TYPES OF SPECIMENS
BloodBlood is a suspension of cells in a protein-salt matrix. The noncellular portion of blood contains a series of proteins, some of which are involved in the coagulation process. This fluid is called plasma. When the coagulation process is allowed to proceed to completion, the non-cellular fluid, which can be separated from the clotted material, is termed serum.
Blood used for biochemical analysis is collected from the veins, arteries or capillaries. For most testing, the site of phlebotomy has no analytical or physiological significance, so that venous blood is utilized because of ease of collection. For a limited number of analytes such as blood gases and lactate, significant difference arises between arterial and venous samples.
Most testing is performed on the liquid or serum fraction of the blood that has been allowed to clot. The assumption is made that the distribution of constituents between cellular and extracellular compartments of blood is roughly equal. For some analytes it may be necessary to prevent the blood clotting process using an anticoagulant and the separated liquid (plasma) will be then used for analysis (see anticoagulants).
Blood is usually drawn from a patient by a syringe, and transferred immediately to a clean plain tube (or a tube containing an anticoagulant) after removal of the needle to prevent breakdown of RBCs. The tube is then centrifuged after clot formation which takes about 10-15 min. (for serum) or immediately (for plasma).
Hemolysis
During sample collection and until the serum or plasma are isolated from RBCs, care must be taken to minimize the opportunities for hemolysis. Hemolysis may arise because of the use of too large or too small needle, moisture in a syringe, vigorous mixing of the blood, rapid expansion of the blood into the tube, or the separation process. Whatever the cause, hemolysis may interfere with a number of chemical procedures and should be avoided. Some constituents are present in high concentrations within the erythrocytes, and so hemolysis will falsely increase the value for those substances in serum such as potassium and enzymes (like glutamate oxaloacetate transaminase, lactate dehydrogenase and acid phosphatase).On the other hand, for those substances that exist at lower concentration in the red cells than outside, hemolysis will result in a dilution effect on the serum constituents such as sodium and chloride, so falsely low result will be obtained. In addition, hemoglobin may directly interfere in a chemical determination by inhibiting an enzyme such as lipase, by interfering with a reaction such as the reaction of diazo with bilirubin, or by yielding a significant color and thus interfering with a colorimetric analysis.
Glucose changes most rapidly of all cells constituents when serum or plasma are left in contact with RBCs. Since glycolysis is an enzyme catalysed reaction, sodium flouride has to be added to the blood sample for glucose estimation to inhibit the enzyme enolase that is involved in glycolysis.
Anticoagulants
Heparin: -mucoitin polysulphuric acid inhibits the formation of thrombin from prothrombin. It is usually available as the Na, K, NH4 and Li salts.EDTA (Ethylene diamine tetra acetate): It chelates calcium ions which are essential for clotting mechanism. Its dipotassium and dilithium salts are most often used.
Oxalate and citrate: Oxalate acts by precipitating the calcium. Potassium oxalate is the most soluble and so it is most commonly used. Sodium citrate does not precipitate calcium but converts it into a non-ionised form.
Sodium flouride: It is also considered as an anticoagulant, but since larger amounts are needed, it is rather used as a preservative for glucose determination by inhibiting red cell metabolism, glycolysis and bacterial action.
Collection of Urine Specimens
Single specimen of urine is used for general urine examination and for most qualitative tests. For quantitative work, 24 hour specimen is best employed, except when collecting specimens as part of tests such as D-xylose test.For collecting a 24h sample, the patient empties the bladder first and the urine is discarded. All specimens passed thereafter during the day and during the following night are saved and the specimen obtained by emptying the bladder at the same time the following morning is added to them. The sample is collected in a clean covered container and kept in a cool place preferably in the refrigerator.
If urine has to be kept, it may be necessary to prevent the effect of bacteria by adding a proper preservative such as hydrochloric acid, chloroform or formalin to the urine.
Other Body Fluids
The laboratory is also called upon to perform a variety of testing on cerebrospinal fluid, amniotic fluid and other body fluids. In all instances, it is essential to ensure that the proper specimen is collected and contamination is avoided. In addition, biochemical analysis may be performed on stool samples, gastric aspiration, and renal or biliary calculi. Stool samples are usually collected over 48-72 h periods. Renal calculi (stones) collection may be a random event depending on when the stone is passed.
URINE
(General Analysis)Urine is one of the biological fluids that is responsible for the removal of toxic substances from the body. The normal quantity of urine that is usually passed daily varies widely from (700-2500)ml depending on the fluid intake and weather. Normally, more urine is excreted during the day than during the night.
A pathological increase in urine output is called (polyuria) in which urinary output becomes more than 2500 ml/day which occurs in:
Diabetes mellitus.
Diabetes insipidus.
Renal failure.
A pathological decrease in the urine output is called (oliguria) in which urine output becomes less than 400 ml/day which occurs in:
Dehydration (diarrhoea, vomiting, fever, severe haemorrhage).
Sudden lowering of blood pressure (hypotension).
Severe heart failure.
Urine examination should include:
Physical Examination
1. APPEARANCEThe color of the urine should be assessed. The color of normal urine varies from clear (dilute) to yellow (concentrated). Macroscopic (gross) hematuria will make the urine appear red. Smoky red or cola-colored urine suggests glomerulonephritis. Dark yellow to orange urine is typical of bilirubinuria. Cloudy urine suggests pyuria or crystalluria (usually phosphates). Milky urine suggests chyluria (lymphatic/urinary fistula).
■ Red urine.
Dipstick positive for blood indicates heme is present
• Red blood cells (RBCs) in urine sediment—hematuria
Other colors
■ Orange—rifampin, phenazopyridine (Pyridium), carotene
■ Yellow—bilirubin
■ White—pyuria, chyluria, amorphous phosphate crystals
■ Green—methylene blue, Pseudomonas infection.
PH:
Urine PH varies with acid-base balance and can range from 5 to 8 in healthy individuals. The urine PH is primarily of interest in limited clinical situations such as metabolic acidosis and with certain types of kidney stones. Low urine ph promotes the formation of uric acid and cystine stones, whereas high urine promote calcium phosphate precipitation.
Specific gravity
Fill a suitable sized cylinder with the urine then place a hydrometer in the fluid, taking care that it floats and does not touch the sides of the cylinder. The normal specific gravity of urine varies from 1.010-1.025. Specific gravity of urine increases in case of dehydration, heavy proteinuria and glucosuria, and it decreased in case of diabetes insipidus
CONSTITUENTS OF NORMAL URINE
Among the inorganic substances present are chlorides, phosphates, and sulphates of sodium, potassium, calcium, and magnesium.
1. Uric acid
This constituent of urine is derived partly from the metabolism of food proteins and tissue proteins.
Follin test for uric acid
Take 2 ml of urine and add few drops of (Follin phosphotungstic acid reagent) and a small amount of anhydrous sodium carbonate, mix. A deep blue colour is produced.2. Creatinine
It arises almost entirely from the breakdown of muscle creatine.Jaffes test for creatinine
To 1 ml of saturated picric acid add 0.5 ml of 10% NaOH, then add 3 ml of urine, mix. A deep reddish-orange colour indicates the formation of creatinine-picrate complex.
3. Ammonium salts
Take 2 ml of urine, boil for 3 min then put a moist red litmus paper at the neck of the test tube, ammonia liberated due to the effect of boiling will change the colour of litmus paper to blue.An average sample of urine has about 4% solids dissolved in it. Of these, approximately 2% is urea, 1% is sodium chloride and all other organic and inorganic constituents make up the remaining 1%. Urea, uric acid, creatinine and ammonium salts are the principal nitrogenous substances.
The Abnormal Urinalysis
Advances in chemistry allowed significant progress in urine testing during the nineteenth century , and the modern era of reagent strip (dipstick) testing began in 1956 .
Urine dipstick testing
Urine dipstick analysis remains one of the few tests commonly performed.It is a narrow plastic strip which has several squares of different colors attached to it. Each small square represents a component of the test used to interpret urinalysis. The entire strip is dipped in the urine sample and color changes in each square are noted. The color change takes place after several seconds to a few minutes from dipping the strip. If read too early or too long after the strip is dipped, the results may not be accurate.The squares on the dipstick represent the following components in the urine:
specific gravity (concentration of urine),
acidity of the urine (pH),
protein in the urine (mainly HYPERLINK "http://www.medicinenet.com/script/main/art.asp?articlekey=2189" \t "_parent" albumin),
HYPERLINK "http://www.medicinenet.com/script/main/art.asp?articlekey=17467" \t "_parent" glucose (sugar),
ketones
blood
HYPERLINK "http://www.medicinenet.com/script/main/art.asp?articlekey=2462" \t "_parent" bilirubin and
Urobilinogen.
Pathologic and Non pathologic causes of abnormal urine dipstick finding
TestNonpathologic causespathologic causes SGLow SG:polydipsia
High SG:inadequate volume intake Low SG: DI, renal tubular dysfunction.
High SG: volume depletion, protienuria, glucose uriaPH
Low PH:high protein diet
High PH:low protein dietLow PH:acidosis High PH:renal tubular acidosisBloodmenses, exerciseGlomerular disordersProteinOrthostatic proteinuria,fever, exercise Glomerular disorders, tubular disorders, UTIGlucoseRenal Glucosuria DM ketonesRestricted carbohydrate intakeDMBilirubinNoneHepatitis, biliary obstructionUrbilinogenSystemic anitiotic theraby Hepatitis,intravascular hemolysiswhich uses a chemically treated strip to check for the following:
The specific gravity (SG):
reagent on the dipstick is sensitive to the number to the number of ions in the urine specimen and is a measure of urine concentration. Usually, it simply reflects recent fluid intake but should be interpreted with the clinical situation in mind. Low urine SG in someone who appears dehydrated can result from a renal concentrating defect.A high urine SG may reflect lack of recent fluid intake in an otherwise healthy-appearing child or dehydration in someone who has been ill.glycosuria and recent intravenous contrast can resuit in a false elevation of urine SG when calculated in the laboratory by a refractometer or urinometer but not on the urine dipstick.
❖ pH is a measure of the amount of acid in the
urine.Normal pH range is 4.5 to 8 (usually 5 to 7).
Low urine pH (<5.3)
• High protein diet (increased endogenous acid production from sulfur-containing amino acids)
Metabolic acidosis (e.g., chronic diarrhea)
High urine pH (usually >7)
Metabolic alkalosis (e.g., vomiting)
An abnormal pH may be a sign of kidney stones, urinary infections, chronic kidney disease or certain disorders that affect growth and development in children.
Glucose :
Glucose is freely filtered at the glomerulus's but is almost completely reabsorbed in the proximal tubule. typically ,glucose dose not appear in the urine until the plasma level exceeds 180 to 200 mg/dl.More specific &more sensitive qualitative test for glucose is the clinistix method .the reagent strip is dipped in the urine ,and the color of the test area is compared with the markers color chart 10 second later.
Protein :
Under normal condition up to 150mg/day in adult of protein in the urine is considered to be within normal limits. In glomerular disease, the primary protien excreted is albumin .in addition ,it is possible to have elevated albuminuria that is still below the threshold of detection by the standard urine dipstick.
Negative or trace protien on the dipstick is considered normal,
A common cause of proteinuria
A benign diagnosis (transient proteinuria)
1-asymptomatic patients is orthostatic protienuria is a benign condition in which protein excretion is normal when the patient is lying down but is elevated when the patient walks or stands erect for any period of time.
2-fever, thyroid disorder ,heart disease and exercise which should be ruled out using a first morning void specimen before pursuing further evaluation.
Persistent proteinuria:
Indicate renal disease
1- acute glomerulus's nephritis ,often associated with recent streptococcal infections, the degree of proteinuria is slight ,usually amounting to less than >2 gm/day
2- chronic glomerulus's nephritis (nephrotic syndrome), protein loss may vary from a few grams to as much as 30 gm/day.
Protein :
Suphosalicylic acid test SSA
It is a specific test for protein
5 ml of urine in the test tube then add 20% SSA drop by drop ,the presence of protein is indicated by cloudy precipitate
Keton bodies:
Keton production is increased when there is altered glucose metabolism .the increased breakdown of fatty acids generates ketones.
ketones in the urine are most commonly seen in patients whose nutritional intake has been compromised by illness or starvation,
Ketone can also be seen with uncontrolled diabetes mellitus,
high fat /low-carbohydrate diet.
liver disease,
and certain forms of glycogen storage diseases.
Ketone bodies are :
Actoacedic acid
B-hydroxy butyric acid
Acetone
Bilirubin and urobilinogen
Bilirubin is a breakdown product of hemoglobin formed in the reticuloendothelial cells. unconjugated Bilirubin such as that observed with hemolysis
is not water soluble, remains tightly bound to albumin ,and is not filtered at the glomerulus's ,after Bilirubin is conjugated to albumin glucuronide by the liver, it is water soluble and can appear in the urine. ln the normal individual ,the amount of bilirubin in the urine is below the threshold of detection of most reagent test strips.
Most conjugated bilirubin is eliminated in bile into the gastrointestinal tract .the appearance of bilirubin in the urine suggests obstruction to bile outflow or hepatitis .the presence of bilirubin in the urine can be confirmed with the diazo test method.
Urobilinogen is formed in the colon when bacterial glucuronidases hydrolyze conjugated bilirubin followed by reduction of free bilirubin.
To urobilinogen ,most urobilinogen is excreted in the feces ,but up to 20% is reabsorbed and enters the portal circulation ,the liver then re-excretes most of this urobilinogen is usually seen in the urine.
COLORIMETRY and SPECTROPHOTOMETRY
The measurement of concentration of coloured substances in solution forms the basis of colorimetric analysis, many substances of biological and medical interest are either coloured or form coloured derivatives when entered into chemical reactions.When a ray of monochromatic light of initial intensity (Io) passes through a coloured solution, some of the light is transmitted with intensity (I) and some is absorbed.
The intensity I/Io (usually expressed as percentage) is called transmittance T of the solution. As the concentration (C) of the compound is increased, the transmittance decreases inversely and logarithmically, as in figure (1):
With modern photoelectric equipments, the colorimeters and spectrophotometers, another property of the coloured solution is measured and is called absorbance A which increases as the concentration of the solution increases.
Concentration C
Fig (2)Transmittance and Absorbance are related to each other by the following relationship:
A = log10 [100/T] = 2 log10 T
Where T = percentage transmittance (%T)
A = absorbance
Beers Lambert law
Under suitable conditions, if a coloured solution is illuminated with monochromatic light, its absorbance (A) will be directly proportional to the concentration (C) of the coloured substance multiplied by the depth (I) of the solution in the light path thus:-
A C x I
Or A = K x C x I where K is constant
This relation is known as the Beer’s-Lambert law which is used to compare the concentration of unknown test solution with a standard solution measured in the same way, then:
Atest = K x Ctest x I
ASt = K x Cst x I
Or CT = x CSt
If at any step of the experiment, the test is treated not exactly the same as the standard (dilution, units etc) the equation should be multiplied by a factor considering the variation.
Wavelength and Choice of Light Colour for Colorimetry
White light (coming from the sun or tungsten lamp) is a mixture of lights of various colors and wavelengths. A red solution absorbs the green component and reflects red so it appears red to the eye. Hence, for the quantitation of a colored compound in a solution, the sample has to be illuminated by a colored light supplied by the proper wavelength to get maximum absorbance of that light.Measurement of absorbance A (which is directly proportional to concentration C) is usually achieved using colorimeter or spectrophotometer. A diagram of the basic arrangement of colorimeter is given in fig. (3).
The major components of a simple spectrophotometer consist of
Light source supplied by a lamp (tungsten, deterium, or U.V.).Monochromator or filter to provide selection of the desired light colour or wavelength for maximum absorbance.
Slit for isolation of a narrow beam and improvement of chromatic purity made of special kind of glass.
Absorption cell or (cuvette) to contain the sample. Here, light is absorbed depending on the concentration and nature of the solution. The remaining light is transmitted to the photocell.
Photocell or phototube which converts transmitted light energy to electric energy.
Meter or recorder to register the electric energy according to the concentration of the compound in the solution.
Solutions Required for Photometric Measurements
In general, it is necessary to prepare three solutions:
Test solution that is made from serum, plasma or blood or other specimen being analysed.
Standard solution that is made from a known quantity of the substance to be measured.
Blank solution containing all the reagents used except of the substance to be measured. The blank solution compensates for non-specific colour already present such as reagent colour. Its absorbance is usually deducted from that of the test and standard respectively.
It is important to avoid cloudiness, turbidity or bubbles which absorb light and introduce error. The solutions should be optically clear.
CALIBRATION CURVE
For accurate work, standard solutions should be included with each determination. Variations in chemical reactions of new batches of reagents and in instrument behavior combine to cause variability in the absorbance A of samples. It is useful to document the successive absorbance A readings of the standard for quality control purposes. Furthermore, when Beer-Lambert law is obeyed, calibration curve is prepared for the range of concentrations intended to be covered in practice. This curve is prepared by taking several dilutions of the substance to be measured, running the experiment as described, then plotting absorbance against concentration on a graph paper. When running the experiment, the concentration of the unknown is found from this curve.Preparation of calibration curve
An alternative procedure to find the concentration of test of unknown protein sample is to prepare a calibration curve, then read the concentration of the unknown.
Procedure
Three known concentrations of protein standard will be provided. Place 3 ml of each concentration in a glass tube and lable (St1, St2 & St3). Place 3 ml of distilled water in another tube and lable B. Add 5 ml of Biuret colour reagent to all tubes, mix, incubate for 30 min. at room temp. or for 10 min. at 37 C water bath. Read the absorbances of the standard and blank by the spectrophotometer at 540 nm against distilled water. Subtract B reading from each reading of the standards, then construct a calibration or standard curve with absorbances on Y-axis and concentrations in g/L ml on X-axis as in fig. (1). Find the concentration of the unknown solution T from the curve.
Concentration C (g/L)
Fig. 1
CT = x CSt
Reagents
Biurt reagent (as for total protein).Protein standards : 2.5, 5.0, 7.5 g/L. They are equivalent to: 37.5, 75.0, 102.5 g/L in serum.
Prepare stock std: 5gm bovine albumin in 100 ml H2O or (50 g/L).
Take 12.5 ml of stock and dilute to 250 ml with DW. This will give
2.5 g/L.
Take 25 ml of stock and dilute to 250 ml with DW. This will give
5.0 g/L.
Take 37.5 ml of stock and dilute to 250 ml with DW. This will
give7.5 g/L.
Unknowns: the above standards can be given as unknowns or any other dilution could be made.
HAEMOGLOBIN
Cyanmethaemoglobin (HiCN) (Drabkins method)Haemoglobin (Hb) or the red pigment of RBCs is the main constituent of erythrocytes. It is a protein that transports oxygen and about 15% of the CO2 in blood. Adult Hb molecule consists of 4 haem groups attached to 2 and 2 globin chains. The essential component of Hb is the iron atome. Each of the haem groups which has one of its coordination valency (6th coordination) is free to bind oxygen, or other ligand.
Clinical significance
Normal values for Hb are 140-200 g/L of blood in infants, 120-150 g/L in adult females and 140-165 g/L in adult males.When Hb value is below normal (less than 120 g/L in F and less than 140 g/L in M), the patient is said to be anaemic. In anaemic patients, concomitant measurements of Hb, hematocrit (PCV), erythrocyte count (and from these data, calculation of the mean corpuscular volume MCV, and mean corpuscular Hb concentration MCHC) usually provide an important clue to the likely causes of the anemia.
Hb values higher than normal are found in polycythemia, erythrocytosis and dehydration.
Principle
When whole blood sample is added to drabkins solution, Hb (haemoglobin), Hi (methaemoglobin) and HbCO (carboxy-haemoglobin) are all oxidized to methaemoglobin by potassium ferricyanide. Then the methaemoglobin is covered into the stable cyanmethaemoglobin (HiCN) by potassium cyanide. The absorbance of cyanmethaemoglobin is measured at 540 nm and compared with a certified cyanmethaemoglobin standard.
Hb
Hi HiCN
HbCO
Specimen
The procedure requires 0.02 ml or (20 l) of whole blood. Venous blood should be mixed with an anticoagulant, or blood may be taken from finger or heel puncture without use of anticoagulant. But it is preferable to use venous blood sample.Procedure
Measure 4 ml of diluent (drabkins solution) with caution into a test tube (T).Add 0.02 ml of well mixed blood to the diluent and cover.
Mix by invertion several times.
Let stand for 10 minutes at room temperature.
Read absorbance of (T) on a spectrophotometer at 540 nm against a blank (drabkins reagent).
Find the concentration of (T) in g/L from a standard curve or table relating absorbance reading to Hb concentration in g/L.
If a standard containing 0.572 g/L cyanmet Hb is used, the following procedure is followed:
Test
4 mlStd4 mlBlank
4 ml0.02 mlLet stand 10 min
Read absorbance at 540 nm
Calculation
Hb in g/L = x Cst (g/L) x Dilution factor *
= x 0.572 x 201
= x 115
V of diluent, 4 + V of blood, 0.02
* Dilution factor = = 201
V of blood, 0.02
Reagents
Diluent (Drabkins solution):
Dissolve 200 mg of potassium ferricyanide, 50 potassium cyanide (with caution) and 140 mg of potassium dihydrogen phosphate in small amount of water. Add 1 ml of triton X 100, then dilute to final volume of 1 liter. Adjust the pH of the solution to (7.0-7.4). The reagent should be clear and pale yellow in colour. Check the absorbance of the solution at 540 nm against water blank, its absorbance must be zero.
Store in brown bottle in the refrigerator (do not freeze); discard the solution if it becomes turbid, if its pH is out of the (7.0-7.4) range or it reads other than zero at 540 nm against water.
Standard solution:
Open an ampoule of HiCN (cyanmethaemoglobin) standard brought to room temperature and measure the absorbance of this solution against a reagent blank.Preparation of standard curve and standard table:
When many blood samples are to be tested, it is convenient to read the results from a standard curve or table relating absorbance reading to Hb concentration in g/L. To do this, open an ampoule of HiCN reference solution brought to room temperature, read its absorbance against a reagent blank (drabkins solution). Make the following dilutions of the standard using drabkins solution:
1 in 2, 1 in 3, 1 in 4, etc.
Translate the Hb values of solutions into terms of g/L.
Hb g/L = x CSt (gm/L) x
SERUM OR PLASMA TOTAL PROTEIN
Human plasma is 97% V/V water. Of the remaining 3% occupied by solids, over 95% is occupied by proteins. There are more than 50 groups of protein known to exist in plasma of which over 95% can be accounted for by 13 proteins. These proteins includes:
(albumin, 1-antitrypsin, 1-acid glycoprotein, – lipoprotein,
transferrin, complements, fibrinogen, IgA, IgG and IgM).
Function of plasma or serum proteins
Individual proteins have specific functions which include:-
Transport, e.g.: bilirubin, metals, hormones.
Colloid osmotic pressure, mainly albumin.
Active enzymes, e.g.: complements, clotting factor.
Enzyme inhibitors, e.g.: antiproteases.
Humoral immunity, e.g.: immunoglobulins, complements.
Endogenous source of aminoacids.
Synthesis and degradation
About (85)% of all plasma proteins are synthesised in the liver. The bulk of the remainder (particularly immunoglobulins) are synthesised by plasma cells and cells of reticuloendothelial system while the site of synthesis of most plasma proteins is known with some certainty; the site of degradation is far from clear. Most proteins are degraded by most tissues.
Important sites of degradation include the: liver, gut, muscle and kidney.
The control of protein synthesis and degradation is complex and often include factors such as dietary status, circulating plasma level (feed back), hormonal and neural.
Clinical significance
Normal range of serum total protein is 64-83 g/L (6.4-8.3 g/dl).
There are two general causes for alteration of serum total protein:
1. Change in the volume of plasma water.
2. Change in the concentration of one or more of the specific proteins.
Decrease in the volume of plasma water (haemoconcentration) as occurs in dehydration due to inadequate water intake or to excessive water loss due to vomiting, diarrhea or burn is reflected as hyperproteinemia (with the concentration of all individual proteins are increased). Haemodilution (increase in plasma water volume) is reflected as hyperproteinemia. This occurs with water intoxication and massive intravenous infusions.
Of the individual serum proteins, albumin is present in such high concentration that low level of this protein alone causes hypoproteinemia. A mild hyperproteinemia may be caused by an increase in the concentration of specific proteins as increase in acute phase proteins and polyclonal immunoglobulins as a result of infection, chronic inflammation, chronic hepatitis and liver cirrhosis.
Marked hyperproteinemia may be caused by high levels of the monoclonal immunoglobulin, produced in multiple myeloma.
Investigations of protein disorders
In general, the following tests are usually requested to characterise the pattern of abnormality. These include:-
Serum total protein.
Serum albumin.
Serum albumin: globulin ratio, where globulin = total protein albumin and normal range for A:G ratio is 1.3-1.8.
Serum protein electrophoresis where different fractions of protein are separated.
Measurement of specific protein concentrations.
Biuret method for measuring total protein
PrinciplePeptide bonds ( C N ) of proteins react with copper ions in alkaline solution to form a violet colour product whose absorbance at 540 nm is proportional to the concentration of protein.
Procedure
Set up a set of the following tubes
Test: 0.2 ml serum + 2.8 ml water.
Standard: 3 ml protein standard solution.
Blank: 3 ml distilled water.
Add 5 ml of Biuret reagent to each tube and mix.
Either leave all tubes for 30 min. at room temperature or incubate at 37 C for 10 min.
Read absorbances of solutions at 540 nm against water.
Calculation
Serum Total protein (g/L) = x protein standard conc. x dilution
factor
= x 5 x 15
= x 75
Reagents
Biuret reagent: Dissolve 9 gm of sodium potassium tartarate in 500 ml of 0.1 M sodium hydroxide. Add 3 gm of copper sulphate (CuSO4 . 5H2O) and dissolve by stirring, then add 5 gm of potassium iodide and make the volume to 1 liter with 0.1 M sodium hydroxide.Protein standard: Prepare 5 g/L of protein standard using Bovine serum albumin standard solution, keep forzen in 3 ml portions.
SERUM ALBUMIN
Albumin is the major plasma protein which is unique in being carbohydrate free. It is a single polypeptide chain containing a solitary thiol group. It is synthesised in the liver at a rate of 10-12 gm/day. Its average half life is 21 days.Functions of albumin
Maintenance of plasma oncotic pressure (colloid osmotic pressure).Transport of lipid soluble anions such as fatty acids, bilirubin, thyroxine, cortisol, aldosterone, calcium, trace elements and drugs.
Source of endogenous amino acids.
Clinical significance
The normal range of serum albumin is 35-52 gm/L (3.5-5.2 g/dl) and for albumin: globulin ratio is 1.3-1.8.The factors affecting normal albumin level are the volume of its distribution, synthetic rate and catabolic rate. In most disease processes a change in more than one of these factors is operating. Hypoalbuminaemia is a common indication of illness which may result from:
Decreased albumin synthesis; e.g.: liver diseases, malnutrition and following acute phase response.
Increased albumin loss; e.g.: nephrotic syndrome, protein lossing enteropathy and burn.
Increased catabolism; e.g.: condition associated with albumin loss, cushings syndrome, thyrotoxicosis, tumors.
Hyperalbuminaemia is not associated with any major clinical situation. Small elevation are seen in patients with severe dehydration.
Bromocresol Green (BCG) Binding Method
Principle
Albumin and BCG are allowed to bind at PH 4.2 and the absorbance of the BCG albumin complex is determined at 625 nm.
At PH 4.2, albumin acts as a cation to bind the anionic dye.
Procedure
Into a set of tubes, labelled for standard (std) and test (T) add 4.0 ml BCG reagent.
Add 20 l. of albumin standard to the (std) tube and 20 l. of serum to the (T) tube and mix. leave for 5 min.
Read absorbance of (std) and (T) at 628 nm against BCG reagent blank.
S. Albumin = x CS (50 gm/L)
Reagents
BCG: Dissolve 105 mg BCG, 8.85 g succinic acid, 100 mg sodium azide and 4 ml Brij-35, 300 g/L in 950 ml distilled water. Adjust the PH of solution to 4.15 4.25 with NaOH 6 mol/L and dilute to 1 L with water.Albumin standard: 50 g/L (5.0 g/dl).
Dissolve 5 gm bovine albumin and 50 mg sodium azide in 100 ml of water.
PROTEIN ELECTROPHORESIS
One of the simple techniques for quantitation of serum proteins is their separation in an electric field, and this procedure is referred to as serum protein electrophoresis (SPE). When an electric field is applied to a medium containing charged particles, the negatively charged particles or molecules migrate toward the positive electrode (anode), while the positively charged particles migrate toward the negative electrode (cathode). The rate of migration of proteins depends on several factors, e.g.: viscosity of the medium, net charge on the protein as well as the PH and ionic strength of the medium.Proteins are amphoteric molecules; that is, they are either uncharged or negatively or positively charged, depending on the PH of the buffer. In an alkaline medium they are negatively charged and migrate in an electric field towards the anode at a speed which depends on the number of charges they carry. The difference in the migration velocities make it possible to separate the serum protein mixture into groups.
Using supporting media such as paper, cellulose acetate or agarose gel: five serum protein bands are observed (albumin, 1, 2, and ( globulins) and plasma will give an additional fibrinogen band between and ( globulins. Albumin has the highest and (-globulin the lowest migration.
The separated fractions are stained with amido black, ponceau S or other stains. After visual inspection of the separated bands, the electrophoresis strips can be cleared to allow semiquantitative evaluation of the bands by densitometry (scanning). The patients serum should always be run in parallel with a normal control serum; the two patterns are then compared by visual inspection and the following changes noted.
Intensely stained band occurring from the to ( regions suggests monoclonal immunoglobulin (paraprotein or M band) as in multiple myeloma.
Decreased albumin and ( - bands together with an increased 2 – band suggest a protein lossing state such as nephrotic syndrome.
An increase in the 1 – band (1 antitrypsin and 1 acid glycoprotein) and 2 – band (haptoglobin) suggests an acute phase reaction.
Fusion of and ( bands suggests an increase in IgA such as occurs in liver cirrhosis, chronic respiratory or skin infection and rheumatoid arthritis.
An increase in the ( - band suggests a polyclonal ( - globulin increase associated with an immune reaction, chronic inflammatory disease, chronic hepatitis or dissiminated neoplasms.
An absent or decrease of the ( - band suggests immune deficiency, either congenital or acquired.
Principle
Proteins are separated by electrophoresis on cellulose acetate membrane at 180 volts for 15 minutes. After electrophoresis, the proteins are stained with Ponceau S, the background cleared and the electrophoresis dried and interpreted.
Method
Cut off the top left hand corner of the strip for future identification of sample positions. Place the plate (s) in the rack provided.Slowly lower the rack into the buffer for about 20 minutes.
Pour 50 ml of working buffer into each of the outer compartments of the Helena electrophoresis chamber. Wet 2 disposable paper wicks in the buffer and drop them over the support bridge ensuring good contact.
Using the microdispenser, fill each well in the sample plate with 3 L of serum. Place a glass slide over the wells if you are not going to use them within 5 minutes.
Load the applicator by depressing the tips into the sample wells. Once the applicator is loaded it must be used within 15 seconds.
Remove the wetted cellulose acetate strip from the buffer with finger tips and blot firmly once. Quickly place the strip in the aligning base, with the cut corner at the top left hand side.
Align the strip with the line marked center application.
Depress the applicator tip once more into the sample wells to ensure maximum pickup. Now promptly transfer the applicator to the aligning base. Press the buttom quickly and hold down for 5 seconds. Quickly place the strip, (cellulose acetate side DOWN), in the electrophoresis chamber.
Place a weight, (2x10p pieces), on the strip. Cover, wait 30 seconds, turn the timer to 15 minutes and the voltage to 180 volts.
After the 15 minutes electrophoresis is up, place the strip in the Ponceau S stain for 6 minutes.
Put into 3 successive washes of 5% acetic acid for 2 minutes each or until the background is white. At this stage the strip can be dried and stored or cleared as follows:
Dehydrate the acetate by transferring to 2 absolute methanol washes for 2 minutes each.
Place the strip in the clearing solution for 7 minutes.
Drain for 1 minute and dry in forced air. Place the acetate side up on a blotter and dry under the microevaporation hood for 3-4 minutes until dry. Store in the plastic envelopes provided.
Reagents
Ponceau S: 1 bottle contains 250 ml of 0.5% Ponceau S in an aqueous solution of 3.5% trichloroacetic acid. This solution is ready for use.Electra HR buffer: This is a tris-barbital-sodium barbital buffer, PH 8.8 with an ionic strength of 0.067.
Clear aid: This consists of polyethylene glycol.
5% acetic acid: Add 50 ml of glacial acetic acid to 950 ml of distilled water.
Absolute methanol reagent grade.
Clearing solution.
Cellulose acetate plates.
Fig (2).
Densitimetric scanning pattern for serum protein electrophoresis
Pattern 1:
Normal serum.
Pattern 2:
Chronic infection (chronic hepatitis): a relative decrease in albumin with a notable elevation in ( globulin.
Pattern 3:
Acute phase reaction: pattern associated with a myocardial infarction with relative increased levels of 1 and 2-globulin.
Pattern 4:
Hypogammaglobulinemia: the considerable drop in (-globulin is readily observed.
Pattern 5:
Nephrotic syndrome: the very low level of albumin is striking, as is the extremely elevated peak for 2-globulin, the moderate rise in -globulin and the fall in (-globulin.
Pattern 6:
Liver Cirrhosis: the elevation in (-globulin, with a partial fusion with band, is present, along with a decrease in albumin.
Pattern 7:
Multiple myeloma: the clone of myeloma protein (paraprotein band) is associated entirely with the -globulin.
Pattern 8:
Multiple myeloma: the myeloma protein (paraprotein band) is entirely in the (-globulin area. The fall in albumin is also apparent.
It is interesting to compare this (-globulin region with pattern 2, 4, 6, and 7. Note the homogeneous peak in pattern 7 and 8 and the heterogenous peak in pattern 2.
CEREBROSPINAL FLUID (CSF)
The surface of the central nervous system is covered by the meninges, three layers referred as the pia, arachnoid and dura mater. The last is the outermost and the CSF is found between the pia and arachnoid. It is formed by secretion from the cells of choroid plexuses, vascular structure lying within the ventricles of the brain and reabsorbed into the blood stream by the arachnoid villi. The total volume of CSF is about 130 ml of which 25 ml is in the ventricles.
Appearance
Normal CSF is clear and colourless and should be compared with water.Colour
Normal CSF is colourless, the presence of blood is the main cause of an abnormal colour. Normally no red blood cells should be present. Some may be introduced as a result of trauma while obtaining the fluid. The question may arise as whether or not the blood in the CSF is due to spinal puncture, this can be determined by collecting CSF in 3 marked test tubes (about 3 ml in each), if blood is due to trauma of spinal puncture; the fluid becomes clearer and less bloody from first to the third tube. If blood is due to subarachnoid hemorrhage, it is mixed with CSF in the 3 test tubes and all the tubes contain an equal amount of blood and show no colour changes.Turbidity
Turbidity is seen when there is an excess of white cells (pus) and so is found in meningitis. Slight turbidity will occur after hemorrhage.Spontaneous clotting
This occurs when there is an excess of fibrinogen in the specimen usually associated with high total protein content.Cell count
In normal CSF, the total cell count is 0-4x106/L present as mononuclear or lymphocyte.Total protein
Normal lumber CSF has a total protein content 0.15-0.45 gm/L.An increase in the total protein is the commonest finding in all types of meningitis especially pyogenic and tuberculous meningitis, this is because of the increase in the cell content and as a consequence of the inflammatory reaction.In blockage of the spinal canal when stasis results in fluid reabsorption, protein concentration is very high.
Procedure
The most common method used is the turbidimetric method using trichloroacetic acid (TCA).
Test (T): mix 1 ml of spinal fluid with 4 ml of 3% TCA.
Standard (St): mix 1 ml of working standard with 4 ml of 3% TCA.
Blank (B): mix 1 ml of distilled water with 4 ml of 3% TCA.
After (10) minutes read the absorbance of T, St and B solutions at
450 nm. Since the test and the standard are treated in the same
manner, then:
CSF protein = X
(gm/L)
Determination of glucose
This test may be carried out by any method used for estimation of glucose in the blood. Glucose concentration in CSF is normally lower than that of the blood (approximately 60-70% that of blood).
Normal CSF glucose is between (2.2-4.2 mmol/L) (40-75) mg/dl. The most important pathological change is the decrease in glucose level in CSF in case of infection as in pyogenic and tuberculous meningitis. In viral meningitis, the glucose level is often normal. In hyperglycemia CSF glucose levels will be high but the CSF figure remains lower than that of the blood.
Reagents
Trichloroacetic acid (TCA), 3%: Dissolve 30 gm of TCA in water to 1 liter volume.Stock protein standard: Dissolve 1 gm of bovine albumin in 1 liter water.
Working protein standard (0.5) gm/L: Dilute the stock protein solution 1:2 dilution with water.
URINARY PROTEIN
Quantitative tests for urinary protein are generally carried out on 24-hour urine collection. Normally small amount of protein is excreted in the urine ranging between (0.1-0.2) gm/24 hour.Proteinuria is said to be present whenever the urinary protein output is greater than that reflected in these normal values, however, not all proteinuria is clinically significant, but persistent abnormal levels of protein in the urine are an indicator of kidney or urinary tract disease.
Proteinuria which is mainly glomerular in origin is often a manifestation of primary renal disease, although transient proteinuria may occur with fever, thyroid disorders, and in heart disease, in the absence of renal disease. Proteinuria may be evident very early in the course of various renal disease states. With such conditions as pyelonephritis and acute glomerulonephritis, often associated with recent streptococcal infections, the degree of proteinuria is slight, usually amounting to less than 2 gm per day. In chronic glomerulonephritis, in the nephrotic syndrome and in some forms of hypertensive vascular diseases, protein loss may vary from a few grams to as much as 30 gm/day.
In healthy individuals, transitory elevation in urine protein output are encountered after intense exercise or work, and after exposure to cold. Orthostatic proteinuria is a benign condition in which protein excretion is normal when the patient is lying down but is elevated when the patient walks or stands erect for any period of time.
Procedure
Test: Mix 1 ml of urine with 4 ml of 3% trichloroacetic acid.St: Mix 1 ml of standard protein solution 0.5 g/L with 4 ml of 3%
trichloroacetic acid.
Blank: Mix 1 ml of distilled water with 4 ml of 3% TCA.
After (10) minutes (mix just before reading) read the absorbance of turbid solution at 450 nm.
Urinary protein = x 0.5
(gm/L)
The amount of urinary protein is then corrected according to the amount of 24 hour urine volume.
Urinary protein = x 0.5 x
Normal range (0.05 0.20 gm/24 hour).
Reagents
Trichoroacetic acid (TCA), 3%: Dissolve 30 gm of TCA in water to 1 liter volume.
Stock protein standard: Dissolve 1 gm of bovine albumin in 1 liter water.
Working protein standard: (0.5) gm/L: Dilute the stock protein solution 1:2 dilution with water.
AMYLASE
The normal level of serum amylase activity is up to 200 somogi units/dl. The clinical significance of this enzyme is almost entirely in the diagnosis of acute pancreatitis in which serum enzyme activity frequently exceeds 1000 units/dl. However levels exceeding 500 units/dl strongly support the clinical diagnosis of pancreatitis. In acute pancreatitis, the rise in activity is rapid and transient, reaching a peak in the first 6 18 hours, returning to normal level usually in 1 2 days after onset of the attack. Other conditions which may lead to high serum amylase activity include perforated peptic ulcer, cholecystitis, calculus obstructing pancreatic duct, postgastrectomy, intestinal obstruction, diabetic coma and after using certain drugs which cause spasm of the sphincter of oddi e.g. morphine. Chronic pancreatitis and carcinoma of the pancreas are rarely associated with a raised serum amylase.Serum amylase activity may also be increased in salivary glands diseases such as mumps, parotitis and calculus obstructing the salivary duct.
Somogi unit: It is the amount of amylase that digests 5 mg of starch under optimum conditions.
Principle
Serum is added to standard solution of starch (substrate) for certain period of time. Some of the starch will be hydrolysed. The remaining starch is treated with iodine solution to form a blue colour. By measuring the intensity of the colour spectrophotometrically and comparing it with a control, the quantity of unhydrolysed starch can be determined and so the activity of serum amylase can be calculated.Procedure
Serum amylase has an optimum PH of 6.5 7. It is activated by chloride ions, and so in the assay, dilution of serum has to be made in normal saline. Use cotton plugs in the mouth piece of all pipettes to avoid traces of saliva causing falsely high amylase value.Test : Dilute serum 1 to 10 with 0.9% normal saline. Pipette 1 ml of buffered starch substrate into a test tube and place in a water bath at 37C for 3 minutes. Add 0.1 ml of diluted serum, mix gently and incubate for exactly 15 minutes. Remove the tube from the bath, add 0.4 ml of working iodine solution, mix well and then add 8.5 ml of water and mix again.
Control : Mix 1 ml of buffered substrate, 0.4 ml of iodine and 8.6 ml of water. Measure immediately the absorbance of the blue colour spectrophometrically.
Calculation :
The control tube contains 0.4 mg of starch.The amount of starch which has been digested is therefore.
= x 0.4 mg
= x somogi units
Serum amylase activity in somogi units/dl
= x x
= x 800
If the test reads less than half the control, the work is repeated using more diluted serum.
Reagents
1- Buffered starch substrate: (PH7)
Dissolve 13.3 g of dry anhydrous disodium hydrogen phosphate (or 33.5 g Na2HPO4.12H2O) and 4.3 g of benzoic acid in 250 ml water, boil. Mix 20 g of soluble starch in 10 ml cold water in a beaker and add it all to the boiling mixture, rinsing out the breaker with additional cold water. Continue boiling for one minute then cool to room temperature and dilute to 500 ml. Keep the solution at 4C, prepare freshly each month.
2- Normal saline solution:
Dissolve 9 g of NaCl in 1 L of water.
3- Stock iodine solution (0.1 N):
Dissolve 13.5 g of pure iodine in a solution of 24 g of potassium iodide in about 100 ml of water and make to 1 L with water.
4- Working iodine solution (0.01 N):
Dissolve 50 g of potassium fluoride in little water, add 100 ml of stock iodine solution and make to 1 L with water. Store at 4C in brown bottle.
معع
مع
معURIC ACID
Uric acid is the end product of purine metabolism. It is a waste product derived from purines of the diet and those synthesized in the body. In human, uric acid arises from ingested nucleoproteins, degradation of nucleoproteins, in nuclear material and by synthesis from simple precursors.Healthy adult human body contains about 1.1 g of uric acid. Normally about one half of uric acid is eliminated and replaced each day, partly by urinary excretion and partly through destruction in the intestinal tract by microorganisms. Serum uric acid is freely filtered by the glomeruli and (98-100%) of it is subsequently reabsorbed in the proximal tubules which is then followed by further secretion in the tubules.
Uric acid concentration in serum is greatly affected by extrarenal as well as renal factors. Its concentration depends upon the net balance achieved between the rate of synthesis of purines or breakdown of nucleoproteins on one hand and the rate of elimination of uric acid on the other.
Clinical significance
Normal levels: Males 0.21 0.42 mmol/L (3.5 7.0 mg/dl)Females 0.15 0.36 mmol/L (2.6 6.0 mg/dl)
In the presence of normally functioning kidney, the ingestion of nucleoproteins has little influence on serum uric acid unless very large amounts are taken.
Hyperuricaemia is most commonly defined by serum uric acid concentration over 0.42 mmol/l (7.0 mg/dl) in men, or over 0.35 mmol/l (6.0 mg/dl) in women.
The major causes of hyperuricaemia are:-
Essential hyperuricaemia (gout).Impaired renal excretion: Renal failure, drug therapy e.g. diuretics, alcohol, ketoacidosis.
Increased nucleic acids turnover: Myeloproliferative disorders e.g. leukaemia, polycythemia.
Specific enzyme defect: Deficiency of HGPRT enzyme in Lesch-Nyhan syndrome.
Principle
Serum proteins are first precipitated using sulfuric acid (0.66 N) and sodium tungstate (10%). Following centrifugation, uric acid in the protein-free filtrate is oxidized to allantoin and carbon dioxide by phosphotungstic acid reagent in alkaline solution supplied by (14%) sodium carbonate. Phosphotungstic acid is reduced in this reaction to tungsten blue which is measured colorimetrically.
Uric acid + Phosphotungstic acid Allantoin + CO2 + Tungsten
Blue
Procedure
In a clean centrifuge tube, place 1 ml of serum. Add 8 ml of water, 0.5 ml of 0.66 N H2SO4 and 0.5 ml of 10% sodium tungstate. Mix after each addition, then centrifuge for 10 minutes or filter.Prepare 3 clean glass test tubes as follows:
Test: 3 ml filterate
Standard: 3 ml of 0.059 mmol/L uric acid standard
Blank: 3 ml of distilled water
To all tubes, add 1 ml of 14% sodium carbonate followed by 1 ml of phosphotungstic acid, mixing after each addition. Allow tubes to stand for 15 minutes then read absorbances at 710 nm.
Calculation
Serum uric acid = x Std concentration x dilution factor
= x 0.059 x 10
Serum dilution factor: 1 + 8 + 0.5 + 0.5 =
Reagents
10% sodium tungstate:Dissolve 100 gm of anhydrous salt in water then complete volume to 1 liter.
0.66 N H2SO4:
Dilute 18.6 ml of concentrated H2SO4 to 1 liter.
14% sodium carbonate:
Dissolve 140 gm of anhydrous salt in water then complete volume to 1 liter.
Stock uric acid standard: 5.9 mmol/L or (1 mg/ml)
Weight out 100 mg of uric acid and 60 mg of lithium carbonate (LiCO3) into 100 ml volumetric flask. Add about 50 ml of water, warm to about 60C, cool then complete the volume to 100 ml with water.
Working uric acid standard: 0.059 mmol/L or (1 mg/dl)
Measure 10 ml of stock uric acid standard and complete volume to 1 liter.
Phosphotungstic acid:
To prepare the stock solution: dissolve 50 gm sodium tungstate (Na2WO4. 2H2O. molybdate free) in about 400 ml water. Add 40 ml of orthophosphoric acid (sp.gr, 1.75) and reflux gently for 2 hrs. Cool, transfer to a 500 ml flask and make to mark with water. Keep in brown bottle in the refrigerator. For use, dilute 1 to 10 and keep in brown bottle.
مع تحيات
مكتب زيـــادللاستنساخ والطباعة الليزرية
موصل مقــــابل كلية طب الموصلBLOOD OR PLASMA GLUCOSE
Many analytical procedures have developed to measure blood glucose. Procedures in common use include enzymatic (glucose oxidase), colourimetric (O – toluidine) or oxidation reaction (copper sulphate). Furthermore, whole blood was the sample of choice for analysis. However, values for glucose in whole blood are less than in plasma since red blood cells contain only about 80% water, compared with 93% in plasma, this despite the identical concentration of glucose in the water phase of both cells and plasma. Therefore, glucose concentration in plasma is about 12% higher than in whole blood (depending on the haematocrit).
Collection and handling of specimens
When venous blood is drawn and permitted to clot, the average rate of decrease in serum glucose is approximately 7% in each hour (0.28 0.56 mmol/L) or 5-10 mg/dl. This decrease is the result of glycolysis. Serum in contact with RBCs without preservative must be separated from the cells or clot as soon as possible, if glucose values within 0.5 mmol/L (10 mg/dl) of the original values are to be obtained. However, it is preferred to prevent reduction in blood glucose as a result of glycolysis by collecting blood into sodium fluoride containing tubes. Fluoride ions prevent glycolysis by inhibiting enolase enzyme. Oxalate, on the other hand, inhibits coagulation by binding calcium.
Cerebrospinal fluid (CSF) is frequently contaminated with bacteria or cellular constituents and should be analysed for glucose without delay or, otherwise, the sample should be preserved with sodium fluoride.
Clinical significance
Normal range (using enzymatic method)Plasma glucose 3.0 6.1 mmol/L (55-110 mg/dl)
Blood glucose 2.8 5.6 mmol/L (50-100 mg/dl)
CSF glucose 2.2 4.2 mmol/L (40-75 mg/dl)
or 60% of plasma value
Hyperglycaemia (raised blood or plasma glucose) is the hallmark of diabetes mellitus. The common underlying defect is a deficiency of insulin action which may be absolute (as in type 1 diabetes) or relative with resistance to insulin action (as in type 2 diabetes). The WHO Expert Committee on Diabetes Mellitus has classified diabetes and other categories of glucose intolerance in 1980-1985 as follows:-
A- Clinical Classes
Diabetes mellitus
Insulin dependent (IDDM) or type 1.
Non insulin dependent (NIDDM) or type 2.
Other types associated with certain diseases e.g. pancreatic disease, endocrine disease, drug induced, insulin receptor abnormalities, malnutrition related and others.
Impaired glucose tolerance
Gestational diabetes. (DM developing during pregnancy).
B- Statistical risk classes (subjects with normal glucose tolerance but with substantially increased risk of developing diabetes).
Previous abnormality of glucose intolerance.
Potential abnormality of glucose intolerance.
The WHO Expert Committee on Diabetes Mellitus had recommended that a fasting plasma glucose (FPG) of ≥ 7.8 mmol/L ( ≥ 140 mg/dl), or a random plasma glucose ≥ 11.1 mmol/L ( ≥ 200 mg/dl) is diagnostic of diabetes. If the results are equivocal i.e. higher than normal but lower than diabetic level, then an oral glucose tolerance test (OGTT) is necessary.
This test is performed by giving the patient 75 g oral glucose dissolved in 250 300 ml water. Blood samples are taken before (fasting) and 2 hr. following the load for the measurement of glucose. A 2 hr. post glucose plasma glucose (2 hPG) level below 7.8 mmol/L ( < 140 mg/dl) is considered as normal, higher than 11.1 mmol/L ( ≥ 200 mg/dl) is diagnostic of diabetes, while 2hPG between 7.8 – 11.1 mmol/L (140 – 199 mg/dl) is indicative of impaired glucose tolerance (IGT).
In 1995, the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus sponsored by the American Diabetes Association (ADA) approved new diagnostic criteria for DM, where FPG value has been lowered to best predict the risk of developing diabetic microvascular complications. The revised criteria include a FPG ≥ 7.0 mmol/L (126 mg/dl), a random or 2h PG (during a standard 75 gm OGTT) ≥ 11.1 mmol/L (200 mg/dl). Random plasma glucose concentration ≥ 11.1 mmol/L.
This change in the diagnostic cut off point for FPG from 7.8 to 7 mmol/L (140 to 126 mg/dl) introduces a new intermediate category; impaired FPG (IFG). This is defined as a FPG concentration of 6.1 6.9 mmol/L (110 125 mg/dl) that refers to a metabolic stage intermediate between normal glucose homeostasis and diabetes. Both IFG and IGT are considered as risk factors for future diabetes and cardiovascular diseases.
The proposed ADA classification of DM includes both clinical and aetiological types of diabetes as well as other types of hyperglycaemia. The terms IDDM and NIDDM are deleted and replaced by the terms type 1 and type 2 respectively.
Clinical staging
Regardless of the underlying cause, DM is subdivided into:Non-insulin requiring.
Insulin requiring for control.
Insulin requiring for survival.
Aetiological classification
Type 1 DM, which is subdivided into:Autoimmune form; which includes both rapidly progressive and slowly progressive forms.
Idiopathic form.
Type 2 DM.
Genetic defects of -cell function.
Diseases of exocrine pancreas.
Hypoglycaemia
On the other hand, hypoglycaemia is defined as plasma glucose less than 2.5 mmol/L (45 mg/dl). It is less common than hyperglycaemia, and it is eventuated in practice in cases including insulinoma, pancreatic tumors, hepatoma, adrenal carcinoma, hypopitutarism, addisons disease, inborn error of metabolism of glycogen storage disease, galactossemia, fructose intolerance and also in essential reactive hypoglycaemia.Enzymatic Method for Measurement of Glucose
PrincipleAldehyde group of glucose molecule is oxidized in the presence of glucose oxidase (GOD) to gluconate with the liberation of hydrogen peroxide, which in turn reacts with phenol and 4-aminophenazone under catalysis of peroxidase enzyme (POD) to form a pink quinoneimine dye as indicator.
Glucose + O2 + H2O Gluconate + H2O2
2H2O2 + phenol + 4-amino-antipyrine Quinoneimine + 4H2O (pink dye)
Procedure (Kit method)Pipette into test tubes
Working reagent (ml) 1 1 1
Standard/R4 (ml) 0.01Sample (ml) 0.01
Mix, incubate at 37C for 10-15 minutes or at room temperature (25C) for 30 minutes. Read absorbance of sample against reagent blank within 60 minutes at 500 nm.
Calculation
Glucose concentration = x 5.55 mmol/L (100 mg/dl)Reagents
Kit enzymatic methodContents:
Reagent/R1: (buffer reagent)
Phosphate buffer, pH 7.5 150 mmol/L
Phenol 7.5 mmol/L
Reagent/R2: (enzyme reagent)
GOD 12000 U/L
POD 660 U/L
3-aminoantipyrine 0.40 mmol/L
4- Reagent/R4: (standard)
Glucose 5.55 mmol/L (100 mg /dl)
Working reagent is prepared by mixing contents of enzyme reagent (R2) with corresponding volume of buffer/R1.
TOTAL CHOLESTEROL
It is generally agreed that the normal range for total cholesterol is rather wide. Serum total cholesterol is slightly higher in men than in women. It is a little lower in persons under 20 years but rises with age. It does not appear to be much altered following meals (unlike triglyceride). In pregnancy there is an increase which may reach 20% above normal at the 30th week.
Recently, for the lipid pattern to be clear, the lipid profile is used as an index of its possible effect in precipitating atherosclerosis. The following table is usually used for its interpretation:
Serum total cholesterol
Desirable < 5 mmol/l (200 mg/dl)
Borderline 5-6 mmol/l (200-240 mg/dl)
High > 6 mmol/l (240 mg/dl)
HDL cholesterol Favourable > 1.4 mmol/l ( > 55 mg/dl)
High risk < 0.9 mmol/l ( < 35 mg/dl)
LDL cholesterol Low risk < 3.4 mmol/l ( < 130 mg/dl)
Moderate risk 3.4-4.1 mmol/l (130-160 mg/dl)
High risk > 4.1 mmol/l (> 160 mg/dl)
VLDL cholesterol Favourable 0-1 mmol/l (0-40 mg/dl)
Total/HDL (cholesterol) Low risk ( < 5)
(Atherogenic index) Moderate risk (5-6)
High risk ( > 6)
Hypercholesterolaemia
Increases are found most characteristically in the primary hypercholesterolaemia (particularly types II, III and IV), nephrotic syndrome, hypothyroidism, obstructive jaundice, primary biliary cirrhosis and diabetes mellitus.
Xanthomatosis is frequently associated with an increase in serum cholesterol. Primary xanthomatosis is divided into two groups, in one of which there is raised serum cholesterol, whereas in the other it is within normal limits, the deposits being composed of other lipids. In secondary xanthomatosis the condition accompanies hypercholesterolaemia arising from one of the conditions listed above.
Raised serum cholesterol signifies hypercholesterolaemia which is considered to be a cardiovascular risk factor that may predispose to coronary thrombosis appearing as angina pectoris or myocardial infarction.
Hypocholesterolaemia
Decreases are not so well defined. Conditions that may create hypercholesterolaemia include hyperthyroidism, malabsorption syndrome malnutrition and pernicious anaemia. Very low values occur in abetalipoproteinaemia. Therapeutic reduction of serum cholesterol is seen during administration of lipid lowering drugs such as clofibrate, cholestyramine and nicotinic acid.
Ratio of free to esterified cholesterol
Free cholesterol normally forms about 30% of the total (range 20 40%). While changes in total cholesterol do not involve any change in this in diabetes, nephrotic syndrome and hypothyroidism The percentage of free cholesterol rises in liver diseases and primary biliary cirrhosis.Measurement of Serum Cholesterol
PrincipleExtraction and oxidation of cholesterol by an acidic solution of ferric chloride and subsequent addition of sulfuric acid to form coloured complex is the basis for total cholesterol estimation.
The direct procedure without extraction does not give good reproducibility owing to the presence of chromogens in serum which may react with sulfuric acid and interfere with the reading of cholesterol complex. The extraction step has increased the accuracy and reproducibility of the method.
Procedure
In a centrifuge tube accurately measure 1.9 ml of ethanol then add 0.1 ml of serum or plasma and shake vigorously for 1 minute. This mixture is centrifuged at 3000 rpm for 3 minutes.
Lable 3 test tubes as test, standard, and blank and make the following additions.
Test tube T: Transfer 0.5 ml of the supernatant fluid from the centrifuge tube.
Test tube St: Add 0.5 ml of cholesterol standard solution (0.259 mmol/L cholesterol).
Test tube B: Add 0.5 ml of ethanol.
To all test tubes (T, St and B) add 2 ml of acidic solution of ferric chloride, mix and carefully add 2 ml of concentrated sulfuric acid to all the 3 tubes with gentle mixing. The tubes are left to cool for at least 10 minutes, transferred to glass cuvettes and the absorbance is measured at 560 nm using spectrophotometer.
Calculation
Serum or plasma total cholesterol (mmol/L).= x conc. of St x
= x x
= x 5.18
Reagents
Absolute alcohol (ethanol).Concentrated sulfuric acid.
Stock cholesterol standard: 200 mg/100ml (5.18 mmol/L) cholesterol dissolved in ethanol (keep in the refrigerator).
Working cholesterol standard: 10 mg/100 ml (0.259 mmol/L) (0.1 mg/ml) prepared by diluting 1 ml of stock standard to 20 ml with ethanol (keep in the refrigerator).
Colour reagent: 100 mg of ferric chloride (FeCL3.6H2O) dissolved in 100 ml of ethyl acetate. Store in a brown bottle at room temperature.
BILIRUBIN
Red cells are broken down at the end of their life in the reticulo-endothelial system mainly in the spleen. The released Hb is split into globin, which enters the general protein pool, and haem, which is converted to bilirubin after removal of iron. The iron is reutilised, 80% of bilirubin is metabolised daily by this process.Hb Biliverdin Bilirubin
Other sources of bilirubin are from the breakdown of immature red cells in the bone marrow and of Hb related compounds such as myoglobin and cytochromes.
Unconjugated bilirubin (free or indirect, that is bilirubin which has not yet been made water-soluble by conjugation with glucoronate) is carried to the liver bound to plasma albumin. At the hepatic cell membrane, bilirubin is removed from the albumin, then conjugated with glucoronic acid forming bilirubin diglucoronide. The reaction is catalysed by the enzyme uridyl diphosphate glucoronyl transferase (UDP). The conjugated or direct bilirubin is water soluble. Normally, about 300 mg of bilirubin reaches the liver and is conjugated daily. The unconjugated (indirect or free) bilirubin is water insoluble and lipid-soluble and can enter and damage brain cells.
Conjugated bilirubin is secreted into bile canaliculus and further to gall bladder for storage, concentration and excretion into small intestine. In the intestine, bacteria deconjugate the bilirubin glucuronate and reduce bilirubin to urobilinogen. Most of this urobilinogen is excreted in the faeces as such or as urobilin after oxidation in air. A small portion is reabsorbed through the enterohepatic circulation to be excreted in the urine. Therefore, normal urine contains urobilinogen but not bilirubin. However, the disappearance of urobilinogen and appearance of bilirubin (direct) in the urine is an abnormal condition that indicate the presence of obstructive jaundice.
Normally, healthy hepatic cells are capable of handling the conjugation and excretion of bilirubin. Jaundice is due to an increase in the concentration of bilirubin in blood and is, a common sign in hepatic or biliary tract disorders.
The abnormal metabolism or retention of bilirubin which results in jaundice can be classified as follows:-
Types of jaundice
A- Pre-hepatic jaundice (hemolytic) ( unconjugated).Acute and chronic hemolytic anemia.
Neonatal physiological jaundice.
B- Hepatic jaundice (mainly unconjugated).
Conjugation failure.
Transport disturbances.
Diffuse hepatocellular damage or necrosis, viral and toxic hepatitis and cirrhosis.
Intrahepatic obstruction (e.g. edema) ( conjugated and unconjugated).
C- Post-hepatic jaundice (obstruction of common bile duct due to stones, tumor or spasm and stricture (mainly conjugated).
Principle of serum bilirubin measurement
Serum bilirubin is present in two forms, conjugated (direct or water soluble) and unconjugated (indirect, free or water-insoluble).This method is a modification of Evelyn-Malloy and is based on the reaction of bilirubin with diazotized sulphanilic acid which produces a purple azobilirubin dye in an acid media. This dye is measured spectrophotometrically and compared with bilirubin standard.
Conjugated bilirubin reacts in aqueous solution directly and hence is called direct, whereas unconjugated bilirubin requires an accelerator or solubilizer to react such as methanol and is therefore termed indirect. The sum of both is referred to as total bilirubin. In the presence of accelerator both direct and indirect react and total bilirubin is therefore measured. In presence of water only direct bilirubin reacts. Subtraction of direct from total will give the indirect bilirubin concentration.
Normal values
Total bilirubin up to 17 mol/L (1 mg/dl)
Direct bilirubinup to 5 mol/L (0.3 mg/dl)
To convent mg/dl to mol/L each 1 mg/dl of bilirubin=17 mol/L
Procedure
Total test
Direct test
Control
Standard
Standard control
Water (ml)
1.81.8
1.8
1.8
1.8
Serum (ml)
0.20.2
0.2
Standard (ml)0.20.2Diazo R (ml)0.50.50.5Diazo B (ml)0.50.5Methanol (ml)2.52.52.5Water (ml)2.52.5Mix all tubes and allow to stand in the dark for 30 min. read the absorbance at 540 nm.
Calculation
Total bilirubin = x 171
Direct (conjugated) bilirubin = x 171
Indirect (unconjugated) bilirubin = Total conjugated
ReagentsMethyl alcohol, absolute.
Diazo blank solution. Dilute 15 ml of conc. HCL to 1 liter with water.
Diazo reagent. Make up fresh as needed from two stock solutions which are prepared as follows.
Solution A
To 985 ml of water add 15 ml of Conc. HCL. Add 1 gm of sulfanilic acid and stir until dissolved.
Solution B
Dissolve 0.5 g of Na-Nitrite and dilute to 100 ml with water. This solution remains stable for several months if kept refrigerated.
Diazo reagent
A fresh solution is prepared before each set of determinations as follows:
Add 0.3 ml of solution B to 10 ml of solution A and mix.
Total and Direct Bilirubin
(Kit method)Principle
Sulfanilic acid reacts with sodium nitrite to form diazotized sulfanilic acid. In the presence of dimethyl sulfoxide, total bilirubin reacts with diazotized sulfanilic acid to form azobilirubin. In the absence of dimethylsulfoxide, only direct bilirubin reacts with diazotized sulfanilic acid to form azobilirubin.
Total bilirubin
Working solution mix 20 R1 vol. with 1 R3 vol.
Wavelength = 555 nm.
StandardTestBlank Reac Blank Reac Standard R450 l
50 l
Sample
50 l
50 lR1
1 ml
1 ml
Working solution1 ml
1 mlMeasurement: against blank.
Mix well and incubate exactly 5 minutes at 37C.Read the absorbance (A) of standard and test against their blanks.
Calculation
[Total bilirubin] = x St. conc.
Direct bilirubin
Working solution: mix 20 vol. R2 with 1 vol. R3.Wave length: 555 nm.
Measurement: against blank.
Standard
TestBlank
ReacBlank
Reac
Standard
50 l50 l
Sample
50 l
50 l
Reagent 2(D)1 mlWorking solution (D)1 ml 1 mlMix well and incubate exactly 5 minutes at 37C
Read the absorbance (A) of standard and test against their blank at 555 nm.
Calculation
[Direct bilirubin] = x St. conc.
ReagentsR1 : Sulfanilic acid 30 mmol/l
Hydrochloric acid 150 mmol/l
Dimethyl sulfoxide 7 mmol/l
R2 : Sulfanilic acid 30 mmol/l
Hydrochloric acid 150 mmol/l
R3 : Sodium nitrite 20 mmol/l
R4 : Standard
ALANINE TRANSAMINASE (ALT)
ASPARTATE TRANSAMINASE (AST)Transamination
Is the process in which an amino group is transferred from -aminoacid to -ketoacid. All naturally occurring -aminoacids can take part in such reaction, different enzymes being involved. Two examples, serum aspartate trasnaminase (AST) and serum alanine transaminase (ALT).
COOH COOH COOH
Clinical significance
These reactions are catalysed by the enzymes (AST) and (ALT) which are intracellular and widely distributed in human tissues. AST is present in tissues like muscles (skeletal and cardiac), liver and erythrocytes. The greater amount of (GPT) (ALT) is in the liver.Normal level of serum AST is up to 20 I.U./L
Normal level of serum ALT is up to 15 I.U./L
Serum AST is greatly increased when there is necrosis of the tissues containing these enzyme, as in myocardial infarction, myopathy, hepatitis and hemolytic anaemia. Serum (AST) level is elevated significantly after myocardial infarction within the 1st (6-12) hours and attains a peak within (24-48) hours and returns to normal within (4-7) days.
For (ALT): higher values are found when there is necrosis of liver tissues, in toxic liver diseases, viral hepatitis and in obstructive jaundice.
Measurement of serum ALT (GPT) activity
Principle
The reagent 2,4 dinitrophenylhydrazine (DNPH) reacts with -ketocarboxylic acid to give brown coloured complex 2,4 – dinitrophenylhydrazone which is measured spectrophotometrically at (510) nm.
Procedure
Warm the substrate by putting in 37C water bath for (3) minutes.GPT (GOT substrate (ml)
Serum 0.1
Incubate for (30) min. in (37C) water bath
Standard (ml) 0.1
D.W (ml) 0.1 0.1
After incubation of the test, to all tubes add
DNPH (ml) 0.5 0.5 0.5 0.5
Serum (ml) 0.1
Wait for (20) min at room temp.
0.4 N (NaOH) (ml) 5 5 5 5
Wait for (10) min at room temp.
Read the absorbance at 510 nm.
The pyruvate formed by the serum is responsible for the difference between the test and the control (T-C). The pyruvate in 0.1 ml of the working standard. (0.4) mol produces the difference between the standard & blank (Std-B).
For ALT: x 0.4 x x
x 133 = Pyruvate per min/L
and convert according to the table to I.U./L
Measurement of serum AST (GOT) activity
The same steps for GPT are followed except thatThe substrate is different (Aspartic & -ketoglutarate).
The incubation time is (60) min instead of (30).(GOT) = x 0.4 x x
x 67and convert according to the table to I.U./L
Table:- The relation of mole of pyruvate per minute, per liter in the colorimetric reaction to international units determined spectrophotometrically.Calculated pyruvate mol/ per min per liter
GPT result in I.U./LCalculated pyruvate mol/ per min. per liter
GPT result in I.U./L
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
501
2
2
3
4
4
5
6
7
7
8
8
9
9
10
11
12
13
14
15
16
17
18
19
20
2152
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98
100
10222
23
24
25
26
27
29
30
31
33
34
35
36
37
38
39
40
42
44
46
48
50
52
56
59
60- For activities over 60 I.U/L, dilute the serum 1/10 with DW and multiply I.U/L x 10
Reagents
Phosphate buffer (PH 7.4): Dissolve 11.3 gm of dry anhydrous disodium hydrogen phosphate and 2.7 gm of dry anhydrous potassium dihydrogen phosphate per liter in water. Check with PH meter, store at 4C.
GPT substrate: Dissolve 9.0 gm of DL-alanine in 90 ml of water with the addition of about 2.5 ml of N-sodium hydroxide to adjust the PH to 7.4. Add 0.146 gm of -ketoglutaric acid, dissolve it by adding a little more sodium hydroxide and adjust the PH to 7.4. make to 500 ml with phosphate buffer. Divide into small portions and store frozen at - 15C.
Stock pyruvate standard: (20 mM) Dissolve 220 mg of sodium pyruvate in 100 ml phosphate buffer. Store at 15C.
Working pyruvate standard: Dilute stock standard 1 in 5 ml with phosphate buffer and store at 15C. Prepare freshly each week.
2,4 dinitrophenylhydrazine (1 mM): Dissolve 0.198 gm (198 mg) of dinitrophenylhydrazine in 10 ml of conc. HCI and make to 100 ml with water. Keep in brown bottle at room temperature.
0.4 N sodium hydroxide: 16 gm of sodium hydroxide per liter in water.
ALKALINE PHOSPHATASE
This is a widely distributed enzyme which releases inorganic phosphate from many organic phosphomonoesters and also pyrophosphates. The enzyme exhibits optimum activity at PH 9 10.The form present in the sera of normal adults originates mainly in the liver or biliary tract and bone (osteoblasts). The respective contribution of these two forms is markedly age-dependent with a high alkaline phosphate (bone) occurring during childhood and adolescence. Minor contribution also comes from intestine, placenta (during pregnancy) and rarely renal tissues.
Although the precise metabolic function of the enzyme is not yet understood, it appears that the enzyme is associated with calcification process in bone and lipid transport in other tissues.
Clinical significance
Serum alkaline phosphatase is of particular interest in the investigation of two groups of conditions, hepatobiliary diseases and bone diseases associated with increased osteoblastic activity.
The elevation tends to be more marked (more than three folds) in extrahepatic obstruction (e.g. by stone or by cancer of the head of pancreas) than intrahepatic.
Liver diseases that principally affect parenchymal cells such as hepatitis, typically has normal or moderately elevated serum alkaline phosphatase activity, the degree of elevation is usually less than three folds and depends on the degree of biliary stasis.
Among bone diseases the highest level of serum alkaline phosphatase activity is encountered in pagets disease. Moderate rises are observed in osteomalacia and rickets (with its activity tends to decrease following Vit. D therapy), primary and secondary hyperparathyroidism with skeletal involvement, very high enzyme activity is also present in patients with osteogenic bone cancer including metastases.
An increase in alkaline phosphatase activity may be observed in women in the third trimester of pregnancy with additional enzyme being of placental origin. Serum alkaline phosphatase should, therefore, be interpreted with caution in pregnant women.
Normal serum level of AP is 20-90 I.U/L (3-13) king. Armstrong units/d (K.A.U./dl) in adults.
Measurement of Serum Alkaline Phosphates Activity
PrincipleThe phenol released by enzymatic hydrolysis from phenyl phosphate substrate at a constant condition of time, temp. and PH is estimated colorimetrically.
Phenyl phosphate = phenol + phosphate
The phenol liberated is measured in the presence of amino-4-antipyrine and potassium ferricyanide. The presence of sodium arsenate in the reagent stops the enzymatic reaction.
Procedure
Test (T)Control Standard (St)Blank (B)Buffer (ml)1.01.01.11.1Substrate (ml)1.01.0Serum (ml)0.1Standard (ml)1.0Water (ml)1.0Mix gently and incubate for exactly (15) minutes
Stop the reaction by the addition of
0.5 N NaOH (ml)0.80.80.80.8Serum (ml)0.10.5 N NaHCO3 (ml)1.21.21.21.2Aminoantipyrene (ml)1.01.01.01.0Potassium ferricyanide (ml)1.01.01.01.0
Mix each tube well after each addition. The successive additions adjust the PH and develop the colour. Failure to mix thoroughly leads to irregular results because colour development is markedly PH dependent.
Compare the reddish-brown colour immediately at 510 nm avoiding exposure to strong sunlight. The amount of phenol present in the standard tube is 10 g.
Thus the phenol produced in 15 minutes in the test is:
x 10 g
Hence 100 ml of serum would liberatex 10 mg of phenol
Since 1 King-Armstrong unit is the production of 1 mg of phenol in 15 minutes under the conditions of the test.Serum alkaline phosphatase (K.A.U./100 ml) = x 10
For activities between 30 and 60 K.A.U./dl the final colour may be diluted with 6 ml of water. For activities greater than this the determination must be repeated with 5 minutes incubation time and the results should be multiplied by 3.
Alkaline Phophatase Activity (Kit method):
Principle: colorimetric determination of alkaline phosphatase activity according to the following reaction:
Reagents
Reagent 1 (substrate buffer)disodium phenyl phosphate Carbonate-bicarbonate Buffer pH = 10 5 mmol/L
50 mmol/LReagent 2 (standard)phenolequal to 20 kind & king UReagent 3
(blocking reagent) amino-4-antipyrine sodium arsenate (toxic reagent)60 mmol/L75 g/LReagent 4
(color reagent) potassium ferricyanide150 mmol/LWavelength: 510 nm.
Reading: against reagent blank
Serum (sample): Hemolysis will interfere.
Procedure
Test (T)Test blank (Tb)St.RbReagent 1 2 ml2 ml2 ml2 mlIncubate for 5 minutes at 37CSerumReagent 2
50 l
50 l
Incubate for exactly 15 minutes at 37CReagent 3
0.5 ml0.5 ml
0.5 ml
0.5 ml
Mix well or preferably vortex.
Reagent 4
SerumDistilled water
0.5 ml
0.5 ml
50 ml0.5 ml
0.5 ml
50 ml
Mix, let stand for 10 minutes in the dark. Read ((A)) at 510 nm against RbCalculation
Alk. ph. Activity = x n where n = 20
Note: one kind and king unit is that amount of enzyme which in the given conditions liberate 1 mg of phenol in 15 minutes at 37C.
Reagents
Buffer PH 10: Dissolve 6.3 gm of anhydrous sodium carbonate and 3.36 gm of sodium bicarbonate per liter in water. Keep at 4C.
Substrate (0.01 M-disodium phenyl phosphate). Dissolve 2.18 gm in one liter of water. Bring the solution quickly to the boiling point to kill any organism.
Cool immediately and preserve with a little chloroform (4 ml/1 liter). Keep at 4C.
Stock phenol standard (1 mg/ml). Dissolve 1.0 gm of pure crystalline phenol per liter in 0.1 N-hydrochloric acid. Keep at 4C in a brown bottle.
Working phenol standard (1 mg/100 ml). Dilute 1 ml of stock phenol standard to 100 ml with distilled water. Preserve with a few drops of chloroform and keep at 4C in a brown bottle.
0.5 N-sodium bicarbonate: 42 gm of sodium bicarbonate NaHCO3 per liter in water.
4-Aminoantipyrine: Dissolve 6 gm of 4-aminoantipyrine per liter water. Store in a brown bottle.
Potassium ferricyanide: Dissolve 24 gm of potassium ferricyanide per liter water. Store in a brown bottle.
TOTAL CALCIUM
Calcium is the most abundant mineral in the body, there being 1 kg (25000 mmol) calcium in the body of which 99% is present in bone. The remaining 1% circulates in blood in two main forms, the albumin bound fraction which accounts for 50% of the total calcium and it is physiologically inactive.
The other part is the free ionized calcium which is physiologically active. The free ionized calcium is essential for coagulation process, neuromuscular activity, membrane permeability, and the activity of many enzymes. Ionized calcium is also very important inside the cell where it acts as a second messenger for many hormones.
There are many factors that control the level of circulating free ionized calcium which include:-
Parathyroid hormone.
1,25 dihyroxy cholecalciferol, 1,25 DHCC (calcitriol).
Calcitonin.
In the lab, we measure the total calcium (free + bound).
Clinical significance
Normal range 2.10 2.60 mmol/L (8.5-10.5 mg/dl).
Serum total calcium increases in hyperparathyroidism, malignancy with metastases, multiple myeloma and vitamin D intoxication. Serum total calcium may be increased as an artifact due to excessive venous stasis during blood collection and therefore correction for albumin or total protein has to be made.
Serum total calcium decreases in hypoparathyroidism, osteomalacia, rickets, chronic renal failure, malabsorption syndrome and hypoproteinemia.
Trinder method
PrincipleThe method involves the formation of coloured complex due to reaction of calcium with ferric nitrate in the colour reagent. Trinder had described the use of naphthalhydroxamic acid as a precipitating agent which is efficient since only a small excess of the reagent is required.
The reaction steps can be summarized as:-
Naphthalhydroxamic acid + Ca+2
Ca-naphthalhydroxamate (orange ppt)
2- Ca-naphthalhydroxamate + EDTA
Ca-EDTA (soluble complex) + naphthalhydroxamic acid
3- Ca-EDTA + Colour reagent Yellow-orange colour complex
Procedure
Serum must be used for estimation of calcium, if plasma is used, heparinized blood sample can be used only, since EDTA or oxalate which are used as anticoagulants will chelate calcium.
Lable two centrifuge tubes as test and standard (std).
TestStSerum 0.2 mlSt (2.5 mmol/L)0.2 mlReagent5 ml5 mlAllow tubes to stand at room temperature (25C) for 30 min. then centrifuge for 10 min.
Decant the supernatant fluid carefully by slowly inverting the tubes and immediately placing them to drain mouth downwards on a filter paper, without returning them to the upright position, let them as such for 5 min.
Add to both tubes 1 ml of EDTA, shake to suspend the precipitate, cover the tubes and heat in boiling water bath for 10 min. with occasional mixing to ensure complete solution of the precipitate. Cool.
Add 3 ml of colour reagent to both tubes. Mix, compare colours against colour reagent (step 4) as a blank B at 450 nm.
Calculation
Serum Ca (mmol/L) = x standard concentration
= x 2.5 mmol/L
Reagents
Calcium reagent: Dissolve (250 mg) of naphthalhydroxamic acid by warming in 100 ml of water containing 5 ml of ethanolamine and (2 gm) of tartaric acid. Add (9 gm) of sodium chloride dissolved in (500 ml) of water and dilute to 1 litre with water. If a ppt. forms, filter through a whatman No. 40 or 43 paper.EDTA solution: Dissolve (2 gm) of disodium EDTA per litre in (0.1 N) NaOH.
Colour reagent: Dissolve (60 gm) of ferric nitrate [Fe(NO3)3.9H2O] in 500 ml of water, add 15 ml of conc. HNO3 and dilute to 1 litre with water.
Calcium standard (2.5 mmol/L): Dissolve (125 gm) of dry CaCO3 in 40 ml of (0.1 N) HCl and dilute to 500 ml with water.
This std. contains 2.5 mmol/l or 10 mg/dl.
Determination of Calcium
Kit method
Principle
Colorimetric determination of Ca, without deproteinization using O-cresolphthalein complexone, interference due to Mg+2 ion is eliminated by 8 hydroxyquinoline.
Procedure
Reagent blank
StandardSample
Sample
20 L
Standard
20 LWorking solution
1 ml1 ml
1 ml
Mix, read absorbance after 5 minutes at 570 nm.
Calculationx conc. of St
x 2.5 mmol/L
Reagents
Reagent 1 (standard): Consists of 2.5 mmol/L of calcium (10 mg/dl).Reagent 2 (colour reagent): Consists of O-cresolphthalein complexone (104 mg/L) and 8-hydroxyquinoline (1.5 g/L).
Reagent 3 (alkaline reagent): Consists of reagent pH > 11 (2 amino-2-methyl-propanol (43.25 gm/L).
INORGANIC PHOSPHATE
Phosphate is an important anion associated with calcium in vivo. Knowledge of its plasma level is needed to interpret disturbances of calcium metabolism. The cellular high-energy compounds like ATP, GTP are of great biological importance.
Clinical significance
There is often a marked variation in plasma phosphate concentration during the day, specially following meals. Reference values relate to specimens collected under fasting conditions very significantly with age; being higher during infancy and childhood. Fasting serum inorganic phosphate normal range of adults is 0.8-1.5 mmol/l (2.4-4.5 mg/dl) and in children it is 1.29-2.25 mmol/L (4-7 mg/dl).
Abnormalities of Serum Phosphate Concentration
Hypophosphatemia: usually due toDisturbances of calcium metabolism as a consequence of excess circulating PTH, as in primary and tertiary hyperparathyroidism.
Renal disorders of phosphate reabsorption (phosphate lost from the body), as in renal tubular acidosis.
During parenteral nutrition with inadequate phosphate and following intravenous glucose therapy.
Chronic or long standing intestinal malabsorption (steatorrhoea).
Hyperphosphatemia
Renal glomerular failure, as in chronic renal failure (CRF).
Hypoparathyroidism.
Catabolic states.
Vit. D intoxication.
Measurement of Serum Inorganic Phosphate
PrincipleThe principle depends on the formation of blue colour by oxidation-reduction reaction. The reactions are summarized in the following steps:-
Serum proteins are precipitated using trichloroacetic acid (TCA).
Ammonium molybdate + perchloric acid molybdic acid.
Molybdic acid + phosphate phopshomolybdic acid
(faint yellowish)
4- Phopshomolybdic acid + Ascorbic acid (Reduced)
phosphomolybdic acid + (oxidized) ascorbic acid (Blue colour).
Procedure
Test: 1 ml serum + 9 ml trichloroacetic acid. Mix, wait 10 min. then centrifuge.
Lable 3 test tubes as Test (T), standard (St) and blank (B).
TSt BSupernatant (ml)5Std solution (ml)5D.W (ml)5Perchloric acid (ml)0.40.40.4Ammonium molybdate (ml)0.40.40.4Ascorbic acid (ml)0.2 0.20.2Mix well after each addition, wait 10 min. then read the absorbance at 700 nm.
Calculation
The conc. of the St is 0.133 mmol/L or (0.4 mg/dl.)
Dilution = 1 + 9 = 10 Total volume
.. is the dilution factor.
Serum inorganic phosphate concentration (mmol/L)
= x conc. of std (mmol/L) x dilution factor
= x 0.133 x 10
Reagents
Trichloroacetic (TCA) 10%: Dissolve 10 gm of TCA in water to 100 ml volume.Perchloric acid 60% use analytical reagent.
Ammonium molybdate: 5 gm/dl in H2O.
Reducing agent (ascorbic acid): 200 mg/L H2O.
Stock phosphate standard: Dissolve 2.194 gm of KH2PO4 in 500 ml H2O.
Working phosphate standard 0.133 mmol/l or (0.4 mg/dl). Dilute 2 ml of stock phosphate standard to 500 ml with water.
Determination of Inorganic Phosphate
Kit methodPrinciple
Inorganic phosphate can be determined without deproteinization using a single reagent which forms a phosphomolybdate complex in the presence of a reducing agent (ferous sulphate).
Procedure
Working solution: is prepared by mixing
Reagent 2 1 volume
Reagent 3 1 volume, and keep in a dark bottle
Pipette into three test tubes:
Reagent Blk
St
Test
Sample
100 lReagent 1 (standard)
100 lD.W
100 l
Working solution (R2+R3)
2.5 ml2.5 ml
2.5 ml
Mix, wait for 10 minutes, and then measure against reagent blank at 690 nm.
CalculationCT (mmol/L) = x CSt 1.61 mmol/L or (5 mg%)
CT (mmol/L) = x 1.61
Reagents
Reagent 1 (standard):Phosphorus 1.61 mmol/L (5 mg%)
Sodium azide 1 g/L
Reagent 2 (reducing agent):
Sulfuric acid 1.06 N
Ferrous ammonium sulphate 100 g/L
Ferrous nitrate 2 g/L
Reagent 3 (color reagent):
Sulfuric acid 1.05 N
Ammonium hepta-molybdate 4.5 g/L
UREA
Urea is the major nitrogen metabolic product of protein catabolism comprising over 75% of the nonprotein nitrogen excreted. The biosynthesis of urea from ammonia is carried out exclusively by hepatic enzymes of the urea cycle
Over 90% of urea is excreted through the kidney. It is filtered freely by the glomerulus and neither actively reabsorbed nor secreted by the tubules. However passive tubular reabsorption occurs to a significant extent, especially at low rates of urine flow with more entering in plasma in slow flow states. Consequently urea clearance underestimates GFR.
Although serum urea concentration is often used as an index of renal glomerular function, measurement of serum creatinine provides a more accurate assessment. Urea production is increased by a high protein intake, in catabolic state and dehydration. Conversely, production is decreased in patients with low protein intake and sometimes in patients with liver diseases.
The normal range of serum urea is 3.3-6.6 mmol/l (20-40 mg/dl). The range is lower in infants than adults and also during pregnancy. There is an increase in serum urea with age particularly over 55 years old. Changes in serum urea particularly increased concentration are feature of renal impairment but it is important to consider possible extra renal influences on urea concentration before ascribing any such changes to an alteration in renal function.
Principle
Blood is mixed with an isotonic salt solution (e.g. sodium sulfate) to prevent haemolysis (because haemolysis will liberate sulfhydryl compounds e.g. glutathione which interfere with nesslerization) and it is treated with urease at 37C. This enzyme (urease) will convert urea to ammonium salt. Then protein of the blood is precipitated and the quantity of ammonium salt is determined by Nesslerization and subsequent spectrophotometric measurement of the yellow colour produced. Standard solution of ammonium chloride is also Nesslerised in the same way and the colour is measured and compared with the test.Procedure
Preparation of test
In a centrifuge tube add 4.4 ml. of isotonic sodium sulfate solution then 0.1 ml. of blood and 0.1 ml of urease suspension. Close the tube with rubber stopper and mix gently and incubate at 37C for 15-20 minutes. After incubation, add 0.2 ml of zinc sulfate solution, mix and add 0.2 ml of 0.5 M NaOH mix (the above two solutions are protein precipitants), Centrifuge for 5-10 minutes
Lable three test tubes as T, St and B and make the following additions:
T: 3 ml of supernatant fluid + 2 ml isotonic solution + 1 ml Nesslers reagent.
St: 2 ml of standard solution + 3 ml isotonic solution + 1 ml Nesslers reagent.
B: 5 ml of isotonic solution + 1 ml Nesslers reagent.
Mix by swirling, leave aside for 5 min. for the colour to develop, then take the spectrophotometric reading at 480 nm.
Calculation
Blood urea (mg%) = x conc. of St x
= x 0.03 x
= x 50
Blood urea mmol/L = x 50 x 0.166 (conversion factor for SI units)
= x 8.3
Note: Blood urea nitrogen (BUN) may be reported. Since 60 g of urea contains 28 g of nitrogen, BUN can be converted into urea by multiplying the value by 2.14 whereby:
Blood urea = BUN x 2.14.
Reagents
Isotonic sodium sulphate:30 gm of crystalline sodium sulphate Na2SO4. 10H2O (or 13.2 gm of anhydrous salt) is dissolved in water and made up to 1 L.
Urease suspension:
Grind 1 tablet of urease in 5 ml of 30% Ethanol. This preparation remains active for about 4 days at room temperature and for longer period in the refrigerator, shake well before use.
Zinc sulphate solution:
10 gm of crystalline zinc sulphate ZnSO4. 7H2O is dissolved in water and made up to 100 ml.
0.5 M NaOH:
10 gm of NaOH is dissolved in 500 ml D.W.
Nesslers reagent:
Weigh 15 gm of KI. Dissolve in 10 ml D.W. Weight 11.3 gm of I2 (iodine crystal) add it to the above solution and shake. Weight 15 gm of mercury (Hg metal) and place in a stoppered conical flask. Pour the iodine solution on mercury then wash with further 5 ml of D.W., then shake vigorously for about two min. with cooling using ice (shake from time to time). The colour of the mixture will be green then change to brownish green or yellow. Leave the mixture in the refrigerator overnight then filter. Prepare 10% NaOH as follows:
50 gm NaOH in 500 ml D.W. Mix together 485 ml of 10% NaOH with 15 ml of the filtered mercury-iodine mixture. The resulting mixture is called Nessler reagent.
6- Urea stock St: Dissolve 267.5 mg of pure ammonium chloride (NH4Cl) in water, then dilute to 1 liter.
Urea working St: (0.015 mg/ml): Dilute 100 ml of stock urea st. + 10 ml of 1 N H2SO4 to 1 liter with distilled water.
CREATININE AND CREATININE CLEARANCE
Creatinine is synthesized in the kidney, liver and pancreas by two enzymatically mediated reactions. In the first, transamination of arginine and glycine form guanidinoacetic acid. The second, methylation of guanidinoacetic acid occurs with S-adenosyl methionine as methyl donar to form creatine. Creatine is then transported to body organs such as muscle and brain where it is phosphorylated to phosphocreatine, a high energy compound. Interconversion of phosphocreatine and creatine is a particular feature of metabolic processes of muscle contraction with some of the free creatine in muscle is spontaneously converted to creatinine, its anhydrous form. About 1-2% of muscle creatine is converted to creatinine daily. Because the amount of endogenous creatinine produced is proportional to muscle mass, the production varies with age, sex and body mass; non-obese adult male excretes about 1.5 g/day, females 1.2 g/day.
Creatinine is excreted mainly by the kidney. Following filtration, no further reabsorption of creatinine will occur through the tubules, small quantity of creatinine is secreted in the tubules (7-10%). As a result, creatinine clearance represents an indicator of glomerular filtration rate (GFR), although the former slightly overestimates the latter. Creatinine has a constant range. Its measurement is used to evaluate renal (particularly glomerular) function. However with mild renal impairment, plasma creatinine remains fairly unchanged until GFR is decreased to 50-60% of its normal value. Therefore, an increased plasma creatinine indicates marked impairment of renal function and for the assessment of mild impairment, calculation of creatinine clearance (a more sensitive index of renal function) is required.
Creatine + ATP Phospho creatine + ADP
Endogenous creatinine clearanceRenal clearance of substance (creatinine in this case) is a figure (ml/min) representing the volume of blood from which the compound is completely cleared each minute by the kidney.
Instructions and method
Hydrate the patient properly with at least 600 ml of water.
Have the patient void and discard the urine.
Note: (record) the time, and from then on collect all urine passed for usually 24 hours.
Collect a blood specimen preferable at mid time of urine collection.
In the laboratory, measure the volume of total urine collected and record both volume and minutes (hx60) of the period in which it was collected. Perform the assays of serum (or plasma) and urine creatinine.
Calculate clearance
Cr. clearance =
Where Ucr = urine creatinine concentration
Pcr = plasma creatinine concentration, in the same units.V = volume of urine flow in ml/min, calculated by dividing
urine volume by collecting time (h x 60).
For children, obese and elderly patients measured clearance should be corrected for the surface area
Corrected clearance = measured clearance x
Where A = surface area in square meter, 1.73 average adults surface area. The surface area can be obtained from knowing the length and weight of the patient.
Clinical significance
An increase in plasma creatinine is likely to be due to a fall in the GFR, the causes of which include:
Any disease in which there is impaired renal perfusion. (e.g. reduced blood pressure, fluid depletion, renal artery stenosis).
Most diseases in which there is loss of functioning nephrons (e.g. urinary tract obstruction due to prostatic enlargement).
Normal range
Serum or plasma creatinine – M 55-150 mol/L (0.5-1.3 mg/dl)
F 55-125 mol/L (0.5-1.1 mg/dl)
Urine creatinine – M 125-225 mol/kg/d
F 100-175 mol/kg/d
Creatinine clearance – M 0.9-1.35 ml/s/m2 (85-125 ml/min)
F 0.7-1.05 ml/s/m2 (175-115 ml/min)
Principle of Serum and Urine Creatinine Measurements
In serum, protein is first precipitated using sodium tungstate and sulfuric acid. Then the creatinine present in the protein free filtrate (PFF) and in the diluted urine is exposed to (Jaffes reaction) where creatinine reacts with alkaline picrate (saturated picric acid and sodium hydroxide) producing an orange red colour.Procedure
In a centrifuge tube, place 1 ml serum. Add 1.0 ml H2O, 1.0 ml of 10% sod. tungstate followed by 1.0 ml of 0.66 NH2SO4. Mix and centrifuge.
Prepare 1 50 urine dilution (1 ml urine + 49 ml water)
or 0.1 ml 5.0 ml
Lable 4 test tubes as T serum, U, Std & B
T serumUrine StdBlankPFF (ml)2(1 50)
diluted urine (ml)2Creat. St (ml)2Dist. H2O (ml)2Alk. Picrate (ml)1111Mix, let stand 10 min. Read A at 520 nm.
Calculation
For serum or plasma creat. (P):
Conc. (P) = x St Conc. x dilution factor
Conc. (P) = x 54 x
Conc. (P) = x 216
For urine (U):-
Conc. (U) = x 54 x
Conc. (U) = x 2700
To calculate creatinine clearance, apply the formula:
CrCl =
ml/min
Reagents
10% sod. tungstate: Dissolve 10 gms of Na2WO4. 2H2O in water then make volume to 100 ml.0.66 NH2SO4: Dilute 18.6 ml of conc. H2SO4 to 1 liter.
Alkaline picrate: Mix 5 vol. of saturated picric acid (11.95 gm/l) solution with 1 volume of 10% NaOH. This solution must be prepared fresh daily
Stock creatinine st. (0.1%): Dissolve 1 gm of purified creatinine in 1 L vol. flask using 0.1 N HCl to complete the volume. This solution is (1 mg/ml), or 0.009 mol/L or 9 mmol/L or 9000 mol/L.
Working creatinine St. (0.006 mg/ml) or 0.054 mmol/L or 54 mol/L: Place 3 ml of stock creatinine St. (0.1%) in 500 ml vol. flask and add 50 ml of 0.1 N HCl, then complete the volume using dist. water.
SI UNITS
The international system of units (systeme international, SI) was adopted in 1960 by the General Conference of Weight and Measures as a logical coherent system based on seven fundamental units: metre, kilogram, second, ampere, kelvin, candela and mole.The system has been adopted generally by international scientific bodies, including the International Federation of Clinical Chemistry (IFCC) and the Section of Clinical Chemistry of the International Union of Pure and Applied Chemistry (IUPAC).
All measurements are expressed in the basic units or in units derived from them. The seven basic units and some of the derived units relevant to medicine with standard abbreviations are:
Physical QuantityName of SI UnitSymbolLengthMetreMMassKilogramKgTimeSecondSElectric currentAmpereATemperatureKelvinKLuminous IntensityCandalaCdAmount of substanceMoleMolEnergyJouleJForceNewtonNPowerWattWPressurePascalPa
Prefixes to indicate fractions of the basic or derived units have been defined:
Fraction
PrefixSymbol10 -1DeciD10 -2CentiC10 -3MilliM10 -6Micro(10 -9NanoN10 -12PicoP10 -15FemtoF10 -18AttoAPrefixes to indicate multiples of the basic or derived units have also been defined:
MultiplePrefixSymbol10DecaDa10 2HectoH10 3KiloK10 6MegaM10 9GigaG10 12TeraTThe major change in clinical chemistry is the reporting of analyses in mmol/l (or (mol/l, etc) in place of the conventional mg/dl. For example, the determinations of substances reported in mmol/l may be converted to mg/dl thus:
Conc. (in mmol/l) Molecular Weight (MW)
10
When the MW of a substance cannot be accurately determined (as in mixture), values are reporterd as weight of substance/litre, e.g. globulin will be reported as g/l. In all reports, the litre (l) is the preferred volume.
Another change is in reporting of pressure with mmHg being replaced by the derived unit the pascal (Nm 2} or in practice since this is too small, the kilopascal (Kpa).
It is important that doctors, nurses and laboratory workers become used to thinking in SI units as rapidly as possible. The conversion scales are provided to help staff to adjust to these new units, and have been deliberately designed to make the new easier than the old! While there is complete agreement on the precise conventions to be used for reporting most tests, there is still uncertainty about some tests, notably drug levels and pH.
Problem areas in the use of SI Units
Problems in the implementation of SI units in laboratory practice have been encountered in the following areas:Expressing Enzyme Units
The proposed base unit katal (kat) is the catalytic amount of any catalyst, including enzymes, that catalyzes a reaction rate of one mole per second in an assay system. There is a constant relationship between the international unit (1 nmol/min) and the katal (1 mol/s): to convert a value in iu to kat, the value is multiplied by 16.67. Note however, that dependence on reaction conditions applies to kat in the same way as to iu: therefore, data reported in the same unit but obtained under different conditions may not be comparable. Replacement of the International Unit for reporting enzyme activity is likely to be slow: even units that antedated the International Unit are still widely used in clinical laboratory practice.
Expressing Protein Concentration
For some proteins whose MW is uncertain, debate continues as to the appropriate unit for reporting results. Nevertheless, because concentration gives a better indication of the relative amount of a protein, substance concentration units are still preferred, even if the MW of the protein is not exactly known. The concentration of hormones should also be reported as substance concentration. Again, where uncertainty in the correct MW of the hormone exists, an approximate value may be used without introducing a major error in the reported result. Even though, the MW of hemoglobin is known, there is no agreement as to whether the monomer Hb(Fe), or the tetramer Hb4(Fe4), should be used to report values as substance concentration. Until agreement is reached, the International Committee for Standardization in Hematology (ICSH) recommends that hemoglobin result be reported in term of mass concentration, that is, grams per litrr (g/L).
Expressing Osmolality
The osmole is not an SI unit, and therefore theoretically is not suitable for reporting results of measurement of the concentration of osmotically active particles. The appropriate alternative would be to report the depression of freezing point, whose unit is kelvin or degree Celsius, or the change in vapor pressure in pascals. But, because the unit would be method dependent, some of the benefit of using the osmoles would be lost. The current recommendation is to report osmolality in moles per kilogram. If osmolarity is reported, the preferred unit remains moles per liter (mol/L).Expressing Drug Concentration
Although it is probable that drugs will be administered in mass units for the immediate future, it is still desirable that the concentration of a drug in a body fluid be expressed in terms of concentration rather than mass concentration. Substance concentration allows the concentration of a drug to be understood in comparison with the concentration of proteins, to which drugs are largely bound, and of other compounds with which drugs compete for protein-binding sites. For drugs, it is essential that units be clearly stated to avoid dangerous misinterpretation of test values, which might occur when drugs are administered and measured in body fluids in different units.Expressing Acidity
Some reports acidity of body fluids in term of hydrogen ion (H+) concentration instead of pH, their rationale being that pH is a measure of chemical potential rather than a measure of the concentration of the ions. Because chemical potential is a function of the activity coefficient, which is not known with certainty, the antilog of pH cannot be assumed to equal the H+ concentration. PH values should be treated as primary variables and reported as measured.مع تحيات
مكتب زياد للاستنساخ والطباعة الليزرية
موصل مقابل كلية طب الموصل
1
97
1.0
0.8
0.6
0.4
0.2
100 200 300 400 500 600
1.00.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
1 2 3 4 5 6 7 8 9
Concentration of T unknownDrabkins
Whole blood
ConcentrationFig (1)
ASt AB
AT ACASt AB
ASt ABAT AB
g/Lalkaline
PH
(oxidized)
(reduced)10 (Total volume)
1 (serum volume)AT AB
ASt ABASt AB
AT ABGOD
1
POD
BLK
STD
TEST
ATASt
AT AB
ASt AB1000
vol. of sampleAT AB
ASt AB
0.259
20001000
0.025ASt AB
AT ABFe
Globulin
ATt ActASt AStc
ADt Act
ASt AStc
AStAT
AT
ASt
(GPT) activity
AT ACASt AB
130
1000
0.1
ASt AB
AT ACASt AB
AT AC60
1
0.1
1000
I Physical examinationColour
Appearance
Reaction (pH)
Specific gravity
Ca mmol/L =
AT
AT AC
ASt ABASt AB
AT ACASt AB
AT ACpH 10
Alkaline phosphataseFig (1). Electrophoretic separation of serum protein on cellulose acetate. Five pattern are normal but 2, 4, 5, 7, 10 show abnormalities:
4: reduced albumin and (-globulin with raised 2-globulin in a
case of nephrotic syndrome.
2 and 5: monoclonal gammopathy in two cases of multiple myeloma.
7 and 10: polyclonal increases in (-globulins with increased 2-
globulin in 10 also-in cases of chronic inflammatory states.
AT - ATb
ASt
level of
AST1 2 4 7
DaysA
Drabkins solution
OH
ASt
AT
ASt AB
gm/24 hr
vol. of bloodUrea
ATdilution factor (e,g, 201)
ASt1000
AT ABAT AB
ASt ABAT AB
ASt AB
AT ABASt AB
ASt ABAT AB
24 urine vol.1000
AC ATAC
AC AT
AC
0.4
5
AC AT
AC5
mmol/L
1000.01
AC ATAC
100
Enzymatic urea cycle
C TProtein
Transamination & oxidativedeamination
Ammonia
AT ABProteolysis
ATAmino acid
ASt
100
0.06
AStAT
Arginine + glycine (in kidney)
Amidino transferaseGuanidinoacetate + ornithine
S-adenosyl methionineN-Methyl transferase
S-adenosyl homocysteine
(in liver)
Creatine
CKH2O
Spontaneous non enzymatic (in muscle)
PiH2O
Creatinine
Ucr x VPcr
(ml/min)
1.73
A
AT AB
ASt ABASt AB
AT AB4
1
ASt AB
AT ABASt AB
AU AB
1
50
ASt AB
AU ABPcr x Time (min)
UrCr x Ur volume (ml)CH2
CHNH2
COOH+
CH2
CH2
C=O
Transaminase
AST (GOT)CH2
C=O
COOH
+CH2
CH2
COOH
CHNH2
COOHAspartic acid
-amino acid-ketoglutaric
acidOxaloacetic
acidGlutamic
acidCOOH
COOHCOOH
C=O
CH2
CH2
CH3
CHNH2
COOH+
ALT (GPT)
TransaminaseC=O
COOH
CH3
COOH
COOHCHNH2
CH2CH2
+
Alanine -amino acid
-ketoglutaric acidPyruvic acid
Glutamic acid
Test
0.50.5
Control
0.4Standard
0.5Blank
AT - ABASt - AB
AT - AB
ASt - AB
ATASt
ASt
AT
10
AT – AB
ASt – ABASt – AB
AT – ABAT
ASt
Fig. (2)
AStThen
=CT
CSt
ASt
AT
lamp
filter
slit
cuvettephotocell
meterASt
AT
AT
ASt
ASt
AT
ASt
AT
0.4
ASt
AT
ASt
AT
Ca mmol/L =
mol/Lmol/L
= mg/100 mlAbsorbance
Protein standard concentration
(mmol/L)(mmol/L)
(ml)AST
activity
AStAT
II Biochemical examination
GlucoseProtein
Ketone bodies
Bilirubin and urobilinogen