NON-PROTEIN NITROGEN COMPOUNDS

NPN COMPOUNDS:
 Urea (45%) Adult
 Amino Acid (20%) Plasma/Serum 6-20 mg/dL 2.1-7.1 mmol/L
 Uric Acid (20%)
Urine, 24 hr 12-20 g/d 0.43-0.71 mol
 Creatinine (5%) urea/d
 Creatine (1-2%)
 Ammonia (0.2%) Reference Range
UREA Plasma Adult 19-60 mg/dL
 Present in high concentrations of the blood (11-35 umol/L)
 Major excretory product of Protein metabolism Ammonia Urine 24hr, Adult 140-1500mg N/d
 Formed in the liver from amino groups. (10-107 umol N/d)
 UREA CYCLE- important pathway to reduce the levels of S/P Children 140-1500mg N/d
ammonia in the blood, as ammonia is toxic to cells. (10-107 umol N/d)
 UREA DETERMINATION- BLOOD Urea Nitrogen (BUN)
o The concentration of urea in the plasma is Pathophysiology
determined by the protein content of the diet,  Normal- 10:1 to 20:1
the rate of protein catabolism, and renal  Decreased renal function = increase in plasma urea
function and perfusion. concentration because of compromised urea excretion.
o Urea-reported in terms of nitrogen concentration  MAJOR CAUSES of decreased plasma urea
rather than urea concentration. (SI-mmol/L) concentration-low protein intake and severe liver disease.
 Azotemia- elevated concentration of urea in blood
Biochemistry o Pre-renal azotemia- reduced renal blood
- Protein metabolism produces amino acids that can be flow (shock, hemorrhage, CHF, dehydration,
oxidized to produce energy or stored as fat and glycogen. increased protein intake)
- During protein metabolism, nitrogen is released, converted  Less blood is delivered to the kidney
to urea, and excreted as a waste product. and so consequently, less urea is
- Most of the urea is excreted in the urine, although some filtered.
urea is reabsorbed by passive diffusion during passage of o Renal azotemia- decreased renal function
the filtrate through the renal tubules (PCT/DCT). (acute and chronic renal failure, glomerular
o Reabsorption depends on the urine flow rate and nephritis, tubular necrosis)
extent of hydration. o Post-renal azotemia- obstruction of urine
o Small quantities of urea (<10% of the total) are flow by renal calculi, tumors of the bladder or
excreted through the gastrointestinal (GI) tract prostate, or severe infection.
and skin.  Uremia (uremic syndrome)- very high plasma urea
Clinical Application concentration with renal failure, fatal if not treated.
- Used to evaluate renal function, hydration status, nitrogen o fatigue, nausea or vomiting, and generalized
balance, adequacy of dialysis and aids in the diagnosis of confusion
renal disease. Clinical Utility
- Measurements of urea- performed on a protein-free filtrate Ratio Interpretation Causes
of whole blood and based on measuring the amount of >20:1
nitrogen.
Methods With normal Prerenal ↓ renal perfusion, ↑
- Enzymatic (urease) methods- catalyzes hydrolysis of urea, creatine protein diet, GI bleed, ↑
ammonium ion (NH4+) reaction is quantified. protein catabolism
o Ammonium from the urease reaction- pH Indicator
(color change) With elevated Postrenal Mechanical obstruction
- Electrode- measure the rate of increase in conductivity as creatinine of urine flow
ammonium ions are produced from urea. 10:1 to 20:1 Normal
- Reference methods using isotope dilution mass <10:1 Loss of renal Tubular necrosis,
spectrometry (IDMS). function, ↓ GFR ↓protein intake, liver
Specimen Requirements disease
- Plasma/ Serum
o In plasma, avoid ammonium ions, high citrate and URIC ACID
fluoride-sodium citrate, and sodium fluoride tubes  Final product of catabolism of purine nucleic acids.
must be avoided, citrate and fluoride inhibit urease  Is filtered by the glomerulus and secreted by the distal
(SST or Lit. Heparin-ideal) tubules into the urine.
o Fasting sample not generally required if minimal o Most uric acid is reabsorbed in the proximal
intake of protein is noted tubules (reused), maintaining a steady
o Hemolyzed samples should not be used. physiological concentration.
- Urine
 Insoluble in plasma
o Susceptible to bacterial decomposition-if it cannot
 High concentrations= deposits in joints and tissue,
be analyzed within a few hours should be
inflammation
refrigerated.
o Timed urine specimens should be refrigerated  Uric acid is usually measured to confirm diagnosis and
during the collection period. monitor treatment of gout- precipitation of uric acid
crystal (sodium urates)deposition in joints and tissues.
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NON-PROTEIN NITROGEN COMPOUNDS
Biochemistry Adult M or F Urine, 24 h 24 h 250–750 mg/dL
- Purines are converted into uric acid in the liver. (1.5–4.4 mmol/dL)
- Uric acid is transported in plasma from liver to kidney and
filtered by glomerulus. Pathophysiology
- Reabsorption of 98% to 100% of the uric acid from the - Hyperuricemia (elevated levels of uric acid) is found in:
glomerular filtrate occurs in the proximal tubules. o Inherited disorders of purine metabolism
o Small amounts of uric acid are secreted by the o Gout (30-50 yrs) renal calculi
distal tubules into the urine.  big toe; ankles, knees, fingers
- 70% is eliminated by renal excretion; remainder passes into o Postmenopausal women
GI tract and is degraded by bacterial enzymes.  deposits of crystalline uric acid and
- At the pH of plasma (pH ~ 7), urate is relatively insoluble; urates called tophi form in tissue, causing
at concentrations greater than 6.8 mg/dL, the plasma is deformities.
saturated. o Increased catabolism of nucleic acids
o Urate crystals may form and precipitate in the o Increased metabolism of cell nuclei
tissues. o Chronic renal disease
- In acidic urine (pH < 5.75), uric acid is the predominant o Hemolytic or megaloblastic anemia
species, and uric acid crystals may form. o Lesch-Nyhan syndrome (males)
Clinical Application - May be exacerbated by a purine-rich diet, drugs, and
- Assessment of inherited disorders of purine metabolism alcohol.
- Confirmation of diagnosis and monitoring of treatment of - Hypouricemia (decreased levels of uric acid):
gout o Liver disease
- Assistance in diagnosis of renal calculi o Defective tubular reabsorption- Fanconi syndrome
- Prevention of uric acid nephropathy in chemotherapy o Chemotherapy
- Detection of kidney dysfunction
Analytical Methods CREATININE/CREATINE
- Uric acid is readily oxidized to allantoin and, therefore, can  Formed in liver, kidneys, and pancreas
function as a reducing agent in chemical reactions.  Formed from creatine and creatine phosphate in muscle
- Caraway method, oxidation of uric acid, lox specificity and is excreted into the plasma
- Primary method- uricase, converts uric acid to allantoin.  Plasma creatinine is inversely related to glomerular filtration
Absorption of uric acid and allantoin are measured and the rate (GFR) and, although an imperfect measure, it is
difference in absorption after incubation with uricase is commonly used to assess renal filtration function.
proportional to the amount of uric acid.
o Peroxidase or catalase- catalyze a chemical Biochemistry
indicator reaction. - Creatine is synthesized mainly in liver from arginine,
o Bilirubin and ascorbic acid- destroy peroxide; glycine, and methionine.
interfere - It is then transported to other tissues (muscles) and
- Other methods: coupled enzyme methods, isotope dilution converted to creatine phosphate, which serves as a high
mass spectrometry (proposed) energy source.
Specimen Collection and Interfering Substances - Creatinine phosphate and creatine form creatinine, which
- Measured in heparinized plasma, serum, or urine. diffuses into plasma and excreted in urine.
- Serum should be removed from cells as quickly as possible o Creatine phosphate undergoes the spontaneous
to prevent dilution by intracellular contents. loss of phosphoric acid while creatine loses water
- Diet can affect uric acid concentration overall, but a fasting Clinical Application
specimen is unnecessary. - Measurement of creatinine concentration is used:
- Gross lipemia (seen in individuals with elevated tri-glyceride o To determine sufficiency of kidney function
levels) should be avoided. o To determine severity of kidney damage
- High bilirubin concentration may falsely decrease results o To monitor progression of kidney disease
obtained by peroxidase methods. - Creatinine clearance- measure amount of creatinine by
- Significant hemolysis, with concomitant glutathione glomerulus from blood by kidneys
release, may result in low values. - Glomerular filtration rate (GFR)- volume of plasma filtered
- Drugs such as salicylates and thiazides have been shown by glomerulus per unit of time
to increase values for uric acid. - Abbreviated modification of diet in renal disease (MDRD)
- Serum samples may be refrigerated for 3 to 5 days. equation- includes 4 variables—serum creatinine.
- Ethylenediaminetetraacetic acid (EDTA) or fluoride Concentration, age, gender, ethnicity
additives should not be used for specimens that will be - New equations are being developed and proposed
tested by a uricase method.
- Urine collections must be alkaline (pH 8) Analytical Methods
- Jaffe Reaction (time consuming, not readily automated)
Patient Sample RR o Creatinine reacts with picric acid in alkaline
solution to form red-orange chromogen
Adult male Plasma or 3.5–7.2 mg/dL - Kinetic Jaffe method (rapid, inexpensive, easy to perform)
Serum (0.21–0.43 mmol/L)) o Serum is mixed with alkaline picrate and rate of
Adult female Plasma or 2.6–6.0 mg/dL change in absorbance is measured
Serum (0.16–0.36 mmol/L) - Coupled enzymatic methods (improved specificity)
Child Plasma or 2.0–5.5 mg/dL - Isotope dilution mass spectrometry (reference method)
Serum (0.12–0.33 mmol/L)
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NON-PROTEIN NITROGEN COMPOUNDS
- Some results from anaerobic metabolic reactions in muscle
Specimen Requirements during exercise
- Maybe measured in plasma, serum, or urine - Ammonia is consumed by the parenchymal cells of the liver
- Avoid hemolyzed and icteric samples in the Krebs-Henseleit or urea cycle to produce urea
- May be refrigerated for 4 days; if longer than 4 days, - At normal physiologic pH, most ammonia in the blood exists
specimen should be frozen as ammonium ion (NH4+).
Sources of Errors - Excreted as ammonium ion by kidney and acts to buffer
- Ascorbate, glucose, a-keto acids, and uric acids may urine
increase creatinine concentration measured by Jaffe Clinical Application
reaction (some of these with enzymatic methods) - Determination of prognosis for severe liver disease
- Bilirubin causes negative bias in both Jaffe and enzymatic - Determination of severity and prognosis of Reye’s
methods syndrome (swelling of brain and liver from certain viral
- Patient use of cephalosporin antibiotics, dopamine, infections)
lidocaine should be noted as these medications interfere o an acute metabolic disorder of the liver, and
with the analytical reactions autopsy findings show severe fatty infiltration of
that organ.
- Diagnosis of inherited deficiency of urea cycle enzymes
- Monitoring of hyperalimentation therapy (anorexia)
Analytical Methods
- Ammonia in plasma is complicated by its low concentration,
instability, and pervasive contamination.
- Two-step approach in which ammonia is isolated from
sample and they assayed
- Direct measurement of ammonia by enzymatic method or
ion-selective electrode

Specimen Requirements
- Whole blood ammonia concentration increases rapidly
following specimen collection because of in vitro amino acid
deamination.
- Venous blood should be obtained without trauma and
placed on wet ice immediately.
- Heparin and EDTA are suitable anticoagulants.
- Commercial collection containers should be evaluated for
ammonia interference before a new lot is put into use.
- Centrifuged at 0 to 4°C within 20 minutes of collection and
the plasma removed.
- Should be assayed as soon as possible or frozen.
- Frozen plasma is stable for several days at –20°C.
- Erythrocytes contain two to three times as much ammonia
as plasma; hemolysis should be avoided.
- Patients do not smoke for several hours before collection
- INCREASE AMMONIA IN PLASMA: Ammonium salts,
Pathophysiology asparaginase, barbiturates, diuretics, ethanol,
hyperalimentation, narcotic analgesics, and some other
Creatinine drugs
 Elevated concentration associated with abnormal renal - DECREASE AMMONIA IN PLASMA: Diphenhydramine,
function, esp as it relates to glomerular function Lactobacillus acidophilus, lactulose, levodopa, and several
 When plasma creatinine is elevated, GFR is antibiotics
decreased, indicating renal damage - Glucose at concentrations greater than 600 mg/Dl (33
Creatine mmol/L) interferes in dry slide methods.
 Elevated concentration associated with muscle Sources of Errors
disease: muscular dystrophy, poliomyelitis, - Tobacco, smoke, urine, and ammonia in detergents,
hyperthyroidism, trauma glassware, reagents, and water
 Not elevated in renal disease
Pathophysiology
AMMONIA - Elevated concentrations are seen in:
 Formed in deamination of amino acids during protein o Severe liver disease (cirrhosis)
metabolism o Encephalopathy- high ammonia is neurotoxic
 Removed from circulation and converted to urea in live o Hyperammonemia-inherited deficiency of urea
 Toxic when free but found low concentrations in plasma cycle enzymes.

Biochemistry Measurement of plasma ammonia is important in the diagnosis

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