ANGELES UNIVERSITY FOUNDATION

College of Allied Medical Professions – Department of Medical Technology

MTCC1 – Clinical Chemistry 1 (Laboratory)
A.Y. 2023-2024

NONPROTEIN NITROGEN COMPOUNDS ▪ Serious disease → death

▪ Survival reaches 100% if plasma NH3
➢ Early days: required the removal of protein from concentration remains below five times
a specimen before analysis normal
➢ The concentration of nitrogen-containing ▪ Ammonia concentration can be correlated
compounds in a protein-free filtrate was then with both the severity of the disease and
quantified spectrophotometrically by converting prognosis
nitrogen to ammonia o As ammonia levels rise, the
➢ The addition of Nessler’s reagent (K2[HgI4]) → prognosis decreases
produce a yellow color ➢ 3. Inherited Deficiency of Urea Cycle Enzymes:
▪ Testing should be considered for any neonate
with unexplained nausea, vomiting, or
neurological deterioration associated with
feeding
➢ Blood ammonia can also be used to monitor
hyperalimentation therapy, and urine ammonia
determination can be used to confirm the ability
of the kidneys to produce ammonia

Specimen Requirements
➢ Whole blood ammonia concentration increases
AMMONIA (NH3)
rapidly following specimen collection because of
in vitro amino acid deamination
➢ Produced in the deamination of amino acids
➢ Heparin and EDTA are suitable anticoagulants
during protein metabolism
➢ Venous blood should be placed on wet ice
➢ Free ammonia is extremely toxic to human cells
immediately
▪ However, ammonia is present in the plasma
➢ Samples should be centrifuged at 0-4C within 20
in low concentrations
minutes of collection and the plasma removed
➢ Ammonia is converted into urea by the liver
➢ Specimens should be assayed as soon as possible
(urea cycle) → urea (nontoxic) is excreted by the
or frozen
kidneys
▪ Frozen plasma is stable for several days at
–20°C
Clinical Application
➢ Erythrocytes contain two to three times as much
➢ Clinical conditions in which blood ammonia
ammonia as plasma
concentration provides useful information:
➢ Cigarette smoking:
▪ Hepatic failure
▪ Significant source of ammonia contamination
▪ Reye’s syndrome
▪ Recommended that patients do not smoke
▪ Inherited deficiencies of urea cycle enzymes
for several hours before a specimen is
➢ 1. Severe Liver Disease:
collected
▪ Is the most common cause of disturbed
ammonia metabolism
Substances
▪ Used to determine prognosis
Increase Decrease
▪ Liver function impaired → ammonia not
removed → blood concentration increases → Ammonium salts,
Diphenhydramine,
asparaginase,
neurotoxic (encephalopathy) Lactobacillus
barbiturates, diuretics,
➢ 2. Reye’s Syndrome: acidophilus, lactulose,
ethanol,
▪ Acute metabolic disorder of the liver levodopa, and several
hyperalimentation,
▪ Occurs most commonly in children antibiotics
▪ Frequently, the disease is preceded by a viral narcotic analgesics
infection and the administration of aspirin
GONZALES JR., Noel (BSMT3D) Exercises 13-15: Nonprotein Nitrogen Compounds | 1
 ANGELES UNIVERSITY FOUNDATION
College of Allied Medical Professions – Department of Medical Technology
MTCC1 – Clinical Chemistry 1 (Laboratory)
A.Y. 2023-2024

Analytical Methods URIC ACID

➢ Chemical Methods:
▪ 1. Digestion Method (Kjeldahl) ➢ Final product of catabolism of purine nucleic
o Nitrogen ion of the specimen is acids
converted to ammonia using hot ➢ Primarily formed in the liver as the end product
concentrated sulfuric acid in the of purine metabolism
presence of catalyst ➢ Relatively insoluble in plasma
➢ At high concentrations → deposited in the joints
and tissue → painful inflammation
➢ Reabsorption of 98% to 100% of the uric acid
from the glomerular filtrate occurs in the
proximal tubules
▪ 2. Nesslerization Reaction
o Yellow end color – N2 low to Clinical Application
moderate ➢ To confirm diagnosis and monitor treatment of
o Orange brown end color – N2 is high gout
▪ An arthritic condition characterized by the
precipitation of uric acid crystal deposition
in joints and tissues
▪ 3. Berthelot Reaction ➢ To assess and prevent uric acid nephropathy
during chemotherapeutic treatment
➢ To assess inherited disorders of purine
metabolism
➢ Enzymatic Methods using Glutamate ➢ To detect kidney dysfunction
Dehydrogenase (GLDH) ➢ To assist in the diagnosis of renal calculi
▪ Most common technique used currently
▪ Accurate and precise Specimen Requirements
▪ 340nm ➢ Can be measured in heparinized plasma, serum,
▪ NADPH: preferred coenzyme or urine
▪ ADP: added to increase the rate of reaction ➢ Serum should be removed from cells as quickly as
and to stabilize GLDH possible to prevent dilution by intracellular
contents
➢ Diet can affect uric acid concentration overall,
but a recent meal has no significant effect;
therefore, a fasting specimen is unnecessary
➢ Uric acid is stable in plasma or serum after red
blood cells have been removed
➢ Serum may be stored (refrigerated) for 3 to 5
days
Reference Ranges ➢ EDTA or fluoride additives should not be used for
specimens to be tested by uricase method
➢ Urine collections must be alkaline (pH 8)

Increase Decrease
High bilirubin
Salicylates Significant hemolysis,
Thiazides with concomitant
glutathione release
Higher concentrations are seen in newborns
GONZALES JR., Noel (BSMT3D) Exercises 13-15: Nonprotein Nitrogen Compounds | 2
 ANGELES UNIVERSITY FOUNDATION
College of Allied Medical Professions – Department of Medical Technology
MTCC1 – Clinical Chemistry 1 (Laboratory)
A.Y. 2023-2024

Analytical Methods UREA

➢ Chemical Methods: Caraway Method
▪ Most common method of this type ➢ NPN compound present in highest concentration
▪ Based on oxidation of uric acid in a protein- in the blood
free filtrate, with subsequent reduction of ➢ Major excretory product of protein metabolism
phosphotungstic acid in alkaline solution to ➢ Formed in the liver from amino groups (-NH2) and
tungsten blue free ammonia generated during protein
▪ Lacks specificity catabolism
▪ In the liver, ammonia is bound with CO2 to
form carbamoyl phosphate, which enters the
urea cycle and ultimately becomes urea
➢ Enzymatic Methods: Uricase Method ➢ Most of the urea in the glomerular filtrate is
▪ Uricase (urate oxidase): catalyzes the excreted in the urine
oxidation of uric acid to allantoin ➢ Concentration of urea in the plasma is
▪ More specific and are used almost exclusively determined by:
in clinical laboratories ▪ Protein content of the diet
▪ Simplest of these methods measures the ▪ The rate of protein catabolism
differential absorption of uric acid and ▪ Renal function and perfusion
allantoin at 293 nm
▪ The difference in absorbance before and Clinical Application
after incubation with uricase is proportional ➢ Evaluate renal function
to the uric acid concentration ➢ Assess hydration status
▪ Interferences: (1) Proteins can cause high ➢ Determine nitrogen balance
background absorbance, reducing sensitivity; ➢ Aid in the diagnosis of renal disease
(2) hemoglobin and (3) xanthine can cause ➢ Verify adequacy of dialysis
negative interference
Specimen Requirement
➢ Measurement in plasma, serum, or urine
▪ If plasma: ammonium ions and high
➢ Isotope Dilution Mass Spectrometry (IDMS): concentrations of sodium citrate and sodium
proposed reference method fluoride must be avoided
o Citrate and Fluoride inhibit urease
Reference Ranges ➢ Although the protein content of the diet
influences urea production, the effect of a single
protein-containing meal on urea concentration is
minimal and a fasting sample is not generally
required
➢ Urea is susceptible to bacterial decomposition,
so, specimens (particularly urine) that cannot be
analyzed within a few hours should be
Pathophysiology refrigerated
➢ Gout is a disease found primarily in men and is ▪ Timed urine specimens should be
usually first diagnosed between 30 and 50 years refrigerated during the collection period
of age
➢ Hyperuricemia: overproduction of uric acid Analytical Methods
➢ Hypouricemia: decreased uric acid excretion; less ➢ Enzymatic Method: used most frequently
common ▪ Hydrolysis of Urea by Urease
o Urease (urea amidohydrolase):
catalyzes hydrolysis of urea in the

GONZALES JR., Noel (BSMT3D) Exercises 13-15: Nonprotein Nitrogen Compounds | 3

 ANGELES UNIVERSITY FOUNDATION
College of Allied Medical Professions – Department of Medical Technology
MTCC1 – Clinical Chemistry 1 (Laboratory)
A.Y. 2023-2024

sample, and the ammonium ion CREATININE

(NH4+) produced in the reaction is
quantified ➢ Formed from creatine and creatine phosphate
➢ Excreted into the plasma at a constant rate
➢ Related to muscle mass
➢ Inversely related to the glomerular filtration rate
(GFR)
▪ Coupled Urease/Glutamate Dehydrogenase ➢ Although an imperfect measure, it is commonly
(GLD) Method used to assess renal filtration function
o Conversion of nicotinamide adenine ➢ Primarily synthesized in the liver from arginine,
dinucleotide (reduced, NADH) at 340 glycine, and methionine
nm is measured ➢ It is removed from the circulation by glomerular
filtration and excreted in the urine
➢ Daily creatinine excretion is reasonably stable

Clinical Application
➢ Determine the sufficiency of kidney function
➢ Determine the severity of kidney damage
➢ Monitor progression of kidney disease
➢ Chemical Method
▪ Diacetyl Monoxime Method Specimen Requirements
o Urea + DAM → Yellow Diazine ➢ May be measured in plasma, serum, or urine
Derivative ➢ Hemolyzed and icteric samples should be avoided
➢ Isotope Dilution Mass Spectrometry (ID-MS) ➢ Lipemic samples may produce erroneous results
▪ Reference method in some methods
▪ Highly specific ➢ A fasting sample is not required
▪ High protein ingestion may transiently
Reference Range elevate serum concentrations
Adult ➢ Urine should be refrigerated after collection or
Plasma/serum 6-20 mg/dL 2.1-7.1 mmol/L frozen if longer storage than 4 days is required
Urine, 24h 12-20 g/d 0.43-0.71 mol urea/d ➢ Ascorbate: interfere in enzymatic methods that
use peroxidase as a reagent
Pathophysiology ➢ Bilirubin: causes negative bias both in Jaffe and
➢ Azotemia: elevated concentration of urea in the enzymatic methods
blood ➢ Dopamine: affect both enzymatic and Jaffe
▪ Prerenal Azotemia: reduced renal blood methods
flow, less urea is filtered ➢ Lidocaine: causes positive bias in some enzymatic
▪ Renal Azotemia: caused by acute and chronic methods
renal failure, glomerular nephritis, tubular
necrosis → compromised urea excretion Analytical Methods
▪ Postrenal Azotemia: due to obstruction of ➢ Enzymatic Methods
urine flow anywhere in the urinary tract by ▪ Creatinine Aminohydrolase-CK Method
renal calculi, tumors of bladder/prostate,
severe infection
➢ Uremia/Uremic Syndrome: very high plasma
urea concentration accompanied by renal failure
➢ Decreased urea: low protein intake, severe liver
disease, late pregnancy, infancy

GONZALES JR., Noel (BSMT3D) Exercises 13-15: Nonprotein Nitrogen Compounds | 4

 ANGELES UNIVERSITY FOUNDATION
College of Allied Medical Professions – Department of Medical Technology
MTCC1 – Clinical Chemistry 1 (Laboratory)
A.Y. 2023-2024

▪ Creatininase-Hydrogen Peroxide Method o Inexpensive, rapid, and easy to

o Enhances the specificity of the Jaffe perform
reaction by utilizing coupled ▪ Disadvantages:
enzymatic methods o Positive bias from a-keto acids and
o A method using creatininase cephalosporine
(creatinine amidohydrolase, o Bilirubin, hemoglobin: negative bias
creatinase (creatine o Required automated equipment
amidinohydrolase), sarcosine ➢ Isotope Dilution Mass Spectrometry (IDMS)
oxidase, and peroxidase was ▪ Reference method
adapted for use on a dry slide ▪ Highly specific
analyzer
Reference Ranges

➢ Chemical Methods based on Jaffe Method

▪ The methods most frequently used to
measure creatinine are based on the Jaffe
reaction
▪ Jaffe Reaction without adsorbent
o First described in 1886
o Creatinine reacts with picric acid in
an alkaline solution to form a red-
orange chromogen
o Nonspecific Creatinine concentration decreases with age
o Positive interferences: beginning in the 5th decade of life
✓ Acetoacetate, Acetone,
Ascorbate BUN:CREATININE RATIO
✓ Glucose, Pyruvate
▪ Jaffe Reaction with adsorbent (Lloyd or ➢ Differentiation of the cause of abnormal urea
Fuller’s Earth Method) concentration is aided by calculation of the urea
o Adsorbent: removes interferences nitrogen/creatinine ratio, normally 10:1 to 20:1
o More accurate results → creatinine ➢ Low Ratio <10:1
in a protein-free filtrate is adsorbed ▪ Low protein diet
onto Fuller’s earth reagent ▪ Acute tubular necrosis
(aluminum magnesium silicate) or ▪ Repeated dialysis
Lloyd’s reagent (sodium aluminum ▪ Hepatic disease
silicate), then eluted and reacted ➢ High Ratio >20:1 with normal creatinine
with alkaline picrate ▪ Prerenal azotemia
o Disadvantages: ▪ Dehydration
✓ Time-consuming ▪ Catabolic states
✓ Not readily automated ▪ GI hemorrhage
➢ Kinetic Jaffe Method ▪ High protein diet
▪ Serum is mixed with alkaline picrate and the ➢ High Ratio >20:1 with increased creatinine
rate of change in absorbance is measured ▪ Postrenal azotemia
▪ Advantages: ▪ Prerenal azotemia with renal disease
o Eliminates some of the interferences ▪ Renal failure

GONZALES JR., Noel (BSMT3D) Exercises 13-15: Nonprotein Nitrogen Compounds | 5