creatinine, Uric Acid, Urea, and ammonia, non protein nitrogen

nonprotein nitrogenous (NPN) compounds

 Nitrogenous compounds
 Not protein in nature
 Derived from:
 Dietary protein
 Nucleic acids
 Muscle mass
 Hepatic deamination
 Are processed in the liver
 Eliminated by the kidney
 Measure
 Renal function
 Hepatic Function

components of non-protein nitrogen

 Urea Nitrogen 45%
 Amino Acid 20
 Uric Acid 20
 Creatinine 5
 Creatine 1-2
 Ammonia 0.2

creatine/creatinine biochemistry

 Synthesized mainly in the liver
 From arginine and glycine
 Transported to muscle
 Converted to phosphocreatine,
 a high energy source
 Spontaneously converts to creatinine
 Is then transported by plasma
 Excreted by kidney
 Excreted at a constant rate
 Proportional to muscle mass
 Removed from the plasma almost entirely by GFR
 Excellent indicator of GFR

structure of creatinine

normal concentrations of creatinine

 Serum or Plasma
 0.6 - 1.2 mg/dl males (53-106 umol/L)
 0.5 - 1.0 mg/dl females (44-88 umol/L)
 ASCP Combined Adult Range: 0.8-1.2 mg/dL
 Urine creatinine: 1-2 g/day

clinical significance of creatinine

 Index of Kidney Function or (GFR)
 Serum elevations:
 Reduction of GFR
 Kidney disease
 Muscular dystrophy (Duchene's type)
 Skeletal muscle atrophy
 Starvation
 Muscular Trauma, crushing injuries
 Gigantism, Acromegaly
 Myasthenia gravis
 Poliomyelitis
 Hyperthyroidism

check creatinine before giving nephrotoxic drugs

 Methotrexate
 Cisplatin
 Cytoxan
 Semustine
 Mithramycin
 Vancomycin

analytical procedures for creatinine

Jaffe Reaction

 Principle reaction
 Developed in 1886
 Oldest Known chemical test principle to
date. OH-
 Creatinine + picric acid  creatinine-picrate
complex (red)
 Measure Absorbance 520 nm
 Original method required a Protein Free
filtrate prepared with TCA

interferences of creatinine

 Non Creatinine Chromogens
 Ascorbic acid
 Pyruvate
 Acetone and aceto-acetic acid
 Alpha Ketoacids - Diabetic
Ketoacidosis

picric acid

 Safety precautions
recommend storing
picric
 Dry picric acid is
relatively sensitive to
shock and friction
 picric acid can easily
form metal picrate salts
that are even more
sensitive and hazardous
 TNT

kinetic Jaffe reaction

 Spectrophotometric
 Measures rate of change in
absorbance
 Requires an initial reading
(baseline) A1
 And an Endpoint reading A2
 Measures increase of Absorbance at
500 nm (Delta Absorbance)

coupled enzymatic methods of creatinine

 Enhanced specificity over Jaffe methods
 Creatininase (creatinine aminohydrolase) Method
 Encorporates enzymes, CK, PK, LD
(Creatininase aminohydrolase)
 Creatinine + H2O  Creatine
CK
 Creatine + ATP  Creatine Phosphate + ADP
PK
 ADP + Phosphoendopyruvate  ATP + Pyruvate
LD
 Pyruvate + NADH  Lactate + NAD+
 Measure the decrease of absorbance as NADH  NAD+
 Requires large sample, not routinely used

other couple enzymatic methods - creatinine

 Creatinine aminohydrolase - H2O2 Methods
Creatininase
 Creatinine + H2O → Creatine
Creatinase
 Creatine + H2O → Sarcosine + Urea
Sarcosine Oxidase
 Sarcosine + H2O + O2  glycine + formaldehyde+H2O2
 H2O2 + phenol derivative + 4-aminophenazone →
benzoquinone immine dye

J & J Vitros CREAT

 Principle
 Creatinine diffuses to the reagent layer, where it is
hydrolyzed to creatine
 The creatine is converted to sarcosine and urea by
creatine amidinohydrolase
 The sarcosine, in the presence of sarcosine oxidase, is
oxidized to glycine, formaldehyde, and hydrogen peroxide
 The final reaction involves the peroxidase-catalyzed
oxidation of a leuco dye to produce a colored product
 Following addition of the sample, the slide is incubated.
During the initial reaction phase, endogenous creatine in
the sample is oxidized. The resulting change in reflection
density is measured at 2 time points
 The difference in reflection density is proportional to the
concentration of creatinine present in the sample

Vitros CREAT

 1. Upper slide mount
 2. Spreading layer
(TiO2)
 3. Reagent layer
 • creatinine
amidohydrolase
 • creatine
amidinohydrolase
 • sarcosine oxidase
 • peroxidase
 • leuco dye
 • buffer, pH 7.0
 4. Support layer
 5. Lower slide mount

Vitros CREAT rxn

Roche Cobas 501

creatinine clearance tests

 Measurement of GFR
 Clearance:
 the amount of plasma that can be cleared of a
substance per unit time
 Procedure
 Hydrate the patient w 600 ml H2O
 Void and discard urine
 Begin 24 hr. collection from this time. Record time.
 Collect urine /unit time
 Collect blood specimen and assay for creatinine

calculation for creatinine clearance

 Clcr = UV/P X 1.73/A
 V = ml/min volume
 U = urine creatinine
 P = Plasma creatinine
 A = Body surface area

reference ranges of creatinine clearance

105 ± 20 ml/min – Males
 95 ± 20 ml/min – Females
 Decreased GFR:
 Impaired GFR
 Kidney disease

uric acid biochemistry

 Catabolism of nucleic acids
 End product of purine metabolism
 Adenosine and Guanine
 Oxidation of xanthine by xanthine Oxidase
 2/3 of uric acid is excreted by the kidneys, 1/3 stool
 96.8 % of uric acid is present as monosodium urate
 There are three disease states associated with
elevated uric acid levels:
 Gout
 Increased nuclear breakdown (leukemia, carcinoma)
 Renal disease

clinical significance of uric acid

 Elevated levels: Hyperuricemia
 Gout
 Leukemia
 Decreased Kidney function - Renal failure
 Lymphomas
 Metastatic cancer
 Multiple myeloma
 Polycythemia
 Hemolytic and Megaloblastic anemia
 Lesch-Nyhan syndrome
 X-linked enzyme deficiency in purine biosynthesis.
 Toxemia of pregnancy and lactic acidosis
 Starvation, increased tissue breakdown
 Purine rich diet
 Alcoholism
 Lead poisoning

significance of uric acid

 Hypouricemia
 Decreased levels
 Secondary to severe liver disease
 Fanconi’s syndrome
 (defective tubular reabsorption)
 Wilson’s disease
 Treatment with Xanthine oxidase
inhibitor drugs
 (Allopurinol)

reference ranges - uric acid

 4.0 - 8.5 mg/dl (males)
 2.7 - 7.3 mg/dl (females)

determination of uric acid

 Two Methods in current use:
 Phosphotungstic acid (PTA)
 Uricase methods

PTA

principle

Na2CO3 /OH-
 Uric Acid +H3PW12O40 + O2 → Allantoin +CO2 + Tungsten Blue
 Measure Absorbance at 710 nm
 Nonspecific, requires protein separation

enzymatic methods - uricase

 Principle:
uricase
 Uric acid +O2 → Allantoin +H2O2 +CO2
 Measure decrease of Absorbance at 293
nm
 As uric acid is converted to allantoin (non
UV absorbing)
 Candidate for reference method
 Hemoglobin and xanthines interfere

couple enzymatic determination of uric acid

 Very popular/ automated
 Enzymatic methods are highly
specific Catalase
 H2O2 +CH2OH → H2CO3 +H2O
 H2CO3 + 3 C5H8O2 + NH3 → 3H2O + Colored
Compound

automated methods

 Differential Absorption
 Uricase Principle
 UA absorbs light @ 293 nm
 Allantoin is non-absorbing
 Measure decreased of
Absorbance/time
 Candidate for reference
methodology

johnson and johnson vitros analyzer

 Dry slide (film)
Technology
 Uric acid migrates
through the scavenger
layer
 Oxidized by uricase to
allantoin and H2O2
 H2O2 reacts with
peroxidase and Dye to
produce a chromogen
 Measured by
Reflectance
colorimetry
 Incorporates
Ascorbate oxidase to
eliminate interference
due ascorbic acid

Roche Cobas 501 principle for uric acid

high performance liquid chromatographic procedure

 Reversed-phase chromatography
 Spectrophotometric detection at
280 or 235 nm
 Ion-exchange separation
 Followed by amperometric detection
 Using thin-layer flow-through
electrochemical cell

interfering substances in uric acid determinations

 Ascorbic acid
 Glucose
 Glutathione
 Acetaminophen
 Caffeine
 Theophylline

urea

 Biochemistry
 Major product of protein metabolism
 Deamination of amino acids to ammonia (NH3 )
 Ornithine or Krebs cycle
 Synthesized in the liver from CO2 and NH3
 Transported by the plasma
 Filter by the glomerulus
 Smaller amounts by skin and GI
 Up to 40% is reabsorbed by the renal tubules

plasma levels

 Affected by renal function
 Protein content of diet and level of protein catabolism
 Historically measured based on nitrogen level, some
assays still measure Urea nitrogen
 Conversion of BUN to urea:
 Atomic Weight of nitrogen is 14 g/mol
 molecular weight of urea=60.06 g/mol (60 Daltons)
 urea contains 2 nitrogen atoms per molecule (2x14=28)
 Mol wt. Urea / At. Wt. N2 = 60/28 = 2.14

 Urea = BUN mg/dl X (2.14)

urea cycle

 Takes place in the mitochondria and the
cytoplasm of the liver cells
 Produced from the conversion of arginine to
ornithine
 Carbamyl Phosphate Synthetase (CPS)
 Enzyme responsible for converting NH3 to Carbamyl
Phosphate used in the urea cycle
CPS
 NH3 + CO2 + H2O + 2ATP → Carbamyl Phosphate

clinical significance of urea (BUN)

 Useful in assessment of Renal Function
 Elevated Urea in the blood is termed azotemia
 Very high Plasma Urea accompanied by renal
failure is termed Uremia or uremic syndrome
 Prerenal azotemia (Reduced renal blood flow)
 Congestive heart failure
 Shock, hemorrhage
 Dehydration
 Renal azotemia
 Acute and chronic renal failure
 Glomerulonephritis, tubular necrosis
 Post renal azotemia
 Blockage of urine flow below the kidney
 Obstruction, calculi, tumors, pregnancy
 Congenital abnormalities, Urinary tract infection

BUN/CREAT ratio

 Aids in differentiating causes of azotemia
 Normally 10-20:1
 Non renal conditions...
 Will elevate urea greater extent than creatinine
 As a result, the BUN/CREAT ratio will be elevated
 Elevation of BUN/CREAT ratio...
 Indicates Pre-renal Azotemia or Urea elevation
 Post renal azotemia
 Decreased BUN Levels
 Primary renal azotemia
 Renal disease
 Acute tubular necrosis

analytical methods for urea

urease/GLDH method

vitros BUN

 Principle:
 A drop of patient sample is deposited on the
slide and is evenly distributed by the spreading
layer to the underlying layers
 Water and nonproteinaceous components then
travel to the underlying reagent layer, where
the urease reaction generates ammonia.
 The semipermeable membrane allows only
ammonia to pass through to the color-forming
layer, where it reacts with the indicator to form
a dye.
 The reflection density of the dye is measured
and is proportional to the concentration of urea
in the sample.

 1. Upper slide mount
 2. Spreading layer
(TiO2)
 3. Reagent layer
 • urease
 • buffer, pH 7.8
 4. Semipermeable
membrane
 5. Indicator layer:
ammonia
 indicator
 6. Support Layer
 7. Lower slide mount

Roche/Hitachi Cobas 501 test principle for urea

reference levels - urea nitrogen

 ASCP Combined Adult Range: 6-20
mg/dl (2.1-7.1 mmol/L)
 Interferences
 Fluoride or citrate inhibits Urease reaction
 Low protein, high carbohydrate diet-falsely
lower
 Urea is quite susceptible to bacterial
decomposition, especially urine

ammonia, NH3 biochemistry

 Arises - deamination of amino acids-protein
 Digestive and bacterial enzymes on proteins
 Release from metabolic reactions occurring in
skeletal muscle
 Consumed by hepatic parenchymal cells in the
production of urea
 In severe liver disease, Parenchymal cells are
damaged
 NH3 levels rise
 Plasma levels of NH3 are not dependent on renal
function, but on liver function

metabolism

 Hepatic Portal vein delivers the ammonia
to the liver
 Enzymes convert the NH3 to urea
 Combines with H+
 Ammonia cannot be excreted by kidney
 Elevations of ammonia are neurotoxic
 Often associated with hepatic
encephalopathy (hepatic coma)

clinical significance of urea

 Hepatic Failure
 Hepatic encephalopathy
 (hepatic coma)
 Reye’s Syndrome
 Viral disease treated with aspirin in young
children and teens can lead to Reye’s disease
 Acute metabolic disorder of the liver
 Severe Liver Disease (most common)
 Impaired Liver circulation
 Genetic enzyme deficiencies
 Involving urea cycle enzymes

specimen handling requirements

 Proper specimen handling is utmost importance
 Ammonia levels can rise rapidly in whole blood
 Venous specimens should be obtained without
trauma
 Place on ice immediately
 Li-Heparin or EDTA plasma is preferred
specimen

ammonia testing considerations

 For best results, assays should be completed
ASAP to prevent in-vitro deamination
 Hemolysis must be avoided, red cells contain 2
- 3 times plasma levels
 Smoking should be avoided for several hours
before sample is drawn
 Glassware and reagents must be free of
ammonia contamination

analytical methods

 Ion Exchange Resin Absorption
 Cation exchange resin (Dowex 50)
 Elution of NH3 with NaCl
 Quantitation by Berthelot reaction
 Manual, time consuming procedure,
not available for automation
 Gives slightly higher results

coupled enzymatic method

 Principle:
 Glutamine dehydrogenase (GLDH)
GLDH
 NH4+ + 2-oxoglutarate + + NADPH → Glutamate + NADP+ + H2O
 Measure decrease of Absorbance at 340 nm
 as NADPH is reduced to NADP+
 Most commonly used on automated systems
 Good precision and accuracy

ammonia ion selective electrode

Diffusion of NH3 through a selective
membrane into NH4Cl solution.
 Measures change of pH as NH3 diffuses
across selective membrane
 Measured potentiometrically
 Problems with membrane stability

reference ranges for urea

 Adult:
 19-60 ug/dl or (11-35 umol/L)
 Newborn:
 68-136 ug/dl or 64-107 umol/L