C - Chapter VIII: NPN Flashcards by Arriane Cyrel (MTI)

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

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Primary:

production of rennin, prostaglandin and erythropoietin

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Secondary:

degradation of insulin, glucagon and aldosterone

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Clinically Significant NPN compounds:

urea – 45%
amino acids – 20%
uric acid – 20%
creatinine – 5 %
creatine – 1-2 %
ammonia – 0.2%

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TOTAL NPN METHODOLOGY: TWO STEPS:

KJELDAHL DIGESTION
MEASUREMENT OF AMMONIA FORMED

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MEASUREMENT OF AMMONIA FORMED

A. NESSLERIZATION
B. BERTHELOT METHOD
C. MONITORING CONSUMPTION OF AMMONIA (Kaplan, Manoukian – Fawaz; Kallet – Cook Reaction)

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 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

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NPN + H2SO4 NH4HSO4

KJELDAHL DIGESTION

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NH4HSO4 + NaOH Na2SO4 + NH3 + H2O

KJELDAHL DIGESTION

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Nessler’s reagent

double iodide salt of potassium & mercury

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– colloidal stabilizer

Gum ghatti

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– yellow to orange brown product

Dimercuric ammonium iodide

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NH3 + HgI2.2KI NH2Hg2I2 + KI + NH4I

NESSLERIZATION

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Reagent: phenol and alkaline hypochlorite

BERTHELOT METHOD

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Catalyst: sodium nitroprusside

BERTHELOT METHOD

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Product: indophenol blue

BERTHELOT METHOD

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NH3 + NaOCl + Phenol Indophenol + NaCl + H2O

BERTHELOT METHOD

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NH3 + α – ketoglutarate + NADH + H Glutamate + NAD

MONITORING CONSUMPTION OF AMMONIA (Kaplan, Manoukian – Fawaz; Kallet – Cook Reaction)

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Catalyst: Glutamate dehydrogenase

MONITORING CONSUMPTION OF AMMONIA (Kaplan, Manoukian – Fawaz; Kallet – Cook Reaction)

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Measure a decrease in the absorbance at 340 nm

Glutamate dehydrogenase

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most abundant NPN compound in plasma

UREA

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major excretory product of protein metabolism

UREA

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synthesized in the liver from CO2 and ammonia that arises from the deamination of amino acids in the reaction of the urea cycle

UREA

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UREA STRUCTURE

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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 :

1. renal function and perfusion 2. protein content of the diet 3. amount of protein catabolism

METHODS FOR UREA DETERMINATION

I. INDIRECT METHOD / ENZYMATIC II. DIRECT METHODS

INDIRECT METHOD / ENZYMATIC

1. Berthelot reaction 2. Nessler’s reaction 3. GLDH-coupled enzymatic method (Dupont ACA Analyzer) 4. Conductimetric method: Beckman BUN Analyzer 5. Urograph or Urastrat strip 6. Indicator dye (uriol): Kodak Ectachem Analyzer

DIRECT METHODS

1. Diacetyl Monoxime (Fearon) 2. 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

1. DIRECT METHOD: JAFFE REACTION 2. INDIRECT / ENZYMATIC METHODS 3. Yatzidis method 4. 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 :

1. ascorbate, glucose, alpha keto acids and uric acid 2. 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

3. 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:

1. Conway and Cook Diffusion Method 2. Forman’s Resin Absorption Method 3. Kunahashi, Ishihora and Euhera Method 4. Van Anken Enzymatic Method 5. Ion Selective Electrode

SOURCES OF AMMONIA CONTAMINATION:

1. Smoking 2. Laboratory atmosphere 3. Poor venipuncture technique 4. 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:

1. Smoking 2. Laboratory atmosphere 3. Poor venipuncture technique 4. 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