammonia levels rise rapidly in whole blood
after drawing because of the deamination
of amino acids.
• Heparin and EDTA are the preferred
anticoagulants.
AMMONIA: Specimen Requirements
and Interfering Substances

• Samples should be centrifuged at 0 to 40C

within 20 minutes of collection
• Because red cells contain 2 to 3 times as
much ammonia as plasma, hemolysis should
be avoided.
AMMONIA: Reference Intervals

CONVERSION FACTOR = 0.587

AMMONIA: Clinical Applications
• HYPERAMMONEMIA – elevated levels
• HYPOAMMOENEMIA – low levels
• Severe liver disease represents the most
common cause of disturbed ammonia
metabolism.
• Arterial ammonia level is a better indicator
for the severity of the disease.
AMMONIA: Clinical Applications
ROLE IN THE MEDICAL MANAGEMENT:
• DIAGNOSTIC – enzyme disorders
• PROGNOSTIC – liver diseases
• MONITORING – hyperalimentation therapy
AMMONIA: Pathophysiology
• Reye's syndrome – the disease is preceded
by a viral infection and the child was given
aspirin.
• Aspirin in children may cause fatty liver –
metabolism of ammonia will be disturbed.
• Ammonia is very neurotoxic.
END