npn (1)
DESCRIPTION
clinical chemistryTRANSCRIPT
NONPROTEIN NITROGEN
COMPOUNDS
Renal function Tests
The urinary system includes kidneys, ureters, the bladder and the urethra. The kidneyis a vital organ that performs three important tasks: Excretory Homeostatic Endocrine function.
Three tests for renal function:1. Assessment of Glomeruli Filtration Rate – Clearnce Tests2. Assessment of Renal Vlood Flow- NPN determination3. Assessment of Tubular Function Tests-(1) measuring the
concentration and dilution of urine, (2) assessment of renal concentrating ability, (3) assessment of renal diluting capacity, and (4) assessment of urinary acidification.
Nonprotein Nitrogen Compounds
Urea
Uric AcidCreatinine
Amino Acid
Nonprotein Nitrogen Compounds
CompoundApproximate Plasma Concentration
(% of Total NPN)
Approximate Urine Concentration (% of Excreted N)
Urea 45-50 86.0Amino Acids 25 ---Uric Acid 10 1.7Creatinine 5 4.5Creatine 1-2 ---Ammonia 0.2 2.8
UREA
1. Physiology2. Clinical application3. Methods4. Specimen Requirement5. Pathophysiology
1. Physiology
Waste product of the protein catabolismExcreted by the kidneys
Reactions involved in Urea cycle:Step 1: Formation of carbamoyl phosphate (catalyzed by Carbamoyl Phosphate Synthetase), in liver mitochondria
Step 2: Formation of Citrulline (catalyzed by Ornithine Trans-Carbamoylase) in liver mitochondria
Step 3: Formation of Argininosuccinate (catalysed by ArgininosuccinateSynthetase) in liver cytoplasm.
Step 4: Cleavage of arginosuccinate to form Arginine (catalyzed by Argininosuccinase, or ArgininosuccinateLyase) in liver cytoplasm.
Step 5: Cleavage of arginine to release urea (catalyzed by Arginase) in liver cytoplasm
Overall equation of the urea cycle2NH3 + CO2 + 3ATP + 3H2O → urea + 2
ADP + 4Pi + AMP
Fate of urea Urea which is the waste product of the urea cycle diffuses
from the liver and is transported in the blood. The kidney filtered the urea from blood to be excreted as a
component of urine. Small portion of urea diffuses from blood into the intestine
where it is converted to carbon dioxide (CO2) and ammonia (NH3)
by bacterial urease. The ammonia is either excreted as a component of the feces
or reabsorbed in the blood.
2. Clinical Application
Clinical Application ConversionEvaluate renal function • Urea N (mg/dL) ↔ urea (mg/dL)
1 urea N 2.14 urea
0.467 urea urea N• 0.357 mg/dL mmol/L
Asses hydration statusDetermine nitrogen balanceDiagnosis of renal diseaseVerify adequacy of dialysis
3. Method of Analysis
Enzymatic Method PrincipleFirst step Urea + 2 H2O –Urease 2 NH4
+ + CO3 2-
i. GLDH coupled enzymatic disappearance of absorption is measured at 340 nm
NH4+ + 2-oxoglutarate + NADH + H+
GLDH Glutamate + NAD+ + H2O
ii. Indicator dye
NH4+ + pH Indicator color change
a. Nessler’s reaction Ammonia + Nessler’s salt –Gum ghatti yellowb. Berthelot reaction Ammonia + alkaline hypochlorite
–Na nitoprusside indophenol blue
iii. ConductimetricConversion of unionized urea to NH4
+ and
CO32- results in increased conductivity
3. Method of Analysis
Chemical Method Principle
i. Fearon’s reaction Urea + DAM (Diacetyl Monoxime Method) yellow solution (Diazine dirivative)
Comment: Non-specific, uses toxic regents
4. Specimen Requirements
Specimen Considerations1. Use fasting blood sample since a high protein diet affects urea2. Avoid fluoride or citrate anticoagulants since they inhibit urease3. Refrigerate samples to avoid bacterial decomposition
5. Pathophysiology
Increased Concentration
Prerenal azotemia
• Caused by reduce blood flow• Congestive heart failure, shock, hemorrhage, dehydration, ↑ protein catabolism, high-protein diet
Renal azotemia
• Damage of filtering structures of the kidney• Renal failure and renal disease (glomerular nephritis, tubular necrosis)
Postrenal azotemia
• Urinary tract obstruction • Renal calculi, tumors of the bladder or protate
Azotemia – ↑ urea in the blood Uremia – ↑ plasma urea accompanied by renal failure
5. Pathophysiology
Decreased Concentration• Low protein intake• Severe vomiting and diarrhea• Liver disease• Pregnancy
URIC ACID
1. Physiology2. Synthesis3. Clinical application4. Methods5. Specimen requirements6. Pathophysiology
1. Physiology1
Major end-product of purine catabolism primarily in the liver.
After the blood is filtered at the glomerulus, the resulting fluid enters the tubules of the kidneys to be secreted as urine.
Degradation of purines to nitrogenous excretory products
Catabolism of uric acid
Two-thirds of the uric acid produced daily is being excreted by the kidneys and the remaining one-third is excreted in the stool.
Overproduction of uric acid and/or under-excretion by the kidneys leads to the excess storage in the joints, tissue and organs causing inflammatory response. This reaction results to a condition characterized by painful joint(s) known as a gout attack. Urate crystals may also appear as kidney stones and lead to painful obstruction of the urinary tract. Uric acid that is not excreted can lead to its catabolism to allantoin, allantoic acid, urea, or ammonia.
2. Clinical Application1
Asses inherited disorders of purine metabolismConfirm diagnosis and monitor treatment of goutDiagnosis of renal calculiPrevent uric acid nephropathy during chemotheraphyDetect kidney disfunction
3. Method
Chemical Method Principle
Phosphotungstic acid(Caraway method)
Uric Acid + H3PW12O4o + O2
-Na2CO3/OH- allantoin + tungsten blue + CO2
3. Method
Enzymatic Methods Principle
First stepUric Acid + O2 +2 H2O –Uricase allantoin + CO2 + H2O2
Spectrophotometric(Blauch and Koch)
Decrease in absorbance at 293 nm is measured (Uric acid v. allantoin)
Coupled enzyme (I)Catalase – catalyzed a chemical indicator reactionH2O2 + + reagent colored compund
Coupled enzyme (II)PeroxidaseH2O2 + indicator dye colored compound
3. Method
Reference Intervals
Adult Male
Plasma or serum
3.5 – 7.2 mg/dL (0.21-0.43 mmol/L)
Adult Female 2.6 – 6.0 mg/dL 0.16-0.36 mmol/L
Child 2.0-5.5 mg /dL 0.12-0.33 mmol/L
Adult Urine/ 24 hour 250-750, mg/day 11.5-4.4
mmol/day
4. Specimen Requirements
Specimen Considerations1. May be measured using heparinized plasma, serum or urine2. Avoid gross lipemia, high bilirubin concentration and hemolysis 3. Avoid EDTA or flouride additives (affects uricase method)
5. Pathophysiology
Increased Concentration (Hyperurecemia)Enzyme deficiencies
Lesch-Nyhan syndrome
Phosphoribosylpyrophosphate synthetase deficiency
Glycogen storage disease type 1 (Glucose-6-phosphatase deficiency)
Fructose intolerance (fructose-1-phosphate aldolase deficiency)
Treatment of myeloproliferative disease w/ cytotoxic drugs
5. Pathophysiology
Increased Concentration (Hyperurecemia)Hemolytic and proliferative process
Chronic renal disease
Toxemia of pregnancy
Lactic acidosis
Drugs and poisons
Purine-rich diet
Increase tissue catabolism or starvation
5. Pathophysiology
Decreased Concentration (Hypourecemia)Liver disease
Defective tubular reabsorption (Fanconi sydrome)
Chemotheraphy with azathioprine or 6-mercaptopurine
Overtreatment with allopurinol
1. Physiology of Creatinine1
Chief product of muscle metabolismNot affected by protein diet
LiverMuscle
2. Clinical Application1
Determine sufficiency of kidney functionDetermine severity of kidney damage
Monitor the progression of kidney disease
Measure completeness of 24-hour urine
2. Clinical Application
Renal Clearance and Glomerular Filtration RateGlomerular filtration rate Volume of plasma filtered
(V) by the glomeruli per unit of time
GFR =UCrVu
PCrt1.73A
X
GFR =Vt
3. Method of Analysis
Chemical Method Principle
Direct Jaffe Reaction Creatinine + picrate red-orange complex
Jaffe-kinetic Detection of color formation timed to avoid interference of noncreatinine chromogens
Jaffe with adsorbent (Lloyd’s method)
Creatine in protein-free filtrate adsorbed onto Fuller’s earth (aluminum magnesium silicate); then reacted with alkaline picrate
Jaffe without adsorbent
Creatine in protein-free filtrate reacts with alkaline picrate to form colored complex
4. Specimen Requirements
Specimen Considerations Specimen Considerations
A. Flasely increase due to
1. Glucose2. α-ketoacids3. Ascorbate4. Uric Acid5. Cephalosporins6. Dopamine
B. Falsely decrease results due to1. Bilirubin2. Hemoglobin3. Lipemic specimens
3. Method of Analysis
Enzymatic Method Principle
Creatininase-CK
Creatinine + H2O —Creatininase Creatine Creatine + ATP CK creatine phosphate + ADPPhosphoenolpyruvate + ADP —PK pyruvate + ATPPyruvate + NADH + H+ LD Lactate + NAD+
Creatininase-H2O2
Creatinine + H2O —Creatininase Creatine Creatine H2O —Creatininase Sarcosine + ADPsarcosine + O2 + H2O ADP —sarcosine oxidase glycine + CH2O + H2O2
H2O2+ colorless substrate —Peroxidase Colored product + H2O
3. Method of Analysis
Specimen Jaffe Method Enzymatic Mtd.
Adult Male
Plasma or serum
0.9 – 1.3 mg/dL (80-115 µmol/L)
0.6 – 1.1 mg/dL (55-96 µmol/L)
Adult Female
0.6 – 1.1mg/dL (53-97 µmol/L)
0.5 – 0.8 mg/dL (40-66 µmol/L)
Child 0.3 – 0.7 mg /dL (27-62 µmol/L)
0.0 – 0.6 mg/dL (0-52 µmol/L)
Adult MaleUrine
24 hour
800 – 2,000 mg/dayAdult Female 600 – 1,800 mg/day
5. Pathophysiology
Increased ConcentrationRenal failure (glomerular function)↑ Plasma Concentration ↓ GFR
1. Physiology of Ammonia1
By product of amino acid deamination.Remove from the circulation and converted to urea in the liver.
2. Clinical Application1
Diagnosis of hepatic failureReye’s syndrome – acute metabolic disorder of the liverInherited deficiencies of urea cycle
3. Method
Chemical Method Principle
Ion-selective electrodeDiffusion of NH3 through selective membrane into NH4Cl causing pH change, which is measured potentiometrically
Spectrophotometric NH3 + bromphenol blue blue dye
Enzymatic Method PrincipleGLDH Decrease in absorbance is measured at 340 nm
NH4+ + 2-oxoglutarate + NADPH + H+ GLDH Glutamate + NADP+ + H2O
3. Method
Specimen Reference ValuesAdult
Plasma19-60 µg/dL 11-35 µmol/L
Child (10 days to 2 yrs) 68-136 µg/dL 40-80 µmol/L
4. Specimen Requirements
Specimen Considerations1. May be measured using heparinized and EDTA tubes2. Samples should be centrifuged at 0°C to 0°C within 20 minutes of
collection and the plasma or serum removed3. Avoid cigarette smoking for several hours
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