Non-Protein Nitrogen Compounds

Urea
Introduction

The major excretory product of protein metabolism. It is formed in the liver from amino groups (−NH2)
and free ammonia generated during protein catabolism. This enzymatically catalyzed process is termed
the urea cycle. Since historic assays for urea were based on the measurement of nitrogen, the term
blood urea nitrogen (BUN) has been used to refer to urea determination. Urea nitrogen (urea N) is a
more appropriate term.

Biochemistry

Produces amino acids that can be oxidized to produce energy or stored as fat and glycogen. These
processes release nitrogen, which is converted to urea and excreted as a waste product. Following
synthesis in the liver, urea is carried in the blood to the kidney, where it is readily filtered from the
plasma by the glomerulus. Most of the urea in the glomerular filtrate is excreted in the urine, although
some urea is reabsorbed by passive diffusion during passage of the filtrate through the renal tubules.
The amount reabsorbed depends on the urine flow rate and extent of hydration. Small quantities of
urea (<10% of the total) are excreted through the gastrointestinal (GI) tract and skin. The concentration
of urea in the plasma is determined by the protein content of the diet, the rate of protein catabolism,
and renal function and perfusion.

Clinical application

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.

Analytical Method

The most common method couples the urease reaction with glutamate dehydrogenase. Ammonium
from the urease reaction can also be measured by the color change associated with a pH indicator. This
approach has been incorporated into instruments using liquid reagents, a multilayer film format, and
reagent strips. A method that uses an electrode to measure the rate of increase in conductivity as
ammonium ions are produced from urea is in use in approximately 15% of laboratories in the United
States. Because the rate of change in conductivity is measured, ammonia contamination is not a
problem as t is in other methods.

Pathophysiology

An elevated concentration of urea in the blood is called azotemia. Very high plasma urea concentration
accompanied by renal failure is called uremia or the uremic syndrome. This condition is eventually fatal
if not treated by dialysis or transplantation. Conditions causing increased plasma urea are classified
according to the cause into three main categories: prerenal, renal, and postrenal.

2 cases:

Increased:

1. Congestive Heart Failure

Congestive heart failure is a syndrome that can be caused by a variety of abnormalities, including
pressure and volume overload, loss of muscle, primary muscle disease or excessive peripheral
demands such as high output failure. In the usual form of heart failure, the heart muscle has reduced
contractility. This produces a reduction in cardiac output, which then becomes inadequate to meet the
peripheral demands of the body

2. Shock

acute widespread reduction in effective tissue perfusion that invokes an imbalance of oxygen supply
and demand, anaerobic metabolism, lactic acidosis, cellular and organ dysfunction, metabolic
abnormalities, and, if prolonged, irreversible damage and death. The pathophysiologic events in the
various types of shock are different and complex with hemodynamic and oxygenation changes,
alterations in the composition of the fluid compartments, and various mediators. Shock results from a
change in one or a combination of the following: intravascular volume, myocardial function, systemic
vascular resistance, or distribution of blood flow. The clinical types of shock include hypovolemic,
cardiogenic, distributive (septic), and obstructive.

Uric Acid
Introduction

Catabolism of the purine nucleic acids. Although it is filtered by the glomerulus and secreted by the
distal tubules into the urine, most uric acid is reabsorbed in the proximal tubules and reused. Uric acid is
relatively insoluble in plasma and, at high concentrations, can be deposited in the joints and tissue,
causing painful inflammation.

Biochemistry

Purines, such as adenine and guanine from the breakdown of ingested nucleic acids or from tissue
destruction, are converted into uric acid, primarily in the liver. Uric acid is transported in the plasma
from the liver to the kidney, where itis filtered by the glomerulus. Reabsorption of 98% to 100% of the
uric acid from the glomerular filtrate occurs in the proximal tubules.

Uric acid in plasma is present as monosodium urate. At the pH of plasma (pH ~ 7), urate is relatively
insoluble; at concentrations greater than 6.8 mg/dL, the plasma is saturated. As a result, urate crystals
may form and precipitate in the tissues. In acidic urine (pH < 5.75), uric acid is the predominant species
and uric acid crystals may form.

Clinical application

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

Analytical Method

Uric acid is readily oxidized to allantoin and, therefore, can function as a reducing agent in chemical
reactions. This property was exploited in early analytic procedures for the determination of uric acid.
The most common method of this type is the Caraway method, which is based on the oxidation of uric
acid in a protein-free filtrate, with subsequent reduction of phosphotungstic acid in alkaline solution to
tungsten blue.
Methods using uricase, the enzyme that catalyzes the oxidation of uric acid to allantoin, are more
specific and are used almost exclusively in clinical laboratories.

Coupled enzyme methods measure the hydrogen peroxide produced as uric acid is converted to
allantoin. Peroxidase or catalase is used to catalyze a chemical indicator reaction. The color produced is
proportional to the quantity of uric acid in the specimen. Enzymatic methods of this kind have been
adapted for use on traditional wet chemistry analyzers and for dry chemistry slide analyzers. Bilirubin
and ascorbic acid, which destroy peroxide, if present in sufficient quantity, can interfere. Commercial
reagent preparations often include potassium ferricyanide and ascorbate oxidase to minimize these
interferences.

HPLC (high-performance liquid chromatography) methods, typically using UV detection, have been
developed. IDMS has been proposed as a candidate reference method.

Pathophysiology

Abnormally increased plasma uric acid concentration is found in gout, increased catabolism of nucleic
acids, and renal disease. Gout is a disease found primarily in men and is usually first diagnosed between
30 and 50 years of age. Affected individuals have pain and inflammation of the joints caused by
precipitation of sodium urates. In 25% to 30% of these patients, hyperuricemia is a result of
overproduction of uric acid, although hyperuricemia may be exacerbated by a purine-rich diet, drugs,
and alcohol. Plasma uric acid concentration in affected individuals is usually greater than 6.0 mg/dL.
Patients with gout are susceptible to the formation of renal calculi, although not all persons with
abnormally high serum urate concentrations develop this complication. In women, urate concentration
rises after menopause. Postmenopausal women may develop hyperuricemia and gout. In severe cases ,
deposits of crystalline uric acid and urates called tophi form in tissue, causing deformities.

2 cases:

1. Gout

Elevated urate concentrations are required: urate overproduction and underexcretion contribute to
total urate balance. Overproduction occurs due to alterations in the purine synthesis and degradation
pathways. Renal under excretion is an important cause of elevated serum urate concentrations
(hyperuricaemia), and occurs through alterations in the urate transporters within the renal tubule
(collectively known as the urate transportasome). Gut under excretion (extrarenal urate
underexcretion) also contributes to development of hyperuricaemia.

Creatinine
Introduction

Formed from creatine and creatine phosphate in muscle and is excreted into the plasma at a constant
rate related to muscle mass. Plasma creatinine is inversely related to glomerular filtration rate (GFR)
and, although an imperfect measure, it is commonly used to assess renal filtration function.

Biochemistry

It is synthesized primarily in the liver from arginine, glycine, and methionine. It is then transported to
other tissues, such as muscle, where it is converted to creatine phosphate, which serves as a high-
energy source. Creatine phosphate loses phosphoric acid and creatine loses water to form the cyclic
compound, creatinine, which diffuses into the plasma and is excreted in the urine.
Creatinine is released into the circulation at a relatively constant rate that has been shown to be
proportional to an individual's muscle mass. It is removed from the circulation by glomerular filtration
and excreted in the urine. Small amounts of creatinine are secreted by the proximal tubule and
reabsorbed by the renal tubules.

Clinical application

This is used to determine the sufficiency of kidney function, to determine the severity of kidney damage,
and to monitor the progression of kidney disease. . The amount of creatinine in the bloodstream is
reasonably stable, although the protein content of the diet does influence the plasma concentration.
Because of the constancy of endogenous production, urinary creatinine excretion has been used as a
measure of the completeness of 24-hour urine collections in a given individual, although the uncertainty
associated with this practice may exceed that introduced by use of the urine volume and collection time
for standardization.

Analytical Method

Most frequently used to measure creatinine are based on the Jaffe reaction first described in 1886. In
this reaction, creatinine reacts with picric acid in alkaline solution to form a red-orange chromogen. The
reaction was adopted for the measurement of blood creatinine by Folin and Wu in 1919.

The reaction is nonspecific and subject to positive interference by a large number of compounds,
including acetoacetate, acetone, ascorbate, glucose, and pyruvate. More accurate results are obtained
when creatinine in a protein-free filtrate is adsorbed onto Fuller's earth (aluminum magnesium silicate)
or Lloyd's reagent (sodium aluminum silicate), then eluted and reacted with alkaline picrate.

This method is time consuming and not readily automated; it is not routinely used. Two approaches
have been used to increase the specificity of assay methods for creatinine: a kinetic Jaffe method and
reaction with various enzymes. In the kinetic Jaffe method, serum is mixed with alkaline picrate and the
rate of change in absorbance is measured. Although this method eliminates some of the nonspecific
reactants, it is subject to interference by α-keto acids and cephalosporins.

Pathophysiology

Elevated creatinine concentration is associated with abnormal renal function, especially as it relates to
glomerular function. Plasma concentration of creatinine is inversely proportional to the clearance of
creatinine. Therefore, when plasma creatinine concentration is elevated, GFR is decreased, indicating
renal damage.

Plasma creatinine is a relatively insensitive marker and may not be measurably increased until renal
function has deteriorated more than 50%.

In muscle disease such as muscular dystrophy, poliomyelitis, hyperthyroidism, and trauma, both plasma
creatine and urinary creatinine are often elevated.Plasma creatinine concentrations are usually normal
in these patients .Measurement of creatine kinase is used typically for the diagnosis of muscle disease
because analytic methods for creatine are not readily available in mostclinical laboratories. Plasma
creatine concentration is not elevated in rena disease.
2 cases:

1. Hyperthyroidism

Most common endocrine disorder. It results from an excessive output of thyroid hormones due to
abnormal stimulation of the thyroid gland by circulating immunoglobulin. This disorder affects women
eight times more frequently than men and peaks between the second and fourth decades of life. It
generally occurs between 20 and 40 years old and is more common in females.

2. Poliomyelitis

Transmitted by faeco- oral route. Virus multiplies initially in

epithelial cells of AC (Oropharynx or intestine) and the lymphatic
tissue ( tonsils / payers patches). Regularly present in throat and
stools before the onset of illness. In the CNS, Virus multiplies
selectively in the neurons destroys the anterior horn cells of spinal
cord. Earliest change is degeneration of Nissils bodies. When
degeneration becomes irreversible, necrotic cell lyses or is
phagocytosed. Viruses don’t multiply in muscle in vivo. Changes
that occur in peripheral nerves and voluntary muscles are
secondary to destruction of nerve cells.

Ammonia
Introduction

This is produced in the deamination of amino acids during protein metabolism. It is removed from the
circulation and converted to urea in the liver. Free ammonia is toxic; however, ammonia is present in
the plasma in low concentrations

Biochemistry

Produced in the catabolism of amino acids and by bacterial metabolism in the lumen of the intestine.
Some endogenous ammonia results from anaerobic metabolic reactions that occur in skeletal muscle
during exercise. Ammonia is consumed by the parenchymal cells of the liver in the KrebsHenseleit or
urea cycle to produce urea, a nontoxic compound that is excreted in the urine. At normal physiologic pH,
most ammonia in the blood exists as ammonium. Ammonia is excreted as ammonium ion by the kidney
and acts to buffer urine.

Clinical application

Provides useful information are hepatic failure, Reye's syndrome, and inherited deficiencies of urea
cycle enzymes. Severe liver disease is the most common cause of disturbed ammonia metabolism. The
monitoring of blood ammonia may be used to determine prognosis, although correlation between the
extent of hepatic encephalopathy and plasma ammonia concentration is not always consistent. Arterial
ammonia concentration is a better indicator of the severity of disease. Ammonia is of use in the
diagnosis of inherited deficiency of urea cycle enzymes. Testing should be considered for any neonate
with unexplained nausea, vomiting, or neurological deterioration associated with feeding. Assay of
blood ammonia can be used to monitor hyperalimentation therapy and measurement of urine ammonia
can be used to confirm the ability of the kidneys to produce ammonia

Analytical Method

One of the first analytic methods for ammonia, developed by Conway in 1935, exploited the volatility of
ammonia to separate the compound in a microdiffusion chamber. Ammonia gas from the sample
diffuses into a separate compartment and is absorbed in a solution containing a pH indicator.

The amount of ammonia is determined by titration. Ammonia can be measured by an enzymatic method
using GLDH. NADPH is the preferred coenzyme because it is used specifically by GLDH; NADH will
participate in reactions of other endogenous substrates, such as pyruvate. Adenosine diphosphate is
added to the reaction mixture to increase the rate of the reaction and to stabilize GLDH.

A dry slide automated system uses a thin-film colorimetric assay. In this method, ammonia reacts with
an indicator to produce a colored compound that is detected spectrophotometrically. Direct
measurement using an ion-selective electrode has been developed.

Pathophysiology

In severe liver disease in which there is significant collateral circulation (as in cirrhosis) or if parenchymal
liver cell function is severely impaired, ammonia is not removed from the circulation and blood
concentration increases. High concentrations of NH3 are neurotoxic and often associated with
encephalopathy.

Ammonia alters metabolic processes in the brain, which results in accumulation of toxic species, and
impairs astrocyte function. Hyperammonemia is associated with inherited deficiency of urea cycle
enzymes.

Measurement of plasma ammonia is important in the diagnosis and monitoring of these inherited
metabolic disorders