Amino Acids Dietary proteins are the primary source of a.a.s for endogenous protein synthesis.
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- Aminoaciduria may be primary or secondary.
Primary disease: due to an inherited enzyme defect, also called inborn error of metabolism.
-The defect is located either in the pathway by which a specific a.a. is metabolized or in the specific renal tubular transport system by which the a.a. is reabsorbed.
-The defect in the enzyme results in substrate accumulation or its diversion into alternative paths. Products of the normal path are not formed at all or formed in smaller amounts.
- Secondary aminoaciduria: could be due to either to disease of an organ such as the liver, which is an active site of a.a. metabolism, or a generalized renal tubular dysfunction.
- It can affect many a.a. simultaneously
Examples of disorders that result in secondary overflow amnioaciduria are acute viral hepatitis and acetaminophen poisoning.
Generalized secondary renal aminoaciduria is due to progressive damage to the renal tubules
It can be caused by poisons (especially heavy metal), or disease, or by congenital conditions such as Wilson’s disease.
Amino acid disorders
Amino acid disorders
Selected disorders of a.a. metabolism
Hyperphenylalaninemias: these are a group of disorders resulting from impaired conversion of phenylalanine to tyrosine due to the defect in phenyalanine hydroxylase that found only in the liver and kidneys.
Defects in this enzyme Hyperphenylalaninemia phenylalanine accumulates in blood, urine and CSF phenylketonuria (PKU)
Untreated PKU results in severe mental retardation.
* Affected children appear normal at birth, and the earliest symptoms are usually nonspecific-delayed development, feeding difficulties and vomiting.
* Children with PKU elicit an unusual but characteristic musty odor in urine or sweat, owing to increased production of phenylpyruvate.
* Early diagnosis is essential to avoid the adverse effects of PKU and consequently neonatal screening has become widespread.
Elevated blood-phenylalanine activates the normal minor metabolic pathways of phenylalanine increased production of phenylketones (e.g., phenylpyruvate) and other metabolites that excreted into the urine.
Treatment of PKU consists of restricting dietary phenyalanine before the onset of brain damage
* Tyrosinemia has several forms, each of which is accompanied by high level of tyrosine and phenolic aciduria.
* Tyrosine is essential for protein synthesis and serves as a precursor for thyroxine, melanin and catecholamines.
* The pigment melanin is derived from tyrosine by the activity of tyrosinase.
* Clinical syndromes resulting from inherited defects in melanin synthesis are collectively known as albinism.
Maple syrup urine disease:
Amino acid disorders
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 of these enzyme are not used as diagnostic tool but used for monitoring the diseases
Many enzymes are common to more than one tissue
This problem may be overcome to some extent in two ways:
A) First, different tissues may contain (and thus release when they are damaged) two or more enzymes in different proportions; e.g. alanine and aspartate aminotransferase are both present in cardiac muscle and hepatocytes, but there is relatively more alanine transaminase in the liver;
B) Second, some enzymes exist in different forms (isoforms), termed isoenzymes.
Individual isoforms are often characteristic of a particular tissue.
So the pattern of increase of different enzymes can indicate the site of problem, e.g. high GGT and high ALP or AST indicates a problem in the liver
While high ALT and CK-MB indicates MI
Factors Affecting Results of Plasma Enzyme Assays
Analytical factors affecting results.
Results of enzyme assays are not usually expressed as concentrations, but as activities.
So the results of such measurements depend on many analytical factors including the concentrations of the substrate and product, the pH and temperature at which the reaction is carried out, the type of buffer, and the presence of activators or inhibitors.
Physiological factors affecting enzyme activities, include for example:
age: plasma aspartate transaminase activity is moderately higher during the neonatal period than in adults;
plasma alkaline phosphatase activity of bony origin is higher in children than in adults.
sex: plasma gama-glutamyltransferase activity is higher in men than in women
physiological conditions: plasma alkaline phosphatase activity rises during the last trimester d pregnancy because of the presence of the placental isoenzyme;
several enzymes, such as the transaminases and creatine kinase, rise moderately in plasma during and immediately after labour or strenuous exercise.
Two important transferases:
Alanine aminotransferas (ALT) called also Glutamate – Pyruvate transferase (GPT) or Serum ALT = SGPT, found in many tissues catalyzes the transfer of amino gp of alanine to produce pyruvate and glutamate.
Aspartate aminotransferase (AST) called also Glutamate–Oxaloacetate transferase (GOT) or Serum AST = SGOT,
- During the catabolism of amino acids AST takes amino group from glutamate to oxaloacetate forming aspartate. Which used as source of NH4 group in Urea synthesis
Aspartate source of amino group of the urea in the urea cycle.
Aspartate Transaminase (AST): 10-45 U/L
AST (glutamate oxaloacetate transaminase GOT) is present in high concentrations in cells of cardiac and skeletal muscle, liver, kidney and erythrocytes.
Damage to any of these tissues may increase plasma AST levels.
AST can be used as indicator of muscle damage.
Causes of Raised Plasma AST Activities
* Artifact: due to in-vitro release from erythrocytes if there is haemolysis or if separation of plasma from cells is delayed.
*Physiological: during the neonatal period (about 1.5 times the upper adult reference limit).
*Marked increase (10 to 100 times the upper adult reference limit):
circulatory failure with 'shock' and hypoxia; myocardial infarction; acute viral or toxic hepatitis.
Aspartate Transaminase (AST)
Cirrhosis (may be normal, but may rise to twice the upper adult reference limit); infectious mononucleosis (due to liver involvement (type of viral infection “mononucleosis” refers to an increase in a special type of white blood cells (lymphocytes); cholestatic jaundice (up to 10 times the upper adult reference limit); malignant infiltration of the liver; skeletal muscle disease; after trauma or surgery (especially after cardiac surgery); severe hemolytic episodes (of erythrocyte origin).
Alkaline Phosphatase (ALP)
-The alkaline phosphatases are a group of enzymes that hydrolyze organic phosphates at high pH.
-They are present in most tissues but are in particularly high concentration in the osteoblasts of boneand the cells of the hepatobiliary tract, intestinal wall, renal tubules and placenta.
-The exact metabolic function of ALP is unknown but it is probably important for calcification of bone.
- In adults plasma ALP is derived mainly from bone and liver in approximately equal proportions; the proportion due to the bone fraction is increased when there is increased osteoblastic activity that may be physiological.
Causes of raised plasma ALP activity
during the last trimester of pregnancy the plasma total ALP activity rises due to the contribution of the placental isoenzyme
Rickets and osteomalacia;
Rickets: is an abnormal bone formation in children resulting from inadequate calcium in their bones
Osteomalacia: softening of the bones, resulting from defective bone mineralization in adults
Paget's disease and (ALP may be very high); Paget's disease is a chronic bone disorder that is due to irregular breakdown and formation of bone tissue.
Primary hyperparathyroidism with extensive bone disease
intra- or extrahepatic cholestasis, lesions, tumor, granulomas, and other causes of hepatic infiltration.
Malignancy: bone or liver involvement or direct tumor production.
Homolactic fermentation: conversion of pyruvate to lactate
Creatine Kinase (CK)
CK is most abundant in cells of cardiac and skeletal muscle and in brain, but also occurs in other tissues such as smooth muscle.
Causes of raised plasma CK activities
*Artifact: due to in vitro haemolysis
*Physiological: neonatal period (slightly raised above the adult reference range).
Circulatory failure and shock; myocardial infarction;
Muscular dystrophies and high breakdown of skeletal muscle.
The muscular dystrophies are the most-known group of hereditary muscle diseases; characterized by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue.
Creatine Kinase (CK)
Muscle injury; after surgery (for about a week); physical effort, moderate exercise and muscle cramp;
an intramuscular injection; hypothyroidism (thyroxine may influence the catabolism of the enzyme); alcoholism (due to alcoholic myositis (inflammation of the muscle)
ISOENZYMES OF CK
CK has 3 isoenzymes:
CK-MM (CK-3) is the predominant isoenzyme in skeletal and cardiac muscle and is detectable in the plasma of normal subjects.
CK-MB(CK-2) accounts for about 35 % the total CK activity in cardiac muscle and less than 5% in skeletal muscle;
Its plasma activity is always high after myocardial infarction.
CK-BB (CK-1) is present in high concentrations in the brain and in the smooth muscle of the gastrointestinal and genital tracts.
Raised plasma activities may occur during labour and child birth.
Non-specific Causes of Raised Plasma Enzyme Activities
Change in plasma enzyme activity could be due to nonspecific causes
-Slight rises in plasma aspartate transaminase activities are common in non-specific findings in many illnesses.
- Moderate exercise, or a large intramuscular injection, may lead to a rise in plasma creatine kinase activity;
- Some drugs, such as the anticonvulsants phenytoin and phenobarbitone, may induce synthesis of the microsomal enzyme, gammaglutamyltransferase, and so increase its plasma activity in the absence of disease.
- Plasma enzyme activities may be raised if the rate of clearance from the circulation is reduced.
Proteins: are polymers of a.a.s that are covalently linked through peptide bonds.
The different R groups found in a.a.s influence the structure, functionality and properties of the individual proteins.
Proteins may be classified as fibrous (mainly structural) or globular.
*Nearly all other proteins of clinical interest are soluble globular proteins such as haemoglobin, enzymes and plasma proteins.
*The complex bending and folding of polypeptide chains is a result of numerous interactions of their R groups.
*Globular proteins are compact and have little or no space of water in the interior of the molecule, where most of the hydrophobic R groups are located.
*Most polar R groups are located on the surface of the protein where influence on protein solubility, acid-base behaviour and electrophoretic mobility
*Most globular proteins are affected with temperature and pH.
- Many of the properties of proteins are used for their separation, identification and assay:
1-Molecular size: Most proteins are macromolecules, so can be separated from smaller molecules by dialysis or ultrafiltration, chromatography and by density-gradient ultracentrifugation
2. Differential solubility: Protein solubility is affected by the pH, ionic strength, temperature and dielectric constant of the solvent.
3. Electrical charge: Separation by electrophoresis, this is based on the capability of a mixture of proteins with various species of different charge/mass ratiosto migrate at different rates in an electrical field.
4.Adsorption on finely divided inert materials: These materials offer a large surface area for interaction with protein, such as charcoal, silica or alumina.
5. Specific binding to antibodies, coenzymes, or hormone receptors: The unique properties of protein to recognize and bind to a complementary compound with high specificity is the basis for immunoassays.
Serum Protein Electrophoresis
*Electrophoresis separates proteins according to their different electrical charges
*It is usually performed by applying a small amount of serum to a strip of cellulose acetate or agarose and passing a current across it for standard time.
Serum Protein Electrophoresis
Electrophoresis separates proteins into five main groups of proteins, albumin and the 1-,2-,- and -globulins,
Principal bands seen after electrophoresis on cellulose acetate of normal adult serum
1. Albumin, usually a single protein, makes up the most obvious band.
2. 1-Globulins consists almost entirely of 1-antitrypsin.
3. 2-Globulins consists mainly of 2-macro-globulin and haptoglobin.
4. -Globulins often separate into two; 1 consists mainly of transferrin with a contribution from LDL and 2 consists of C3 complement.
5. -Globulins are immunoglobulins. Some immunoglobulins are found also in the 2 and regions.
- If plasma rather than serum is used, fibrinogen appears as a distinct band in the - region. This may make interpretation difficult; blood should be allowed to clot and serum used if electrophoresis is to be performed.
Many other important proteins are present in only very low concentration
Serum protein electrophoresis: applied to the serum not plasma
*Electrophoresis separates proteins into five main groups of proteins, albumin and the 1-,2-,- and -globulins, may be distinguished after staining and may be visually compared with those in a normal control serum.
*Each of the globulin fractions contain several proteins.
*Changes in electrophoretic patterns are most obvious when:
1. The concentrations of protein, such as albumin, which is usually in high concentration, are abnormal.
2. There are parallel changes in several proteins in the same fraction.
3. New band that is not seen in normal serum.
Total plasma protein (62-80 gm/L)
- Alterations in plasma protein can be due to
A) Change in the concentration of a specific protein in plasma (due to changes in the rate of synthesis or removal)
B) Change in the volume of distribution (plasma water).
*Decrease in the volume of plasma water (haemoconcentration) as relative hyperproteinaemia concentrations of all plasma proteins are increased to the same degree
*Hyperproteinaemia is caused by:
1) dehydration (haemoconcentration) due to inadequate water intake or excessive water loss, as in sever vomiting, diarrhoea, diabetic acidosis.
2) an increase in the concentration of specific protein normally present in relatively low concentration, as, for example, increases in APRs and polyclonal or monoclonal immunoglobulins as a result of infection.
*Hypoproteinaemia: caused by a) decreased synthesis, b) Haemodilution and c) protein redistribution.
Haemodilution (increase in plasma water volume) hypoproteinaemia; concentrations of all the individual plasma proteins are decreased to the same degree. Haemodilution occurs with water intoxication or salt retention syndromes, during massive intravenous infusions.
Plasma protein can be divided into
1) Acute phase reactants (APR) these proteins have specific role in inflammatory response so they will increase in inflammatory conditions
2) Negative acute phase reactants these proteins have no role in in inflammation but number of these proteins decrease in inflammatory conditions.
Like albumin, prealbumin, transferrin ….
* Low albumin concentration may be due to dilution or redistribution.
* True albumin deficiency may be caused by a decreased rate of synthesis, or by an increased rate of catabolism or loss from the body.
Consequences of hypoalbuminaemia
1. Fluid distribution.
The decreased plasma oncotic pressure disturbs the equilibrium between plasma and interstitial fluid there will be a decrease in the movement of the interstitial fluids back into the blood accumulation of interstitial fluid (edema) relative decrease in plasma volume fall in renal blood flow stimulates the secretion of renin, and aldosterone through the formation of angiotensin sodium retention and thus an increase in ECF volume which potentiates the edema.
2. Binding functions.
Albumin is a high capacity, low affinity transport protein for many substances, such as thyroid hormones, calcium, bilirubin and fatty acids.
Many drugs are bound to albumin in the blood stream as salicylates, penicillin and sulphonamides.
The drug fraction that is bound to albumin is physiologically and pharmacologically inactive A reduction in plasma albumin, may increase the plasma free concentration of those drugs cause toxic effects
* TRF is a -globulin which is the major iron-transporting protein in the plasma.
* It reversibly binds numerous cations iron, copper, zinc, cobalt and calcium-although only iron binding appear to have physiological significance.
* TRF is normally about 30-40% saturated with iron and its half life 7 days.
* Its concentration correlates with the total iron-binding capacity of serum.
Transferrin (TRF) : (2.1-3.6 g/L)
*TRF Plasma levels are regulated by availability of iron iron deficiency, TRF rise and, upon successful treatment with iron, it returns to normal level.
* In common iron deficiency, the TRF level is increased due to increases in synthesis this guarantee that any amount of iron absorbed will be transported and bound to TRF directly and in this case the % of saturation will be less than 30% the protein is less saturated with iron because plasma iron levels are low
*If the anemia is due to failure to incorporate iron into erythrocytes, Vit B12 or folic acid deficiency the TFR level is normal or low but the protein is highly saturated with iron.
High levels of TRF occur in pregnancy and oestrogen administration.
Neural tube defect (NTD):
Anencephaly: congenital absence of all or a major part of the brain
Acute phase reactants (APR)
Are protein synthesized in the liver in response to inflammatory mediators causethe non-specific changes in plasma protein concentrations, in response to acute or chronic tissue damage and other inflammatory responses
The acute-phase reactants include:
1) Activators of other inflammatory pathways such as C-reactive protein, so called because it reacts with the C-polysaccharide of bacteria. During this response, the concentrations of C-reactive protein may increase as much as thirty-fold.
2) Inhibitors for enzymes released in inflammation such as 1-antitrypsin, so they will protect body cells from the attack from these enzymes.
3) Scavengers such as haptoglobin which binds haemoglobin released by local in vivo haemolysis during the inflammatory response.
- It is a substance in the sera of acutely ill patients.
- It binds and complexes with the polysaccharides present in many bacteria, fungi and protozoal parasites and becomes an activator of the classic complement pathway.
* C-reactive protein, dramatically increases following myocardial infarction, trauma, infections, surgery or neoplastic proliferation.
* C-reactive protein is test of choice in monitoring the acute phase response, in monitoring patients with inflammatory joint disease such as rheumatoid arthritis.
- The most widely used parameters in inflammation monitoring are C-reactive protein and ESR (erythrocyte sedimentation Rate) both will increase in inflammation.
Acute phase proteins:
1. 1-Antitrypsin (AAT)
- It is an acute phase reactant with antiprotease activity.
- Plasma concentrations rise two to three days after trauma or acute infection.
- Its deficiency is associated with lung and liver disease.
- As a protease inhibitor, AAT acts against chymotrypsin, renin, urokinase, plasmin and possibly thrombin, but the inhibition of greatest clinical significance is directed against neutrophil elastase and collagenase.
- The function of AAT is to neutralise lysosomal elastase released on phagocytosis of particles by polymophonuclear leukocytes.
-AAT, being a relatively small molecule, can pass from capillaries into tissue fluid, bind protease, and pass back into the intravascular fluid.
2. Haptoglobin acute phase protein
- Its function is to bind free haemoglobin released into the plasma during intravascular haemolysis.
- The haemoglobin-haptoglobin complexes formed are removed by the reticuloendothelial system
- Its components are metabolised to free a.a.s and iron
- Haptoglobin thus prevents loss of haemoglobin to urine and conserves iron.
- Thus a low plasma haptoglobin concentration can be indicative of intravascular haemolysis, or haemoglobin turnover, as occur in haemolytic anaemias, transfusion reactions and malaria.
- Low concentrations due to decreased synthesis are seen in chronic liver disease, metastatic disease and severe sepsis.
- Haptoglobin is an acute phase protein and its concentration also increases in burns, nephrotic syndrome
- Caeruloplasmin is a late APR
- It is the principal copper-containing protein in plasma. It is a copper donor, another possible role of CER is as an antioxidant
- Once CER is synthesised, it neither gains nor loses copper unless metabolised.
-Plasma copper consists of a non-dialysable fraction (95%) attached to CER and of dialyzable (free) fraction (5%) loosely bound to albumin and histidine.
-Copper is transported in the dialysable form from the gut to the liver; it is incorporated into the CER apoprotein, which is released into the bloodstream.
- Increased absorption of copper leads to increased synthesis of CER and increased excretion of copper-protein complexes in the bile.
-Synthesis of CER thereby provides a first-line reaction to potential copper toxicity.
Wilson’s disease: CER deficiency
* It is rare condition where the plasma CER is typically reducedfree copper concentration is increased.
* Unless treated with copper chelators such as penicillamine, the disease is always progressive and fatal and causes liver dysfunction.
The two fundamental disturbances could occur:
I) a gross decrease in the rate of incorporation of copper into apoprotein
II) a marked reduction in the biliary excretion of copper.
- Copper is deposited in the kidneys, in the liver (where it causes cirrhosis) and in brain (where it damages the basal ganglia).
- Low plasma levels of CER are also found in malnutrition, malabsorption, nephrosis and sever liver disease.
Exercise, pregnancy and by oestrogen-containing oral contraceptives increase CER concentration.
*Most plasma proteins are synthesised in the liver.
*Immunoglobulins are synthesised and secreted by plasma cells (mature B-lymphocyte, immunoglobulin producing cells),
-These cells develop numerous receptor immunoglobulins on their surface membranes.
Upon encountering antigen, these B-lymphocytes proliferate and develop into plasma cells, each of which secretes into the blood a highly specific antibody capable of binding additional antigen.
*The stimulating antigen are normally foreign but may be on host cell surfaces and cause autoimmune disease
* All Immunoglobulins share similar basic structure, consisting of two identical ‘heavy’ polypeptide chains and two identical ‘light’ chains, linked by disulphide bridges.
* There are five types of heavy chain ( Gamma, Alpha, Mu, Delta, Epsilon) and two types of light chain ( Kappa, Lambda),
* The immunoglobulin class is determined by the type of heavy chains.
* Light chains, are produced independently and in slight excess of their incorporation into immunoglobulins, their constant regions have different structures.
There are five types of heavy chain ( Gamma, Alpha, Mu, Delta, Epsilon) and two types of light chain ( Kappa, Lambda),
*The immunoglobulin class is determined by the type of heavy chains.
Excretion and detoxification function of the liver:
2. Amino acids, which are deaminated in the liver. Amino groups, and the ammonia produced by intestinal bacterial action and absorbed into the portal vein are converted to urea.
3. Cholesterol, which is excreted in the bile either unchanged or after conversion to bile acids.
4. Steroidal hormones, which are metabolised and inactivated by conjugation with glucuronate or sulphate and excreted in the urine in these water-soluble forms.
5. Many drugs.
6. Toxins, the reticuloendothelial Kupffer cells in the hepatic sinusoids are well placed to extract toxic substances which have been absorbed from the GIT.
Efficient excretion of the end-products of metabolism and of bilirubin depends on:
1. Normally functioning liver cells.
2. Normal blood flow through the liver.
3. Normal biliary ducts.
- Bilirubin is derived from the haem moiety of the haemoglobin, myglobin and cytochrome molecules
Haem & Pophyrins
Porphyrins: are cyclic compounds that bind metal ions usually Fe+2,Fe+3.
-Heme: one ferrous ion coordinated in the center of porphyrins.
-Heme is highly turned over: 6–7 gm is synthesized and destroyed daily
Structure of porphorins
-Ring structure of 4 pyrrole rings linked with methylenyl bridge.
-Side chains: different porphyrins vary of the side chain that are attached to pyrrole rings.
Reabsorbed,go to kidneys and converted excreted as urobillinogen & urobillin (yellow color)
Most are oxidizedby bacteria to stercobillin (brown color)
Excretion of Billirubin into bile
- Conjugated bilirubin is water-soluble and is secreted actively into the biliary canaliculi, eventually reaching the small intestine via the ducts of the biliary system.
Secretion into the biliary canaliculi is the rate limiting step in bilirubin metabolism
-Billirubin diglucuronide is hydrolyzed and reduced by bacteria in the gut to yield urobillinogen.
Jaundice can be classified to:
An increased rate of bilirubin production exceeds normal excretory capacity of the liver (prehepatic jaundice)(Hemolytic jaundice: sickle cell anemia, or malaria).
The normal load of bilirubin cannot be conjugated and/or excreted by damaged liver cells (hepatic jaundice). Hepatocellular Jaundice: liver damage, cirrhosis, hepatitis.
The biliary flow is obstructed, so that conjugated bilirubin cannot be excreted into the intestine and is returned into the systemic circulation (posthepatic jaundice) Obstructive jaundice: bile duct obstruction.
Jaundice in newborns
Diseases of the Liver
-Alkaline phosphatase activity is the most sensitive test for cholestasis.
*Increased synthesis of ALP in the affected ducts increases the activity of this enzyme in plasma.
* Patients with prolonged, cholestasis may present with severe jaundice and itching due to deposition of retained bile salts in the skin;
* Rarely there is bleeding due to malabsorption of vitamin K, with consequent prothrombin deficiency.
* Cholesterol retention may cause hypercholesterolaemia.
* Dark urine and pale stools suggest biliary retention of conjugated bilirubin,
* Cirrhosis can be the cause of biochemical abnormalities decreased albumin synthesis, decreased cholesterol synthesis, insulin resistance and increased prothrombin time,
Prothrombin time (PT) is a blood test that measures how long it takes blood to clot. It can be used to check for bleeding problems and for monitoring the anticoagulant activities.
Prothrombin time, clotting time acute diseases
* Most of clotting factors are synthesized in the liver (Factor VII is an important one) if there is any liver problem clotting time will be increased
Factor VII is vit K dependent so in certain cases, the high prothrombine time is not due to liver diseases but due to vit K deficiency
* Due to the great functional capacity of the liver, metabolic and clinical abnormalities may not become apparent until late in the course of the disease;
There are no reliable, simple biochemical tests to diagnose subclinical liver disease.