Non-protein Nitrogen Compounds

The determination of nonprotein nitrogenous substances in the blood has traditionally been used to monitor renal function. Nitrogen containing compounds that are not proteins or polypeptides Useful clinical information is obtained from individual components of NPN fraction

Clinically Significant NPN

The

NPN fraction comprises about 15 compounds Majority of these compounds arise from catabolism of proteins and nucleic acids

Urea Nitrogen (Blood) BUN

Highest concentration of NPN in blood Major excretory product of protein metabolism These processes release nitrogen, which is converted to ammonia Synthesized in the liver from CO2 and Ammonia that arises from deamination of amino acids

Urea Nitrogen (Blood) BUN

Assays for urea were based on measurement of nitrogen, the term blood urea nitrogen (BUN) has been used to refer to urea determination. Excreted by the kidneys 40% reabsorbed <10% of the total are excreted through the gastrointestinal tract and skin. Concentration is determined by:

Renal function Dietary intake

Clinical Application
Measurement of urea is used to: evaluate renal function, to assess hydration status, to determine nitrogen balance, to aid in the diagnosis of renal disease, and to verify adequacy of dialysis.

Disease Correlations
Azotemia: elevated conc. of urea in blood Very high plasma urea concentration accompanied by renal failure is called uremia, or the uremic syndrome

Disease Correlations
Causes

of urea

plasma elevations are:

Prerenal Renal Postrenal

Pre-Renal Azotemia

Reduced renal blood flow is delivered to the kidney filtered

Less blood less urea

Anything that produces a decrease in functional blood volume, include:

Congestive heart failure, shock, hemorrhage, dehydration

High protein diet or increased catabolism (Fever, major illness, stress)

Renal Azotemia
Decreased

renal function causes increased blood urea due to poor excretion

Acute & Chronic renal failure Glomerular nephritis Tubular necrosis & other Intrinsic renal disease

Post-Renal Azotemia
Obstruction Tumors

of urine flow

Renal calculi

of bladder or prostate Severe infections

Decreased Urea Nitrogen

Low

protein dietary intake Liver disease (lack of synthesis) Severe vomiting and/or diarrhea (loss) Increase protein synthesis

Analytical methods
Assays for urea were based on measuring the amount of nitrogen in the sample (BUN) Current analytic methods have retained this custom and urea often is reported in terms of nitrogen concentration rather than urea concentration (urea nitrogen). Urea nitrogen concentration can be converted to urea concentration by multiplying by 2.14

Analytical methods

Urease hydrolysis of urea to ammonium ion , then detect ammonium ion (NH4+)

Enzymatic
The most common method couples the urease reaction with glutamate dehydrogenase

Analytical methods

Indicator dye NH4+ + pH indicator color change Conductimetric

Conversion of unionized urea to NH4+ and CO32- results in increased conductivity

Reference range of Urea N:

Serum or plasma: 6-20 mg/dl 24 hours Urine: 12-20 g/day

Creatinine/ Creatine
Creatine is synthesized in Liver from arginine, glycine & methionine Converted to Creatine Phosphate = high energy source for muscle tissue Creatinine is produced as a waste product of creatine and creatine phosphate.

Creatine Phosphate phosphoric acid = Creatinine

Creatinine production

Creatinine/Creatine
Creatinine is released into circulation at stable rate proportional to muscle mass Filtered by glomerulus Excreted in urine Plasma creatinine concentration is a function of:

relative muscle mass, rate of creatine turnover and renal function

Daily creatinine excretion is fairly stable.

Its a very good test to evaluate renal function

Disease Correlations

Elevated Creatinine is found with abnormal renal function (i.e. GFR)

Measurement of creatinine concentration is used to determine:

sufficiency of kidney function and the severity of kidney damage and to monitor the progression of kidney disease.

Disease Correlations

GFR is the volume of plasma filtered (V) by the glomerulus per unit of time
GFR is used to estimate renal function

Creatinine Clearance
A measure of the amount of creatinine eliminated from the blood by the kidneys per unit time

Plasma concentration of creatinine is inversely proportional to clearance

Therefore increased plasma levels mean decreased

Analytic Methods
Jaffe reaction Most frequently used, was first described in 1886

Creatinine reacts with picric acid in alkaline solution red-orange chromogen

Kinetic

Rate of change in absorbance is measured Using creatininase, creatine kinase, pyruvate kinase and lactate

Jaffe Reaction

Enzymatic

Method

Analytic Methods
creatininase

Analytic Methods

Creatine

Elevated in plasma and urine in

Muscular dystrophy, hyperthyroidism, trauma,

Plasma creatinine levels usually normal, but urinary is elevated Specialized testing not part of routine lab

Assay of creatine
Analyzing

the sample for creatinine before and after heating in acid solution using an endpoint Jaffe method. Heating converts creatine to creatinine and the difference between the two samples is the creatine concentration.

Uric Acid
Uric

acid is a final breakdown product of purine metabolism (adenosine/guanine) in liver Most other mammals degrade it further to allantoin

Uric Acid
Uric acid is a final breakdown product of purine metabolism (adenosine/guanine) in liver Most other mammals degrade it further to allantoin Uric acid is transported to kidney and filtered (70%)

98% reabsorbed in PCT Some secreted by DCT Net amount 6-12% of filtered amount

Remaining 30% by GIT

Uric Acid
Present in plasma as monosodium urate At plasma pH relatively insoluble Conc. > 6.8 mg/dl plasma saturated urate crystals may form & precipitate in tissue Uric acid is measured to:

assess inherited disorders of purine metabolism, to confirm diagnosis and monitor treatment of gout, to assist in the diagnosis of renal calculi, to prevent uric acid nephropathy during chemotherapeutic treatment, and to detect kidney dysfunction

Disease Correlations
Gout Primarily in men Onset 30-50 years UA greater than 6.0 mg/dL Pain & inflammation of joints by precipitation of sodium urates in tissues Increased risk of renal calculi hyperuricemia due to overproduction of uric acid in 25-30%

Disease Correlations

Increased catabolism
occurs in patients on chemotherapy for diseases such as leukemia & multiple myeloma. Allopurinol inhibits xanthine oxidase, an enzyme in the uric acid synthesis pathway, is used to treat these patients.

Chronic renal disease

causes elevated levels of uric acid because filtration and secretion are hindered.

Disease Correlations
Hypouricemia Secondary to severe liver disease Defective renal tubular reabsorption
Fanconis Syndrome

Chemotherapy with 6mercaptopurine or azathioprine inhibit purine synthesis Over treatment with allopurinol

Analytic Methods

Primary method uses enzyme uricase (urate oxidase) to convert uric acid to allantoin

Differential absorption at 293 nm

uric acid has a uv absorpance peak at 293 nm. Whereas allantoin does not

Analytic Methods

Newer methods couple uricase with catalase or peroxidase action on hydrogen peroxide product from allantoin production Some interferences from reducing agents

Reference range: Males 0.5-7.2, Females: 2.6-6.0 mg/dl

Ammonia
Comes from deamination of amino acids Digestive & bacterial enzymes in intestine Also released from muscle during exercise Consumed by parenchymal cells of liver and converted to urea Free ammonia is toxic;

however, ammonia is present in the plasma in low concentrations

Disease Correlations

Severe liver disease

Most common cause of abnormal ammonia levels Ammonia is not removed from circulation & not converted to urea

Elevated ammonia levels are neurotoxic and are often associated with encephalopathy.

Disease Correlations

Reyes Syndrome
Most commonly seen in children Often preceded by viral infection treated with aspirin Severe fatty infiltration of liver May be fatal if ammonia levels remain high 100% survival if ammonia stays below 5x normal

Disease Correlations
Ammonia is of use in the diagnosis of inherited deficiencies of urea cycle enzymes Measurement of ammonia used to diagnose and monitor treatment

Analytic Methods
Low concentration, volatile nature, instability, easy contamination testing difficult Historical Methods

Conway 1935 volatilize, absorbed then titrated Dowex 50 cation-exchange column + Berthelot reaction

Analytic Methods

Glutamate dehydrogenase
Decrease in absorbance at 340 as NADPH is consumed (oxidized)

Direct ISE
Change in pH of solution as ammonia diffuses through semi-permeable membrane

Reference Interval: Adult Plasma 19 60 g / dl