Nonprotein Nitrogen Compounds

To convert to urea concentration in mols per liter

▪ removal of protein from a specimen before analysis. multiply by 0.36
▪ Converting nitrogen to ammonia and subsequent reaction
with Nessler's reagent (K2 [HgI4 ])
▪ arise from the catabolism of proteins and nucleic acids.

▪ (urea amidohydrolase, EC 3.5.1.5) – catalyzes

hydrolysis of urea in the sample
▪ ammonium ion (NH+4)- produced in the reaction which
is quantified
▪ GLUTAMATE DEHYDROGENASE (GLDH, EC 1.4.1.3)-
common method couples the urease
▪ Highest concentration in the blood ▪ (reduced, NADH) at 340 nm is measured.
▪ Major excretory product of protein metabolism
▪ Formed in liver amino groups s (−NH2 ) and free
ammonia generated during protein catabolism
▪ BUN) has been used to refer to urea determination.
▪ Urea nitrogen (urea N)- more appropriate

▪ Protein- amino acids (oxidized)= energy/ stored fat

and glycogen = release nitrogen -> converted to ▪ Isotope dilution mass spectrometry (IDMS)- reference
urea= excreted as a waste product method
▪ Most urea= excreted in the urine / some are
reabsorbed by passive diffusion ( renal tubules)
▪ <10% of the total urea- excreted GIT tract and skin
▪ Concentration of urea in the plasma- determined by
protein content of diet, rate protein catabolism , renal
function and perfusion

▪ 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.
▪ Urea N concentration can be converted to urea
concentration by multiplying by 2.14,
▪ International System of Units (SI),- units of millimoles
per liter
▪ Urea N concentration- miligrams per deciliter
▪ Measured in plasma, serum/ urine
▪ PLASMA- ammonium ions + high concen. Of sodium
citrate + sodium fluoride MUST BE AVOIDED! BECAUSE
IT INHIBIT UREASE
▪ FASTING IS NOT REQUIRE
▪ NONHEMOLYZED SAMPLE is req
▪ Should be refrigerated to avoid bacterial
contamination
▪ Time urine should be ref during collect period
▪ PLASMA/ SERUM- req modification due to high urea
concentration + presence of endogenous ammonia
 ▪ High concentrations, can be deposited in the joints
and tissue, causing painful inflammation

▪ AZOTEMIA- urea in the blood

▪ Uremia/ uremic syndrome - plasma urea concn +
renal failure
▪ three main categories: prerenal, renal, and postrenal
▪ Pre- renal azotemia-reduced renal blood flow
o Less blood is delivered to the kidney- less
urea filtered
o Congestive heart failure, shock, hemorrhage,
dehydration, and other factors resulting in a
significant decrease in blood vol.
o May increase in the urea con- high-protein
diet or increased protein catabolism, in stress,
▪ Purines- adenine and guanine -> breakdown of
fever, major illness, corticosteroid therapy,
and GI hemorrhage ingested nucleic acid/ tissue destruction ->
o Renal function= increse plasma urea con= converted = URIC ACID
compromised urea excretion ▪ Reabsorption of 98% to 100%- proximal tubules
o Plasma creatinine- NORMAL- high N/ ▪ Small amounts- distal
CREATININE RATIO ▪ 70% of uric acid elimination- renal excretion
▪ GI tact -> degraded by bacterial
▪ RENAL- elevated urea include acute and chronic renal ▪ Monosodium urate
failure, glomerular nephritis, tubular necrosis, and ▪ Ph 7 plasma – insoluble + high con > 6.8 mg/DL,
other intrinsic renal disease plasma is saturated = urate crystals form and
▪ POST RENAL AZOTEMIA -obstruction of urine flow precipitate in the tissue
anywhere in the urinary tract by renal calculi, tumors
▪ acidic urine- pH <5. 75, uric acid is predominant + uric
of the bladder or prostate, or severe infection.
crystals
▪ Decrease plasma urea con.- low protein intake and
severe liver disease.
o Low urea N/ crea rataio
▪ urea nitrogen/creatinine (urea N/creatinine) ratio- ▪ detection of gout
calculation to differentiate abnormal urea ▪ prevent uric acid nephropathy during chemotherapeutic
concentration; 10:1- 20:1 treatment
o high UREA N/creatinine ratio wth elevated ▪ assess inherited disorder of purine metabolism
creatinine ▪ kidney dysfunction
▪ diagnosis of renal calculi

▪ other mammals - catabolize purine to allantoin (water

▪ product of catabolism of the purine nucleic acids
▪ reabsorbed in the proximal tubules and reused
▪ insoluble in plasma
soluble end product)
▪ Uric acid – oxidized to allantoin= reducing agent in
chemical reactions = CARWAY METHOD most common
method
▪ Caraway method- oxidation of uric acid in a protein-free
filtrate, with subsequent reduction of phosphotungstic
acid in alkaline solution to tungsten blue; LACKS
SPECIFICITY
▪ URICASE method ( urate oxidase EC 1.7.3.3)- oxidation of
uric acid to allantoin, are more specific and are used
almost exclusively in clinical laboratories
o differential absorption of uric acid and
allantoin at 293 nm
o proportional to the uric acid concentration
o negative interference: hemoglobin and
xanthine
▪ Coupled enzyme methods- measure the hydrogen peroxide
produced as uric acid is converted to allantoin
o Peroxidase or catalase (EC 1.11.1.6-catalyze
a chemical indicator reaction.
o color produced is proportional to the
quantity of uric acid
o bilirubin and ascorbic – destroys peroxide
o commercial reagent- potassium ferricyanide
and ascorbate oxidase to minimize interf.
▪ HPLC (high-performance liquid chromatography) methods
– use uv detection
SUMARY OF ANALYTIC METHODS OF URIC ACID
▪ Measured in heparinized plasma, serum/ urine
▪ Serum- should be removed from cells TO PREVENT DILUTION
by intracellular contents
▪ No fasting require but diet may affect uric acid
concentration
▪ High bilirubin concentration- false decrease obtain by
peroxidase methods
▪ Significant hemolysis + glutathione release- low values
▪ Drugs- salicylates + thiazides – increase values for uric
acid
▪ Uric acid- stable in plasma/ serum after rbc have been
removed
▪ Serum sample- stored ref for 3-5 days
▪ EDTA/ fluoride additives - should not be used for uricase
method
▪ Urine- should be alkaline ph 8
▪ Results expressed in conventional units of milligrams
per deciliter can be converted to SI units using the
molecular mass of uric acid (168 g/mol).
▪ Gout- INC. plasma uric acid concentration ,
increase catabolism of nucleic and renal disease
o Men- 30-50 yrs of age
o Pain, inflammation of joints –
precipitation of sodium urates
o HYPERURICEMIA – 25-30% over
production of uric acid
o Renal calculi
o Uric acid con > 6.0
o Women- urate concentration rise after
menopause
 o Post menopausal women- develop
hyperuricemia ang gout
o TOPH- deposits of crystalline uric acid and
urates= deformities
▪ formed from creatine and creatine phosphate in
▪ Elevated plasma uric concentration in chemo-
muscle and is excreted into the plasma at a constant
ALLUPORINOL treatment which inhibits xanthine rate related to muscle mass
oxidase 1.1.3.22), ▪ PLASMA CREATININE- inversely related to GFR
▪ HEMOLYTIC OR MEGALOBLASTIC ANEMIA-may
exhibit elevated uric acid concentration
▪ Lesch-Nyhan syndrome-X-linked genetic disorder
(seen only in males) caused by the complete ▪ Creatine- synthesized primarily in the liver from
deficiency of hypoxanthine–guanine arginine, glycine, and methionine
phosphoribosyltransferase (EC 2.4.2.8) (IMPORTANT ▪ Tissue-> muscle = creatinine phosphate ( high
enzyme in purines) energy source
o Neurologic symptoms, mental retardation, ▪ Creatine phosphate loses phosphoric acid
and self- mutilation characterize this ▪ creatine loses water to form the cyclic compound,
extremely rare disease. creatinine, which diffuses into the plasma and is
▪ Hyperuricemia- decreased uric acid excretion excreted in the urine.
(common feature of toxemia of pregnancy

▪ Endogenous production- urinary creatinine

excretion has been used as a measure of the
completeness of 24-hour urine collections.
▪ Creatinine clearance (CrCl)- ) a measure of the
amount of creatinine eliminated from the blood
by the kidneys, and GFR are used to gauge renal
function
▪ The GFR is the volume of plasma filtered (V) by the
glomerulus per unit of time (t):

▪ the volume of plasma filtered would be equal to

the mass of S filtered (MS ) divided by its plasma
concentration (PS ):

▪ The mass of S filtered is equal to the product

of its urine concentration (US ) and the urine
volume (VU):

▪ If the urine and plasma concentrations of S,
the volume of urine collected, and the time
over which the sample was collected are
known, the GFR can be calculated:
▪ The formula for CrCl is given as follows, where
UCr is urine creatinine concentration and PCr
is plasma creatinine concentration
▪ o enhance the specificity of the Jaffe
▪ measurement of plasma creatinine does not
provide sufficient sensitivity for the
detection of mild renal dysfunction.
▪ Modification of Diet in Renal Disease (MDRD)
equation was advocated.
o The equation includes four variables—
serum creatinine concentration, age,
gender (sex), and ethnicity—
o When serum creatinine is measured using an
IDMS-traceable method, the MDRD equation
for estimated glomerular filtration rate
(eGFR) is
o More accurate- creatinine in a protein-free
filtrate is adsorbed onto Fuller's earth
(aluminum magnesium silicate) or Lloyd's
reagent (sodium aluminum silicate) +
ALKALINE PRICRATE
▪ Two approaches have been used to increase the
specificity of assay methods
A. Jaffe method
o serum is mixed with alkaline picrate and
the rate of change in absorbance is
measured.
o used routinely despite these problems
because it is inexpensive, rapid, and
easy to perform.
o method eliminates some of the
nonspecific reactants, it is subject to
interference by α-keto acids and
cephalosporins
o negative bias- hemoglobin and bilirubin
reaction: coupled enzymatic methods h
✓ (creatinine amidohydrolase, EC
3.5.2.10), creatinase (creatine
amidinohydrolase, EC 3.5.3.3),
sarcosine oxidase (EC 1.5.3.1),
and peroxidase (EC 1.11.1.7)
was adapted for use on a dry
slide analyzer.
B. IDMS- is used as a reference method.

▪ most frequently used to measure creatinine are based

on the Jaffe reaction first described in 1886
o reacts with picric acid in alkaline solution to
form a red-orange chromogen.
o measurement of blood creatinine by Folin
and Wu in 1919.
o acetoacetate, acetone, ascorbate, glucose,
and pyruvate

Requirements
▪ Creatinine may be measured in plasma, serum, or urine
▪ Jaffe method is used - Hemolyzed and icteric samples
should be avoided
▪ Lipemic samples- erroneous results in some methods
▪ A fasting sample is not required
▪ elevate serum concentrations- high-protein ingestion
▪ increase creatinine concentration measured by the
Jaffe reaction-
o Ascorbate, glucose, α-keto acids, and uric
acid 30°C
o decreased when kinetic measurement is
applied.
▪ Bilirubin causes a negative bias in both Jaffe and
enzymatic methods
▪ cephalosporin antibiotics - elevated results when the
Jaffe reaction is used.
▪ Dopamine- known to affect both enzymatic and Jaffe
methods.
▪ Lidocaine- positive bias in some enzymatic methods
▪ Jaffe method- heated in acid solution.
= creatinine-> creatinine
=difference is the creatine concentration
▪ High temperatures formation of additional methods
▪ Measured by HPLC
Reference Intervals
▪ Vary with assay type, age, and gender
▪ decreases with age beginning in the 5th decade of
life.
Pathophysiology
▪ Elevated creatinine concentration is associated with
abnormal renal function, especially as it relates to
glomerular function.
▪ Plasma concentration of creatinine inversely
proportional to the clearance of creatinine.
= plasma concentration is high- GFR decrease= renal
damage
▪ .Plasma concentration of creatinine s a relatively
insensitive marker and may not be measurably
increased until renal function has deteriorated more
than 50%
▪ plasma creatine and urinary creatinine are often
elevated
o muscle disease such as muscular dystrophy,
poliomyelitis, hyperthyroidism, and trauma
o Plasma creatinine concentrations are usually
normal in these patients.
▪ Plasma creatine concentration is not elevated in renal
disease.
▪ typically for the diagnosis of muscle disease
▪ produced in the deamination of amino acids during
protein metabolism
▪ removed in the circulation
▪ Present in plasma in low concentration
▪ Endogenous ammonia-anaerobic metabolic reactions
that occur in skeletal muscle during exercise
▪ consumed by the parenchymal cells of the liver in the
Krebs Henseleit or urea cycle to produce urea, a
nontoxic compound that is excreted in the urine.
▪ excreted as ammonium ion by the kidney and acts to
buffer urine
▪ USE FOR are hepatic failure, Reye's syndrome, and
inherited deficiencies of urea cycle enzyme
▪ severe liver disease- most common cause of disturbed
ammonia metabolism
▪ Arterial ammonia concentration- a better indicator of
the severity of disease
▪ Reye's syndrome- most commonly in children
o Acute metabolic disorder of liver, and
autopsy = fatty infiltration of organ
o Survival reaches 100% if plasma NH3
concentration remains below five times
normal
▪ diagnosis of inherited deficiency of urea cycle
enzymes
▪ considered for any neonate with unexplained nausea,
vomiting, or neurological deterioration associated
with feeding
▪ Assay of blood ammonia can be used to monitor
hyperalimentation therapy
 ▪ Measurement of urine ammonia - confirm the ability
of the kidneys to produce ammonia
Two approaches have been used for the measurement of
plasma ammonia
a. a two-step approach- ammonia is isolated from the
sample and then assayed.
b. The second- involves direct measurement of ammonia
by an enzymatic method or ion-selective electrode.
Assays detect NH3 or NH+4
Analytic method
Conway in 1935,- exploited the volatility of ammonia to
separate the compound in a microdiffusion chamber.
o Ammonia gas from the sample diffuses into a separate
compartment and is absorbed in a solution containing
a pH indicator.
o amount of ammonia is determined by titration.
enzymatic method using GLDH
o convenient and the most common technique used
currently
o decrease in absorbance at 340 nm as nicotinamide
adenine dinucleotide phosphate (reduced, NADPH) is
consumed in the reaction is proportional to the
ammonia concentration in the specimen.
o NADPH is the preferred coenzyme, participate in
reactions of other endogenous substrates, such as
pyruvate.
o ADP is added = INCRERASE the rate of the reaction
and stabilize gldh
o many automated systems and is available as a
prepared kit from numerous manufacturers.
o dry slide automated system uses a thin-film
colorimetric assay= colored compound detected
spectrophotometrically
▪ Venous blood should be obtained without trauma and
placed on ice immediately.
▪ Heparin and EDTA are suitable anticoagulants.
▪ Commercial collection containers should be evaluated
for ammonia interference before a new lot is put into
use
▪ Centrifuged at 0 to 4°C within 20 minutes of collection
and the plasma removed.
▪ Frozen plasma is stable for several days at −20°C.
▪ Erythrocytes contain two to three times as much
ammonia as plasma; hemolysis should be avoided
▪ Cigarette smoking by the patient is a significant
source of ammonia contamination.
Substances influence the in vivo ammonia concentration.
o Ammonium salts, asparaginase, barbiturates,
diuretics, ethanol, hyperalimentation, narcotic
analgesics, and some other drugs may increase
ammonia in plasma. Diphenhydramine, Lactobacillus
acidophilus, lactulose, levodopa,
o several antibiotics decrease concentrations. Glucose
at concentrations greater than 600 mg/dL (33
mmol/L) interferes in dry slide methods
INTERFERENCE: tobacco smoke, urine, and ammonia in
detergents, glassware, reagents, and water.
Reference Intervals
Pathophysiology
o severe liver disease - ammonia is not removed from
the circulation and blood concentration increases.
o High concentrations of NH3 are neurotoxic and often
associated with encephalopathy
o Hyperammonemia- associated with inherited
deficiency of urea cycle enzymes