Trans by : nahaeminrmt

Introduction: Nonprotein Nitrogen Compounds

 Analytical Methods
 Nonprotein Nitrogen - originated in the early days of  Enzymatic Methods
clinical chemistry 1. Enzyme Urease
 Analytic methodology that requires the removal of  Hydrolyzes urea to produce ammonium
protein for specimen analysis ion (NH4) → quantitated
 Nessle’s Reagent = Nitrogen → Ammonia 2. GLDH (coupled enzymatic method)
(measured spectrophotometrically)  Most common method
 Majority of the these compounds arise from  Urease reaction + glutamate
catabolism of proteins and nucleic acids dehydrogenase
 Analyte measured: the rate of
Urea
disapperance of NADH to NAD at 340
 Introduction nm
 NPN compound present in highest concentration
in the blood.
 Major excretory product of protein metabolism
 Synthesized by the Liver
 Urea Cycle = product from the catabolism of
amino groups (-NH2) and free ammonia. 3. Indicator Dye
 Blood Urea Nitrogen - term used to refer to urea  NH4+ + pH indicator = color change
determination. Although, Urea Nitrogen (Urea N)  Incorporated using liquid reagents,
is a more appropriate term. multilayer film, and reagent strips
4. Conductimetric
 Biochemistry  It uses electrode to measure the rate of
 Protein metabolism - produces amino acids that increased conductivity as NH4+ are
can be oxidized to produce energy or stored fat, produced
and glycogen  Rate of change in conductivity is
 Protein metabolism release nitrogen and measured
converted to urea and excreted as a waste product.  Ammonia contamination don’t interfere
 Liver (synthesis of urea) → blood (plasma) → 5. Isotope Dilution Mass Spectrometry
kidney (for filtration in the glomerulus)  Detection of characteristic
 Most of the Urea is excreted in the urine, fragmentations following ionization
however a small amount of urea is  Quantification using isotopically labeled
reabsorbed by passive diffusion. compound
 Amount Reabsorbed depends on: urine flow  Proposed reference method
rate and extent of hydration
 Small amount of Urea (<10% of the total urea) is  Specimen Requirements
excreted through GI tract and skin.  Samples: plasma, serum, or urine
 Concentration of Plasma in urea is determined by:  Plasma = avoid ammonium ions and ↑
 Protein content of the diet concentrations of sodium citrate and sodium
 Rate of protein catabolism fluoride
 Renal function and perfusion  Citrate and Fluoride can inhibit urease
 Fasting sample is not required (diet can influence
 Clinical Application urea but not a single protein containing meal)
 Measurement of urea is used to:  Specimens (esp. urine) that are delayed in testing
 Evaluate renal function should be refrigerated
 Asses hydration status  Methods for plasma or serum should be modified
 Determine nitrogen balance when tested on urine due to ↑ urea concentration
 Aid in the diagnosis of renal disease and presence of endogenous ammonia
 Verify the adequacy of dialysis REFERENCE INTERVAL OF UREA NITROGEN
 Urea is often reported in terms of nitrogen Adult Plasma or Serum Urine, 24h
concentration rather than urea concentration. 6 - 20 mg/dL or 12 - 20 mg/dL or
Nitrogen Concentration × 2.14 (mg/dL) = urea concentration 2.1 - 7.1 mmol/L 0.43 - 0.71
mmol/L
Urea N (mg/dL) × 0.36 = urea concentration in mmol/L

1
  30% is degraded by bacterial enzymes in
 Pathophysiology GI tract
 Azotemia - ↑ concentration of urea in the blood  Monosodium urate - uric acid in plasma
 Uremic Syndrome or Uremia - ↑ plasma urea  pH of plasma (pH ~7) = uric acid is INSOLUBLE
concentration accompanied renal failure  pH of > 6.8 = uric acid is SATURATED
 Fatal in not treated with dialysis  Acidic urine (pH <5.75) = uric acid is predominant
 ↑ plasma urea: pre-renal, renal, and post renal and uric acid crystals may form
I. Pre-renal Azotemia
 ↓ renal blood flow = ↓ blood flow in the  Clinical Application
kidneys = ↓ urea is filtered  Confirm diagnosis and monitor treatment for gout
 Causative factors: CHF, shock, hemorrhage,  To prevent acid nephropathy during chemotherapy
dehydration, and other factors resulting in a  Assess inherited disorders of purine metabolism
significant ↓ in blood volume  To detect kidney dysfunction
 Amount of protein metabolism  To assist in the diagnosis of renal calculi
 High-protein diet
 Increased protein catabolism: stress, fever,  Analytical Methods
major illness, corticosteroid therapy, and GI  Uric acid is the final breakdown product of purine
hemorrhage metabolism in human and apes
II. Renal Azotemia  Other mammals have the ability to catabolize
 Compromised urea excretion uric acid to allantoin (water-soluble product)
 Acute and chronic renal failure  Allantoin
 Glomerular nephritis  Uric acid is readily oxidized to allantoin
 Tubular necrosis  Acts as a reducing reagent
 Other intrinsic renal disease 1. Caraway Method
III. Post-renal Azotemia  Most common method
 Obstruction in urine flow  Oxidation of uric acid in a protein-free filtrate, with
 Renal calculi subsequent reduction of phosphotungstic acid in
 Tumors of the bladder or prostate alkaline solution to tungsten blue
 infection  uric acid + phosphotungstic acid + O2 → allantoin +
 ↓ plasma urea concentration tungsten blue + CO2
 Low protein intake 2. Enzyme Uricase
 Severe liver disease  Specific and used almost exclusively in clinical
 Pregnancy and infancy laboratories
 Urea N/Creatinine Ratio I. Spectrophotometric
 Normal = 10:1 to 20:1  Measures the differential absorption of uric
 Pre-renal conditions = ↑ plasma urea; normal acid and allantoin at 293 nm
plasma creatinine (↑ urea N/creatinine ratio)  The difference in absorbance before and after
 Post-renal conditions = ↑ urea N/creatinine incubation with uricase is proportional to the
ratio with ↑ creatinine uric acid concentration
 ↓ urea production = ↓ urea N/creatinine  Interferences:
ratio (low protein intake, severe liver dse.,  Protein - ↑ background absorbance
acute tubular necrosis) reducing sensitivity
 Hemoglobin and Xanthine - negative
Uric Acid
interference
 Introduction II. Coupled-Enzyme Methods
 Product of catabolism of the purine nucleic acids  It measures the hydrogen peroxide produced
 Filtered but mostly reabsorbed (PCT) and reused as uric acid is converted to allantoin
 Insoluble in plasma  H2O2 is used to catalyze chemical indicator
 ↑ concentrations can be deposited on joint and reaction
tissue = painful inflammation  Color produced = quantity of uric acid in
specimen
 Biochemistry  Adapted for use on traditional wet chemostry
 Purines → uric acid analyzers and dray chemistry slide analyzers
 Purines - adenine and guanine from the  Commercial reagent preparations -
breakdown of ingested nucleic acids potassium ferricyanide and ascorbate oxidase
 Primarily synthesized in the Liver (to minimize interferences)
 uric acid → plasma → kidneys (filtration) III. Isotope Dilution Mass Spectrometry
 Kidneys  Proposed as reference method (see urea)
 PCT (98% - 100%) reabsorption of UA
 DCT small amounts are secreted into the  Specimen Requirements
urine  Specimen: HEPARINIZED plasma, serum, or urine
 70% renal excretion
2
  Serum - should be removed from cells as  Allopurinol - inhibits xanthine oxidase and an
quickly as possible to prevent dilution by enzyme in the uric acid synthesis pathway
intracellular contents which is used for treatment
 Diet - may affect UA concentration overall but not  Hemolytic or Megaloblastic Anemia
the recent meal; FASTING IS NOT REQUIRED  Elevated uric acid concentration
 Gross lipemia - should be AVOIDED  Increased urate concentration
 ↑ bilirubin - falsely ↓ results obtained by  Ingestion of a diet-rich in purines (e.g., liver,
peroxidase methods kidney, sweetbreads, and shellfish)
 Significant hemolysis with glutathione release -  ↑ tissue catabolism due to inadequate
may result to ↓ values dietary intake (starvation)
 Drugs (salicylates and thiazides) - ↑ uric acid  Enzyme Deficiency
 Preservation  Lesch-Nyhan Syndrome
 Uric acid is stable in plasma or seum after  X-linked genetic disorder (males)
RBCs have been removed  Complete deficiency of hypoxanthine-
 Serum samples may be stored refrigerated for guanine phosphoribosyltransferase, an
3 - 5 days important enzyme in the biosynthesis of
 EDTA and Fluoride should NOT be used purines
 Urine collections- must be alkaline (pH of 8)  It prevents the utilization of purine
REFERENCE INTERVALS OF URIC ACID bases in the nucleotide salvage
Plasma or Serum pathway
Adult Male 3.5 - 7.2 0.21 - 0.43  ↑ de novo synthesis of purine
mg/dL mmol/L  ↑ plasma and urine concentrations
Female 2.6 - 6.0 0.16 - 0.36 of uric acid
mg/dL mmol/L  Symptoms include: Neurologic
Child 2.0 - 5.5 0.12 - 0.33 symptoms, mental retardation, and self-
mg/dL mmol/L mutilation
Urine, 24h  Mutation in Phosphoribosylpyrophosphate
Child 250 - 750 1.5 - 4.4 synthetase
mg/d mmol/d  It is the first enzyme in the purine synthesis
Conversion of mg/dL to SI unit = 168 g/ml (molecular mass pathway
of uric acid)  ↑ uric acid
 Glycogen storage disease
 Pathophysiology  Deficiency of glucose-6-phosphate
 Abnormally ↑ plasma uric acid concentration:  Fructose Intolerance
 Gout  Deficiency of fructose-1-phosphate aldolase
 ↑ catabolism of nucleic acids  Increased lactate and triglycerides
 Renal disease  They compete with urate for renal excretion
 Gout in these diseases
 Found primarily in men aging 30 - 50 years old  Toxemia of pregnancy (Preeclampasia)
 Characterized by the pain and inflammation  Result of decreased excretion of uric acid
of joints due to precipitation of sodium urates  Lactic acidosis
 Hyperuricemia *lactic acidosis and preeclampasia result of a
 Exacerbated by: purine-rich diet, drugs, competition for binding sites in the renal tubules
and alcohol  Chronic Renal Disease
 > 6.0 mg/dL of plasma uric acid  ↑ uric acid concentration because filtration
concentration and secretion are impaired
 Patients are susceptible in forming renal  Uric acid nephrolithiasis
calculi  In acidic urine, the relatively insoluble uric
 Women - urate concentration rise after acid precipitates to form calculi which can
menopause cause intense flank pain.
 Postmenopausal women - may develop  Stones may be dissolved by alkalinization of
hyperuricemia and gout urine
 Severe cases - formation of tophi, deposits of  Treated by increased fluid intake
crystalline uric acid and urates in tissue which  Administration of xanthine and oxidase
will cause deformities inhibitors - reduce uric acid production
 Increased metabolism of cell nuclei  Hypouricemia
 Elavated plasma uric acid concentration  Severe liver disease
 Occurs in patients on chemotherapy for such  Defective tubular reabsorption (Fanconi
proliferative diseases: leukemia, lymphoma, Syndrome)
multiple myeloma, and polycythemia  Chemotherapy with 6-mercaptopurine or
aziathioprine
 Inhibitors of de novo purine synthesis
3
  Overtreatment with allopurinol  Units of mL/min with correction for the body
surface area.
Creatinine/Creatine
 It provides a reasonable approximation of
 Introduction of Creatinine GFR
 Creatinine - is formed from creatine and creatine  Plasma creatinine does not provide sufficient
phosphate in muscle and is excreted into the sensitivity for the detection of mild renal
plasma at a constant rate related to muscle mass. dysfunction
 Plasma creatinine is inversely related to  Estimated GFR (eGFR)
glomerular filtrate rate (GFR)  Modification of Diet in Renal Disease (MDRD)
 Commonly used to assess renal filtration function  Serum creatinine concentration, age,
gender, and ethinicity
 Biochemistry of Creatinine  Useful when serum creatinine results are
produced in an assay that has been
Creatine (liver) → Creatine Phosphate (muscle) → loss of calibrated to be traceable to an IDMS
phosphoric acid and creatine loss water to form the cyclic
method
compound → Creatinine → diffuses into the plasma and is
excreted in the urine
 Results are normalized to a standard
body surface area (1.73 m2)
 Creatinine is released into the circulation at a  Valid for individuals older than 18 years
relatively constant rate that has been shown to be and younger than 70 years
proportional to an individual’s muscle mass  Chronic Kidney Disease Epidemiology (CKD-
 circulation → glomerular filtration → urine EPI)
 Small amounts of creatinine are excreted by the  Its equation is used to report higher
proximal tubule and reabsorbed by the renal values (>60 mL/min/1.73 m2)
tubules  Modified Shwartz equation
 Daily creatinine excretion is reasonably stable  eGFR (mL/min/1.73m2) = (0.41 x
height)/Scr
 Clinical Application  Equation is used for children < 18 y/o
 Importance of measuring creatinine concentration:  Analytical Methods
 To determine the sufficiency of kidney  Creatinine
function  Jaffe Reaction - creatinine reacts with picric
 To determine the severity of of kidney acid in an alkaline solution to form a red-
damage orange chromogen
 To monitor the progression of kidney disease  Folin and Wu - Jaffe reaction but measure the
 Plasma creatinine concentration blood creatinine
 Function of relative muscle mass, the rate of  Positive interference: acetoacetate,
creatine turnover, and renal function acetone, ascorbate, glucose, and
 Amount of creatinine in blood is reasonably pyruvate
stable  Jaffe with Adsorbent
 Protein content of diet influence plasma  Adsorbent - Fuller’s earth (aluminum
concentration magnesium silicate) or Lloyd’s reagent
 Urinary constituents may be expressed as a ratio to (sodium aluminum silicate)
create quantity rather than mass excreted per day  Eluted and reacted with alkaline picrate
 Creatinine Clearance (CrCl)  Time consuming and not readily
 It is a measurement of the amount of automated
creatinine eliminated from the blood to the  Kinetic Jaffe Method
kidneys  Serum is mixed with alkaline picrate
 Glomerular Filtration Rate (GFR)  The rate of change in absorbance is
 Unit of plasma filtered (V) by the measured
glomerulus per unit of time (t)  Interferences: α-keto acids and
 GFR = V/t cephalosporins
 V = Ms/Ps where; S = substance Ms  Negative bias: bilirubin and hemoglobin
= mass of S filtered, Ps = plasma (as a result of their strong base used)
concentration  Routinely used (inexpensive, rapid, and
 Ms = UsVs where; Us = urine easy to perform)
concentration, Vs = urine volume  Enzymatic Methods - for dry slide analyzers
 GFR = UsVs/Pst  Creatinininase (creatinine
 Formula of Creatinine Clearance amidohydrolase)
Ucr Vu  Creatinase (creatine amidinohydrolase)
CrCl =
Pcr t  Sarcosine oxidase
 Ucr = urine creatinine concentration  Peroxidase
 Pcr = plasma creatinine concentration

4
 Specimen Requirements Adult Urine, 24h
 Plasma, serum, urine may be used Male 800 - 2000 mg/d (7.1 - 17.7 mmol/d)
 Hemolyzed and icteric samples should be avoided Female 600 - 1800 mg/d (5.3 -15.9 mmol/d)
 Lipemic samples may produce erroneous results in
some methods  Pathophysiology
 Fasting is not required  Creatinine
 High-protein ingestion may transiently  ↑ creatinine concentration
elevate serum concentrations  Abnormal renal function (glomerular
 Urine should be refrigerated after collection or filtration
frozen if longer storage than 4 days is required  Plasma concentration of creatinine is
 Sources of Errors inversely proportional to the clearance of
 ascorbate, glucose, a-keto acids, and uric acid creatinine
 ↑ creatinine concentration measured by the  ↑ plasma creatinine = ↓ GFR = renal
Jaffe reaction (>30°C) damage
 Significantly decreased when kinetic  Plasma creatinine is a insensitive marker
measurement is applied  It may not be measurable until 50% of
 Interference of a-keto acids may interfere renal function has detoriorated
depending on the concentrations and  Creatine
measuring time  Muscular dystrophy, poliomyelitis,
 bilirubin hyperthyroidism, and trauma = both plasma
 Negative bias in both Jaffe and enzymatic and urinary creatinine are often ↑
methods  Measurement of creatine kinase is used
 ascorbate typically for the diagnosis of muscle disease
 It will interfere in enzymatic methods that use  Plasma creatine concentration is not elevated
peroxidase as a reagent in renal disease
 cephalosporin antibiotics
 Falsely ↑ results in Jaffe reaction Ammonia
 drugs  Introduction of Ammonia
 Dopamine - affects both Jaffe and enzymatic  Deamination of amino acids during protein
methods metabolism
 Lidocaine - positive bias in some enzymatic  It is removed from the circulation and converted to
methods urea in the liver
 Free ammonia is toxic - ammonia is present in the
 Creatine plasma in low concentrations
 Traditional method for measurement of creatine  Biochemistry
relies on the analysis of the sample using an end-  Ammonia (NH3) - it is produced in the catabolism
point Jaffe method for creatinine before and after of amino acids and by bacterial metabolism in the
heated in acid solution lumen of the intestine
 Heating converts creatine to creatinine and  Endogenous ammonia - anaerobic metabolic
the difference between the two sample reactions that occur in skeletal muscle during
measurements is the creatine concentration exercise
 High temperature may result in the formation of  NH3 is consumed by the parenchymal cells of the
additional chromogens will lead to poor precision liver in the production of urea
 Enzymatic assay  At normal physiologic pH most ammonia in blood
 The initial enzyme is omitted and creatine exists as ammonium ion (NH4)
kinase, pyruvate kinase, and LDH are coupled  Ammonia is excreted as ammonium ion (NH4) by
to produce a measurable colored-product the kidney and acts as a buffer urine
 Creatine can be measured by HPLC  Clinical applications
 Reference Intervals  For the diagnosis of hepatic failure, Reye’s
 Vary with assay type, age, and gender Syndrome, and inherited deficiencies of urea cycle
 Creatinine concentration - decreases with age enzymes
beginning in the 5th decade of life  Severe Liver Disease
REFERENCE INTERVALS - CREATININE  Most common cause of disturbed ammonia
Adult Plasma or Serum metabolism
Jaffe Method Enzymatic Method  Monitoring of blood ammonia - for
Male 0.9 - 1.3 mg/dL 0.6 - 1.1 mg/dL determination of prognosis
(80 - 115 μmol/L) (53 - 97 μmol/L)  Correlation between the extent of
Female 0.6 - 1.1 mg/dL 0.5 - 0.8 mg/dL hepatic encephalopathy and plasma
(53 - 97 μmol/L) (44 - 71 μmol/L) ammonium concentration is not always
Child 0.3 - 0.7 mg/dL 0.0 - 0.6 mg/dL consistent
(27 - 62 μmol/L) (0 - 53 μmol/L)

5
  Arterial ammonia concentration - a  Frozen aliquotes of human serum albumin
better indicator of the severity of the containing known amounts of ammonium chloride
disease or ammonium sulfate may be used
 Reye’s Syndrome
 Commonly occur in children  Reference Intervals
 Preceded by a viral infection or  Vary in methods used
administration of aspirin  Higher concentrations are seen in newborns
 Acute metabolic condition of liver REFERENCE INTERVALS - AMMONIA
 Severe fatty infiltration of the liver Plasma or Serum
 Diagnosis of inherited deficiency of urea cycle Adult 19 - 60 μg/dL 11 -35 μmol/L
enzymes Child (10 d to 2 y) 68 - 134 μg/dL 40 - 80 μmol/L
 Neonate with unexplained nausea, vomiting, Urine, 24 hr 68 - 134 μg/dL 40 - 80 μmol/L
or neurological deterioration associated with
feeding  Pathophysiology
 Assays of blood ammonia - monitor  Severe liver disease
hyperalimentation therapy  Severe colateral circulation or parenchymal
 Measurement of urine ammonia - confirm the liver cell function is severely impaired
ability of the kidneys to produce ammonia  Ammonia is not removed from the circulation
and blood concentration increases
 Analytical Methods  ↑ concentrations of ammonia - neurotoxic
 Accurate laboratory measurement is complicated and often associated with encephalopathy
due to ammonia in plasma is unstable, low in  ↑ extracellular glutamate
concentration, and pervasive contamination concentration
 Two approaches  Subsequent depletion of ATP in brain
 Ammonia is isolated from the sample then  Hyperammonemia
assayed  Associated with inherited deficiency of urea
 Direct measurement of ammonia thru cycle enzymes
enzymatic methods of ion-selective electrode
(basahin mo nalang hahahsakdsd)

 Specimen requirements
 Whole blood ammonia concentration increases
rapidly following specimen collection of in vitro
amino acid deamination
 Venous blood should be obtained without trauma
and placed on ice immediately
 Heparin and EDTA - suitable anticoagulants
 Samples should be centrifuged at 0 - 4°C within 20
mins of collection and the plasma or serum should
be removed
 Frozen plasma is viable for several days at -20°C
 Erythrocytes contain 2-3× as much ammonia in
plasma therefore, hemolysis should be avoided
 Cigarette smoking could be a cause of ammonia
contamination
 Patients should not smoke for several hours
 Substances that may ↑ increase ammonia in
plasma
 Ammonium salts, asparginase, barbiturates,
diuretics, ethanol, hyperalimentation,
narcotic analgesics
 ↓ ammonia concentrations
 Diphenhydramine, Lactobacillus acidophilus,
lactulose, levodopa, and several antibiotics
 Glucose at concentrations >600 mg/dL - interferes
in dry slide methods

 Sources of Errors
 Tobacco smoke, urine, and ammonia in detergents,
glasswares, reagents, and water

6
7