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CLINICAL CHEMISTRY CHAPTER 9. NON - PROTEIN NITROGEN. 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.

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clinical chemistry chapter 9

CLINICAL CHEMISTRYCHAPTER 9

NON - PROTEIN NITROGEN

introduction
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
key terms
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

Key Terms
objectives
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
slide5
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
slide6
BUN ( Blood Urea Nitrogen )
    • Blood Urea Nitrogen = BUN = 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

Urea

slide7
BUN disease correlations
    • Azotemia = Elevated plasma BUN
    • Prerenal 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
    • Prerenal  BUN( Not related to renal function )
      • Decreased dietary protein
      • Increased protein synthesis ( Pregnant women , children )
slide8
Renal causes of  BUN
    • Renal disease with decreased glomerular filtration
      • Glomerular nephritis
      • Renal failure form Diabetes Mellitus
  • Post renal causes of  BUN ( not related to renal function )
    • Obstruction of urine flow
      • Kidney stones
      • Bladder or prostate tumors
      • UTIs
slide9
BUN / Creatinine Ratio
    • Normal BUN / Creatinine ratio is 10 – 20 to 1
    • Creatinine is another NPN
    • Pre-renal increased BUN / Creat ratio
    • BUN is more susceptible to non-renal factors
    • Post-renal increased ratio BUN / Creat ratio
    • Both BUN and Creat are elevated
    • Renaldecreased BUN / Creat ratio
    • Low dietary protein or severe liver disease
slide10
BUN analytical methods
    • BUN 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 )

Urease

2 NH4+ + HCO3-

UREA

GLDH

NH4+ + 2-OXOGLUTARATE

GLUTAMATE

NADH

NAD

Measure the rate of decreased absorbance at 340 nm

NADH absorbs … NAD does not absorb

Reference range : 10 – 20 mg / dl

slide11

Liver Amino Acids Creatine

Muscles Creatine Phosphocreatine

Muscles Phosphocreatine Creatinine

  • 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

slide12
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
slide13
Creatinine analytical techniques
    • Jaffee Method ( the Classic technique )

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

slide14
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

slide15
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 )
slide16

Uricase

Uric acid + O2 + H2O

Allantoin + CO2

+

H2O2

  • Uric acid analysis

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

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

slide17
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
slide18

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

slide19
Creatinine Clearance
    • Calculated measurement of the rate at which creatinine is removed from the plasma by the kidneys
    • 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
slide20

24 Hour Urine collection

Container.

The volume can be measured

directly off the container.

slide21
Creatinine Clearance specimens
      • 24 hour urine specimen
      • Plasma / serum creatinine collected during the urine collection
      • 24 Hour Creatinine Clearance Formula
  • CREATININE CLEARANCE =

U = Creatinine concentration of the 24 hour urine ( mg / dl )

V = 24 hour urine volume ( mls ) 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

slide22

Example of a 24 Hour Creatinine Clearance calculation

24 hour urine volume = 1000 mls

24 hour urine creatinine = 20.0 mg / dl

Plasma creatinine = 5.0 mg / dl

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

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

slide23
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 timeinto the urine container. This completes the collection.
    • If possible, keep the urine specimen refrigerated.
slide24
Reference range
    • 97 - 137 ml / min ( male)
    • 88 - 128 ml / min (female)
    • Let’s remember 90 - 130 ml / min
npn top 10
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

Don’t forget to divide V by 1440 !

reference ranges
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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