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. 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 Pre-renal Post- renal Purines Renal absorption 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

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

Urea 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

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 )

Renal disease with decreased glomerular filtration 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

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 Renal decreased BUN / Creat ratio Low dietary protein or severe liver disease

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

Liver Amino Acids Creatine Muscles Creatine Phosphocreatine CREATININE Liver Amino Acids Creatine Muscles Creatine Phosphocreatine Muscles 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

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

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 )

+ Uric acid analysis Uricase Uric acid + O2 + H2O Allantoin + CO2 H2O2 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

Ammonia is very toxic - The liver converts ammonia into urea 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

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 is an endogenous substance ( not affected by diet ) 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

24 Hour Urine collection Container. The volume can be measured directly 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 = 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

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 !!!

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.

Reference range 97 - 137 ml / min ( male) 88 - 128 ml / min (female) Let’s remember 90 - 130 ml / min

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 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