Experiments in Clinical Biochemistry-2019, Study notes of Biochemistry

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Mustansiriyah University Chemistry Department

Experiments in

Clinical Biochemistry

(A hands-on approach) by Assist Prof Dr. Zahraa S. Al-Garawi Created in 2003 and updated in 2019

Preface Clinical Biochemistry brings together knowledge and skills from a variety of scientific disciplines including Biochemistry, Chemistry and Physiology. It applies these to detect, diagnose, treat and prevent diseases in human beings. On the basis, this Clinical Biochemistry handbook has been prepared for two main reasons:

1. To provide the required information about Biochemical basis and clinical significance of human disorders.
2. To supply accurate and specific methods for clinical determination of blood and urine contents. The results should offer clinical diagnosis and management of related diseases. The present manual is designed for one-year course in Clinical Biochemistry for fourth year Science students at the Mustansyriah University, Department of Chemistry. The availability of chemicals and the use of simple equipment were the main reason to the select experiments here. The Chemical and Biochemical principles are involved. These clinical tests are standard and done in clinical lab. in Iraqi hospitals. This handbook has been firstly prepared in 2003 and authorized by the Chemistry Department-Mustansiriyah University. Zahraa Baghdad 2019

Biochemical specimens 3

The great majority of clinical chemistry analysis performed on blood and urine These analyses required the whole blood, serum or plasma to be collected from patients. When blood is drawn and allowed to clot, a clear liquid (serum) exuded from the clotted blood. Plasma on the other hand, separates from the cells only when blood is prevented from clotting (uncoagulated). The clotting processes The blood clot is formed by a protein (fibrinogen) which is present in a soluble form in the plasma and which is transformed to an insoluble network or fibrous material (fibrin). The change of fibrinogen to fibrin is caused by thrombin, which in blood fluid exists as prothrombin. The conversion of prothrombin to thrombin depends on the cation of thromboplastin and Ca+2. These stages may be diagrammed as follows: Stage I Ca+ Stage II prothrombin Stage III fibrinogen fibrin (clot) Thromboplasti n Thrombin

Biochemical specimens 4

Anticoagulants: When the whole blood or plasma is required, an anticoagulant is required. The most commonly used are: Heparin= is a normal blood constituent, but its physiological concentration is not high enough to prevent clotting in freshly down blood. it is a polysaccharide derivation and apparently function by inhibiting thrombin formation (anti-thrombin). Oxalate= act by precipitating Ca+2^ from the blood and prevents clotting. Potassium oxalate are used most commonly, also Li, Na and ammonium salts are used. EDAT= acts similarly to oxalate, except that it chelates Ca+ rather than precipitates it and therefore prevent clotting process. Blood collection and handling The general considerations relevant to the accuracy of subsequent chemical analysis are:

1. Stasis (stop the blood flow by a tourniquet) should be used for a minimum period of time since prolonged stasis may result in alteration of some chemical values.
2. Blood should not be taken while intravenous solutions are being administered, since these solutions may influence the chemical assay.
3. Syringes or evacuated tubes use to collect blood should be valid (not expire) to avoid contamination or hemolysis.

Calorimetric analysis 6

Calorimetric analysis Most methods in clinical Biochemistry are based on quantitative measurement of a colored compound produced when a test sample is mixed with appropriate reagents. Such methods are referred to as calorimetric methods and measurements are made with instruments called photoelectric calorimetric or spectrophotometer in the visible region (400-750nm), where colors are normally visible. Furthermore, the amount of color produced should be proportional to the amount of substance being measured. Light intensity and Beer's law: Light intensity is of principle interest in spectrophotometry. When light is absorbed, its intensity is reduced, the amount of the absorbed light depends on the concentration of the absorbed substance and on the depth of the solution which the light pass through. This has been formalized into Beer's law: ! = #, %, & Where: A= the absorbance of the light by the solution. a= the molar absorptivity (constant). b= the thickness of solution in cm. c= the molar concentration of the absorbing sub. This is illustrated in the figure below:

Calorimetric analysis 7

b Incident Light c Light It is reasonable that more concentration solution or longer light path should absorb more light, since in either case there are more absorbing molecules placed in the path of light. The quantitative application of Beer's is illustrated in the figure below: Another measurement of change in light intensity is transmittance (T), which measures the amount of the light, passes through the solution. The (T) measurement is out of the scope of this handbook, however, the actual relationship between A and T is logarithmic one,! = − log +. A

Calorimetric analysis 9

2. Wave length selector: This device is used to isolate specific wavelengths or wavelength bands of light from the source. The principle component is either transmission or interference types, and monochormators are composed of either prisms or grating. 3. Cuvette: it is a transport container for samples which made of glass or plastic in a range of (320-1000nm), but quartz cuvette (silica) is for measurement below 320nm. 4. Detector: The detector measures light intensity by converting the light into an electrical signal. The more intense the light, the stronger the electrical signal. The most common detectors are barrier layers cells and photomultiplier tubes. Photo tubes and photoconductive tubes also are used. 5. Readout device: The electrical signal from the detector may be readout in terms of %Transmittance T or Absorbance A. this readout may be a digital display, a needle reading on a meter, galvanometer scale, or an ink signal on the chart paper of a recorder.

Blood Glucose 10

Experiment No. Determination of Blood Glucose Glucose the Greek word for sweet, is one of the simple sugars product of carbohydrates or starches by digestive enzymes in the liver. Glucose is synthesized in the liver when dietary sources of glucose are not available from glycogen (glycogenolysis) or protein (gluconeogenesis). All cells use glucose as an important energy source. Some tissues (e.g. the central nrvous system, the brain and spinal cord) depend entirely on glucose. Glucose is stored in muscle and liver as glycogen by insulin hormone or it nay converted into keto acids, amino acids, and proteins or, it stored in adipose tissues, see Fig 1. Fig 1. The glucose sources and metabolism pathways There are many hormones affected blood sugar level, these include:

1. Insulin:

Blood Glucose 12

depends upon a large number of factors which are regarded as physiologic factors (not disease). These include age, how long it has been fast since the last meal, what sort of diet the patient has eaten for several days before the test, weather he has exercised gust before the test…etc. After collecting the blood sample from patients, it is important to separate the serum from cells within a reasonable short time, since red and white blood cells continue to metabolize glucose after they have been removed from the body, which causes false results. Plasma glucose is higher than whole blood glucose by roughly ( 12 - 13)% due to the lower water content of the cells(73%) than the water content of plasma(93%), although the concentration of glucose in both is the same. Plasm sample collect when blood added to a tube containing an anticoagulant such as NaF (to avoid glycolysis) mix with potassium oxalate K$C$O' in a ratio (2mg oxalate/2.5mg florid per 1 ml of blood). Clinical Significance of (. glucose Hyperglycemia: (a) High values of F.B.S cause diabetes mellitus, in which it may vary from normal up to 500mg/dl and over, according to the severity of the condition. As the !- glucose rises above 500mg/dl, there is an increasing possibility of proteins and lipids metabolism disturbance, ketosis, and some degree of diabetic coma will be present. (b) Gushing's Syndrome: excess production of hydrocortisone, which increased gluconeogenesis.

Blood Glucose 13

(c) Acromegaly: over production of growth hormone (GH) from pituitary gland. (d) Hyperadrenalinism: over production of adrenaline (epineph-rine) which causes glucogenolysis by the liver. Hypoglycemia: The hypoglycemia may be considered to be present when the !- glucose is below(40mg/dl), and this occurs most frequently as a result of: (a) Over dosage with insulin in the treatment of diabetes. (b) Starvation also tend to lower the !- glucose starving person do not die from hypoglycemia. (c) Myxedema: low production of thyroxine gland hormones(hypothyroidism). (d) Hypopituitarism: low production of pituitary gland hormones. Methods used of (. glucose determination Enzymatic method : Principle: Glucose Glucuronic acid + *+,+

Glucosoxidase Peroxidase O - toluidine *+, + ./010231 O-toluidine Color

G.T.T 15

Experiment No.( 2 ) Glucose Tolerance test (G.T.T) Following eating a carbohydrate male, there is a temporary rise in the blood glucose, where the extent and duration of increasing glucose depends on the type of food. Blood glucose does not increase in normal people more than (160-180) mg/dl and then return to the normal fasting levels within 2 - 3hr after taking the food. This effect of carbohydrates can be studied under standard conditions by means of the glucose tolerance test G.T.T. The G.T.T is a body response to an oral administration of a standard amount of glucose. It is of considerable use in investigating the abnormalities of carbohydrate metabolism, where glucose is found in urine. The natural rate of increasing blood glucose is determined by two forces, the rate of gastric emptying and the rate of intestinal absorption. A rise in blood glucose level normally stimulates the release of insulin from the pancreas, either directly or by intermediary gut hormones. The insulin response determines the rate of fall of the blood glucose. The normal F.B.S is (6mmol/L) or less The diabetic F.B.S is (8mmol/L) and more If the glucose concentration after 2hr after an oral dosage of (75gm) glucose is (8mmol/L) or less, the result is normal. If it is (11mool/L) or more, the result is diabetic. Between ( and 11 mmol/L), the patient has impaired glucose tolerance.

G.T.T 16

Oral G.T.T :

1. The patient should be fasting overnight (at least 10- 16hr). Only water is permitted during the time course of the test (2.5-3hr). The patient should not smoke and remain seated.
2. Initially, a fasting blood sample and urine specimens are collected.
3. Fifty gm of 75 gm of glucose dissolved in (25-300) mL of water is then feed it to the patient (children 1.75gm/kg body weight). This is best followed by about (50-100)mL of water to take away the sweet taste and decrease the risk of the patient vomiting.
4. Blood samples are collected at 1/2 hour intervals for a period of 3hr (0.5, 1.0, 1.5, 2.0, 2.5, 3 hrs) after the glucose has been drunk. Urine specimens are collected at the similar intervals. Table 1. A typical normal response of GTT. Time/hrs. 0 0.5 1.0 1.5 2.0 2. F.B.S(mg/dL) 70 130 - 150 120 90 70 80 Urine glucose Negative tests throughout Table 1 showed the normal response of GTT, the initial blood glucose is within normal fasting limits (70mg/dl), The maximum level appeared after (0.5- 1 hr) of taking the glucose (130-150mg/dL). Insulin will secret in a large amount and the blood glucose will return rapidly to the normal fasting levels which are often reached in 1.5.-2 hrs. Glucose should not appear in urine in at all time points, as glucose appears in urine only when its level in blood rise over (180mg/dL).

G.T.T 18

after 1hr of taking the glucose dosage(140mg/dl). After that, the glucose level is little raised over the normal limits. This curve is seen in patients with impaired absorption of carbohydrates. Curve (5): Flattened : The F.B.S may sometime be under the usual normal limits, and there is a little rise in blood glucose. This curve is frequently observed in endocrine hypo activity as in hypothyroidism, hypoadrenocorticalism (Addison's disease), and hypopituitarism (Simmond's disease). Factors influence the G.T.T. :

1. Previous diet. : no special restrictions are necessary and a normal diet for (3-4) days. If, however, the test performed after a period of carbohydrate restriction, there may be abnormal G.T.T. 2. Time of the test: most G.T.T. are performed in the morning. There is an evidence that tests performed in the afternoon indicates higher plasma glucose level and that the accepted normal values may not be applicable. 3. Drugs: Drugs such as steroids, diuretics, hormones (ACTH, glucose, and thyroxine) may impaired G.T.T.

The Lipid Profile 19

Experiment No.(3) Determination of Lipid Profile Lipid profile test includes serum total cholesterol, triglycerides, chylomicrons, high-density lipoproteins, low- density lipoproteins and very low-density lipoproteins.

1 - Total Cholesterol :

Cholesterol is a derivative lipid has a chemical formula !"#$%&'$ and its structural formula is shown below: It is formed from steroids unit consist of 4 rings. Cholesterol is a bile solid alcohol, where Chole means bile and sterol means solid. Cholesterol is found in all cells of the body. The brain and spinal cord contain large amount of cholesterol, which is form part of the lipid "insulation". It is a precursor of the adrenal and sex hormones. Cholesterol may be synthesized from 2-carbon units (acetyl CoA) in many body tissues, particularly liver, intestine, and skin. The liver is the main endogenous source of cholesterol, it synthesized about 1gm of cholesterol daily while other tissues synthesized only 1 2 gm. Measurement of total cholesterol includes both the esterified and free forms of the steroid. In plasma or serum, 2/3 of the