Non-Protein Nitrogenous

(NPN) Substances
(Kidney Function Tests)
 NPN’s
▪ Substances in the blood which contain
nitrogen but are not considered as proteins
▪ End products of the metabolism of nucleic
acids, amino acids and proteins
▪ The determination of NPNs in the blood
(specifically UREA and CREATININE) has
traditionally been used to monitor renal
function and thus test for NPNs is part of
KIDNEY FUNCTION TEST (KFT)
 The Kidney
• Paired, bean-shaped organ located retroperitoneally
• Each kidney is comprised of 1-1.5 million nephrons -
the functional unit of the kidney
• Receives approximately 20-25% of the total cardiac
output
• Functions:
[Link] of waste products
[Link] of Blood Volume
[Link] of electrolyte balance
[Link] of acid-base balance
[Link] of erythropoietin
[Link] of Vitamin D
 1. UREA CO(H2N)2
▪ Major end product of PROTEIN metabolism.
It has the molecular weight of 60 Daltons.
▪ Urea is 90% excreted and 10% reabsorbed
(PCT, collecting ducts)
▪ Synthesized solely in the liver from
ammonia, CO2 and H2O through the urea or
Krebs-Henseleit cycle.
2 NH3 + CO2 + 3 ATP + H2O → urea + 2 ADP +
4 Pi + AMP
 UREA
▪ The concentration of urea in the plasma is
determined by the
▪ protein content of the diet
▪ the rate of protein catabolism
▪ renal function and perfusion
▪ Measured by its nitrogen content (Urea = BUN x
2.14)
▪ First to increase in a renal/kidney disease although it
requires 70-80% glomerular damage before the
concentration of urea is increased in the blood.
▪ Screening procedure for kidney function
▪ Easily removed by dialysis
▪ Urea results are effective for diagnosis if
combined with creatinine results
 DISEASE CORRELATIONS
▪ Azotemia- elevated concentration of NPN's in the
blood.
▪ Classified into pre-renal, renal or post-renal
depending on the etiology
1. Pre-renal azotemia - caused by
▪ Reduced renal blood flow
▪ congestive heart failure
▪ shock
▪ hemorrhage
▪ dehydration
▪ High protein diet
▪ Increased protein catabolism
▪ stress
▪ fever
▪ major illnesses
▪ corticosteroid therapy
▪ GI hemorrhage
 DISEASE CORRELATIONS
2. Renal azotemia- caused by damage within the kidneys
such as
▪ acute and chronic renal failure
▪ glomerulonephritis
▪ tubular necrosis
▪ other intrinsic renal disease
3. Post-renal azotemia- caused by obstruction of urine flow
anywhere in the urinary tract by
▪ renal calculi
▪ tumors of the bladder or prostate
▪ severe infection
 DISEASE CORRELATIONS
▪ Urea levels in the blood are decreased in
the following conditions:
1. Poor nutrition
2. High fluid intake / excessive intravenous
(IV) fluids
3. Pregnancy
4. Severe liver diseases
5. Severe vomiting and diarrhea
 DISEASE CORRELATIONS
• Causes of decreased urea concentration
– Low protein intake
– Severe liver disease
– Late pregnancy
– Infancy
 UREA: CREA RATIO
▪ Normal Urea: Crea Ratio
▪ 10:1 / 20:1
▪ Pre-renal azotemia
▪ Increased plasma urea
▪ Normal plasma crea
▪ Increased Urea N: Crea Ratio
▪ Post-renal azotemia
▪ Increased plasma urea
▪ Increased plasma crea
▪ Increased Urea N: Crea Ratio
▪ Decreased Urea N:Crea Ratio
▪ Decreased urea production
 DISEASE CORRELATIONS
▪ Uremia
▪ Ve r y h i g h p l a s m a u r e a c o n c e n t r a t i o n
accompanied by renal failure
▪ The kidneys fail to eliminate waste products of
metabolism.
▪ Clinical findings:
▪ Normocytic normochromic anemia
▪ Uremic frost (dirty skin)
▪ Generalized edema
▪ Foul-breath
▪ Urine-like sweat
▪ Presence of BURR cells in the PBS
 Laboratory Methodologies/
Analytical Techniques
A. Direct Methods- directly measure urea
B. Indirect methods- measure the nitrogen
content of Urea (Blood Urea Nitrogen- BUN)
BUN x 2.14 = UREA

UREA = 60 moles
Nitrogen = 28 moles
60/28 = 2.14
 Indirect methods
• Answers must always be multiplied by 2.14
1. Micro-Kjeldahl method
▪ Uses sulfuric acid to convert nitrogen into
ammonia (Kjeldahl process), then measure
ammonia using
▪ Nessler's reaction: NH3 + NH2Hg2I2 (di mercurc
potassium iodide)
▪ Bethelot's reaction: NH3 + Phenol hypochlorite =
indophenol blue
 Indirect methods
1. Urease Method
•Uses urease to convert urea into ammonia, then ammonia is
measured
• Urease-Nessler: ammonia is reacted to di mercuric
potassium iodided (yellow)
• Urease-berthelot: ammonia is reacted to phenol
hypochlorite (blue)
2. Coupled-Enzymatic
– Urease employed to convert urea into ammonia then
ammonia reacted to glutamate dehydrogenase
(GLDH). Conversion of NADH to NAD is then
measured at 340nm
 Direct Methods
1. Diacetyl Monoxime (DAM) - employed in
autoanalyzers
•Urea + DAM = yellow diazine derivative
(540 nm)
•Ferric ions and thiosemicarbazide may be
employed to intensify the reaction
•No ammonia interference
2. Isotope Dilution/Mass Spectrometry
 PRECAUTIONS/ SPECIMEN
CONSIDERATIONS

▪ Urea is highly affected by protein diet BUT the

effect of a recent protein meal is minimal and
a fasting sample is NOT required usually.
▪ NaF and citrate is an inhibitor of UREASE
enzyme (gray-top tubes not suitable for BUN
determination)
▪ Avoid contamination with ammonium salts
(can become ammonia)
▪ Avoid prolonged standing of sample (because
urea will be converted to ammonia)
Reference Interval / Range
▪ Reference Range for BUN:
▪ Conventional:
▪ 8 – 23 mg/dL
▪ SI
▪ 2.9 – 8.2 mmol/L
▪ Conversion Factor:
▪ Conventional to SI unit ! 0.357
▪ Values are lower in children
▪ Males have slightly higher values
compared to females
 2. CREATINE AND CREATININE
▪ Creatinine is derived from creatine and creatine
phosphate in a nonenzymatic process in the
muscles
▪ Creatine is synthesized primarily in the liver from
arginine, glycine, and methionine
▪ Creatine is transported to muscle tissues where it
is converted to creatine phosphate.
▪ Plasma creatinine concentration is a function of
▪ Muscle mass
▪ Rate of creatine turnover
▪ Renal function
▪ Creatine phosphate loses phosphoric acid and
creatine loses water to form the cyclic compound,
CREATININE, which diffuses into the plasma and
is excreted in the urine.
 CREATININE
▪ Major end product of MUSCLE metabolism
▪ An additional source of creatinine is creatine
contained in ingested meat or dietary
supplements. Convertion of creatinine from
creatine in meat is enhanced by high
temperature and low pH
▪ Not reused by the body; solely a waste product
(100% excreted; maximum of 1% reabsorbed)
▪ NOT easily removed by dialysis
▪ Can be used to check for the completion of a 24-
hour urine
 CREATININE
▪ The amount of creatinine is directly proportional
to creatine-creatine phosphate pool which is
directly related to muscle mass.
▪ Creatinine is also used to evaluate fetal
maturity, as gestation progresses, more
creatinine is excreted by the fetus into the
amniotic fluid (2mg/dL)
▪ Insensitive marker. Creatinine may not be
increased until renal function has deteriorated
more than 50%.
 Laboratory Methodologies/
Analytical Techniques
• 1. Direct JAFFE Reaction Method (1886)
• Creatinine (PFF) + alkaline picrate -------> red-orange
tautomer of creatinine picrate
• Alkaline picrate is made up of picric acid dissolved in 10%
NaOH.
▪ Picric acid is not specific to creatinine. Ketones, glucose,
fructose, protein, urea, ascorbic acid and cephalosporins
also react with the test (falsely increased) whereas bilirubin
and hemoglobin falsely decrease the results.
▪ LLOYD’S (Method of Hare) and FULLER’S EARTH reagent
are employed to remove the interferences to make the
method more specific and sensitive.
 Laboratory Methodologies/
Analytical Techniques
2. Kinetic Jaffe Method
▪ Differential rate of color development eliminating
interferences with Jaffe chromogens
▪ Requires automated equipments
▪ Do not use PFF, serum used directly
 Laboratory Methodologies/ Analytical
Techniques
3. ENZYMATIC methods
▪ No interference from glucose and other Jaffe chromogens
▪ Creatinine Iminohydrolase Method
Creatinine ---(creatinine iminohydrolase)---> NH4 + N-methylhydantoin
NH4 + 2-oxoglutarate + NADH ---(glutamate dehydrogenase)---> glutamate
and NAD+ (340nm)
***Decrease in absorbance at 340nm reflects the concentration of creatinine
▪ Creatinine Amidohydrolase Method
Creatinine --(creatinine amidohydrolase)--> Creatine
Creatine --(imidinohydrolase and sarcosine oxidase)--> H202
2,4-dichlorophenolsulfonate --(H202 + horseradish peroxidase)--> colorless
polymer (510 nm)
 Laboratory Methodologies/
Analytical Techniques
4. ISOTOPE DILUTION MASS SPECTROMETRY
(IDMS)
• Ultimate reference standard for creatinine
measurement
 Reference Interval / Range
▪ Serum or plasma
▪ Adult: 0.6–1.2 mg/dL (53–106 µmol/L)
▪ Children <2 yr): 0.3–0.6 mg/dL (27–53 µmol/L)
▪ Conversion Factor:
▪ Conventional to SI unit ! 88.40
▪ Values are lower in children
▪ Males have slightly higher values compared
to females
 3. URIC ACID / URATE
▪ Major end product of PURINE (adenine, guanine)
NUCLEIC ACID metabolism
▪ Formed from xanthine by the action of xanthine
oxidase in the liver and intestine.
▪ At a pH of 7.4 ; more than 95% of uric acid in the body
fluids exist as monosodium urate
▪ In humans and higher primates, uric acid is the final
oxidation (breakdown) product of purine metabolism
and is excreted in urine. In most other mammals, the
enzyme uricase further oxidizes uric acid to allantoin
 3. URIC ACID / URATE
▪ The loss of uricase in higher primates parallels the
similar loss of the ability to synthesize ascorbic acid.
▪ Both uric acid and ascorbic acid are strong reducing
agents (electron donors) and potent antioxidants. In
humans, over half the antioxidant capacity of blood
plasma comes from uric acid
▪ Uric acid is relatively insoluble in plasma and, at high
concentrations, can be deposited in the joints (tophi) and
tissue, causing painful inflammation
▪ In acidic urine (pH <5.75), uric acid crystal is the
predominant species
BLOOD URIC ACID (BUA) / URATE

▪ Two major sources:

▪Endogenous- nucleic acid
metabolism
▪Exogenous- uric acid in the diet
(vegetables and legumes)

▪ 90% reabsorbed; 10% excreted

 Laboratory Methodologies/
Analytical Techniques
1. Cyanide Method (REDOX) (odor of bitter almonds)
(Folin, Brown, Newton, Benedicts)
NaCN
Uric Acid + PTA tungsten blue
(reducing property) phosphotungstic acid (NaCN is the color
stabilizer; toxic; not readily available)

2. Sodium Carbonate Method (Archibald, Henry,

Caraway)
Uric Acid in serum + PTA Na2CO3 tungsten blue
*** Sodium carbonate- not toxic
 Laboratory Methodologies/ Analytical
Techniques
3. Enzymatic Method
▪ URICASE METHOD
▪ Simplest and most specific method
▪ Candidate reference method
▪ Uric Acid has a UV absorbance peak at 293 nm; allantoin does not have
a UV peak at that wavelength
▪ 290-293 nm
▪ Uric acid absorbance

uricase

290-293 nm
Allantoin no absorbance
(derived comp)
*** Upon addition of uricase, allantoin will be formed. The decrease in
absorbance is proportional to the concentration of uric acid present in
 Reference Interval / Range
▪ Reference Range for Uric Acid Using PTA:
▪ Conventional :
▪ Males! 4 - 8.5 mg/dL
▪ Females! 2.7 – 7.3 mg/dL
▪ SI
▪ Males ! 0.24 – 0.51 mmol/L
▪ Females ! 0.16 – 0.43 mmol/L
▪ Conversion Factor:
▪ Conventional to SI unit ! 0.059
▪ Males have slightly higher values compared to
females
 PRECAUTIONS/ SPECIMEN
CONSIDERATIONS
▪ Diet can affect blood uric acid levels but recent
consumption has no immediate effect on uric acid
levels therefore fasting is unnecessary.
▪ Gross lipemia should be avoided.
▪ High bilirubin concentration may falsely decrease
results obtained by peroxidase methods
▪ (EDTA) or fluoride additives should not be used for
specimens that will be tested through uricase method.
 CLINICAL APPLICATION
1. Confirm diagnosis and monitor treatment of
gout
2. Prevent uric acid nephropathy during
chemotherapeutic treatment
3. Assess inherited disorders of purine
metabolism
4. Detect kidney dysfunction
5. Assist in the diagnosis of renal calculi
 HYPERURICEMIA
▪ Hyperuricemia- increased uric
acid concentration in the
blood (> 7 mg/dL). This is
associated with:
▪Increased uric acid production
▪Decreased renal excretion
 HYPERURICEMIA
Causes of hyperuricemia
1. Gout
▪ Degenerative disorder; commonly found in men between
30 and 50 years of age and women after menopause
▪ Affected individuals have pain and inflammation of the
joints caused by precipitation of sodium urates
▪ In 25% to 30% of these patients, hyperuricemia (>6.0 mg/
dL) is a result of overproduction of uric acid
▪ Patients are susceptible to the formation of renal calculi
 HYPERURICEMIA
2. Increased catabolism of nucleic acids
– Chemotherapy for proliferative diseases such
as leukemia, lymphoma, multiple myeloma,
and polycythemia
– Treatment: Allopurinol - inhibits xanthine
oxidase (EC [Link]), an enzyme involved in
uric acid synthesis
3. Renal disease
4. Hemolytic or megaloblastic anemia
 HYPERURICEMIA
5. Ingestion of diet rich in purines (liver, kidney, sweetbreads,
and shellfish) or various disease processes.
6. Starvation due to increased tissue catabolism
7. Decreased uric acid excretion
8. Inherited disorders of purine metabolism
• Lesch-Nyhan syndrome - complete deficiency of
hypoxanthine-guanine phosphoribosyltransferase
which is involved in the de novo sythesis of uric acid
(salvage pathway)
 HYPERURICEMIA
8. Inherited disorders of purine metabolism
• Lesch-Nyhan syndrome
– An X-linked genetic disorder (seen only in males)
caused by the complete deficiency of hypoxanthine-
guanine phosphoribosyltransferase (EC [Link]),
an important enzyme in the biosynthesis of purines.
– Lack of this enzyme prevents the reutilization of
purine bases in the nucleotide salvage pathway
and results in increased de novo synthesis of purine
nucleotides and high plasma and urine concentrations
of uric acid
 4. AMMONIA
▪ Ammonia (NH 3 ) is produced from the
catabolism of amino acids and by bacterial
metabolism in the lumen of the intestine
▪ Because the liver is the sole site of ammonia
detoxification via the urea cycle, and because,
in this condition, loss of hepatocytic function
occurs, serum ammonia levels are elevated
▪ At normal physiologic pH, most ammonia in
the blood exists as ammonium ion (NH4+).
▪ Ammonia is excreted as ammonium ion by
the kidney and acts to buffer urine
 4. AMMONIA
• Free ammonia is neurotoxic and often associated with
encephalopathy. Toxicity may be partly a result of increased
extracellular glutamate concentration and subsequent
depletion of adenosine triphosphate in the brain
• Increased blood ammonia is seen in
– Hepatic failure (test for ammonia is a part of the LIVER
FUNCTION TESTS)
– Reye's syndrome
– Inherited deficiencies of urea cycle enzymes
• Although classified as an NPN, it is NOT a good indicator of
kidney function.
 4. AMMONIA
Reye's syndrome
• A serious, fatal disease
common in children
frequently preceded by a
viral infection and the
administration of aspirin
• An acute metabolic
disorder of the liver and
brain in which there is
fatty infiltration of the
organs.