practical biochemistry

Amylase
Amylase is enzyme that catalyzes the breakdown of starch, glycogen, and some oligosaccharides. Alpha amylases, break down the alpha-1,4 glycosidic linkages in these substrates, producing glucose, maltose, and dextrins, which contain the branched products of degradation.
Amylase is produced in the pancreas and salivary glands.
The finding of increased concentrations of amylase in blood and urine is clinically significant to the diagnosis of pancreatitis, salivary gland tumor, mumps, perforated peptic ulcer, renal insufficiency, and diabetic ketoacidosis.
Low levels of serum amylase may indicate pancreatic insufficiency such as found in cystic fibrosis.
Normal serum value is 50-120 IU/L. The value is increased about 1000 times in acute pancreatitis which is a life-threatening condition. Moderate increase in serum levels are seen in chronic pancreatitis, mumps (parotitis) and obstruction of pancreatic duct.
Blood Glucose
Glucose is the most important carbohydrate fuel in the body. In the fed state, the majority of circulating glucose comes from the diet; in the fasting state, gluconeogenesis and glycogenolysis maintain glucose concentrations. Very little glucose is found in the diet as glucose; most is found in more complex carbohydrates that are broken down to monosaccharides though the digestive process. Glucose is classified as a monosaccharide because it cannot be broken down further by hydrolysis. It is further classified as a hexose because of its six-carbon skeleton and as an aldose, because of the presence of an aldehyde group on carbon1.high blood glucose levels is referred to as hyperglycemia; low levels are referred to as hypoglycemia. Diabetes mellitus is characterized by persistent hyperglycemia.
The international standard way of measuring blood glucose levels are in terms of a molar concentration, measured in mmol/L (millimoles per litre; or millimolar, abbreviated mM). In the United States, West-Germany and other countries mass concentration is measured in mg/dL (milligrams per decilitre).
Since the molecular weight of glucose C6H12O6 is 180, for the measurement of glucose, the difference between the two scales is a factor of 18, so that 1 mmol/L of glucose is equivalent to 18 mg/dL. Ordinarily the concentration of glucose in the blood is maintained at a relatively stable concentration from 80 to 120 mg/dl (4.4-6.6 mmol/L).
The body s homeostatic mechanism keeps blood glucose levels within a narrow range. It is composed of several interacting systems, of which hormone regulation is the most important.
There are two types of mutually antagonistic metabolic hormones affecting blood glucose levels:
• Catabolic hormones (such as glucagon, cortisol and catecholamines) which increase blood glucose;
• Anabolic hormone (insulin), which decreases blood glucose.

Conditions associated with hyperglycaemia
1-Diabetes mellitus
2-Cushing s syndrome (Adrenal cortical hyperactivity)

3-Hyperthyroidism

4-Acromegaly (increase growth hormone)

5-Obesity

6-Pheochromocytoma (increase catecholamines)

Measuring blood glucose level
Glucose concentration may be determined in whole blood, plasma, or serum samples.
Rapid separation of the sample or cooling will prevent glycolysis and will allow the sample to be used for other determinations. Unhemolyzed samples that have been separated within 30 minutes of drawing are generally considered adequate. Rapid cooling of the sample followed by centrifugation is even more effective in preventing glycolysis. If the specimen has been promptly centrifuged, it is reasonable to ask the laboratory to measure the glucose concentration even though a sodium fluoride (green top) tube was not used.
Glucose oxidase is widely used method for measuring blood glucose level. In the glucose oxidase assay, the glucose is first oxidized by glucose oxidase to produce gluconate and hydrogen peroxide. The hydrogen peroxide is then be used to oxidize a chromogen to produce a red colored compound which can measured by spectrophotometer. For example, hydrogen peroxide together with 4 amino-antipyrene (4-AAP) and phenol in the presence of peroxidase yield a red quinoeimine dye that can be measured at 505 nm. The absorbance at 505 nm is proportional to concentration of glucose in the sample.




Diagnostic criteria (adults) of diabetes mellitus
1. Classic symptoms and random glucose >200 mg/dl
(more than once)
2. Classic symptoms and Fasting glucose ?126 mg/dl
(more than once)
3. OGTT† ?200 mg/dl (11.1 mmol/L) for the two hour sample

Oral Glucose Tolerance Testing (OGTT)
The test should be done in the morning, after a 8- to 12-hours fast in which the patient is permitted only water. Patients are not allowed to smoke during the test. A fasting blood sugar is obtained. Adults are then given a drink containing 75 g of glucose. Timing is begun when the patient begins to drink the glucose solution. Blood samples are obtained every 30 minutes for 2 hours.
Interpretation:
Diagnosis Fasting plasma glucose 2hours

Normal ? 6 mmol/L ? 7.8 mmol/L
Diabetes mellitus ? 7.0 mmol/L ? 11.1 mmol/L

Hemoglobin A1c
A test that reflects long-term blood glucose control in diabetics is the concentration of hemoglobin A1c.
Hemoglobin A1c is formed by the irreversible attachment of glucose to the hemoglobin in a two-step reaction. The percentage of hemoglobin glycosylated depends on the average glucose concentration the red cell is exposed to over time. Since the average life of the red cell is 120 days, the percentage of glycosylated hemoglobin gives a good indication of the degree of blood sugar control over the preceding weeks.
RENAL FUNCTION TESTS
Blood Urea
Urea is an organic compound with the chemical formula CO(NH2)2. Urea serves an important role in the metabolism of nitrogen-containing compounds (protein ) by animals, and is the main nitrogen-containing substance in the urine of mammal so called blood urea nitrogen.
Amino acids from ingested food that are not used for the synthesis of proteins and other biological substances or produced from catabolism of muscle protein, are oxidized by the body, yielding urea and carbon dioxide, The oxidation pathway starts with the removal of the amino group by a transaminase; the amino group is then fed into the urea cycle. The first step in the conversion of amino acids from protein into metabolic waste in the liver is removal of the alpha-amino nitrogen, which results in ammonia. Because ammonia is toxic, it is immediately converted into urea by mammals. The urea is then transported to the kidneys where it is excreted. The overall urea formation reaction is:
2 Ammonia + carbon dioxide + 3ATP ---> urea + water + 3 ADP
It is filtered from the blood at the glomerulus but significant tubular reabsorption occurs through passive diffusion.
Although plasma urea concentration is often used as an index of renal glomerular function, measurement of plasma creatinine provides a more accurate assessment.
Urea production is increased by a high protein intake, in catabolic states, and by the absorption of amino acids and peptides after gastrointestinal haemorrhage. Conversely,
production is decreased in patients with a low protein intake and sometimes in patients with liver disease. Tubular reabsorption increases at low rates of urine flow (e.g. in fluid depletion) and this can cause increased plasma urea concentration even when renal function is normal.
Changes in plasma urea concentration are a feature of renal impairment but it is important to consider possible extrarenal influences on urea concentrations before ascribing any changes to an alteration in renal function.
Causes for Increased Blood Urea
1. Pre-renal causes
Dehydration: Severe vomiting, intestinal obstruction, diarrhea, severe burns,Fever.
2. Renal causes
Acute glomerulonephritis.
3. Post-renal causes
Stones in the urinary tract, Enlarged prostate, Tumors of bladder, Medications.

Causes for Decreased Blood Urea
Urea concentration in serum may be low in late pregnancy, in starvation, in diet grossly deficient in proteins and in hepatic failure.
Creatinine
Creatine constitutes about 0.5% of total muscle weight. It is synthesized from 3 amino acids, glycine, arginine and methionine. Synthesis primarily takes place in the kidney and liver, then being transported to the muscles via the blood.
Creatine phosphate, a high-energy compound is formed by creatine kinase (CK) and is the first source of metabolic fuel used in muscle contraction. . Phosphocreatine can anaerobically donate a phosphate group to ADP to form ATP during the first 2 to 7 seconds following an intense muscular or neuronal effort. Conversely, excess ATP can be used during a period of low effort to convert creatine to phosphocreatine. The phosphorylation of creatine is catalyzed by creatine kinases. Every day, up to 20% of total muscle creatine (and its phosphate) spontaneously dehydrates and cycles (irreversible, non enzymatic dehydration and loss of phosphate) to form the waste product creatinine. Therefore, creatinine levels are a function of muscle mass and remain approximately the same in an individual from day-to-day unless muscle mass or renal function changes. Creatinine has a molecular weight of 113 Da and is, therefore, readily filtered by the glomerulus. Unlike urea, creatinine is not reabsorbed by the tubules. However, a small amount of creatinine is secreted by the kidney tubules at high serum concentrations.
Plasma creatinine concentration is the most reliable simple biochemical test of glomerular function. The 24-hour urinary excretion of creatinine is proportionate to muscle mass.Endogenous creatinine produced is proportional to muscle mass. Dietary fluctuations of creatinine intake cause only minor variation in daily creatinine excretion of the same person.
Causes of increased serum creatinine:-
1-kidney impairment (renal failure)
2-diabetes mellitus
3-infections
4-hypothyriodism
Causes of decrease serum creatinine:-
1-advance renal disease
2-anemia
3-leukemia
4-muscular dystrophy





creatine kinase( CK)
Plasma enzymes as diagnostic tools
Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma. The activities of many of these enzymes are routinely determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues see table (1). The level of specific enzyme activity in the plasma frequently correlates with the extent of tissue damage. Thus, determining the degree of elevation of a particular enzyme activity in the plasma is often useful in evaluating the prognosis for the patient. Some enzymes show relatively high activity in only one or a few tissues. The presence of increased levels of these enzymes in plasma thus reflects damage to the corresponding tissue. For example, the enzyme alanine aminotransferase is abundant in the liver. The appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue. Increases in plasma levels of enzymes with a wide tissue distribution provide a less specific indication of the site of cellular injury. This lack of tissue specificity limits the diagnostic value of many plasma enzymes.
Isoenzymes and Diseases
? Most isoenzymes (also called isozymes) are enzymes that catalyze the same reaction. However, they do not necessarily have the same physical properties because of genetically determined differences in amino acid sequence. For this reason, isoenzymes may contain different numbers of charged amino acids and may, therefore, be separated from each other by electrophoresis. Different organs frequently contain characteristic proportions of different isoenzymes. The pattern of isoenzymes found in the plasma may, therefore, serve as a means of identifying the site of tissue damage. For example, the plasma levels of creatine kinase (CK)
• CK is a dimer; their subunits are called B for brain and M for muscle. Three isoenzymes of CK present, BB, MM and MB, occur.
• CK-MM--------Skeletal muscle
• CK-MB---------heart (myocardium)
• CK-BB----------Brain &smooth muscle
• Causes of an increased plasma creatine kinase activity:
polymyositis ,myocardial infarction ,skeletal muscle trauma .