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CLINICAL CHEMISTRY (MLT 301)

NONPROTEIN NITROGEN(NPN)

LECTURE ONE

Dr. Essam H. Jiffri

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Nonprotein Nitrogen

- The determination of nonprotein nitrogenous substances in the blood has traditionally been used to monitor renal function.

- The term nonprotein nitrogen (NPN) originated in the early days of clinical chemistry when analytical methodology required that protein be removed from the sample before analysis.

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Nonprotein Nitrogen

- The NPN fraction is made up of about 15 compounds of clinical interest, the majority of these compounds rise from the catabolism of proteins and nucleic acids.

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Components of the Nonprotein- Nitrogen Fraction

Approximate Plasma Concentration

Compound (% of Total NPN)

Urea 45

Aminoacids 20

Uric Acid 20

Creatinine 5

Creatine 1-2

Ammonia 0.2

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UREA

- Urea constitutes nearly half the nonprotein nitrogen substances in the blood.

- It is synthesized in the liver from Co2 and the ammonia arising from the deamination of amino acids by means of the ornithine.

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Structure of Urea

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UREA

- Urea constitutes the major excretory product of protein metabolism, following synthesis in the liver, urea is transported by the plasma to kidney, where it is readily filtered from the plasma by the glomerulus.

- Most of the urea in the glomerular filtrate is

excreted in the urine, and up to 40-50 % is reabsorbed by passive diffusion during passage of the filtrate through the renal tubules.

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UREA

- An elevated level of urea in the blood is called azotemia.

- Very high levels of plasma urea accompanied by renal failure is called uremia or the urermic syndrome this is eventually fatal if not treated by dialysis.

- The major causes of decreased plasma urea

levels include decreased protein intake and severe liver disease.

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Analytical Methods

- Because measurements of urea were initially done on a protein-free filtrate of whole blood, and early analytical methods were based on measuring the amount of nitrogen, this assay is commonly called a BUN or blood urea nitrogen determination.

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Analytical Methods

Two analytical approaches have been used to assay for urea:

The oldest and most often used involves the hydrolysis of urea by the enzyme urease and quantitation of the NH+

4.

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Analytical Methods

Early colorimetric methods were based on using Nesslers reagent or the Berthelot reaction to detect the NH+

4 produced.

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Specimen Requirementsand Interfering Substances

- Fluoride or citrate will inhibit the urease utilized in the coupled enzymatic methods.

- When collecting plasma, NH+4 ions and high

concentrations of sodium fluoride or sodium çitrate must be avoided.

- Urea concentration may be readily measured in serum, plasma, or urine.

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Specimen Requirementsand Interfering Substances

- Fasting sample is usually not required.

- A non-hemolyzed sample is recommended.

- Urea is quite susceptible to bacterial decomposition, so samples (especially urine) that cannot be analyzed within a few hours should be refrigerated.

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Reference Interval

Urea nitrogen 7 to 18 mg/dL (2.5-6.4 mmol/L urea)

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CREATININE

- Creatine is synthesized mainly in the liver from arginine, glycine, and methionine.

- It is then transported to other tissues, such as muscle, where it is converted to: phosphocreatine, which serves as a high energy source.

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CREATININE

- Either creatine phosphate or creatine, under physiologic conditions, spontaneously loses phosphoric acid or water, respectively, to form its anhydride, creatinine which excreted into the plasma.

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Analytical Methods for Creatinine

- The methods most frequendy used to measure creatinine are based on the Jaffe reaction first described in 1886, in this reaction, creatinine reacts with picric acid in alkaline solution to form a red-orange chromogen.

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Specimen Requirements and Interfering Substances

- Creatinine can be measured in serum, plasma, or urine.

- Hemolysis should be avoided, since there are a significant number of nonspecific chromogens in the red cells.

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Specimen Requirements and Interfering Substances

- Fasting is not require.

- A heavy protein ingestion may elevate levels of creatinine.

- Urine should be refrigerated after collection or frozen if storage longer than four days is required.

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Reference Interval

- Reference intervals vary with age and sex and may decrease in the elderly.

Cord blood 0.6-1.2 mg/dl (53-106 mmol/L)

Children 0.3-0.7 mg/dl (27-62 mmol/L)

Adult

Male 0.6-1.2 mg/dl (53-106 mmol/L)

Female 0.5-1.1 mg/dl (44-97 mmol/L)