CLINICAL CHEMISTRY

NON - PROTEIN NITROGEN1

OPTOM FASLU MUHAMMED

INTRODUCTION NPN ( Non - Protein Nitrogen ) is a “funky” term that can

be used for a bunch of different substances that have the element nitrogen in them, but are not proteins.

This is a little unusual, because most of the body’s nitrogen is associated with proteins.

There are many different unrelated NPNs, but we are only interested in 4 of them:

Creatinine , Blood Urea Nitrogen ( BUN ) , Uric Acid and Ammonia

In general, plasma NPNs are increased in renal failure and are commonly ordered as blood tests to check renal function 2

KEY TERMS

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Allantoin Ammonia Azotemia BUN / Creat Ratio Creatinine Clearance Creatine Creatinine GFR Glomerulus Gout Hyper ( hypo ) uricemia NPN

Pre-renal Post- renal Purines Renal absorption Renal secretion Uric acid Urea Uremic syndrome Reyes Syndrome

OBJECTIVES List the origin and principle clinical significance of BUN,

Creatinine, Uric Acid and Ammonia

List the reference ranges for the 4 principle NPNs

Discuss why creatinine is the most useful NPN to evaluate renal function

Calculate Creatinine Clearance

Discuss the common methodologies used to measure BUN, Creatinine, Uric Acid and Ammonia

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General ideas about the NPNs

Antiquated term when protein – free filtrates were required for testing

The NPNs were used for evaluating renal function

The NPNs include about 15 different substances

Most NPNs are derived from protein or nucleic acid catabolism

Most important NPNs BUN ( Blood Urea Nitrogen ) Creatinine Uric acid Ammonia

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BUN ( Blood Urea Nitrogen )BUN ( Blood Urea Nitrogen ) Blood Urea Nitrogen = BUNBUN = Urea 50% of the NPNs

Product of protein catabolism which produces ammonia Ammonia is very toxic – converted to urea by the liver Liver converts ammonia and CO2

Filtered by the glomerulus but also reabsorbed by renal tubules ( 40 % )

Some is lost through the skin and the GI tract ( < 10 % )

Plasma BUN is affected by

Renal function Dietary protein Protein catabolism

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Urea

BUN disease correlationsBUN disease correlations

Azotemia = Elevated plasma BUN

PrerenalPrerenal BUN BUN ( Not related to renal function )

Low Blood Pressure ( CHF, Shock, hemorrhage, dehydration )

Decreased blood flow to kidney = No filtration Increased dietary protein or protein catabolism

PrerenalPrerenal BUN BUN ( Not related to renal function ) Decreased dietary protein Increased protein synthesis ( Pregnant women ,

children )

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RenalRenal causes of causes of BUN BUN

Renal disease with decreased glomerular filtration Glomerular nephritis Renal failure form Diabetes Mellitus

Post renalPost renal causes of causes of BUN ( not related to renal BUN ( not related to renal function )function )

Obstruction of urine flow Kidney stones Bladder or prostate tumors UTIs

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BUN / Creatinine RatioBUN / Creatinine Ratio

Normal BUN / Creatinine ratio is 10 – 20 Normal BUN / Creatinine ratio is 10 – 20 to 1to 1

Creatinine is another NPNCreatinine is another NPN

Pre-renal increased BUN / Creat ratio BUN is more susceptible to non-renal

factors

Post-renalPost-renal increased ratio BUN / Creat ratio

Both BUN and Creat are elevated

RenalRenal decreased BUN / Creat ratio Low dietary protein or severe liver

disease

Increased BUNNormal Creat

Increased BUNIncreased Creat

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Decreased BUNNormal Creat

BUN analytical methodsBUN analytical methods

BUN is an old term, but still in common useBUN is an old term, but still in common use Specimen : Plasma or serum To convert BUN to Urea : BUN x 2.14 = Urea

( mg / dl )

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UREA 2 NH4+ + HCO3

-Urease

NH4+ + 2-OXOGLUTARATE

GLDHGLUTAMATE

NADH NAD

Measure the rate of decreased absorbance at 340 nmNADH absorbs … NAD does not absorb

Reference range : 10 – 20 mg / dl

CREATININE

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Liver Amino Acids CreatineMuscles Creatine PhosphocreatineMuscles Phosphocreatine Creatinine

Creatinine formed at a constant rate by the muscles as a function of muscle mass

Creatinine is removed from the plasma by glomerular filtration

Creatinine is not secreted or absorbed by the renal tubules

Therefore : Plasma creatinine is a function of glomerular filtration

Unaffected by other factors

It’s a very good test to evaluate renal function

Creatinine disease correlations

Increased plasma creatinine associated with decreased glomerular filtration ( renal function )

Glomerular filtration may be 50 % of normal before plasma creatinine is elevated

Plasma creatinine is unaffected by diet

Plasma creatinine is the most common test used to evaluate renal function

Plasma creatinine concentrations are very stable from day to day - If there is a delta check , its very suspicious and must be investigated

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Creatinine analytical techniques

Jaffee Method ( the Classic technique )

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Creatinine + Picrate Acid Colored chromogen

Specimen : Plasma or serum

Elevated bilirubin and hemolysis causes falsely decreased results

Reference range : 0.5 - 1.5 mg / dl

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URIC ACID

Breakdown product of purines ( nucleic acid / DNA )

Purines from cellular breakdown are converted to uric acid by the liver

Uric acid is filtered by the glomerulus ( but 98 – 100 % reabsorbed )

Elevated plasma uric acid can promote formation of solid uric acid crystals in joints and urine

Uric acid diseases Gout

Increased plasma uric acid Painful uric acid crystals in joints Usually in older males ( > 30 years-old ) Associated with alcohol consumption Uric acid may also form kidney stones

Other causes of increased uric acid

Leukemias and lymphomas ( DNA catabolism ) Megaloblastic anemias ( DNA catabolism ) Renal disease ( but not very specific )

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Uric acid analysis

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Uric acid + O2 + H2O Allantoin + CO2

Uricase

Uric acid absorbs light @ 293 nm , Allantoin does not.The rate of decreased absorption is proportional to the uric acid concentration.

Specimen : Plasma or serum

+ H2O2

Reference range : 3.5 - 7.2 mg/dl (males) 2.6 - 6.0 mg/dl (females)

Let’s remember 3.0 - 7.0 mg/dl

AMMONIA Produced from the deamaination of amino acids in the

muscle and from bacteria in the GI tract Ammonia is very toxic - The liver converts ammonia

into urea Urea is less toxic and can be removed from the

plasma by the kidneys In severe hepatic disease, the liver fails to convert

ammonia into urea, resulting in increased plasma ammonia levels

Increased plasma ammonia concentrations in : Liver failure Reye’s Disease

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Ammonia analytical techniques

NH4+ + 2-OXOGLUTARATE + NADPH L-GLUTAMATE +

NADP+ There is a decreasing absorbance @ 340 nm, proportional to the ammonia concentration.

Specimen : EDTA or Heparinized Whole Blood on ice Must be tested ASAP or plasma frozen

Delayed testing caused false increased values

Reference range : 20 – 60 µg / dl

Creatinine Clearance

Calculated measurement of the rate at which creatinine is removed from the plasma by the kidneys

Measurement of glomerular filtration ( renal function )Measurement of glomerular filtration ( renal function )

A good test of glomerular filtration because

Creatinine is an endogenous substance ( not affected by diet )

Creatinine is filtered by the glomerulus, but not secreted or re-absorbed by the renal tubules

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24 Hour Urine collection Container.

The volume can be measureddirectly off the container.

Creatinine Clearance specimens

24 hour urine specimen Plasma / serum creatinine collected during the

urine collection

24 Hour Creatinine Clearance Formula

CREATININE CLEARANCE =

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AU 73.1PV

U = Creatinine concentration of the 24 hour urine ( mg / dl )V = 24 hour urine volume ( mls ) per minute - V / 1440 = mls / minute per minute - V / 1440 = mls / minute P = Plasma creatinine concentration ( mg / dl )

A = Correction factor accounts for differences in body surface area obtained from a height – weight chart

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Example of a 24 Hour Creatinine Clearance calculation

24 hour urine volume = 1000 mls24 hour urine creatinine = 20.0 mg / dlPlasma creatinine = 5.0 mg / dl

Patients height / weight = 6’00 / 190 lbs ( see pg. 680 )

100020.01.73 1.7314405.0 2.05

UVCreat Cl

P A

Creat Cl = 2 ml / min …. Very poor clearance !!!

Procedure for 24 Hour Urine Collection

Have the patient empty his / her bladder ( discard this urine ).

Note the time . For the next 24 hours, have the patient collect and save all urine in an appropriate container.

At the end of the 24 hour period have the patient void one last time into the urine container. This completes the collection.

If possible, keep the urine specimen refrigerated.

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Reference range

97 - 137 ml / min ( male) 88 - 128 ml / min (female)

Let’s remember 90 - 130 ml / min

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NPN TOP 10 Increased Creatinine associated with renal failure Increased BUN associated with renal failure and protein catabolism Increased Uric Acid associated with Gout Increased Ammonia is associated with liver disease

Creatinine derived from cellular creatine … very constant from day to day

Delta checks on plasma Creatinine must be investigated !!!

BUN ( Urea ) is derived from protein catabolism Protein Ammonia Urea Uric Acid is derived from purine( a component of DNA ) catabolism

Decreased Creatinine Clearance associated with decreased Glomerular Filtration

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APUV 73.1 Clearance Creatinine

Don’t forget to divide V by 1440 !

REFERENCE RANGES BUN 10 - 20 mg / dl

Creatinine 0.5 - 1.5 mg /dl

Uric Acid 3.0 - 7.0 mg / dl

Creatinine Clearance 90 - 130 ml / min

Ammonia 20 - 60 ug / dl

BUN / Creat Ratio 10 - 20 to 1

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