C - Chapter VIII: NPN Flashcards
FUNCTIONS of the KIDNEYS:
1. Elimination of (?)
2. Elimination of (?) (urea and creatinine)
3. Elimination of (?)like drugs
4. Retention of substances necessary for (?) (proteins & amino acids, glucose)
5. Regulation of (?) of the body fluids)
6. (?) function:
excess body water
waste products of metabolism
foreign substances
normal body function
electrolyte balance and osmotic pressure
Endocrine
Primary:
production of rennin, prostaglandin and erythropoietin
Secondary:
degradation of insulin, glucagon and aldosterone
Clinically Significant NPN compounds:
- urea – 45%
- amino acids – 20%
- uric acid – 20%
- creatinine – 5 %
- creatine – 1-2 %
- ammonia – 0.2%
TOTAL NPN METHODOLOGY: TWO STEPS:
- KJELDAHL DIGESTION
- MEASUREMENT OF AMMONIA FORMED
MEASUREMENT OF AMMONIA FORMED
A. NESSLERIZATION
B. BERTHELOT METHOD
C. MONITORING CONSUMPTION OF AMMONIA (Kaplan, Manoukian – Fawaz; Kallet – Cook Reaction)
The nitrogen in a pff of the specimen is converted to ammonia using hot conc. H2SO4 with copper sulfate, mercuric sulfate or selenium oxide as the catalysts.
KJELDAHL DIGESTION
NPN + H2SO4 NH4HSO4
KJELDAHL DIGESTION
NH4HSO4 + NaOH Na2SO4 + NH3 + H2O
KJELDAHL DIGESTION
Nessler’s reagent
double iodide salt of potassium & mercury
– colloidal stabilizer
Gum ghatti
– yellow to orange brown product
Dimercuric ammonium iodide
NH3 + HgI2.2KI NH2Hg2I2 + KI + NH4I
NESSLERIZATION
Reagent: phenol and alkaline hypochlorite
BERTHELOT METHOD
Catalyst: sodium nitroprusside
BERTHELOT METHOD
Product: indophenol blue
BERTHELOT METHOD
NH3 + NaOCl + Phenol Indophenol + NaCl + H2O
BERTHELOT METHOD
NH3 + α – ketoglutarate + NADH + H Glutamate + NAD
MONITORING CONSUMPTION OF AMMONIA (Kaplan, Manoukian – Fawaz; Kallet – Cook Reaction)
Catalyst: Glutamate dehydrogenase
MONITORING CONSUMPTION OF AMMONIA (Kaplan, Manoukian – Fawaz; Kallet – Cook Reaction)
Measure a decrease in the absorbance at 340 nm
Glutamate dehydrogenase
- most abundant NPN compound in plasma
UREA
- major excretory product of protein metabolism
UREA
- synthesized in the liver from CO2 and ammonia that arises from the deamination of amino acids in the reaction of the urea cycle
UREA
UREA STRUCTURE
UREA MW
60 g/mole
UREA
MW = 60 g/mole
C =
H =
O =
N =
C = 1 x 12 = 12
H = 4 x 1 = 4
O = 1 x 16 = 16
N = 2 x 14 = 28
From urea mass units to urea nitrogen (28/60) =
0.467
From urea nitrogen to urea mass units (60/28) =
2.14
- [?] excreted through the kidneys
90%
- excreted through the skin and GIT
10%
- [?] reabsorbed in the renal tubules by passive diffusion
40 – 70%
Urea concentration depends on the following :
- renal function and perfusion
- protein content of the diet
- amount of protein catabolism
METHODS FOR UREA DETERMINATION
I. INDIRECT METHOD / ENZYMATIC
II. DIRECT METHODS
INDIRECT METHOD / ENZYMATIC
- Berthelot reaction
- Nessler’s reaction
- GLDH-coupled enzymatic method (Dupont ACA Analyzer)
- Conductimetric method: Beckman BUN Analyzer
- Urograph or Urastrat strip
- Indicator dye (uriol): Kodak Ectachem Analyzer
DIRECT METHODS
- Diacetyl Monoxime (Fearon)
- ortho – phthaldehyde: adapted by automated methods
measures Blood Urea Nitrogen
INDIRECT METHOD / ENZYMATIC
Based on the preliminary hydrolysis of urea with urease followed by some process that quantitates the ammonium ion
INDIRECT METHOD / ENZYMATIC
Decrease in absorbance of NAD at 340 nm
➢ Based on the measurement of the conductivity generated from the reaction of urease on urea producing ammonium ions & bicarbonates
Conductimetric method: Beckman BUN Analyzer
➢ Physical principle: based on chromatography
Urograph or Urastrat strip
➢ Chemical principle: Conway Microdiffusion method
Urograph or Urastrat strip
➢ Dye is added to NH4 ions from urea hydrolysis & the color change is measured
Indicator dye (uriol): Kodak Ectachem Analyzer
➢ Used in multilayer film reagents, dry reagent strips and automated systems
Indicator dye (uriol): Kodak Ectachem Analyzer
➢ Direct condensation reaction
DIRECT METHODS
➢ Diacetyl – very toxic
DIRECT METHODS
Urea + OP Isoindoline
ortho – phthaldehyde: adapted by automated methods
Isoindoline + Naphthylethylenediamine colored compound
ortho – phthaldehyde: adapted by automated methods
– a biochemical abnormality pertaining to increase NPN compounds especially creatinine and urea defining GFR defect
Azotemia
– due to reduced renal blood flow
a. prerenal
– decreased renal function
b. renal
– obstruction of urine flow
c. postrenal
Azotemia Three categories:
a. prerenal
b. renal
c. postrenal
> calculi, tumors of bladder or prostate
c. postrenal
> kidney diseases: glomerular nephritis
b. renal
> hemorrhage, dehydration, increased protein catabolism
a. prerenal
– a clinical syndrome characterized by increased BUN accompanying renal failure seen in metabolic acidosis, hyperkalemia and edema
Uremia/ Uremic syndrome
Decreased Urea: -
decreased protein intake, severe vomiting and diarrhea
Specimen Requirements and Interfering Substances
1. plasma, [?], or [?]
2. plasma : [?] and high concentrations of [?] and [?] must be avoided
3. [?] is acceptable
4. [?] is recommended
5. urine sample guarded against bacterial decomposition of [?]
serum or urine
ammonium ions; sodium citrate and sodium fluoride
non fasting sample
nonhemolyzed sample
urea
Reference Interval:
− adult serum/plasma
6-20 mg/dL 2.1-7.1 mmol/L
Reference Interval:
− conversion factor
mg/dL —> mmol/L :
0.357
Reference Interval:
- urine, 24hr
12-20 g/day 0.43-0.71 mol/day
- principal waste product of muscle metabolism derived mainly from Creatine (alphamethylguanidinoacetate
CREATININE
Creatine is produced from two enzymatic processes:
o transamination of arginine & lysine forming guanidinoacetic acid in the kidneys, small intestines, pancreas and probably the liver
o methylation of guanidinoacetic acid in the liver
is the muscles’ energy source
Creatine PO4
RENAL HANDLING of CREATININE:
1. Glomerular filtration
2. Excreted without being reabsorbed. Thus, excretion is relatively constant. Creatinine output is sometimes used to measure the completeness of a 24-hour urine sample collection
3. When serum creatinine is elevated, it is secreted in the renal tubules
ANALYTICAL METHODS
- DIRECT METHOD: JAFFE REACTION
- INDIRECT / ENZYMATIC METHODS
- Yatzidis method
- High Performance Liquid Chromatography (HPLC)
Creatinine + alkaline picrate Creatinine picrate (red orange/yellow) 510 nm
DIRECT METHOD: JAFFE REACTION
Alkaline picrate:
1 part 10% NaOH and 5 parts sat. picric acid (2,4,6 trinitrophenol)
lacks specificity
Jaffe reaction
Non-creatinine Jaffe-reacting chromogens:
- Proteins
- Glucose
- Ascorbic acid
- Guanidine
- Acetone
- Cephalosporins
- α-ketoacids (acetoacetate and pyruvate)
INDIRECT / ENZYMATIC METHODS
a. F. Lim – Creatininase or creatinine iminohydrolase
b. G.A. Moss – Creatinine Amidohydrolase
Creatinine —————-→ N-methylhydantoin + NH3 (Creatininase)
F. Lim – Creatininase or creatinine iminohydrolase
NH3 + α-ketoglutarate + NADH —————–→ glutamate + NAD + H+ (Glutamate DH)
F. Lim – Creatininase or creatinine iminohydrolase
Creatinine ———————————–→ Creatine (Creatinine amidohydrolase)
G.A. Moss – Creatinine Amidohydrolase
Creatine + ATP ——————→ CreatinePO4 + ADP (Creatine kinase)
G.A. Moss – Creatinine Amidohydrolase
ADP + PEP ——————→ Pyruvate + ATP (Pyruvate kinase)
G.A. Moss – Creatinine Amidohydrolase
Pyruvate + NADH + H+ —————→ Lactate + NAD (Lactate DH)
G.A. Moss – Creatinine Amidohydrolase
Creatinine reacts with alkaline picrate at two different pH levels
Yatzidis method
: protein & other interfering materials will reacts w/ picrate but creatinine does not
pH 10
: both creatinine & proteins react
pH 11
High Performance Liquid Chromatography (HPLC)
Sources of error :
- ascorbate, glucose, alpha keto acids and uric acid
- drugs
false increase
ascorbate, glucose, alpha keto acids and uric acid
cephalosporin and dopamine intake
lidocaine intake
CREATININE
Specimen Requirements and Interfering Substances
3. plasma, [?]
4. avoid [?] especially for the Jaffe reaction
5. [?] cause errors
6. [?] acceptable
7. [?] may transiently elevate serum concentrations
8. [?] : refrigerated after collection or frozen if longer storage than 4 days is required
serum or urine
hemolyzed and icteric sample
lipemic samples
non-fasting sample
high protein ingestion
urine
- overestimates but gives a reasonable approximation of glomerular filtration rate
Creatinine Clearance
expressed in mL/minute
Creatinine Clearance
plasma concentration of creatinine is inversely proportional to creatinine clearance (elevated creatinine clearance = decreased GFR)
Creatinine Clearance
insensitive marker: >50% renal dysfunction = abnormal plasma creatinine
Creatinine Clearance
Creatinine Clearance =
U x V / P
− used in the diagnosis of muscle diseases
Creatine kinase
: CREATININE RATIO
BUN
NORMAL CREATININE RATIO :
10–20:1
a. Acute tubular necrosis
BUN:CREA ratio <10:1
b. Low protein intake; starvation
BUN:CREA ratio <10:1
c. Severe liver disease
BUN:CREA ratio <10:1
d. Repeated dialysis
BUN:CREA ratio <10:1
e. Severe vomitting or diarrhea
BUN:CREA ratio <10:1
a. Catabolic states w/ tissue breakdown
BUN:CREA ratio >10:1 with normal creatinine
b. Pre-renal azotemia
BUN:CREA ratio >10:1 with normal creatinine
c. High protein intake
BUN:CREA ratio >10:1 with normal creatinine
d. After GIT hemorrhage
BUN:CREA ratio >10:1 with normal creatinine
High ratio with elevated creatinine levels
✓ Post-renal obstruction
✓ Pre-renal azotemia superimposed on renal disease
is MORE SPECIFIC for the diagnosis of renal disease
Creatinine determination
is MORE SENSITIVE for the diagnosis of renal disease
BUN determination
ASSOCIATED MYOPATHIES:
− muscular dystrophy
− familial periodic paralysis
− myasthenia gravis
− dermatomycosis
Reference Interval
(1) plasma/serum
5. Jaffe: adult female
0.6-1.1 mg/dL (53-97 µmol/L)
Reference Interval
(1) plasma/serum
5. Jaffe: adult male
0.9-1.3 mg/dL (80-115 µmol/L)
Reference Interval
(2) 24h urine
adult female
600-1800 mg/day 5.3-15.9 mmol/day
Reference Interval
(2) 24h urine
adult male
800-2000 mg/day 7.1-17.7 mmol/day
Reference Interval
(1) plasma/serum
6. Enzymatic: adult male
0.6-1.1 mg/dL (53-97 µmol/L)
Reference Interval
(1) plasma/serum
6. Enzymatic: adult female
0.5-0.8 mg/dL (44-71 µmol/L)
− major product of the catabolism of purine nucleosides: adenosine & guanosine
URIC ACID
− formed in the liver & intestinal mucosa from xanthine
URIC ACID
− The bulk of purines ultimately excreted as uric acid in the urine arises from degradation of endogenous nucleic acids.
URIC ACID
− Reutilization of the major purine bases (adenine, hypoxanthine and guanine) is achieved through “salvage” pathways
URIC ACID
[?] of the free bases causes re-synthesis of the respective nucleotide monophosphates
Phosphoribosylation
• 75% is excreted through the urine
URIC ACID
The remainder is secreted into the GIT, where it is degraded to allantoin & other compounds by bacterial enzymes.
− Glomerular filtration ‘
− Tubular reabsorption in the PCT: 98 – 100%
− Active secretion
− Reabsorption in the DCT
− Net excretion: 10%
RENAL HANDLING of URIC ACID FACTORS
1. Diet: [?]
2. Age & gender: increase w/ age; higher in [?]
3. 2x greater concentration in [?] than in plasma 4. Avoid the use of [?] because it forms salts that cause turbidity
5. UA is stable in [?] for several days at RT and longer at ref. temp.
6. [?] increases its stability
legumes, seeds, internal organs
males
RBC
K oxalate
serum
Thymol
Uric Acid + PTA ——- OH——– Allantoin + CO2 + Tungsten blue (710 nm)
DIRECT METHOD: Phosphotungstic Acid (PTA)
DIRECT METHOD: Phosphotungstic Acid (PTA)
Alkaline solution:
NaCN: Folin
Na2CO3: Caraway
Brown Henry Benedict Archibald Newton
Uric Acid Allantoin + CO2
Blaunch and Koch (UV test with uricase)
The decrease in the UA concentration is determined by measuring the absorbance in the range of 290 – 300 nm
Blaunch and Koch (UV test with uricase)
Uric Acid + O2 + 2H2O Allantoin + CO2 + H2O2
Trinder – Uricase method
H2O2 + DHBS + PAP Quinoneimine derivative (480 – 550 nm)
Trinder – Uricase method
DHBS: 3,5 – dichloro – 2- dihydroxy benzene sulfonic acid
Trinder – Uricase method
PAP: 4 – aminophenazone
Trinder – Uricase method
Uric Acid Allantoin + CO2 + H2O2
Uricase – catalase system
H2O2 + methanol —————→ formaldehyde + H2O (Catalase)
Uricase – catalase system
Formaldehyde + acetylacetone + NH3 3H2O + 3,5-diacetyl-1,4- dihydrolutidine (410 nm)
Uricase – catalase system
H2O2 + ethanol —————→ Acetaldehyde + H2O (Catalase)
Uricase – catalase system
Acetaldehyde + NAD —————→ Acetate + NADH (increase in Abs at 340 nm) (Aldehyde DH)
Uricase – catalase system
Cupric ions —–UA—→ Cuprous ions
Bittner method
Cuprous ions + neocuproine copper neocuproine complex (yellow to orange)
Bittner method
Ferric ions —–UA—→ Ferrous ions
Ferrous ions + TPTZ blue colored complex (590 nm)
TPTZ Method by Morin
TPTZ : 2,4,6- tripyridyl – 5 – triazine
TPTZ Method by Morin
TPTZ : 2,4,6- tripyridyl – 5 – triazine
TPTZ Method by Morin
OTHER METHODS:
A. HPLC
B. Amperometric Principle: Polarographic method
URIC ACID DISEASE CORRELATIONS:
(1) HYPERURICEMIA
(2) HYPOURICEMIA
HYPERURICEMIA
A. Increased Formation
Primary:
- Idiopathic
- Inherited metabolic disorders
HYPERURICEMIA
A. Increased Formation
Secondary:
- Excess dietary purine intake
- Increased nuclear breakdown (e.g. Leukemia)
- Psoriasis
- Altered ATP metabolism
- Tissue hypoxia
- Pre-eclampsia
- Alcohol
HYPERURICEMIA
B. Decreased Formation
Primary:
- Idiopathic
HYPERURICEMIA
B. Decreased Formation
Secondary:
- Renal failure
- Drug therapy: salicylate
- Poisons: heavy metal
- Pre-eclampsia
- Organic acids
- Trisomy 21 (Down syndrome)
Hereditary Hyperuricemia:
Lesch-Nyhan syndrome
Abnormal phosphoribosyl pyroPO4 synthetase
: x-linked genetic disorder; deficiency of hypoxanthine guanine phosphoribosyl transferase (muricase)
Lesch-Nyhan syndrome
: prevents reutilization of purine bases in the nucleotide salvage pathway
Abnormal phosphoribosyl pyroPO4 synthetase
: monosodium urate precipitates from supersaturated body fluids
GOUT
: o Atrophy of the liver
HYPOURICEMIA
Specimen Requirements and
Interfering Substances
− heparinized plasma, serum or urine
− immediate separation from red cells to prevent dilution by intracellular contents
− non-fasting sample acceptable
− gross lipemia should be avoided
− high bilirubin may cause false decrease
− significant hemolysis will lower results
− drugs : salicylates and thiazides : false increase
Reference Interval (uricase method)
Adult female
plasma or serum
2.6-6.0 mg/dL (0.16-0.36 mmol/L)
Reference Interval (uricase method)
Adult male
urine, 24h
250-750 mg/day (1.48-4.43 mmol/day)
Reference Interval (uricase method)
Child
plasma or serum
2.0-5.5 mg/dL (0.12-0.33 mmol/L)
Reference Interval (uricase method)
Conversion factor:
0.059
Reference Interval (uricase method)
Adult male
plasma or serum
2.5-7.2 mg/dL (0.21-0.43 mmol/L)
− from deamination of amino acids thru the action of digestive and bacterial enzymes on proteins in the GIT
AMMONIA
− used in the liver for urea production
AMMONIA
− level in circulation is extremely low (15 – 45 µg/dL)
AMMONIA
− increased in concentration in the blood in cases of severe liver damage
AMMONIA
− most ammonia in the blood exists as ammonium ion
AMMONIA
− concentration is not dependent on renal function
AMMONIA
− high ammonia :
neurotoxic — encephalopathy
METHODS for AMMONIA DETERMINATION:
- Conway and Cook Diffusion Method
- Forman’s Resin Absorption Method
- Kunahashi, Ishihora and Euhera Method
- Van Anken Enzymatic Method
- Ion Selective Electrode
SOURCES OF AMMONIA CONTAMINATION:
- Smoking
- Laboratory atmosphere
- Poor venipuncture technique
- Metabolism of nitrogenous constituents
• specimen is alkalinized to convert NH4 ions to NH3
Conway and Cook Diffusion Method
• NH3 is trapped in acid medium of diffusion cell
Conway and Cook Diffusion Method
• Quantitated by titration or colorimetry
Conway and Cook Diffusion Method
• Time consuming with poor accuracy and precision
Conway and Cook Diffusion Method
• Uses cation-exchange resin
Forman’s Resin Absorption Method
• NH3 absorbed by the resin and eluted
Forman’s Resin Absorption Method
• Quantitated by Berthelot reaction or by Nesslerization
Forman’s Resin Absorption Method
Forman’s Resin Absorption Method N.V.:
16 – 33 µmol/L
• NH3 is obtained through the use of a Dowax column
Kunahashi, Ishihora and Euhera Method
• Assayed using the Berthelot method
Kunahashi, Ishihora and Euhera Method
2-oxoglutarate + NH4+ + NADPH Glutamate + NADP + H2O
Van Anken Enzymatic Method
Van Anken Enzymatic Method N.V.:
11 – 35 µmol/L
• Based on the diffusion of NH3 through a selective membrane into NH4 chloride causing pH change which is determined potentiometrically
Ion Selective Electrode
• Good precision and accuracy
Ion Selective Electrode
SOURCES OF AMMONIA CONTAMINATION:
- Smoking
- Laboratory atmosphere
- Poor venipuncture technique
- Metabolism of nitrogenous constituents
Metabolism of nitrogenous constituents
Minimized by:
o placing the specimen in ice water
o centrifuging w/o delay
o performing the assay immediately
Use of heparin lock
Poor venipuncture technique
Probing for a vein
Poor venipuncture technique
Partial fill of the evacuated tube
Poor venipuncture technique
Drawing blood into a syringe & transferring it into an anti-coagulated tube
Poor venipuncture technique
Blood collection & NH3 analysis must be done in a lab w/ restricted traffic
Laboratory atmosphere
Glassware: soaked in hypochlorite solution (52.5g/L)
Laboratory atmosphere
AMMONIA CLINICAL SIGNIFICANCE
I. PRIMARY OR INHERITED HYPERAMMONEMIA
II. ACQUIRED HYPERAMMONEMIA
A. Enzyme defects in the Kreb’s Henseleit Cycle
PRIMARY OR INHERITED HYPERAMMONEMIA
B. Defects in the metabolism of amino acids: Lysine & Ornithine
PRIMARY OR INHERITED HYPERAMMONEMIA
C. Defects in the metabolism of:
Propionic acid
Methylmalonic acid
Isovaleric acid
PRIMARY OR INHERITED HYPERAMMONEMIA
A. Severe liver disease
ACQUIRED HYPERAMMONEMIA
B. Impaired venous drainage (from intestine to liver by portal vein)
ACQUIRED HYPERAMMONEMIA
C. Impaired renal excretion
ACQUIRED HYPERAMMONEMIA
Severe liver disease:
o Acute –
o Chronic –
toxic or fulminant viral hepatitis & Reye’s syndrome
cirrhosis
from intestine to liver by portal vein
Impaired venous drainage
Impaired renal excretion
Decreased (?)
Increased (?)
increased excretion of (?) into intestines converted to (?)
urine output
BUN reabsorbed
urea; ammonia
AMMONIA
Reference Interval
adult plasma
19-60 ug/dL 11-35 umol/L
AMMONIA
Reference Interval
adult urine,24h
140-1500 mg N/day 10-107 mmol N/day
Reference Interval
child
10days to 2y
68-136 ug/dL 40-80 umol