CC1_Finals_Lec: NPN and Kidney Function Test

What are Non-Protein Nitrogen

• The are compounds that doenst have a protein component in them (CHONS)

• They are byproducts of protein catabolism

• a product of nucleic acid catabolism

A rgt used to produce a yellow color

Nessler’s reagent

Component of Nessler’s rgt.

Potassium tretaiodomercurate

Major excretory product of protein metabolism

Urea

Where is Urea synthesized?

Liver

Illustrate how Urea is formed and excreted

Amino Acid undergoes deamination → AA becomes → Free ammonia → Urea → carried in blood → filtered out of the Glomerulus

This compund is the first to increase in kidney dsx.

Blood Urea Nitrogen (BUN)

Urea vs. BUN

• Urea is the metabolite for protein catabolism

• BUN is the NITROGEN content of Urea

Fill up:

_% Urea is excreted; _% remain in the blood

90% , 10%

Oldest method

MICRO-KJELDAHL NESSLER

Micro-Kjeldahl Nessler

What is the compound of the yellow color produced when Ammonia reacts with Nessler’s rgt.

Dimercuric Ammonium iodide

What does Urease-Nessler Method use?

• Uses Urease enzyme

• Nessler’s rgt.

Chemical reaction of Micro-Kjeldahl Nessler

N → NH4 + alk. K2HgI4 → NH2HgI3

• This method uses enzymes such as Urease and Glutamate Dehydrogenase (GLDH)

• have greater specificity but more expensive

GLDH-coupled enzymatic Method

Wavelength used for GLDH-coupled enzymatic method

340 nm

Tip: NAD = 340 nm

• This method is

• Urea → CO2+NH3

• NH3+ phenol + sodium hypochlorite → indophenol blue

Urease Berthelot Method

end product of Urease Berthelot Method

indophenol blue

a compound e is added to enhance color formation and to exclude protein interference (in DAM)

Arsenic thiosemicarbazide

Urea is made to react with diacetyl monoxime to produce a yellow diazine derivative

Diacetyl Monoxime Method (DAM)

Name of Reaction used in DAM

Fearon's reaction

BUN Normal Values

6-20 mg/dL

(2.1 – 7.1 mmol/L)

CF for Urea N (mg/dL) → Urea(mg/dL)

x2.14

Urea N (mg/dL) → Urea (mmol/L)

x0.36

T/F:

Urea is highly affected by protein diet

True

principal waste product of muscular metabolism derived mainly from creatine

Creatinine

metabolite used for assay for renal blood flow

Creatinine

Creatinine Aminohydrolase Method

• Uses Creatininase + CK

Direct Jaffe Reaction Method

• End point:

• reagent used:

• End point: red orange

• reagent used: Alkaline picrate

Components of Alkaline picrate

• NaOH

• Picric Acid

Adsorbents used in Direct Jaffe reaction method

• Lloyd’s rgt - Na Al silicate

• Fuller Earth’s rgt - Al Mg Silicate

CF for Creatinine

88.4

Note

mg/dl to mmol/L = x88.4

mmol/L to mg/dL = /88.4

reference method for Urea, Crea

Isotope Dilution-Mass Spectrometry (ID-MS)

Give conditions associated with BUN:Creatinine Ratio of

LOW <10:1

LOW Protein Diet

Repeated Dialysis

Hepatic disease

Give conditions associated with BUN:Creatinine Ratio of

High >20:1(normal creatinine)

Pre renal Azotemia, Dehydration, CHF

Catabolic states

GI hemorrhage

High Protein Diet

Give conditions associated with BUN:Creatinine Ratio of

High >20:1(Increased creatinine)

Post renal Azotemia, Urinary tract obstruction, Renal calculi, tumor

Renal disease

Renal Failure

Azotemia vs Uremia

• Azotemia - Increase in BUN and Crea in Blood due to low GFR

• Uremia - Increase in BUN and Crea with symptoms related to renal failure

Give Pre-renal causes of Azotemia

 Hemorrhage (blood loss)

 Cardiac decompression

 Increased protein catabolism

 Heatstroke (Dehydration)

 Burns (Fluid loss)

Give renal causes of Azotemia

 Chronic nephritis

 Acute glomerulonephritis (AGN)

 Polycystic kidney

 Nephroschlerosis

 Tubular necrosis

Give post-renal causes of Azotemia

 Stones

 Prostatic enlargement

 Tumors

is the major end product of purine metabolism (Adenine and Guanine)

Blood Uric Acid (BUA)

What does NPN stand for?

Non-Protein Nitrogen

Why was the term “nonprotein nitrogen” originally used?

Analytic methods required removal of proteins before analysis

What reagent was historically used to detect ammonia in NPN analysis?

Nessler’s reagent (K2[HgI4])

What produces the yellow color in Nesslerization?

Dimercuric ammonium iodide

Most NPN compounds arise from what process?

Catabolism of proteins and nucleic acids

Major excretory product of protein metabolism

Urea

Where is urea formed?

Liver

Urea is formed from what during protein catabolism?

free ammonia

Factors affecting plasma urea concentration

Renal function, renal perfusion, protein diet, rate of protein catabolism

What is BUN?

Blood Urea Nitrogen

What happens to BUN in severe liver damage?

Decreases

First metabolite to increase in kidney disease

Urea/BUN

Major organic solid in urine

Urea

Percentage of urea excreted in urine

90%

Clinical applications of BUN

Evaluate renal function, hydration status, nitrogen balance, dialysis adequacy

Oldest method for BUN determination

Micro-Kjeldahl Nessler method

What does the Kjeldahl process use?

Sulfuric acid and phosphoric acid

Principle of urease-Nessler method

Urea → CO2 + NH3; NH3 reacts with Nessler reagent

What enzyme is commonly coupled with urease in enzymatic BUN methods?

GLDH (Glutamate dehydrogenase)

What wavelength is monitored in GLDH-coupled BUN assay?

340 nm

What happens to NADH in the GLDH method?

It decreases/oxidizes to NAD

Advantage of GLDH-coupled enzymatic method

Greater specificity

Disadvantage of GLDH-coupled urease method

More expensive

Principle of Urease-Berthelot method

Ammonia reacts with phenol and hypochlorite forming indophenol blue

Color produced in Berthelot reaction

Indophenol blue

What is titrated in Van Slyke Cullen method?

Ammonia produced from urea

A Direct method measuring urea, this doesnt use an enzyme

Diacetyl Monoxime (DAM) method

Fearon’s reaction refers to what method?

Diacetyl Monoxime method

Color produced in DAM method

Yellow diazine derivative

Advantage of DAM method

Inexpensive

Disadvantage of DAM method

Lacks specificity

Why should whole blood be deproteinized in BUN testing?

To eliminate hemoglobin interference

Anticoagulant contraindicated in enzymatic BUN methods

Ammonium oxalate

Why are sodium fluoride and citrate avoided in BUN testing?

They inhibit urease

Effect of prolonged standing on ammonia concentration

Increases 2–3× due to deamination

Normal BUN value

6–20 mg/dL

Conversion factor for urea nitrogen mg/dL to urea mmol/L

0.36

How do you convert urea nitrogen concentration to urea concentration?

Multiply by 2.14

Principal waste product of muscular metabolism

Creatinine

Creatinine is derived mainly from what?

Creatine

Amino acids needed to synthesize creatinine

Methionine, arginine, lysine

Main use of creatinine testing

Monitor renal function

Creatinine production is related to what?

Muscle mass

Relationship of plasma creatinine to GFR

Inversely related (this means that the higher plasma creatinine, the lower the GFR which is bad)

T/F: Creatinine is easily removed by dialysis

False

Method using creatininase, creatinase, sarcosine oxidase, and peroxidase

Creatininase method

What analyzer commonly uses the creatininase method?

Ektachem analyzer

Principle of creatinine aminohydrolase method

Creatinine hydrolyzed to creatine followed by coupled enzyme reactions

What happens to NADH in creatinine aminohydrolase method?

Oxidized

Principle of Jaffe reaction

Creatinine reacts with alkaline picrate forming red-orange tautomer

Color formed in Jaffe reaction

Red-orange

Substances causing falsely elevated Jaffe results

Ascorbate, glucose, uric acid, alpha-ketoacids

Substances causing falsely decreased Jaffe results

Hemoglobin and bilirubin

What is Lloyd’s reagent?

Sodium aluminum silicate

What is Fuller Earth’s reagent?

Aluminum magnesium silicate

Creatinine conversion factor

88.4

Simple but nonspecific creatinine method

Jaffe endpoint

Rapid and more specific creatinine method

Jaffe kinetic

Enzymatic creatinine methods measure what?

Ammonia