BIOCHEMICAL INVESTIGATION OF BLOOD PLASMA PROTEINS
CLINICAL ENZYMOLOGY
Total blood volume is about 4.5 to
If blood is mixed with an anticoagulant and centrifuged, the cell components (RBC and WBC) are precipitated. The supernatant is called plasma. About 55-60 % of blood is made up of plasma.
If blood is withdrawn without anticoagulant and allowed to clot, after about two hours liquid portion is separated from the clot. This defibrinated plasma is called serum, which lacks coagulation factors including prothrombin and fibrinogen.
Plasma contains about hundred of different proteins.
Total protein content of normal plasma is 6 to 8 g/ 100 ml (65-85 g/l). As a first step of study, the plasma proteins may be separated into Albumin (35-50g/l), Globulins (25-35 g/l) and fibrinogen (2-4 g/l).
The albumin : globulin ratio is usually between 1.2:1 to 1.5:1.
Almost all plasma proteins, except immunoglobulins are synthesized in liver.
In clinical laboratory, total proteins in serum or plasma of patients are estimated by Biuret method.
In clinical laboratory, electrophoresis is employed regularly for separation of serum proteins.
The term electrophoresis refers to the movement of charged particles through an electrolyte when subjected to an electric field.
The positively charged particles (cations) move to cathode and negatively charged ones (anions) to anode. Since proteins exist as charged particles, this method is widely used for the separation of proteins in biological fluids. The technique was first used by Tiselius in 1937; named it as moving boundary or frontal electrophoresis (Nobel Prize in 1948).
Factors Affecting Electrophoresis
The rate of migration (separation of particles) during electrophoresis will depend on the following factors:
1. Net charge on the particles
2. Mass and shape of the particles.
3. The pH of the medium.
4. Strength of electrical field.
5. Properties of the supporting medium.
6. Temperature.
Types of Electrophoresis
There are mainly two types of electrophoresis—horizontal and vertical.
Different types of support media are used in horizontal electrophoresis, e.g. filter paper, cellulose acetate, agar gel, agarose gel, starch gel, etc. The vertical electrophoresis mainly uses polyacrylamide gel. The nature of the supporting medium will also influence the mobility.
Electrophoresis Apparatus
The electrophoresis system basically consists of the electrophoresis tank to hold the buffer and fitted with the electrodes, as well as a power pack to supply electricity at constant current and voltage. When the electrophoresis is carried out, the buffer is chosen in such a way so as to ensure effective separatum of the mixture of proteins. The pH and ionic strength and nature of the buffer may be varied according to the proteins to be separated, e.g. serum proteins are separated at a pH of 8.6 using barbitone buffer. At this pH all serum proteins will have a net negative charge and will migrate towards the anode.
Support Medium for Electrophoresis
Filter Paper
Cellulose Acetate Membrane
Agar or Agarose
Starch Gel
Polyacrylamide gel electrophoresis (PAGE)
Visualisation of Protein Bands
After the electrophoretic run is completed, the proteins are fixed to the solid support using a fixative such as acetone or methanol. Then it is stained by using dyes
(Amido Schwartz, naphthalene black, Ponceau S or Coomassie Blue) and then destained by using dilute acetic acid. The electrophoretogram can be scanned using a densitometer and each band quantitated. In the densitometer, light is passed through the agar gel plate; the absorption of light will be proportional to the quantity of protein present on a band. Another method is that the stain may be eluted from the support and each fraction quantitated colourimetrically.
Normal Values and Interpretations
In agar gel electrophoresis, normal serum will be separated
into five bands. Their relative concentrations are given
below:
Albumin : 55-65%
Alpha-1 -globulin : 2-4%
Alpha-2-globulin : 6-12%
Beta-globulin : 8-12%
Gamma-globulin : 12-22%
ALBUMIN
The name is derived from the white precipitate formed when egg is boiled (Latin, albus = white). Albumin constitutes the major part of plasma proteins. It has one polypeptide chain with 585 amino acids and 17 disulfide bonds. It has a molecular weight of 69,000. It is synthesised by hepatocytes; therefore, albumin level in blood is decreased in liver cirrhosis. Estimation of albumin is a liver function test. Half-life of albumin is about 20 days. Liver produces about
Functions of Albumin
1. It contributes to the colloid osmotic pressure of plasma.
The total osmolality of serum is 278-
2. Another major function of albumin is to transport
various hydrophobic substances.
3. Being a watery medium,
blood cannot solubilise lipid components. Bilirubin and
non-esterified fatty acids are specifically transported by
albumin. Drugs (sulpha, aspirin, salicylates, dicoumarol,
phenytoin), steroid hormones, thyroxine, calcium, copper
and heavy metals are non-specifically carried by albumin.
Only the unbound fraction of drugs is biologically active.
The histidine residue at position 3 of albumin binds
copper.
4. All proteins have buffering capacity. Because of its high concentration in blood, albumin has maximum buffering capacity. Albumin has a total of 16 histidine residues which contribute this buffering action.
5. All tissue cells can take up albumin by pinocytosis. It is then broken down to amino acid level. So albumin may be considered as the transport form of essential amino acids from liver to extrahepatic cells.
Clinical Applications
1. Albumin-fatty acid complex cannot cross blood-brain barrier and hence fatty acids cannot be taken up by brain. The bilirubin from albumin maybe competitively replaced by aspirin and such other drugs. Iewborns, bilirubin is already high, and if such drugs are given, there is a probability that free bilirubin is deposited in brain leading to kernicterus and mental retardation.
2. When two drugs having high affinity to albumin are administered together, there may be competition for the available sites, with consequent displacement of one drug. Such an effect may lead to clinically significant drug interactions, e.g. phenytoin -dicoumarol interaction.
3. Protein-bound calcium is lowered in hypoalbuminemia. Thus, even though total calcium level in blood is lowered, ionised calcium level may be normal, and so tetany may not occur. Calcium is lowered by 0.8 mg/dl for a fall of 1 g/dl of Albumin.
4. Human albumin is therapeutically useful to treat burns, hemorrhage and shock..
Normal Value and Interpretation:
Hypoalbuminemia
1. In cirrhosis of liver and in chronic liver failure, albumin
synthesis is decreased and so blood level is lowered.
2. In malnutrition and in malabsorption syndrome the availability of amino acids is reduced and so albumin synthesis is affected.
3. In nephrotic syndrome, kidney glomerular filtration is defective so that albumin is excreted in large quantities. Increased loss, to a certain extent, is compensated by increased synthesis; but blood level of albumin is decreased.
4. Presence of albumin in urine (albuminuria) is always pathological. Large quantities (several grams per day) of albumin is lost in urine iephrotic syndrome. Small quantities are lost in urine in acute nephritis, and other inflammatory conditions of urinary tract. Detection of albumin in urine is done by heat and acetic acid test. In microalbuminuria or minimal albuminuria or pauci-albuminuria, small quantity of albumin (50-300 mg/ d) is seen in urine (Paucity = small in quantity). However, microalbuminuria is also clinically important, as it is a predictor of future renal diseases.
5. In burns, albumin is lost through the unprotected skin surface.
6. In protein losing enteropathy large quantities of albumin is lost through intestines.
7. Hypoalbuminemia will result in tissue edema. It may be seen in malnutrition, where albumin synthesis is depressed {generalized edema), iephrotic syndrome, where albumin is lost through urine {facial edema) or in cirrhosis of liver (mainly ascites). In chronic congestive cardiac failure, venous congestion will cause increased hydrostatic pressure and decreased return of water into capillaries and so pitting edema of feet may result.
8. Albumin is a negative acute phase protein; level of albumin falls mildly in presence of inflammatory cytokines such as interleukin-6.
9. Analbuminemia {absence of albumin) is a very rare genetically determined condition.
Albumin-Globulin Ratio: In all the above mentioned conditions of hypoalbuminemia, there will be a compensatory increase in globulins which are synthesised by the reticuloendothelial system. Albumin-globulin ratio (A/G ratio) is thus altered or even reversed. This again leads to edema.
Hypoproteinemia: Since albumin is the major protein present in the blood, any condition causing lowering of albumin will lead to reduced total proteins in blood (hypoproteinemia). So it is observed in cirrhosis, nephrotic syndrome, malnutrition and malabsorption syndromes.
Hyper-gamma -globulinemias
1. When albumin level is decreased, body tries to compensate it by increasing the production of globulins from reticuloendothelial system. Thus, all causes for
hypoalbuminemia will result in albumin : globulin ratio reversal, and corresponding increase in percentage in globulins.
2. In chronic infections, the gamma globulins are increased, but the increase is smooth and widebased.
3. Drastic increase in globulins are seen in paraproteinemias, when a sharp spike is noted in electrophoresis. This is termed as M-band. This is due to monoclonal origin of immunoglobulins in multiple myeloma.
Hyper-beta-globulinemia: It is associated with hyperlipoproteinemia, atherosclerosis and other hyperlipidemic conditions.
Hyper-alpha-globulinemia: Iephrotic syndrome, small molecular weight proteins (including albumin) leak out through urine. But proteins with larger molecular weight remains in blood; so there is an increase in alpha globulin fraction, which contains alpha-2-macroglobulin.
TRANSPORT PROTEINS
Blood is a watery medium; so lipids and lipid soluble substances will not easily mix in the blood. Hence such molecules are carried by specific carrier proteins. Albumin is an important transport protein, which carries bilirubin, free fatty acids, calcium and drugs.
1. Pre-albumin is so named because of its faster mobility in electrophoresis than albumin. It is more appropriately named asTransthyretin or Thyroxin binding pre-albumin (TBPA), because it carries thyroid hormones, thyroxin (T4) and tri-iodothyronine T3). It can bind loosely with all substances which are carried by albumin. Its molecular weight is lesser than that of albumin. It is rich in tryptophan. Its half-life in plasma is only one day.
2. Retinol binding protein (RBP) carries vitamin A. It is a low molecular weight protein, and so is liable to be lost in urine. To prevent this loss, RBP is attached with pre-albumin; the complex is big and will not pass through kidney glomeruli. It is a negative acute phase protein. Zinc is required for RBP synthesis, and so RBP level and vitamin A level may be lowered in zinc deficiency.
3. Thyroxine binding globulin (TBG) is the specific carrier molecule for thyroxine and tri-iodo thyronine. TBG level is increased in pregnancy; but decreased iephrotic syndrome.
4. Transcortin, otherwise known as Cortisol binding globulin (CBG) is the transport protein for Cortisol and corticosterone..
5. Haptoglobin (for haemoglobin), Hemopexin (for heme) and Transferrin (for iron) are importantto prevent loss of iron from body.
6. Cholesterol in blood is carried by lipoproteins, HDL and LDL varieties.
POLYMORPHISM
The term polymorphism is applied when the protein exists in different phenotypes in the population; but only one form is seen in a particular person. Haptoglobin, transferrin, ceruloplasmin, alpha-1-antitrypsin and immunoglobulins exhibit polymorphism. For example, Haptoglobin (Hp) exists in three forms, Hp1-1, Hp2-1, and Hp2-2. Two genes, designated Hp1 and Hp2 are responsible for these polymorphic forms. Their functional capabilities are the same. These polymorphic forms are recognised by electrophoresis or by immunological analysis. Study of polymorphism is useful for genetic and anthropological studies.
ACUTE PHASE PROTEINS
The level of certain proteins in blood may increase 50 to 1000-folds in various inflammatory and neoplastic conditions. Such proteins are acute phase proteins. Interleukins (ID, especially IL-1 and IL-6, released by macrophages and lymphocytes, are the primary agents which cause induction and release of these acute phase proteins. Important acute phase proteins are C-reactive protein, ceruloplasmin, haptoglobin,a1 -acid glycoprotein, a-1-anti-trypsin and fibrinogen.
C-reactive Protein (CRP):
It is thus named because it reacts with C-polysaccharide of capsule of pneumococci. CRP consists of five polypeptide subunits to form a disc-shaped cyclic polymer. It has a molecular weight of 115-140 kD. It is synthesised in liver. It can stimulate complement activity and macrophage phagocytosis. When the inflammation has subsided, CRP quickly falls. CRP level has a positive correlation in predicting the risk of cardiovascular disease.
Ceruloplasmin
Ceruloplasmin is blue in colour (Latin, caeruleus=blue). It is an alpha-2 globulin with molecular weight of 160,000. It is synthesised in liver. It contains 6 to 8 copper atoms per molecule. Ceruloplasmin is also called Ferroxidase, an enzyme which helps in the incorporation of iron into transferrin. Ninety per cent of copper content of plasma is bound with ceruloplasmin, and 10% with albumin. Copper is bound with albumin loosely, and so easily exchanged with tissues. Hence transport protein for copper is Albumin. Ceruloplasmin is an enzyme. It is an important antioxidant in plasma.
Clinical Application:
Normal blood level of ceruloplasmin is 25-50 mg/dl. It is estimated either by its oxidative property on phenylene diamine, or by radial immunodiffusion. This level is reduced in
Lowered levels of ceruloplasmin is also seen in malnutrition, nephrosis, and cirrhosis. Ceruloplasmin is an acute phase protein. So its level in blood may be increased in all inflammatory conditions, collagen disorders and in malignancies.
Alpha-1 Anti-trypsin (AAT)
AAT is otherwise called a-anti-proteinase or protease inhibitor (Pi). It inhibits all serine proteases (proteolytic enzymes having a serine in their active centre), such as plasmin, thrombin, trypsin, chymotrypsin, elastase, and cathepsin. Serine protease inhibitors are abbreviated as Serpins. Binding of this inhibitor to protease is very tight; once bound it is not released. Normally, about 95% of the anti-protease activity in plasma is due to AAT. It is synthesised in liver. It has a molecular weight of 50,000 and has 3 polypeptide chains. It forms the bulk of moleculesjn serum having a-1 mobility. It is estimated by radial immuno-diffusion method. Normal serum level is 75-200 mg/dl. Electrophoretically, multiple allelic forms can be separated, the most common variety is PiMM determined by the genotype MM. More than 75 variants are known, out of which about 30 genetic variants show decreased or very low serum concentrations. Gene is located on the small arm of chromosome number 14.
AAT deficiency causes the following conditions:
1. Emphysema: The deficiency is inherited as a co-dominant trait. The incidence of AAT deficiency is
2. Cirrhosis: AAT deficiency is also seen in persons with PiZZ genes. This genetic make up is associated with cirrhosis of liver. The ZZ protein has a substitution of glutamic acid by lysine at position 342. The protein is unsialylated and is not released from hepatocytes, causing death of cells with consequent fibrosis and cirrhosis.
3. In Nephrotic syndrome, AAT molecules are lost in urine, and so AAT deficiency is produced.
Alpha-2-Macroglobulin (AMG): AMG is a tetrameric protein with molecular weight of 725,000. It is the major component of a-2 proteins. Gene is located in the long arm of chromosome number 12. It is synthesised by hepatocytes and macrophages. AMG inactivates all proteases, and thus it is an important in vivo anti-coagulant. Proteases cleave the “bait” region of AMG, releasing a small unit, to provoke conformational changes in AMG, which then “traps” the enzyme. So proteolytic enzymes cannot function. AMG-protease complexes are internalised by a receptor mediated endocytosis by macrophages, and then degraded. AMG is the carrier of many growth factors such as platelet derived growth factor (PDGF). Normal serum level is 130^300 mg/dl. AMG contributes about 1% of all total plasma proteins. Its concentration is markedly increased (up to 2-3 g/d I) in Nephrotic syndrome, because other proteins are lost through urine in this condition.
Alpha-1-Acid Glycoprotein: It is otherwise known as Orosomucoid. It has a molecular weight of 44,000 and has a high content of about 45% of carbohydrates. Its isoelectric pH is 0.7-3.5. It is synthesised by hepatocytes. It binds lipophilic substances and various drugs. It binds with progesterone tightly. Normal serum level is 55-140 mg/dl, and its half-life is five days. It is increased in pregnancy. It is also an acute phase protein. It is a reliable indicator of clinical activity of ulcerative colitis.
NEGATIVE ACUTE PHASE PROTEINS
During an inflammatory response, some proteins are seen to be decreased in blood; these are called negative acute phase proteins. Examples are albumin, transthyretin (prealbumin), retinol binding protein and transferrin.
Transferrin: It is a specific iron binding protein. It is a negative acute phase protein; so the blood level is decreased in acute diseases. It has a half-life of 7-10 days and is used as a better index of protein turnover than albumin.
CLINICAL ENZYMOLOGY
Measurements of the activity of enzymes in plasma are of value in the diagnosis and management of a wide variety of diseases. Most enzymes measured in plasma are primarily intracellular, being released into the blood when there is damage to cell membranes, but many enzymes, for example renin, complement factors and coagulation factors, are actively secreted into the blood, where they fulfil their physiological functions. Small amounts of intracellular enzymes are present in the blood as a result of normal cell turnover. When damage to cells occurs, increased amounts of enzymes will be released and their concentrations in the blood will rise. However, such increases are not always due to tissue damage. Other possible causes include:
· increased cell turnover
· cellular proliferation (e.g. neoplasia)
· increased enzyme synthesis (enzyme induction)
· obstruction to secretion
· decreased clearance.
Little is known about the mechanisms by which enzymes are removed from the circulation. Small molecules, such as amylase, are filtered by the glomeruli but most enzymes are probably removed by reticuloendothelial cells. Plasma amylase activity rises in acute renal failure but, in general, changes in clearance rates are not known to be important as causes of changes in plasma enzyme levels.
Enzyme activity
Enzyme assays usually depend on the measurement the catalytic activity of the enzyme, rather than the concentration of the enzyme protein itself. Since each enzyme molecule can catalyze the reaction of many molecules of substrate, measurement of activity provides great sensitivity. It is, however, important that the conditions of the assay are optimized and standardized to give reliable and reproducible results. Reference ranges for plasma enzymes are dependent on assay conditions, for example temperature, and may also be subject to physiological influences. It is thus important to be aware of both the reference range for the laboratory providing the assay and the physiological circumstances when interpreting the results of enzyme assays.
Enzyme Units
One international unit is the amount of enzyme that will convert one micromole of substrate per minute per litre of sample and is abbreviated as U/L. The SI Unit (System Internationale) expression is more scientific, where or Katal (catalytic activity) is defined as the number of mole of substrate transformed per second per litre of sample. Katal is abbreviated as kat or k (60 U = 1 μkat and 1 nk = 0.06 U).
Disadvantages of enzyme assays
A major disadvantage in the use of enzymes for the diagnosis of tissue damage is their lack of specificity to a particular tissue or cell type. Many enzymes are common to more than one tissue, with the result that an increase in the plasma activity of a particular enzyme could reflect damage to any one of these tissues. This problem may be obviated to some extent in two ways:
first, different tissues may contain (and thus release when they are damaged) two or more enzymes in different proportions; thus alanine and aspartate aminotransferases are both present in cardiac and skeletal muscle and hepatocytes, but there is only a very little alanine aminotransferase in either type of muscle;
second, some enzymes exist in different forms (isoforms), colloquially termed isoenzymes (although, strictly, the term ‘isoenzyme’ refers only to a genetically determined isoform). Individual isoforms are often characteristic of a particular tissue: although they may have similar catalytic activities, they often differ in some other measurable property, such as heat stability or sensitivity to inhibitors.
After a single insult to a tissue, the activity of intracellular enzymes in the plasma rises as they are released from the damaged cells, and then falls as the enzymes are cleared. It is thus important to consider the time at which the blood sample is taken in relation to the insult. If taken too soon, there may have been insufficient time for the enzyme to reach the blood- stream and if too late, it may have been completely cleared. As with all diagnostic techniques, data acquired from measurements of enzymes in plasma must always be assessed in the light of whatever clinical and other information is available, and their limitations borne in mind.
Alkaline phosphatase (ALP)
This enzyme present in high concentrations in the liver, bone (osteoblasts), placenta and intestinal epithelium. These tissues each contain specific isoenzymes (strictly, isoforms) of ALP. Pathological increases in ALP activity are most frequently seen in cholestatic liver disease and in bone diseases in which there is an increase in osteoblastic activity (e.g. Paget’s disease and osteomalacia).
The causes of an increase in plasma ALP activity
Physiological increases are been in pregnancy, due to the placental isoenzyme, and in childhood (when bones are growing), due to the bone isoenzyme. Plasma ALP activity is high at birth but falls rapidly thereafter. However, it remains two to three times the normal adult level and rises again during the adolescent growth spurt before falling to the adult level as bone growth ceases. Plasma ALP activity,is slightly higher thaormal in apparently healthy elderly people. This may reflect the high incidence of mild, sub-clinical Paget’s disease in the elderly. Levels of ALP as high as ten times the upper limit of normal (10 x ULN) may be seen in severe Paget’s disease of bone, rickets and osteomalacia and occasionally in cholestatic liver disease. Lesser increases are, however, more common in these conditions. Note that ALP activity is not increased in uncomplicated osteoporosis, unless the condition has been complicated by a fracture.
Plasma ALP is frequently elevated in malignant disease: it may be of bony or hepatic origin and associated with the presence of either primaly or secondary turnouts in these tissues. A number of apparently turnout-specific
Causes of an increased plasma alkaline phosphatase
Physiological: pregnancy (last trimester), childhood
Pathological: often >5 x ULN
Paget’s disease or bone osteomalacia, rickets, cholestasis (intra- and extrahepatic),
cirrhosis
usually <5 x ULN
bone tumours (primary and secondary),
primary hyperparathyroidism with bone involvement, healing fractures,
osteomyelitis, hepatic space-occupying lesions (tumour, abscess), infiltrative hepatic disease, hepatitis, inflammatory bowel disease
ULN = upper limit of normal.
ALP is frequently measured as part of a biochemical profile and it is not uncommon to find a raised activity in the absence of clinical evidence of bone or liver disease, and in the absence of other biochemical abnormalities. In establishing the cause of such an increase it is clearly helpful to determine the tissue of origin. This can be done by measuring tissue-specific isoenzymes of ALP. These can be separated and quantitated using various techniques, including electrophoresis and differential heat inactivation. A simpler but less reliable alternative is to measure plasma γ-glutamyl transferase. This enzyme is found in the liver but not in bone. Its plasma activity is often (but not always) increased when there is an excess of hepatic ALP in the plasma.
Aminotransferases
Two aminotransferases are used in diagnosis and management: aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Both enzymes are widely distributed in body tissues, but ALT is present in only small amounts except in the liver. Even here, there is more than three times as much AST; in cardiac and skeletal muscle, there is twenty times as much AST as ALT.
ASPARTATE AMINO TRANSFERASE (AST)
It is also called as serum glutamate-oxaloacetate transaminase (SGOT). AST needs pyridoxal phosphate as co-enzyme. AST is estimated by taking aspartate, α-ketoglutarate, pyridoxal phosphate (vitamin B6) and patient’ serum as the source of AST. The oxaloacetate formed may be allowed to react with dinitrophenyl hydrazine to produce a colour which is estimated colorimetrically at 520 nm.
Normal serum level of AST is 5-30 U/L. It is significantly elevated in myocardial infarction. It if moderately elevated in liver diseases. However, a marked increase in AST may be seen in primary hepatoma. AST has two iso-enzymes; cytoplasmic and mitochondrial. In mile degree of tissue injury, cytoplasmic form is seen in serum. Mitochondrial type is seen in severe injury.
ALANINE AMINO TRANSFERASE (ALT)
It is also called as serum glutamate-pyruvate transaminase (SGPT). ALT needs pyridoxal phosphate as co-enzyme.
Normal serum level of ALT is 8-40 U/L. Very high values (100 to 1000 U/L) are seen in acute hepatitis, either toxic or viral in origin. Both ALT and AST are increased in liver diseases, but ALT >AST. Moderate increase (25 to 100 U/L) may be seen in chronic liver disease such as cirrhosis, and malignancy in liver. A sudden fall in ALT level in cases of hepatitis is a very bad prognostic sign.
Causes of an increased plasma aspartate aminotransferase
often > 10 x ULN
acute hepatitis and liver necrosis
major crush injuries
severe tissue hypoxaemia
(levels may sometimes exceed 100 x ULN in these conditions)
5-10 x ULN
myocardial infarction
following surgery or trauma
skeletal muscle disease
cholestasis
chronic hepatitis
usually <5 x ULN
physiological (neonates)
other liver diseases
pancreatitis
haemolysis (in vivo and in vitro)
Levels of up to 2 x ULN are sometimes found in patients who have no clinical evidence of tissue damage. Alcohol abuse and non-alcohollc steatohepatitis should be considered as posslble causes in such cases. There are no tissue- specific isoenzymes of AST and if there are no other biochemical changes, nor any readily apparent cause of the raised level, the wisest procedure is to repeat the analysis after an interval of one or two weeks.
Glutamyl transferase (GGT)
This enzyme is present in high concentrations in the liver, kidney and pancreas. Measurement of its plasma activity provides a sensitive indicator of hepatobiliary disease although it is of no value in distinguishing between cholestatic and hepatocellular disease. In biliary obstruction, plasma GGT activity may increase before that of alkaline phosphatase. Plasma GGT is raised in the absence of liver disease in many patients taking the anticonvulsant drugs phenytoin and phenobarbital; rifampicin, used in the treatment of tuberculosis, can have a similar effect. This is an example of enzyme induction. The increased plasma GGT is not
due to cell damage but to an increase in enzyme production within cells with the result that an increased amount is released during normal cell turnover. Plasma GGT activity is frequently very high in patients with alcoholic liver disease but can be elevated, due to enzyme induction, in heavy alcohol drinkers in the absence of other evidence of liver damage. Up to 70% of such people may have elevated levels of the enzyme but it could be appreciated both that similar increases may be seen in other conditions and that a significant number of people who abuse alcohol have a normal plasma enzyme activity. Plasma GGT activity can remain elevated for up to 3-4 weeks following absfinence from alcohol, even in the absence of liver damage.
Some causes of an increased plasma γ-glutamyl transferase activity.
often > 10 x ULN
cholestasis
alcoholic liver disease
5-10 x ULN
hepatitis (acute and chronic)
cirrhosis (without cholestasis)
other liver diseases
pancreatitis
usually <5 x ULN
excessive alcohol ingestion
enzyme-inducing drugs
congestive cardiac failure
Lactate dehydrogenase (LDН)
This enzyme exists in body tissues as a tetramer. Two monomers, H and M, can combine in various proportions with the result that five isoenymes of LD are known. Increases in plasma LD activity are seen in a wide variety of conditions including acute damage to the liver, skeletal muscle and kidneys, and also in megaloblastic and haemolytic anaemias. In patients with lymphoma, a high plasma LD activity indicates a poor prognosis.
There is a correlation between enzyme activity and turnout bulk and so serial measurements may be useful in following response to treatment. In both cardiac muscle and red blood cells LD 1 is the predominant isoenzyme. An increased plasma activity (due to release from red blood cells) occurs in haemolytic crises in sickle cell anaemia, and measurement of the enzyme may be of value when this diagnosis is suspected.
Isoenzymes of LDH
LDH enzyme is a tetramer with four subunits. But the subunit may be either H (heart) or M (muscle) polypeptide chains. These two are the products of two different genes.
Although both of them have the same molecular weight (32 kD), there are minor amino acid variations. So five combinations of H and M chains are possible; H4, H3M, H2M2, M3H and M4 varieties, forming five iso-enzymes. All these five forms are seen in all persons. M4 form is seen in skeletal muscles; it is not inhibited by pyruvate. But H4 form is seen in heart and is inhibited by pyruvate. Normally LDH-2 (H3M1) concentration in blood is greater than LDH-1 (H4); but this pattern is reversed in myocardial infarction; this is called flipped pattern. The iso-enzymes are usual ly separated by cellulose acetate electrophoresis at pH 8.6. They are then identified by adding the reactants finally producing a colour reaction. . Lactate dehydrogenase isoenzymes (as percentage of total):
LDH1 14-26 %
LDH2 29-39 %
LDH3 20-26 %
LDH4 8-16%
LDH5 6-16 %
Normal value of LDH in serum is 100-200 U/L. Values the upper range are generally seen in children
Creatine kinase (CK)
The enzymatically active CK molecule is a dimer; there are two monomers, M and B. Three isoenzymes, BB, MM and MB, occur. BB is confined mainly to the brain. The CK normally present in plasma is mainly the MM isoenzyme. Even in severe brain damage, the contribution of the BB isoenzyme to plasma activity is minimal. Increases in plasma CK activity are usually the result of skeletal or cardiac muscle damage. The CK in skeletal musde is almost entirely MM; in cardiac muscle, up to 30% is the MB isoenzyme. When plasma CK activity is increased, the demonstration that more than 5% of the total CK is due to the MB isoenzyme is highly suggestive of its being cardiac in origin.
CK-MB can be measured either by measurement of enzyme activity in the presence of an antibody that inhibits the M subunit, or by measurement of enzyme mass using an immunoassay. In the plasma, the terminal lysine residue of the CK-M polypeptide is removed by a carboxypeptidase. This does not affect enzyme activity but alters the charge on the polypeptide and hence the dectrophoretic mobility of the enzyme. The three possible forms of CK-MM are termed isoforms: CK- MM3 is composed of two intact CK-M polypeptides; in CK-MM1, both polypeptides have had their terminal lysines removed; CK-MM2 has one polypeptide of each type. An increase in the ratio CK-MM3:CK-MM1 occurs earlier than other enzyme changes following myocardial infarction (2-5 h after the onset of chest pain). However, the measurement of CK isoforms is technically demanding and few laboratories offer it routinely.
Causes of an increased plasma creatine kinase activity.
often >10 x ULN
polymyositis
rhabdomyolysis (e.g. trauma, malignant hyperpyrexia)
Duchenne muscular dystrophy
myocardial infarction
5-10 x ULN
following surgery
skeletal muscle trauma
severe exercise
grand mal convulsions
myositis
carriers of Duchenne muscular dystrophy
usually <5 x ULN
physiological (Afro-Caribbeans)
hypothyroidism
drug (statin) treatment
Amylase
This enzyme is found in the salivary glands and exocrine pancreas, and tissue-specific isoenzymes can be distinguished by means of dectrophoresis or the use of
inhibitors. Plasma amylase activity is usually increased, often to 5 x or even to more than 10 x ULN, in acute pancreatitis. Its use in the diagnosis of patients presenting with an acute abdomen.
Alkaline phosphatase (ALP)
It is a non- specific enzyme which hydrolyses aliphatic, aromatic or heterocyclic compounds. The pH optimum for the enzyme reaction is between 9 and 10. It is prodused by osteoblasts of bone, and localized in cell memmbranes (ecto-enzyme).
Normal serum level of ALP is 40-125 U/L.
In children the upper level of normal value may be more, becouse of the increased osteoblastic activity. Mild increase is noticed during pregnancy, due to production of placental isoenzyme.
Moderate (2-3 times) increase in ALP level is seen in hepatic diseases such as hepatitis, alcoholic hepatosis or hepatocellular carcinoma. Very high levels of ALP (10-12 times of upper limit) may be noticed in extrahepatic obstructions or cholestasis. ALP is produced by epithelial cells of biliary canaliculi and obstruction of bile with consequent irritation of epithelial cells leads to secretion of ALP into serum.
Drastically high levels of ALP (10-25 times of upper limit) are also seen in bone diseases where osteoblastic activity is enhanced such as Paget’s disease, rickets, osteomalacia, osteoblastoma, metastatic carcinoma of bone and hyperparathyroidism (Paget’s disease or osteitis deformans was described in 1877 by Sir James Paget).
Iso-enzymes of Alkaline Phosphatase
1. α-1 ALP moves in α -1 position, it is synthesised by epithelial cells of biliary canaliculi. It is about 10% of total activity and is increased in obstructive jaundice and to some extent in metastatic carcinoma of liver.
2. α -2 heat labile ALP is stable at
3. α -2 heat stable ALP will not be destroyed at
4. Pre-ß ALP is of bone origin and elevated levels are seen in bone diseases. This is the most heat labile (destroyed at
5. γ-ALP is inhibited by phenylalanine and originates from intestinal cells. It is increased in ulcerative colitis. About 10% of plasma ALP are of intestinal variety.
6. The leucocyte alkaline phosphatase (LAP) is significantly decreased in chronic myeloid leukemia. It is increased in lymphomas.
ALP has different isoforms. Although ALP is a monomer, depending on the number of sialic acid residues, the charged groups differ. Such different forms are detected in agar gel electrophoresis.
NUCLEOTIDE PHOSPHATASE (NTP)
It is also known as 5′ nucleotidase. This enzyme hydrolyses 5′ nucleotides to corresponding nucleosides at an optimum pH of 7.5. It is a marker enzyme for plasma membranes and is seen as an ecto-enzyme (enzyme present on the cell membrane).
Usually, AMP is used as substrate, which is hydrolysed to adenosine and inorganic phosphate. The latter reacts with ammonium molybdate to produce the yellow ammonium phosphomolybdate, which is estimated colorimetrically. However, ALP will also catalyse the same reaction. Serum samples contain both ALP and NTP. These are distinguished by Nickel ions which inhibit NTP but not ALP.
Normal NTP level in serum is 2-10 U/L. It is moderately increased in hepatitis and highly elevated in biliary obstruction. Unlike ALP, the level is unrelated with osteoblastic activity and therefore is unaffected by bone diseases.
GAMMA GLUTAMYL TRANSFERASE (GGT)
The old name was gamma glutamyl transpeptidase. It can transfer γ-glutamyl residues to substrate. In the body it is used in the synthesis of glutathione. GGT has 11 iso-enzymes. It is seen in liver, kidney, pancreas, intestinal cells and prostate gland.
Normal serum value of GGT is 6-45 U/L.in male and 5-30 U/L in female. It is slightly higher in normal males, due to the presence of prostate gland. This value is moderately increased in infective hepatitis and prostate cancers. The GGT level is highly elevated in alcoholism, obstructive jaundice and neoplasm’s of liver. GGT-2 is positive for 90% of hepatocellular carcinomas. It is not elevated in cardiac or skeletal diseases.
GGT is a microsomal enzyme. Its activity is induced by alcohol, phenobarbitone and rifampicin. GGT is clinically important because of its sensitivity to detect alcohol abuse. GGT is increased in alcoholics even when other liver function tests are withiormal limits. GGT level is rapidly decreased within a few days when the person stops to take alcohol. Increase in GGT level is generally proportional to the amount of alcohol intake.
ACID PHOSPHATASE (ACP)
It hydrolyses phosphoric acid ester at pH between 4 and 6. Methods for assay are the same as described for ALP; but the pH of the medium is kept at 5 to 5.4.
Normal serum value for ACP is 2.5-12 U/L . ACP is secreted by prostate cells, RBC, platelets and WBC. Isoenzymes of ACP are described. Erythrocyte ACP gene is located in chromosome 2; osteoclast ACP gene is on chromosome 19; lysosomal gene is on 11 and prostate ACP gene is on 13. The prostate iso-enzyme is inactivated by tartaric acid. Cupric ions inhibit erythrocyte ACP. Normal level of tartrate labile fraction of ACP is 1 U/L.
ACP total value is increased in prostate cancer and highly elevated in bone metastasis of prostate cancer. In these conditions, the tartrate labile iso-enzyme is elevated. This assay is very helpful in follow up of treatment of prostate cancers. ACP is therefore an important tumour marker.
Since blood cells contain excess quantity of ACP, must be taken to prevent hemolysis while taking blood from the patient. Prostate massage may also increase to value. So blood may be collected for ACP estimation before per rectal examination of patient. ACP is present in high concentration in semen, a finding which is used in forensic medicine in investigation of rape.
PROSTATE SPECIFIC ANTIGEN (PSA)
It is produced from the secretory epithelium of prostal gland. It is normally secreted into seminal fluid, where it is necessary for the liquefaction of seminal coagulum. It is a serine protease, and is a 32 kD glycoprotein; encoded in chromosome number
Normal value is 1 -5 µg/L. It is very specific for prostate activity. Values between 4-10 µg/L is seen in benign prostate enlargement; but values above 10 µg/L is indicative of prostate cancer.
CHOLINESTERASE (ChE)
Acetyl cholinesterase or true ChE or Type 1 ChE can act mainly on acetyl choline. It is present ierve endings and in RBCs. About 25 allelic forms are reported. Normal serum range is 2-12 U/ml. Newly formed RBC will contain good quantity of ChE which is slowly reduced according to the age of the cell. Therefore, ChE level in RBCs will be proportional to the reticulocyte count. Organophosphorus
insecticides (Parathione) irreversibly inhibit ChE in RBCs. Measurement of ChE level in RBCs is useful to determine the amount of exposure in persons working with these insecticides.
Pseudocholinesterase or type II ChE is non-specific and can hydrolyse acyl esters. It is produced mainly by liver cells. Normal serum level is 8-18 U/ml. Succinyl choline is a widely used as muscle relaxant. It is a structural analogue of ACh, and so competitively fix on post-synaptic receptors of ACh. Succinyl choline is hydrolysed by the liver ChE within 2-4 minutes. But in certain persons the ChE activity may be absent; this is a genetically transmitted condition. In such individuals when succinyl choline is given during surgery, it may take hours to get the drug metabolised. Very prolonged scoline apnoea may result in ‘nightmare of anaesthetist’. The pseudocholinesterase level in serum is reduced in viral
hepatitis, cirrhosis, hepatocellular carcinoma, metastatic cancer of liver and in malnutrition.
GLUCOSE-6-PHOSPHATE DEHYDROGENASE
GPD is a dimer with identical subunits. This is an important enzyme in the hexose monophosphate shunt pathway of glucose. It is mainly used for production of NADPH . It has a special role in the RBC metabolism. Due to the presence of oxygen, hydrogen peroxide is continuously formed inside the RBC. Peroxide will destroy biomembranes, and RBCs are lysed. Normal value of GPD in RBC is 125-250 U/1012 cells/ Nearly 400 variants (isoforms) of GPD are described.
LIPASE
It will hydrolyse triglyceride to β-monoglyceride and fatty acid. Molecular weight is 54,000. The gene is in chromosome 10. The enzyme is present in pancreatic secretion. Normal serum range is 0.2-1.5 U/L. It is highly elevated in acute pancreatitis and this persists for 7-14 days. Thus, lipase remains elevated longer than amylase. Moreover, lipase is not increased in mumps. Therefore, lipase estimation has advantage over amylase. It is moderately increased in carcinoma of pancreas, biliary diseases and perforating peptic ulcers.
Aldolase (ALD)
It is a tetrameric enzyme with A and B subunits; so there are 5 iso-enzymes. It is a glycolytic enzyme. Normal range of serum is 1.5-7 U/L. It is drastically elevated in muscle damages such as progressive muscular dystrophy, poliomyelitis, myasthenia gravis and multiple sclerosis. It is a very sensitive early index in muscle wasting diseases.
Enolase
It is a glycolytic enzyme. Neuron-specific enolase (NSE) is an iso-enzyme seen ieural tissues and Apudomas. NSE is a tumour marker for cancers associated with neuro-endocrine origin, small cell lung cancer, neuroblastoma, pheochromocytoma, medullary carcinoma of thyroid, etc. It is measured by RIA or ELISA. Upper limit of NSE is 12 μg/ml.
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