PLAZA, GEORGE CARR, G.

– Nonprotein Nitrogenous Compounds – Assignment

Clinical Chemistry 1- Nonprotein Nitrogenous (NPN) Compounds

Urea Creatinine Uric Acid Ammonia

Description/  Highest concentration in  Formed from creatine  Major product of  Produced in the
Bio- the blood among NPNs - and creatine phosphate catabolism of the deamination of
chemistry most abundant (45-50% of in muscle purine nucleic acids amino acids during
total NPNs)  Formed as BY-PRODUCT  Not for kidney function protein
 Major excretory product of muscle activity test metabolism
of protein metabolism  Excreted into the plasma  Mostly reabsorbed in  Removed from the
 Formed/synthesized in at a constant rate the proximal tubules circulation and
the liver from amino groups  INVERSELY related to and reused converted to urea
and free ammonia glomerular filtration rate  Filtered by glomerulus in the liver
generated during protein (GFR) and secreted by distal  FREE AMMONIA is
catabolism  Better renal function tubules into the urine toxic
 Carried in the blood to marker than urea (in small amounts)  Low ammonia
the kidney  Insoluble in plasma concentration is
 Readily filtered from the Creatine  Deposited in tissues present in the
plasma by the - synthesized in the liver and joints plasma
glomerulus from Arginine, Methionine,  Also produced by
 90% --> excreted in the Glycine  Purines (adenine and bacterial
urine - transported to other tissues guanine) are converted metabolism in the
 10--> reabsorbed via (muscles) to uric acid in the LIVER intestinal lumen
passive diffusion - converted to CREATINE  Transported to the  Some endogenous
 Its concentration in the PHOSPHATE (serves as a kidney (filtered by the ammonia results
plasma is determined by high-energy source) glomerulus & from anaerobic
- Protein consumption reabsorbed) metabolic
- Rate of protein Creatine phosphate (loses  Reabsorbed (98-100%) reactions that
catabolism phosphoric acid) + Creatine mostly in the proximal occur in skeletal
- Renal (loses water) = creatinine tubules muscle during
Function/perfusion  Monosodium urate: exercise
-Removed by glomerular most common form of  Consumed by the
 PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

filtration and excreted in uric acid in the plasma parenchymal cells

the urine  Relatively insoluble (pH of the liver in the
-Small amounts of creatinine of plasma) Krebs-Hanseleit or
are:  Become saturated and urea cycle to
-Secreted by proximal crystals may form and produce urea
tubules precipitate in the (nontoxic
-Reabsorbed by renal tissues if the compound that is
tubules concentrations exceed excreted in the
- Daily creatinine excretion is 6.8 mg/dL urine)
reasonably stable  At normal
physiologic pH,
most ammonia in
the blood exists as
ammonium ion
(NH4+)
 Excreted as
ammonium ion and
acts as BUFFER in
the urine
Clinical  Evaluate renal  Determine the Uric acid is measured to: Blood ammonia
Application function sufficiency of kidney  Confirm diagnosis and concentration provides
 Assess hydration function monitor treatment of useful information of
status  Determine severity of gout these following clinical
 Determine nitrogen kidney damage  Prevent uric acid conditions
balance  Monitor progression of nephropathy during  Hepatic
 Aid in the diagnosis kidney disease chemotherapy failure/severe liver
of renal diseases Plasma creatinine  Assess inherited disease (most
 Verify the adequacy concentration is dependent disorders of purine common cause of
of dialysis on: metabolism disturbed
- Often reported as nitrogen 1. Relative muscle  Detect kidney ammonia
concentration (BUN) mass dysfunction metabolism)
2. Rate of creatinine  Assist in the diagnosis  Hepatic coma
turnover of renal calculi  Reye’s syndrome
3. Renal function Commonly in
 PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

children
 Not heavily affected by  Preceded by a
diet (reason why viral
creatinine is preferred infectionand
over urea) the
 Endogenously produced administration
by the muscles of aspirin
 Concentration is An acute
relatively stable metabolic
 Used as a measure of disorder of the
completeness of 24-hour liver
urine samples Autopsy
 Creatinine clearance = a findings show
measure of creatinine severe fatty
eliminated from the infiltration of
blood by the kidneys and liver
Glomerular Filtration  Inherited
Rate (GFR) are used to deficiencies of urea
gauge renal function cycle enzymes
= reported in units of
mL/min - Assay of blood
ammonia - monitor
hyperalimentation
therapy
- Measurement of
URINE ammonia-
confirm the ability of
the kidneys to
produce/secrete
ammonia
Pathophy-  Impaired renal function  Elevated creatinine Abnormal increased  Ammonia is
siology  buildup of concentration --> plasma uric acid not removed in
urea in the abnormal renal function concentration can be severe liver
circulation (glomerular filtration found in disease
PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

(increase function)  Gout- uric acid  Leads to

amount of urea  Plasma creatinine deposition in JOINTS increase
in the blood) concentration is  Found primarily ammonia levels
 Increased plasma urea INVERSELY proportional in men in the
concentration can be to creatinine clearance  Pain and circulation
classified into:  Renal damage is inflammation of the  High levels of
1.Pre-renal - due to suspected when plasma joints (caused by ammonia leads to
reduced renal blood flow creatinine is elevated & precipitation of neurotoxicity and
or increases in protein GFR is decreased sodium urates) encephalopthy
diet  Plasma creatine  Hyperuricemia-  Hyperammone
Causative factors: concentration is not result of mia - caused by
Congestive heart elevated in renal overproduction of deficiencies of
failure, shock, diseases uric acid; may enzymes in the
hemorrhage,  Plasma creatine is exacerbated by a urea cycle
dehydration, increased ELEVATED in muscles purine-rich diet,
protein catabolism & diseases (muscular drugs and alcohol
high protein diet dystrophy, poliomyelitis, --> Increase uric
2. Renal- due to hyperthyroidism, acid
decreased renal function trauma) Patients are
or damaged kidneys susceptible to the
Causative factors: formation of calculi
Acute/chronic renal  Tophi- uric acid
failure, Renal diseases crystalline deposition in
3. Post-renal- due to TISSUES
obstruction of urine flow  Causes
@ urinary tract deformities
 Decreased urea  Patients on
concentration can be chemotherapy
caused by  Increased
1. Low protein metabolism of cell
intake nuclei
2. Severe vomiting  Occurs in
3. Liver disease patients with
4. Pregnancy proliferative
PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

diseases (leukemia,
lymphoma,
multiple myeloma &
polycythemia)
 Allopurinol -
inhibits xanthine
oxidase (enzyme in
the uric acid
synthesis pathway
used for treatment
in decreasing uric
acid in the plasma
 Megaloblastic/Hemolyti
c anemia
 Ingestion of diet rich in
purines
 Disorders in purine
metabolism
 Lesch-Nyhan
syndrome -
hypoxanthine
guanine
phosphoribosyl
transferase
deficiency
 Lack of this
enzyme prevents
utilization of
purine bases in
the nucleotide
salvage pathway
 X-linked genetic
disorder (seen
only in males)
PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

 Mental
retardation and
self-mutilation-
characterize this
extremely rare
disease
 Phosphoribosyl
pyrophosphate
synthetase
deficiency
 Glycogen
storage disease
type I-
glucose-6-phosph
ate deficiency
 Fructose
intolerance-
fructose-1-phosph
ate aldolase
deficiency
 Uric acid nephrolithiasis
 Formation of kidney
stones (renal calculi)
 Insoluble uric acid
precipitates in acidic
urine to form calculi
which can cause
FLANK pain
 Stones may be
dissolved by
alkalinization of the
urine/ treated by
increased fluid/water
intake and
 PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

administration of
xanthine oxidase
inhibitors (reduces
uric acid production)

HYPOURICEMIA
- usually secondary to
severe liver disease or
defective tubular
reabsorption, as in
Fanconi syndrome (a
disorder of reabsorption in
the proximal convoluted
tubules of the kidney)
- caused by chemotherapy
with 6-mercaptopurine
or azathioprine,
inhibitors of de novo
purine synthesis, and as
a result of overtreatment
with allopurinol
- have shown association
with neurodegenerative
conditions such as
Alzheimer’s and
Parkinson’s diseases

Methods Specimen used: Serum/ Specimen used: Specimen used: Specimen used:
Plasma (also urine) Serum/Plasma/Urine Serum/Plasma Venous whole blood
 Should not be  Hemolyzed, icteric & (HEPARINIZED)  Heparin and EDTA
hemolyzed lipemic samples should  Serum samples may be are suitable
 Avoid using sodium be avoided stored in refrigerator anticoagulants.
citrate and sodium  Urine should be for 3-5 days  Must be placed on
fluoride as refrigerated after  Serum should be ICE immediately
PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

anticoagulants since collection or FROZEN if removed from cells as  Must be

they inhibit urease longer storage than 4 quickly as possible to centrifuged at 0 to
 Fasting sample is not days is required prevent dilution by 4 degrees Celsius
usually required intracellular contents within 20 minutes
 Specimens should be METHODS  Fasting is unnecessary of collection and
refrigerated if analysis is 1. Chemical Methods  Gross lipemia should be the plasma
not done within a few 1.1. Jaffe reaction avoided removed
hours  Most frequently  High bilirubin immediately
 Urea is used to measure concentration may yield  Should be assayed
susceptible to creatinine false-decrease results as soon as possible
bacterial  Principle:  EDTA or fluoride or FROZEN
composition Creatinine reacts additives should not be  Frozen plasma is
(especially urine) with picric acid in used for specimens that stable for several
alkaline solution will be tested for days at -20
METHODS (alkaline picrate) uricase method degrees Celsius
1. Direct Method to form a  Urine collections must  Hemolysis should
- Directly red-orange be alkaline (pH= 8) be removed
measures urea chromogen  Erythrocytes
-Known as  Reagent:Picric Uric acid is readily oxidized contain 2-3x as
“Rosenthal acid in alkaline to allantoin (a more soluble much ammonia as
method” solution (10% end product) and can plasma
-Reagent: Diacetyl NaOH) function as a reducing  Cigarette
monoxime  Nonspecific and agent in chemical reactions smoking is a
- End product: subject to positive significant source
Yellow diazine interferences METHODS of ammonia
2. Indirect Method (ascorbate, 1. Chemical Method contamination.
- Measures acetoacetate, 1.1 Caraway Method Patients should not
nitrogen acetone, glucose  Principle: smoke for several
- Known as & pyruvate) Oxidation-reduction hours
“Microkjedahl reaction  Substances that
method” 1.2 Jaffe Kinetic  Oxidation of uric acid in influence the in
3. Enzymatic Methods  Jaffe reaction a protein-free filtrate vivo ammonia
- Most commonly performed with subsequent concentration
used method in directly on sample reduction of Ammonium salts
PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

measuring urea  Detection of Color phosphotungstic acid in Asparaginase

1. Catalyzed by formation timed alkaline solution to Barbiturates
urease (urea to avoid tungsten blue Diuretics
amidohydrolas interference of  Reagent: Ethanol
e) noncreatinine Phosphotungstic acid Hyperalimentatio
- enzymatic chromogens  Produces allantoin & n
production of  Requires tungsten blue Narcotic
ammonium automated complex Analgesics
ion from urea equipment for  However, it lacks Drugs (increases
2. Ammonium ions precision specificity/ requires ammonia
can be measured:  Subject to positive protein removal concentration)
- Berthelot’s interference by  Diphenhydr
method (direct alpha-ketoacids Uric acid + phosphotungstic amine
measurement and acid + O2 --> Allantoin +  Lactobacillu
of ammonia) cephalosporins tungsten blue + CO2 s
- Glutamate  Bilirubin and acidophilus
dehydrogenase hemoglobin can 2. Enzymatic methods  Lactulose
- coupled cause negative (most commonly used)  Levodopa
enzymatic bias 2.1 Uricase-
-Requires  Used routinely catalyzes the Sources of Error in
NADH (rapid, oxidation of uric acid Ammonia
-Rate of inexpensive and to allantoin Contamination
disappearance easy to perform) more specific 1. Tobacco smoke
of NADH  Measures the 2. Urine
measured at 1.3 Jaffe with differential absorption 3. Ammonia in
340 nm adsorbent of uric acid and detergent, glasswares,
- Used on many  Creatinine in allantoin at 293 nm reagents and water
automated protein-free  Difference in
instruments; filtrate adsorbed absorbance before METHODS
best as a onto and after incubation 1. Titration method
kinetic  Fuller’s earth with uricase is  “Conway
measurement reagent: proportional to the method”
aluminum uric acid  Uses volatility of
magnesium concentration ammonia to
PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

- Indicator dye silicate  Proteins- cause separate the

-Ammonium  Lloyd’s reagent: high background compound in a
ion with pH sodium absorbance (reduces microdiffusion
indicator aluminum specificity) chamber
proceeds to a silicate  Hemoglobin and  Ammonia gas from
color change  Then reacted with xanthine- cause the sample
-Used in alkaline picrate to negative interference diffuses into a
automated form colored separate
systems, complex 2.2 Coupled compartment and
multilayer  Adsorbent enzymatic methods is absorbed in a
film reagents improves  Measure the solution containing
and dry specificity hydrogen peroxide pH indicator
reagent strips produced as uric acid
2. Enzymatic Methods is converted to 2. Berthelot
Conductometri  Coupled enzymatic allantoin Reaction (chemical
c methods  Uses method)
-Conversion of  Uses following peroxidase/catalase  Reagent: Sodium
unionized urea enzymes for dry to catalyze a chemical Nitroprusside
to ammonium slide analyzers: indicator reaction  Positive Color:
ions and  Creatininase  Color produced is Indophenol blue
carbonate ions  Creatinase proportional to the  NH3 + phenol +
(CO32-)  Sarcosine oxidase quantity of uric acid in hypochlorite -->
- Specific and  Peroxidase the specimen (sodium
rapid  Readily automated nitroprusside)-->
Creatine Analytical  Interference: Indophenol blue
Methods Bilirubin and ascorbic
 Traditional method: acid (reducing agents 3. Enzymatic
relies on the analysis of that can destroy method
the sample using an peroxide)  Most commonly
endpoint Jaffe method used/ convenient
for creatinine before and  Uses GLUTAMATE
after it is heated in acid DEHYDROGENASE
solution (GLDH)
 Heating --> creatine  Measures decrease
PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

converts to creatinine in absorbance at

 Difference of the two 340nm
samples measurements  NADPH consumed
= creatine concentration is proportional to
 High temp = formation of ammonia
additional chromogens concentration
and poor precision  NADPH is the
preferred
Creatininase-H2O2 coenzyme because
 In a series of it is used
enzymatically catalyzed specifically in
reactions, creatinine is GLDH
hydrolyzed to  Adenosine
creatine,which is diphosphate is
converted to sarcosine added to the
and urea. Sarcosine is reaction mixture to
oxidized to glycine, increase the rate of
CH2O, and H2O2. reaction and to
Peroxidase-catalyzed stabilize GLDH
oxidation of a colorless
substrate produces a 4. Colometric Assay
colored product + H2O  Dry slide
automated system
Creatininase-CK uses thin-film
 In a series of reactions colometric assay
catalyzed by the  Ammonia reacts
enzymes creatininase, with an indicator to
creatine kinase, pyruvate produce a colored
kinase, and lactate compound that is
dehydrogenase, NAD+ is detected
produced and measured spectrophotometri
as a decrease in cally
absorbance
 Can be measured by 5. Ion-selective
 PLAZA, GEORGE CARR, G. – Nonprotein Nitrogenous Compounds – Assignment

HPLC electrode
 Electrode
measures the
change in pH of a
solution of
ammonium
chloride as
ammonia diffuses
across a
semipermeable
membrane
 Good accuracy and
precision
 Membrane stability
may be a problem
REFERENCE
METHOD
ISOTOPE DILUTION MASS SPECTROMETRY
CONVERSIO 0.357 88.4 0.059 0.59
N FACTOR

Reference:
Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2018). Clinical chemistry: principles, techniques, and correlations. 8th ed.
Philadelphia: Wolters Kluwer Health/Hippincott Williams & Wilkins.