Clinical Chemistry 2

Our Lady of Fatima UniversityValenzuela CityCollege of Medical Laboratory ScienceClinical Chemistry 2 (Special Chemistry)

Book Summary:Clinical Chemistry, Immunology andLaboratory Quality Controlby Amitava Dasgupta, and Amer Wahed

As a Requirement for the SubjectClinical Chemistry 2 Special Chemistry

Presented by:Francis Cedrick J. VictorinoMD 3Y2 2

Submitted to:Mr. Billy Joe AfricaClinical Chemistry 2 InstructorChapter 1Instrumentation and Analytical Methods1.1 INTRODUCTIONMajor analytical methods used in the clinical chemistry laboratory include spectrophotometry, chemical sensors, gas chromatography with various detectors, gas chromatography combined with mass spectrometry, high-performance liquid chromatography, and liquid chromatography combined with mass spectrometry or tandem mass spectrometry.1.2 SPECTROPHOTOMETRY AND RELATED METHODSSpectrophotometric measurement are based on Beers law (sometimes referred to as Beer-Lamberts Law) which states that the concentration of substance is directly proportional to the amount of light absorbed and inversely proportional to the logarithm of transmitted light. In spectrophotometry, transmittance is often measured as absorption because of its linear relationship with absorbance and concentration of analyte in the solution.1.3 ATOMIC ABSORPTIONIn atomic absorption spectrophotometry (used for analysis of various elements, including heavy metals), components of gaseous samples are converted into free atoms. A hollow cathode lamp containing an inert gas at a very low pressure is used as a light source. The metal cathode contains the analyte of interest. A part of the light beam is absorbed and results in a net decrease in the intensity of the beam that arrives at the detector. Applying the principles of Beers Law, the concentration of the analyte of interest can be measured. Cold vapor atomic absorption can only be used for analysis of mercury since it is already vaporized at room temperature.1.4 ENZYMATIC ASSAYSEnzymatic assay often use spectrophotometric detection of signal at a particular wavelength. For example, an enzymatic assay of blood lactate utilizes lactate dehydrogenase to catalyze the conversion of lactate to pyruvate, and in this process NAD is converted to NADH and measured spectrophotometrically at 340 nm. 1.5 IMMUNOASSAYSImmunoassays are based on the principle of antigen-antibody reactions. In many immunoassays, the final signal generated is measured using spectrophotometric principle via a suitable spectrophotometer.1.6 NEPHELOMETRY AND TURBIDIMETRYThere is a complete difference between nephelometry and turbidimetry. Nephelometry measure the amount of scattered light by a particulate matter suspended in a turbid solution, on the other hand, Turbidimetry determines the amount of light blocked by the particulate matter1.7 CHEMICAL SENSORSChemical sensors are capable of detecting various chemical species present in the biological matrix. Chemical sensors capable of detecting selective ions can be classified under three broad categories: ion-selective electrodes, redox electrodes, and carbon dioxide-sensing electrodes.1.8 BASIC PRINCIPLES OF CHROMATOGRAPHIC ANALYSISChromatography is a separation method developed in the 19th century. The first method developed was column chromatography, where a mixture is applied at the top of silica column (solid phase) and a non-polar solvent is passed through the column (mobile phase). The differential interaction of a component in the mixture with the solid phase and mobile phase is the basis of chromatographic analysis. There two major form of chromatography used in clinical laboratory namely gas chromatography which can be used in toxicology laboratories for analysis of volatiles and liquid chromatography which can be used for analysis of both polar and non-polar molecules. In addition, thin-layer chromatography (TLC) is sometimes used in a toxicological laboratory to screen for illicit drugs in urine. High performance liquid chromatography is used in clinical laboratory in order to achieve better separation.1.9 MASS SPECTROMETRY COUPLED WITH CHROMATOGRAPHYMass spectrometry is a very powerful detection method that can be coupled with a gas chromatography or a high-performance liquid chromatography analyzer. During mass spectrometric analysis, analyte molecules in the gaseous phase are bombarded with high-energy electrons (electron ionization) or a charged chemical compound with low molecular weight. The mass spectrometric detector can detect ions with various molecular mass and construct a chromatogram. Most commonly, an electron ionization mass spectrometry is coupled with a gas chromatography. However, gas chromatography combined with chemical ionization mass spectrometry is gaining more traction in toxicological laboratories. One advantage of chemical ionization mass spectrometry is that it is soft ionization method, and usually a good molecular ion peak as adduct can be observed. Electrospray ionization is the most common mass spectrometric method used in liquid chromatography combined with mass spectrometric method.1.10 EXAMPLES OF THE APPLICATION OF CHROMATOGRAPHIC TECHNIQUES IN CLINICAL TOXICOLOGY LABORATORIESChromatographic techniques are used in toxicology laboratory for therapeutic drug monitoring, legal blood alcohol determination and GC/MS or LC/MS for confirmation of drug abuse for legal drug testing.1.11 AUTOMATION IN THE CLINICAL LABORATORYAutomated analyzers are widely used in clinical laboratories for speed and ease. The most common configuration of automated analyzers is random access analyzers where multiple specimens can be analyzed for a different selection of tests. Automated analyzers can be broadly classified under two categories: open systems where a technologist is capable of programming parameters for a test using reagents prepared in-house or obtained from a different vendor than the manufacturer of the analyzer, and closed systems where the analyzer requires that the reagent be in a unique container or format that is usually marketed by the manufacturer of the instrument or a vendor authorized by the manufacturer.1.12 ELECTROPHORESIS (USING CAPILLARY ELECTROPHORESIS)Electrophoresis is a technique that utilizes migration of charged analytes in a liquid medium under the influence of an applied electrical field. This is a very powerful technique for analysis of proteins in serum or urine, as well as analysis of various hemoglobin variants.

Chapter 3Pre-analytical Variables3.1 LABORATORY ERRORS IN PRE-ANALYTICAL, ANALYTICAL, AND POST-ANALYTICAL STAGESErrors in clinical laboratory can occur in pre-analytical, analytical and post-analytical steps. About two-third of all errors that occurs in clinical laboratory are associated in pre-analytical steps which involves patient identification error, tube filling error, inappropriate container, improperly stored specimen, contamination of culture tube, etc. The worst pre-analytical error is incorrect patient identification where a physician may act of test results from the wrong patient.3.2 ORDER OF DRAW OF BLOOD COLLECTION TUBESCorrect order of drawing blood: (1) microbiological blood culture tubes (yellow top), (2) royal blue tube (no additive) if trace metal analysis is desired, (3) citrate tube (light blue), (4) serum tube (red top) or tube with gel separator/clot activator (gold top or tiger top), (5) heparin tube (green top), (6) EDTA tube (purple/lavender top), and (7) oxalate-fluoride tube (gray top). Tubes with additives must be thoroughly mixed by inversion as per manufacturers protocol.3.3 ERRORS WITH PATIENT PREPARATIONThere are some important issues regarding patient preparation in obtaining accurate results. For example, glucose and lipid testing requires a patient to fast overnight. Physiologically, blood volume differs significantly in relation with body posture. Blood volume of a normal adult in upright position is 600-700 mL less that when the person is lying and this shift directly affects certain analytes due to dilution effects. It is vital for a laboratory requisition to specify the need for supine samples when these analytes are requested. Several analytes shows diurnal variation thus time of collection of specimen may affect test results.3.4 ERRORS WITH PATIENT IDENTIFICATION AND RELATED ERRORSAccurate patient and specimen identification is required for providing clinicians with correct results. Patient and specimen misidentification mostly occurs during pre-analytical phase. To ensure correct identification, the specimen should be collected and labeled in front of the patient and then send to the laboratory with the test request. Although errors in patient identification usually occur in pre-analytical phase, errors can also occur during analytical and post-analytical phase. Delta checks are a simple way to detect mislabels. This is a process of comparing a patients result to his/her previous results for any one analyte over a specified period of time.3.5 ERROR OF COLLECTING BLOOD IN WRONG TUBES: EFFECT OF ANTICOAGULANTBlood specimens must be collected using the right tube in order to obtain accurate results. It is important to have the correct anticoagulant in the tube. In an optimal anticoagulant, blood to anticoagulant ratio is important to preserve analytes and prevent clots. Proper anticoagulant for various test are as follows: (1) Ethylenediamine tetraacetic acid (EDTA) is used for CBC, blood bank pre-transfusion testing, flow cytometry, hemoglobin A1C and most common immunosuppressive drugs, (2) Heparin is used for the determination of pH blood gases, electrolytes, and ionized calcium, (3) Citrate is used for coagulation studies, (4) Potassium oxalate, in combination with sodium fluoride inhibits enzymes involed in glycolytic pathway, therefore should be used for glucose testing.3.6 ISSUES WITH URINE SPECIMEN COLLECTIONUrinalysis remains one of the key diagnostic tests in modern clinical laboratory. Examination of urine may take several forms: physical, chemical and microscopic examination. Different timings of collection are commonly encountered, however first morning voided urine is the best sample because it is generally more concentrated and contains the highest concentration of sediments. For most urine testing, midstream clean-catch specimen is optimal. For some point of care urinalysis (like pregnancy tests) any clean and dry container is acceptable but in tests like urine cultures, sterile containers are used. Storage of urine specimens at room temperature is generally accepted for up to two hours. After this time results to degradation of cellular and some chemical elements occurs. Therefore, if more than two hours will elapse between collection and testing, sample must be refrigerated for a maximum of twelve hours.3.7 ISSUES WITH SPECIMEN PROCESSING AND TRANSPORTATION Appropriate preparation of specimen prior to centrifugation is required to ensure accurate laboratory results. Serum specimens must be allowed ample time to clot before centrifugation. Tubes with clot activators requires sufficient mixing and at least 30 minutes clotting time, plasma specimens must be gently mixed according to the manufacturers protocol. After collection, specimen requires transportation to the clinical laboratory. If specimens are transported to clinical laboratory, care must be taken in shipping specimens. Ice packs are especially useful for preserving specimen at lower temperature because analytes are more stable at lower temperature.3.8 SPECIAL ISSUES: BLOOD GAS AND IONIZED CALCIUM ANALYSISIdeally, all blood gas specimens should be measured immediately and never stored. A plastic syringe, transported at room temperature, is recommended if analysis will occur within 30 minutes of collection. Otherwise, a specimen must be stored in ice. Glass syringes are recommended for delayed analysis because glass does not allow the diffusion of oxygen or carbon dioxide. Bubbles must be completely expelled from the specimen prior to transport, as the pO2 will be significantly increased and pCO2 decreased within 2 minutes. Ionized calcium is often measured with ion-sensitive electrodes in blood gas analyzers. Ionized calcium is inversely related to pH: decreasing pH decreases albumin binding to calcium, thereby increasing free, ionized calcium. Therefore, specimens sent to the lab for ionized calcium determinations should be handled with the same caution as other blood gas samples since pre-analytical errors in pH will impact ionized calcium results.

Chapter 4Laboratory Statistics and Quality Control4.1 MEAN, STANDARD DEVIATION AND COEFFICIENT OF VARIATIONWhen measuring the value of an analyte, the same should be produced over and over again. However, in reality, same value is not produced by the instrument, but a similar value is observed. Therefore, the most basic statistical operation is to compute the mean, standard deviation and the coefficient of variation. The term mean refers to the average of a given values. After computing for the mean, standard deviation can be easily computed. Standard deviation represents the average difference of an individual value from the mean. Coefficient of variation is an important parameter because it expresses the precision of both mean and standard deviation.4.2 PRECISION AND ACCURACYPrecision is a measure of how reproducible values are in series of measurements, while accuracy is indicates the closeness of each values to the target value. Accuracy can be determined for a particular test where in the target value is known. An ideal analytical method has both accuracy and precision, but a good precision of a test may not have a good accuracy. 4.3 GAUSSIAN DISTRIBUTION AND REFERENCE RANGESGaussian distribution, or normal distribution, is a bell-shaped curve. it is assumed that any measurement values follows a normal distribution with measurements below or above the mean value. In order to understand Gaussian distribution, it is important to know the terms mean, median, and mode. If a distribution is normal, the value of the mean, median, and mode is the same. However, the value of the mean, median, and mode may be different if the distribution is skewed. Other characteristics of Gaussian distribution, the mean SD contains 68.2% of all values, the mean SD contains 95.5% of all values, and the mean SD contains 99.7% of all values in the distribution.Reference range is determined by measuring the values of an analyte in a large number of normal subjects. The reference range when determined by measuring an analyte in at least 100 healthy people and the distribution of values in a normal Gaussian distribution is calculated as mean SD.4.4 SENSITIVITY, SPECIFICITY AND PREDICTIVE VALUEA test cannot be 100% sensitive or specific because of the overlap between values of a biochemical parameters observe in normal individual and a patient with particular disease. For calculating sensitivity, specificity, and predictive value of a test, the following formulas can be used, where TP = true positive, FP = False positive, TN = True negative, and FN = False negative: (a) Sensitivity (individuals with disease who show positive test results) = (TP/(TP1FN)) x 100; (b) Specificity (individuals without disease who show negative test results) = (TN/(TN + FP)) x 100; and (c) Positive predictive value = (TP/(TP + FP)) x 100. When test results are positive, results are a combination of TP and FP, and when assay results are negative, results are combination of TN and FN.4.5 RANDOM AND SYSTEMATIC ERRORS IN MEASUREMENTSRandom and systematic errors are important issues in clinical laboratory. Random errors occur due to the imprecision of analytical method. Systematic errors, on the other hand, are often due to errors in measurement using a particular test. The goal of quality control is to minimize systematic errors since random errors are unavoidable.

4.6 LABORATORY QUALITY CONTROL: INTERNAL AND EXTERNALGood quality control is the heart of a good clinical operation. Because the value of the patients analyte is unknown, clinical laboratory professionals rely on producing accurate results using controls. Control is defined as a substance that contains an analyte with a known concentration. There are three types of control materials used: assayed control where the value of the analyte is predetermined, un-assayed control where the target value is not predetermined and homemade control where the control material is not easily commercially available.Quality control in the laboratory may be internal or external. Internal quality control is essential and results are plotted in a Levey-Jennings chart. Most common example of external quality control is analysis of CAP proficiency samples for most tests offered in a clinical laboratory.4.7 LEVEY-JENNINGS CHART AND WESTGARD RULESA Levey-Jennings chart is a graphical representation of all control values for an assay during an extended period of laboratory operation. In this graphical representation, values are plotted with respect to the calculated mean and standard deviation. If all controls are within the mean and SD, then all control values were within acceptable limits and all runs during that period have acceptable performance. A Levey-Jennings chart must be constructed for each control (low and high control, or low, medium, and high control) for each assay the laboratory offers. The laboratory director or designee must review all Levey-Jennings charts each month and sign them for compliance with an accrediting agency.Usually Westgard rules are used for interpreting Levey-Jennings charts, and for certain violations, a run must be rejected and the problem must be resolved prior to resuming testing of patients samples. Various errors can occur in Levey-Jennings charts, including shift, trend, and other violations. Usually 12s is a warning rule and occurs due to random error; other rules are rejection rules.4.8 DELTA CHECKSDelta check is a quality control where a value is flagged if the value deviate more than a predetermined limit from the previous value in the patient. The basis of delta check is that the value of an analyte in a patient should not deviate from the previous value unless certain intervention is done.4.9 METHOD VALIDATION/EVALUATION OF A NEW METHODComparison of a new method with an existing method is a very important step in method validation. For this purpose, at least 100 patient specimens must be run with the existing method in the laboratory at the same time as the new method. Then values are plotted and a linear regression equation determines the line of best fit as expressed by the equation y5mx1b, where m is the slope of the line and b is the intercept. The computer calculates the equation of the regression line using a least squares approach. The software also calculates r, the correlation coefficient, by using a complicated formula. The ideal value of m is 1, while the ideal value of b is zero. In reality, if slope is less than 1.0, it indicates negative bias with the new method compared to the old method, and if the slope is over 1.0, it indicates positive bias. 4.10 HOW TO INTERPRET THE REGRESSION EQUATION?The regression equation (y = mx + b) provides a lot of information regarding how a new method compares with the reference method. Interpretation of linear regression equation includes: (1) Ideal value: m = 1, b = 0, and y = x. In reality this never happens. (2) If the value of m is less than 1.0, then the method shows negative bias compared to the reference method. (3) If the value of m is over 1.0, it indicates positive bias in the new method. (4) the intercept b can be a positive and negative value and must be a relative small number and (5) the ideal value of r is 1, but any value above 0.95 is acceptable, and a value of 0.99 is considered excellent.4.11 BLAND-ALTMAN PLOTA bland-altman plot compares two methods by plotting the difference between two measurement of y-axis, and the average of the two measurements on the x-axis. The difference between two methods can be expressed as a percentage difference between two methods or a fixed difference. It is easier to see bias between two methods using bland-altman plot.

4.12 RECEIVER-OPERATOR CURVEA receiver-operator curve (ROC) is often used to make an optimal decision level for a test. ROC plots the true positive rate of a test (sensitivity) either as a scale of 0-1 (1 is highest sensitivity) or as percent on the y-axis versus a false positive rate (1-specificity).4.13 WHAT IS SIX-SIGMA?Six sigma goals are achieved if the error rate is only 3.4 out of one million processes, or error rate is only 0.00034%.4.14 ERRORS ASSOCIATED WITH REFERENCE RANGEReference range only accounts for 95% of the values observed in healthy individuals for the particular tests, and statistically 5% of the values of the normal population should fall outside the reference range. The likelihood of n test results falling within the reference range can be calculated from the formula % results falling within normal range = 0.95n x 100. Therefore % results falling outside the reference range in normal people is (120.95n) x 100.4.15 BASIC STATISTICAL ANALYSIS: STUDENT T-TEST AND RELATED TESTSThe Student t-test is useful for determining if one set of values is different from another set of values based on the difference between mean values and standard deviations. This statistical test is also useful in clinical research to see if values of an analyte in the normal state are significantly different from the values observed in a disease state. The F-test is a measure of differences in variances and can also be used to see if one set of data is different from another set of data. The F-test can be used for analysis of multiple sets of data, when it is called ANOVA (analysis of variance). If the distribution of data is non-Gaussian, then neither the t-test nor the F-test can be used. In this case, the Wilcoxon rank sum test (also known as the Mann-Whitney U test) should be used.

Chapter 5Water, Homeostasis, Electrolytes and Acid-base Balance5.1 DISTRIBUTION OF WATER AND ELECTROLYTES IN THE HUMAN BODYWater is the major constituent of the body that represents approximately 60% of the total body weight. Two-thirds of the total water in the body is associated with intracellular fluid and one-third is found in the extracellular fluid. Extracellular fluid is composed mostly of plasma and interstitial fluid. Electrolytes are classified either as cations or anions. Electrolytes play an important role in human physiology including maintenance of water osmolality, acid-base balance, and as co-factor for enzymes to name a few. Sodium is the major extracellular electrolyte, and potassium is the major intracellular electrolytes. The balance between intracellular and extracellular electrolytes is maintained by sodium-potassium ATPase ion pump present in cell membranes.5.2 PLASMA AND URINE OSMOLALITYOsmolality is a measure of osmoles of solutes per kilogram of a solution where osmolarity is a measure of osmoles per liter of solvent. Plasma osmolality measurement is a way to determine electrolyte balance of the body. Normal plasma osmolality is 275-300 mOsm/kg of water while urine osmolality is 50-1000 mOsm/kg of water.5.3 HORMONES INVOLVED IN WATER AND ELECTROLYTE BALANCEAntidiuretic hormone (ADH) and aldosterone play an important role in maintenance of water and electrolyte balance. ADH secretion is regulated by plasma osmolality. If plasma osmolality increases, ADH secretion is increased which cause reabsorption of sodium in the collecting duct of the nephron. ADH at high concentration causes vasoconstriction, thus raising blood pressure. 5.4 RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEMWith low blood pressure, the juxtaglomerular apparatus of kidney secretes renin. Renin converts angiotensinogen to angiotensin I, which is then converted to angiotensin II in the lungs by angiotensin-converting enzyme. Angiotensin II is a vasoconstrictor and also stimulates release of aldosterone. Aldosterone causes reabsoption and retention of water and sodium. Retention of water and sodium increases plasma volume and blood pressure.5.5 DIABETES INSIPIDUSDiabetes insipidus is a condition that occurs when the kidneys are unable to concentrate urine properly. This is caused by lack of ADH or inability of ADH to work at the collecting duct of the nephron. There are two types of diabetes insipidus: Hypothalamic diabetes insipidus and nephrogenic diabetes insipidus. In hypothalamic diabetes insipidus, there in low to zero concentration of ADH and in nephrogenic diabetes insipidus, there is normal level of ADH but it is unable to work at the collecting duct.5.6 THE SYNDROME OF INAPPROPRIATE ANTIDIURETIC HORMONE SECRETION (SIADH)The syndrome of inappropriate antidiuretic hormone or Schwartz-Bartter syndrome is due to excessive and inappropriate release of ADH. Usually reduction of plasma osmolality causes reduction of ADH secretion, but in SIADH reduced plasma osmolality does not inhibit release from the pituitary gland, causing water overload.

5.7 HYPONATREMIA, SICK CELL SYNDROME AND HYPERNATREMIAHyponatremia can either be absolute hyponatremia and dilutional hyponatremia. In absolute hyponatremia, total sodium concentration of the body is low. In dilutional hyponatremia, total body sodium is not low, rather, total body sodium may be increased. The patient may be volume overloaded resulting to dilution of sodium levels. Sick cell syndrome is a hyponatremia seen in individual with acute or chronic illness where cell membranes leak, allowing solutes normally inside the cell escape into extracellular fluid. Sick cell syndrome also produces a positive osmolar gap. Hypernatremia is due to elevated serum sodium levels. Hypernatremia can be hypovolumic and hypervolumic. Hypervolumic hypernatremia may be observed in patients receiving sodium bicarbonate or hypertonic solution. 5.8 HYPOKALEMIA AND HYPERKALEMIAHypokalemia is defined as a serum potassium concentration higher than the reference range. Hypokalemia may occur due to loss of potassium from the gastrointestinal tract, loss of potassium from the kidney, and intracellular shift due to drug therapy with beta-2 agonist. Most of potassium of the body resides intracellularly. Hyperkalemia presents as elevated serum or plasma potassium levels. Causes of hyperkalemia include lysis of cell, intracellular shift, renal failure and pseudohypernatremia.5.9 INTRODUCTION TO ACID-BASE BALANCEAn acid is defined as a compound that donates hydrogen ions, and a base is a compound that accepts hydrogen ions. In order to determine if a solution is acid or base, the pH scale is used. pH is equal to the negative log of hydrogen ion concentration in solution. Neutral pH is 7.0. If pH is lower than 7.0, it is acidic. If it is higher than 7.0, it is basic. Buffer is a substance that regulates pH, it can act as an acid or base depending on the situation. For instance, the normal pH of the blood (7.35-7.45) is regulated by carbonic acid and bicarbonate. The pH of the blood can be calculated by Henderson-Hasselbach equation which states that the pH of the blood is equal to the pKa of the blood plus the logarithm of quotient of weak acid and conjugate base. Normal blood pH can also be regulated by the lungs (respiratory compensation) and kidneys (renal compensation). Respiratory compensation correct acid-base balance is the first compensatory mechanism. It is effective immediately, but it may take longer time for initiation of renal compensatory mechanism. Renal compensation corrects acid-base balance by reabsorbing bicarbonate from the glomerular filtrate in the proximal convoluted tubule.5.10 DIAGNOSTIC APPROACH TO ACID-BASE DISTURBANCEMajor acid-base disturbance can be divided into four categories: metabolic acidosis, metabolic alkalosis, respiratory acidosis, and respiratory alkalosis. Generally, metabolic acidosis or alkalosis is due to abnormal regulation of bicarbonate and other buffers in blood, while abnormal removal of carbon dioxide may cause respiratory acidosis or alkalosis. In diagnosing acid-base disturbance, the first question is to know whether the pH is higher or lower than normal. If the pH is lower than normal, the next question is to ask whether the acidosis is respiratory or metabolic. If the pH is basic, the question is whether the alkalosis is metabolic or respiratory. Metabolic acidosis is a condition where the value of pH is decreased along with the decreases in the value of pCO2 and bicarbonate. Respiratory acidosis is where the value of pH is decreased but values of both pCO2 and bicarbonate is increased from normal. Metabolic alkalosis is where the value of pH is increased along with the values of both pCO2 and bicarbonate. Respiratory alkalosis is a condition where the value of pH is increased while pCO2 and bicarbonate are decreased.

Chapter 6Lipid Metabolism and Disorders6.1 LIPIDS AND LIPOPROTEINSLipids, along with other macromolecules are also building blocks of life. It is essential because lipids are integral structural part of cell membranes of humans and animals. All lipids are insoluble to water. Carbohydrates and lipids are major sources of energy. Steroids are also lipids, and many steroids also act as hormone. Major lipids are as follows: Triglycerides, fatty acids, phospholipids, and cholesterol. Triglycerides are formed when three fatty acid molecules are esterified into one glycerol molecule. Fatty acid is one of the major energy sources of the body. Phospholipids is an integral part of cell membrane where two hydroxyl groups are esterified with a fatty acid but the third hydroxyl group is esterified with a phosphorus-containing ester. Cholesterol is an integral part of the cell membrane and is a precursor of steroid hormones and bile acids. Most cholesterol in the circulation is in the form of cholesterol ester, where the hydroxyl group is esterified with a fatty acid. Another lipid found in cell membranes is sphingolipid, this are formed when amino alcohol sphingosine is esterified with fatty acids. When sphingosine is bound to one fatty acid molecules containing 18 or more carbons, it is called ceramide. When a ceramide is bound to phosphocholine, it forms sphingomyelin. Sphingolipids are complex molecules that play a role in cell-to-cell communication.When a lipoprotein is void of lipids, they are called apolipoprotein (Apo) and this can be classified into various groups: Apo A, Apo B, Apo C, Apo D and Apo E. Apo A is composed of Apo AI and Apo AII. The most abundant is large Apo B called Apo B-100, while the less abundant is the smaller particle known as Apo B-48. Apo C has three forms, Apo CI, Apo CII and Apo CIII.6.2 CLASSES OF LIPOPROTEINSLipoproteins are classified based on their density following ultracentrifugation of serum. Ultracentrifugation is the gold standard for separation and analysis of plasma lipoprotein fractions. The major lipoproteins found in plasma are as follows (arranged in decreasing density): chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL).6.3 LIPID METABOLISMThere are two sources of lipids in the human body: exogenous lipids from diet and endogenous lipids that are synthesized mostly in the liver. Dietary triglycerides are broken down into fatty acids, glycerol, and monoglycerides in the small intestine. Triglycerides are resynthesized and are then incorporated into chylomicrons to enter systematic circulation. Troglycerides found in VLDL are hydrolyzed in the liver to form IDL. Chylomicrons, which are the major transport form of exogenous triglycerides, contain very small amounts of lipoproteins. In the circulation, lipoprotein lipase breaks down the triglyceride component of chylomicron into glycerol and free fatty acids. Apo CII, which is present in chylomicron, plays an important role in activating lipoprotein lipase. The resultant particle is a chylomicron remnant that is quickly removed by hepatic lysosomes. Free fatty acids generated during catabolism of chylomicrons are taken up by cells for oxidation to produce energy or can be utilized for re-synthesis of triglycerides for storage. In this process chylomicron particles are converted into chylomicron remnants. If chylomicrons are present, they are found to float at the top of serum of plasma as a creamy layer. Synthesized cholesterol is released into circulation as lipoprotein and 70% of it is esterified because it can be more readily transported by lipoproteins.6.4 LOW DENSITY LIPOPROTEIN METABOLISMLow density lipoprotein has the highest amount of cholesterol. It is taken up by tissues with LDL receptors. Apo B-100 interacts with LDL receptors present mostly in liver. If Apo B-100 is defective, uptake of LDL by LDL receptor is impaired. Patients with familial hypercholesterolemia have a defect in the gene that codes for the LDL receptor thus unable to uptake LDL causing elevated plasma cholesterol level. 6.5 HIGH DENSITY LIPOPROTEIN METABOLISMHigh density lipoprotein is produced by the liver. It can be classified into sub-classes, HDL2 and HDL3. The major role of HDL is to remove cholesterol from the peripheral cells and then return it to the liver for excretion, a pathway called reverse cholesterol transport. 6.6 LIPID PROFILE AND RISK OF CARDIOVASCULAR DISEASECardiovascular diseases are the leading causes of morbidity and mortality throughout the world. Several studies have demonstrated the link between elevated cholesterol levels and risk of cardiovascular diseases. Un-modifiable risk factors of developing cardiovascular diseases include sex, age (male > 45 years, female > 55 years), postmenopausal woman, family history and genetic factor. Modifiable risk factors comprise abnormal lipid profile, hypertension, diabetes, smoking, obesity (> 20% of ideal body weight), physical inactivity, excessive alcohol intake, poor diet, and excessive stress. According to WHO, majority of cardiovascular diseases can be prevented by risk factor modification and a change in lifestyle. The link between high total cholesterol and low HDL cholesterol and risk for cardiovascular disease, but elevated triglyceride was thought to play little role in elevating risks of cardiovascular disease. However, later reports observed the link between elevated triglycerides and a risk of heart disease. Although desirable total cholesterol level of less than 200 mg/dL is universally accepted, guidelines for desirable LDL level have changed significantly overtime. Some reports tell that the desirable LDL cholesterol in high-risk patient is around 70 mg/dL because LDL cholesterol level is the most important in predicting the risk for cardiovascular diseases. Atherosclerosis has been observed in individuals with low LDL cholesterol levels (90130 mg/dL). The most recent guidelines consider serum triglycedride concentrations of 150 mg/dL or less. When triglycerides levels are greater than 200 mg/dL, risk of developing cardiovascular disease also increased. Increased triglyceride levels are mainly caused by overweight and obesity, excessive alcohol intake, very high carbohydrate diet, type 2 diabetes mellitus and nephritic syndrome, certain drug therapies and genetic factors. Hypertriglyceridemia is also at risk for acute pancreatitis. Epidemiological studies have shown a correlation between low HDL cholesterol and higher risk of cardiovascular disease; high HDL cholesterol is associated with lower risk. Low HDL cholesterol is encountered in individuals with high triglycerides. Low HDL cholesterol is usually caused by high serum triglycerides, obesity and physical activity, cigarette smoking, type 2 diabetes mellitus, certain drug therapies and genetic factors. Non-HDL cholesterol is equal to the difference between total cholesterol and HDL cholesterol. All non-HDL cholesterol includes all lipoproteins that contain Apo B, the major atherogenic lipoprotein. Total cholesterol-to-HDL cholesterol ratio is also used for calculating risk factors for cardiovascular disease. Usually if the ratio is above 5, it is considered high. Comparably, Apo B/Apo A1 ratio has also been used for calculating risk of cardiovascular disease. Lipoprotein (a), also known as Lp(a) is structurally related to LDL because both particles contain Apo B. It is a modification of LDL with the addition of the lipoprotein angtigen.6.7 VARIOUS TYPES OF HYPERLIPIDEMIAHyperlipidemia is also called as hyperlipoproteinemia and can be primary or secondary in origin. Several primary hyperlipidemia include: Familial hypercholesterolemia, polygenic hypercholesterolemia, familial hyperytriglyceridemia, familial hyperchylomicronemia, familial dysbetalipoproteinemia and familial combined hyperlipidemia. Secondary hyperlipidemia is common, and is caused by diabetes mellitus, hyperthyroidism, nephritic syndrome, cholestasis, and alcohol abuse. Lipid disorders are also classified according to the Fredricksons classification. There are five types of hyperlipidemia in this classification: Type I (elevated chylomicrons due to lipoprotein lipase or Apo CII deficiency), Type IIa (elevated LDL cholesterol and total cholesterol due to familial hypercholesterolemia, polygenic hypercholesterolemia, familial combined hyperlipidemia, nephritic syndrome and hypothyroidism), Type IIb (elevated LDL and VLDL due to familial combined hyperlipidemia), Type III (elevated IDL called dysbetalipoproteinemia), Type IV (elevated VLDL seen in hypertriglyceridemia or familial combined hyperlipidemia), and Type V (elevated VLDL and chylomicrons causing elevated triglycerides).6.8 VARIOUS TYPES OF HYPOLIPIDEMIAHypolipidemia, also known as hypolipoproteinemia, are also classified as primary and secondary. Secondary hypolipidemia can be seen in severe liver disease, protein malabsorption, and energy and malnutrition states. Primary hypolipidemia includes Tangier disease, abetalipoproteinemia, familial hypobetalipoproteinemia, and chylomicron retention disease.6.9 NEWER LIPID PARAMETERS AND OTHER FACTORS RELATED TO RISK FOR CARDIOVASCULAR DISEASEIn addition to traditional lipid parameters such as cholesterol, triglycerides, HDL cholesterol and Lp(a), there are also lipid markers and non-lipid markers that can be use for assessing risk of cardiovascular disease. These markers include lipoprotein-associated phospholipase, LDL particle size, C-reactive protein, homocysteine and myeloperoxidase. Lipoprotein-associated phospholipase catalyzes oxidized phospholipid found in LDL to lysophosphatidylcholine and oxidized fatty acid. C-reactive protein is also a predictor for risk of cardiovascular disease, 3 mg/L with high risk for cardiovascular disease.6.10 LABORATORY MEASUREMENTS OF VARIOUS LIPIDSLipid profiles that consist of total cholesterol, triglyceride, HDL, and LDL are measured routinely, Blood specimens should be collected after an overnight fast of 10-12 hours. In serum, the majority of cholesterol exists as cholesterol ester. Therefore, the first step cholesterol ester is hydrolyzed by cholesterol ester hydrolase enzyme. HDL is usually measured as HDL cholesterol after precipitating out other lipoprotein fractions. For serum triglyceride measurement, lipase enzyme is used, which converts triglyceride into glycerol and free fatty acids. Elevated chylomicrons cause the plasma to appear as milky, and when plasma is allowed to stand, a creamy layer is visible at top. Elevated triglyceride causes the entire plasma to appear turbid. 6.11 DRUGS FOR TREATING LIPID DISORDERSSeveral drugs are available for treating lipid disorders, including statins, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, nicotinic acid, fibrates and bile acid sequestering agents.

Chapter 7Carbohydrate Metabolism, Diabetes and Hypoglycemia7.1 CARBOHYDRATES: AN INTRODUCTIONCarbohydrates are important for the human physiology because glucose provides more than half of the total energy requirements of the human body. Glucose is a breakdown product of dietary carbohydrates. Carbohydrates are often referred to as saccharides and can be sub-divided into four categories: Monosacharides simplest carbohydrates that cannot be further hydrolyzed Disaccharide when two monosacchride are joined together and it can be hydrolyzed to monosaccharides. Polysaccharides complex carbohydrates that contains 200-2500 monosacchride molecules. Oligosaccharides less complex molecules than polysaccharides that contains less than ten monosacchrides.7.2 REGULATION OF BLOOD GLUCOSEGlucose concentrations in the blood are tightly controlled by the biochemical processes of the body. Increase blood glucose concentration is encountered in patients with diabetes. The major biochemical processes involved in the regulation of blood glucose concentration includes glycolysis (breakdown of glucose to pyruvate and lactate), Glycogenesis (conversion of glucose into glycogen), Glycogenolysis (breakdown of glycogen to glucose), and gluconeogenesis (formation of glucose from non-carbohydrate sources). The major hormones involved in blood glucose regulation are insulin and glucagon. Insulin is secreted by the beta cells of the islets of langerhans in the pancreas. Proinsulin is cleaved to form insulin and c-peptide. The normal ratio of c-peptide to insulin is 5:1. Insulin has several functions in regulation of blood glucose level which includes cellular uptake of glucose, glycogen and protein synthesis and synthesis of fatty acid and triglycerides and it inhibits glycogenolysis, gluconeogenesis, proteolysis and lipolysis. Glucagon is secreted by the alpha cells of the islets of langerhans in the pancreas. It has the exact opposite function to that of insulin. Thus, it increases gluconeogenesis, glycogenolysis and lipolysis. Somatostatin is a hormone found in various organs but mostly in hypothalamus and in delta cells of islets of langerhans in the pancreas; its function is to regulate secretion of insulin and glucagon.7.3 DIABETES MELLITUS: BASIC CONCEPTDiabetes mellitus is a condition characterized by hypoglycemia due to relative insulin deficiency or insulin resistance. It can be primary or secondary in nature. Primary diabetes mellitus can be monogenic or polygenic. Monogenic diabetes mellitus is characterized by impaired insulin secretion by beta cells of the pancreas. It accounts for 2-5% of all diabetes mellitus and is less common than type1 and type2 diabetes mellitus, which are the most common forms of diabetes mellitus encountered in clinical practice. Polygenic diabetes mellitus can either be type 1 and type 2. Type 1 diabetes mellitus is characterized by an absolute deficiency of insulin due to islet cell destruction while type 2 diabetes mellitus is characterized by insulin resistance and beta cell dysfunction. There is also secondary diabetes, which may be drug-related or due to various diseases.7.4 MONOGENIC DIABETES MELLITUSMaturity onset diabetes of the young (MODY) is the most common form of monogenic diabetes. It is characterized by young age onset of diabetes and a marked family history of diabetes in every generation due to autosomal dominant inheritance. Many genetic mutation have been reported in patients with MODY, but most commonly encountered mutation are due to mutation is gene encoding the enzyme glucokinase (GCK) and mutations of genes encoding nuclear transcription factors of the hepatocyte nuclear factor (HNF). MODY can further be classified as MODY 1, 2 and 3, with MODY 3 the most commonly encountered. Neonatal Diabetes Mellitus is a type of monogenic diabetes mellitus that occurs up to the age of six months due to mutation of different genes involved in organogenesis.

7.5 TYPE 1 DIABETES MELLITUSType 1 diabetes mellitus, formerly known as insulin-dependent diabetes mellitus or juvenile onset diabetes, is encountered in 5-10% of all patients with diabetes mellitus and is characterized by polyuria, polydipsia and rapid weight loss. This type of diabetes mellitus is due to autoimmune destruction of pancreatic beta cells by T-lymphocytes. Usually 85-90% of patients with type 1 diabetes mellitus have one or more autoantibodies. Individuals with antibodies are classified as Type 1A, and other individuals with type 1 diabetes mellitus without any autoantibodies or any known etiology of islet cell destruction are classified as type 1B. Children and adolescents may first present with ketoacidosis as the manifestation of the disease. Patients are usually dependent on insulin as very little or no insulin is produced.7.6 TYPE 2 DIABETES MELLITUSType 2 diabetes mellitus, formerly called as non-insulin dependent diabetes mellitus is the most common form of diabetes mellitus and account for over 90% of all cases. It is described by insulin resistance, and may also be accompanied by beta cell dysfunction causing insulin deficiency. Ketoacidosis seldom occurs in type 2 diabetes mellitus, is usually occurs secondary to stress factors such as infection.7.7 METABOLIC SYNDROME OR SYNDROME X Metabolic syndrome or syndrome X is characterized by insulin resistance, hyperinsulinemia, hyperglycemia, dyslipidemia, and arterial hypertension. It was first described in 1988 by Gerald Raeven. Individuals with this syndrome have increased risk of developing coronary heart disease, stroke and type 2 diabetes mellitus. Blood institute (AHA/NHBLI) criteria for metabolic syndrome may include three or more of the following risk factors which are central obesity, insulin resistance, elevated triglycerides, reduced high density lipoprotein cholesterol and elevated blood pressure. 7.8 COMPLICATIONS OF DIABETESDiabetes complication can be divided into two broad categories: acute and chronic complications. Acute complications include diabetic ketoacidosis, hyperosmolar non-ketosis, and lactic acidosis. Chronic complications can either be macrovascular or microvascular. Diabetes ketoacidosis may be the presenting feature of type 1 diabetes mellitus which is fatal is not treated on time. Absence of insulin also leads to release of fatty acids, mostly from adipose tissue causing generation of excess fatty acids that result in the formation of ketone bodies, including acetoacetic acid, beta hydroxybutyric acid and acetone. Diabetic ketoacidosis is more commonly encountered in patients with type 1 diabetes mellitus, although under certain circumstances, severe stress diabetic ketoacidosis may also be observed in patients with type 2 diabetes. Hyperosmolar non-ketosis is usually seen in patients with type 2 diabetes mellitus. It is characterized by hyperglycemia with high plasma osmolality and dehydration. Macrovascular complications of diabetes mellitus are related to atherosclerosis and diabetic patients are at risk for cardiovascular disease.7.9 SECONDARY CAUSES OF DIABETES MELLITUSGestational diabetes mellitus is typically seen during the second and third trimester of pregnancy. It is most likely due to increased levels of hormones such as estrogen, progesterone, cortisol etc., which counteract the action of insulin.7.10 DIAGNOSTIC CRITERIA FOR DIABETESThe classic clinical presentation of diabetes mellitus in patients includes polyuria, polydipsia and weight loss. The American Diabetes Association (ADA) recommends screening for diabetes mellitus for any individual over 45 years of age. Fasting blood glucose and glycosylated hemoglobin A1C are the best criteria for diagnosis of diabetes mellitus. Normal fasting glucose level should be 70-99 mg/dL. Individuals with fasting glucose level between 100 and 125 mg/dL are classified as having impaired fasting glucose. The criteria for diagnosis of diabetes mellitus are fasting plasma glucose level of 126 mg/dL or higher on more than one occasion with no calorie intake in the last eight hours. In addition, a glucose tolerance test after an oral dose of 75 gm of glucose, with two-hour plasma glucose level of 200 mg/dL or higher, indicates diabeted mellitus. For diagnosis of gestation diabetes mellitus, typically glucose tolerance test is performed using 75 g of oral anhydrous glucose during week 24-28 of gestation in pregnant women and glucose tolerance test is typically performed in the morning after at least eight hours of overnight fasting. 7.11 HYPOGLYCEMIAHypoglycemia is generally defined as blood glucose level below 50-60 mg/dL. Epinephrine is mostly responsible for symptoms of hypoglycemia, including trembling, sweating, lightheadedness, hunger and possibly epigastric discomfort. Hypoglycemia can be related to prolonged fasting, but other disease correlations may participate in such condition. Postprandial or reactive hypoglycemia may be related to insulin therapy, inborn error of metabolism, or other causes. Hypoglycemia can occur in type 1 or type 2 diabetes mellitus but is more common in type 1 diabetic patients receiving insulin. Individuals with insulinomas can also result to hypoglycemia. Insulinomas are tumor of the insulin-secreting beta cells of the islet of langerhans in the pancreas where in insulin and C-peptides are high.7.12 LABORATORY METHODSIn the clinical laboratory, glucose concentration is usually measured in serum or plasma. The best method in collecting sample for glucose determination is to aspirate venous blood in tubes containing sodium fluoride and potassium oxalate because sodium fluoride inhibits glycolysis and the glucose level is stable for upto three days at room temperature. Measurement of glucose can be done using either the hexokinase method or the glucose oxidase method. The hexokinase method is considered as the reference method. 7.13 GLUCOSE METERSSince the 1980s, glucometers have been available for blood glucose monitoring both in point of care testing sites and in home monitoring of glucose. A sample of blood is placed on the test pad and then this test strip is inserted into the meter, after a short period of time, a digital reading is taken. Glucometers also utilize glucose oxidase, hexokinase or glucose dehydrogenase with pyrroquinoline quinine cofactor or glucose oxidase combined with nicotinamide adenine dinucleotide for glucose measurement. Major limitations of glucometers include inaccuracy of measurement compared to the reference glucose method. Maltose falsely increases glucose value if the glucometer is based on the glucose oxidase method.

Chapter 10Liver Disease and Liver Function Test10.1 LIVER PHYSIOLOGYLiver is the largest internal organ of the body, approximately 1.2 to 1.5 kg in weight. A functioning normal liver produced 10-12 g of albumin per day. In addition to it, all clotting factors are also produced in the liver except factor VIII. Therefore, if liver function is impaired, production of albumin and clotting factors is also impaired that may result to severe hypoalbuminuria and prolonged prothrombin time (PT). The liver also stores about 80 g of glycogen. Liver release glucose into the circulation by gluconeogenesis and glycogenolysis thus liver disease may cause hypoglycemia due to decreased glucose supply. Other functions of liver include lipoprotein synthesis, cholesterol synthesis, bile acid synthesis, and bilirubin metabolism.10.2 LIVER FUNCTION TESTS AND INTERPRETATIONSThe conventional liver function test (LFT) consists of determination of blood levels of bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT). Prolonged prothrombin time indicates significant impairment of liver function. Most of serum bilirubin is unconjugated (indirect bilirubin). However, unconjugated bilirubin may be increased in hemolytic anemia. Breakdown of hepatocytes results in the release of ALT and ALP into the blood. ALP levels are increased during cholestasis which results in elevated activity of ALP in the blood. Measurement of GGT or 5-nucleotidase levels can be used to determine if the source of ALP is the liver or not because GGT and 5-nucleotidase are only produced by biliary epithelium. GGT is also a well-established marker for alcohol abuse. In acute liver disease both total protein and serum bilirubin concentrations are unaltered. In chronic liver disease total protein may be low or high. If total protein is high it is more likely due to polyclonal hypergammaglobulinemia. Prothrombin time (PT) reflects extrinsic and common pathway activity of coagulation cascade.10.3 JAUNDICE: AN INTRODUCTIONJaundice is defined as yellow discoloration of skin and sclera. It is associated with hyperbilirubinemia. Jaundice is commonly caused by congenital hyperbilirubinemia, hemolytic jaundice, hepatocellular jaundice, and cholestatic jaundice.10.4 CONGENITAL HYPERBILIRUBINEMIACongenital hyperbilirubinemia may be caused by Gilberts syndrome, Crigler-Najjar syndrome, Dubin-Johnson syndrome and Rotors syndrome. Gilberts syndrome is associated with reduced level of uridyldiphosphoglucoronyl transferase (UDPGT) leading to elevated unconjugated bilirubin that increases with fasting. Crigler-Najjar syndrome can be classified in to two types: Crigler-Najjar Type I and type II. Crigler-Najjar Type I is characterized by total absence of UDGPT while Crigler-Najjar Type II is described by a reduced level of UDGPT but can be treated with enzyme inducers. Dubin-Johnson syndrome is associated with impaired excretion of conjugated bilirubin, causing conjugated hyperbilirubinemia. Rotors syndrome is also characterized by impaired excretion of conjugated bilirubin.10.5 HEMOLYTIC (PREHEPATIC) JAUNDICEHemolytic jaundice is due to hemolytic anemias. In hemolytic jaundice, increased unconjugated bilirubin and reticulocytosis are accompanied by normal liver enzyme levels. However, lactate dehydrogenase (LDH) levels may be high due to increased destruction of red blood cells. It may be observed due to hematoma absortion, or after blood transfusion.10.6 HEPATOCELLULAR JAUNDICEHepatocellular jaundice may be seen in patients with acute hepatitis or chronic liver disease.

10.7 CHRONIC LIVER DISEASECommon causes of chronic liver disease are chronic alcohol intake, chronic infection with hepatitis B and D, or hepatitis B or C alone. Other causes include autoimmune liver disease, primary liver cirrhosis, hemochromatosis, Wilsons disease, and alpha-1 antitrypsin deficiency. In chronic liver disease, moderate to severe hypoalbuminemia is commonly observed, but the other liver function tests may be normal or abnormal depending on the severity of illness.10.8 CHOLESTATIC JAUNDICECholestatic jaundice can be classified into two broad categories: intrahepatic and extrahepatic. Intrahepatic cholestatic jaundice is due to impaired hepatobiliary production and excretion of bile causing bile components to enter the circulation. Extrahepatic cholestasis may be related to gallstones, a malignancy such as a pancreatic tumor, pancreatitis, or can be secondary to surgical procedure. 10.9 ALCOHOL- AND DRUG-INDUCED LIVER DISEASEAlcohol abuse can produce a spectrum of liver diseases that include fatty changes in the liver, alcoholic hepatitis and eventually liver cirrhosis. Cytoplasmic inclusions called Mallory bodies may be seen. Heavy drinking for even only a few days can produce fatty changes in the liver, which can be reversed after abstinence. Women are more susceptible to alcoholic liver disease than men.Liver is the major site of drug metabolism. Drugs are converted into more water-soluble forms through drug metabolism so that drug metabolites can be excreted in bile or urine. Drugs that cause liver damage may do so in a dose-dependent or dose-independent manner. An example of drug that causes dose-dependent hepatotoxicity is acetaminophen. Other drugs may also cause liver damage, such as azathioprine, methotrexate, chlorpromazine, erythromycin, and statins. Reyes syndrome is a potentially fatal disease observed mostly in children. If aspirin is given to infant or a child, it could cause Reyes syndrome due to inhibition of beta-oxidation of fatty acids in the mitochondria and uncoupling of oxidative phosphorylation. There is a microvesicular fatty infiltration of the liver. Mortality rate is high.10.10 LIVER DISEASE IN PREGNANCYHyperemesis gravidarum is a condition during pregnancy that is associated with nausea, vomiting and dehydration. The cause of this condition is still controversial, but it may be related to hormonal changes during pregnancy. Intrahepatic cholestasis of pregnancy is a liver-specific disorder characterized by maternal pruritus observed in the third trimester. The etiology of this disease is not fully elucidated, but may occur due to cholestatic effects of reproductive hormones. Acute fatty liver of pregnancy is a rare but potentially life-threatening condition that usually occurs in the third trimester with a mean gestational age of 35-36 weeks or may also be seen in early postpartum period. This disease may be linked to an abnormality in fetal fatty acid metabolism. HELLP syndrome (H, hemolysis; EL, elevated liver enzyme; LP, low platelet count) is a complication of pregnancy that occurs mostly on patients with severe preeclampsia or eclampsia.10.11 LIVER DISEASE IN NEONATES AND CHILDRENPhysiological jaundice is observed in neonates due to decreased activity of UDGPT leading to unconjugated hyperbilirubinemia. Neonatal hepatitis can occur due to infection with cytomegalovirus, rubella, or toxoplasma. Biliary atresia can also cause jaundice in infants. Metabolic disorders such as tyrosinemia and galactosemia also cause jaundice. Alagille syndrome is a genetic disorder affecting the liver, heart, kidney, and other organs, and problems associated with this disorder first appear in infancy or early childhood.10.12 MACRO LIVER ENZYMESIsolated and unexplained elevated levels of liver enzymes such as AST are observed due to AST binding with serum IgG. This bound enzyme is referred to macro AST. Binding of ALT with IgG has also been reported. Another enzyme that can bind to IgG is creatinine kinase (CK), giving rise to macro CK. Macro AST may also be detected in patients with chronic hepatitis or malignancy.

10.13 LABORATORY MEASUREMENT OF BILIRUBIN AND OTHER TESTSFor determination of bilirubin, it is important to protect the specimen from light because conjugated and unconjugated bilirubin is photooxidized. For measuring conjugated bilirubin, serum or plasma is acidified then mixed with diazotized sulfanilic acid to produce azobilirubin then the reaction is stopped and alkalinized the solution, the azobilirubin produces more intense blue color, which is measured colorimetrically. The difference between total bilirubin and direct bilirubin is equal to indirect bilirubin. Liver biopsy is often done to establish a diagnosis of cirrhosis. One such test measures levels of procollagen type (III) peptide (PIIINP) in blood. Levels are increased in cirrhosis. However, levels can also be increased in inflammation and necrosis.

Chapter 11Renal Function Tests11.1 BASIC FUNCTIONS OF KIDNEYSKidneys are a paired organ system located in the retroperitoneal space. Renal blood supply represents roughly 25% of cardiac output. Nephron is the functional unit of the kidneys and it composes of glomerulus, proximal tubules, loop of henle, distal tubule and collecting duct. Kidneys have three vital functions namely excretory function, regulatory function and endocrine functions. It is also in charge for urine formation and excretion of end products of metabolism. Glomerulus is the site of filtration. The basement membrane of the capillaries serves as the barrier to passage of large proteins. Approximately two-thirds of the filtrate volume is reabsorbed in the proximal tubules. Loop of henle is the site where urine is concentrated. At the distal tubules, sodium and chloride are reabsorbed while potassium and hydrogen are excreted. The collecting duct is the site of further reabsorption which is influenced by antidiuretic hormone. The kidneys also produce two hormones: erythropoietin and renin. Erythropoietin is produced secondary to hypoxia and acts primarily on the bone marrow to stimulate erythropoiesis. Renin is produced by the juxtaglomerular apparatus which converts angiotensinogen to angiotensin I, which will further be converted to angiotensin II by angiotensin-converting enzyme. Angiotensin II is a vasoconstrictor and stimulator of aldosterone production by adrenal cortex. Aldosterone is a hormone that promotes water and sodium reabsorption. The kidneys also produce vitamin D that is essential for absorption of calcium.11.2 GLOMERULAR FILTRATION RATEGlomerular filtration is one of the major functions of the kidneys. Glomerular filtration rate is a measure of the function capacity of the kidneys and is an important parameter to assess kidney function. Estimation of glomerular filtration rate by insulin clearance is considered to be the gold standard. In order to compute glomerular filtration rate based on the creatinine clearance, a 24-h urine collection is recommended which should be from one morning to void the next days morning void. 11.3 CREATININE CLEARANCESCreatinine is a waste product derived from creatine and phosphocreatine. Its production is related to muscle mass of an individual. It is transported into the blood to the other organs, especially the brain and muscles, where it is phosphorylated to phosphocreatine. Phosphocreatine is a high-energy compound and interconversion of phosphocreatine to creatinine is important for muscular function. Women usually excrete 1.2 g of creatinine per day while men excrete 1.5 g/day.11.4 CHRONIC KIDNEY DISEASEKidney function depends on age. Renal function declines after the age of 40 and declines ever further after the age of 65. Early diagnosis of kidney disease may delay or even prevent end-stage renal disease. The following criteria are adopted to describe chronic kidney disease: creatinine clearance more than 90mL/min/1.73 m2, presence of kidney damage for three months, and glomerular filtration rate below 15 mL per minute. Fractional excretion of sodium, a measure of percent of filtered sodium that is excreted in urine, is also useful in diagnosing renal failure. Fractional excretion of sodium level less than 1% is indicative of pre renal disease, a velue of 3% is indicative of acute tubular narcosis or urinary tract obstruction.11.5 CYSTATIN C Cystatin C is a low-molecular weight protein that can be used in calculating glomerular filtration rate. It has been recognized that plasma cystatin C is a better marker of kidney failure than creatinine. The reference value of plasma cystatin C is 0.5-1.0 mg/L.

11.6 UREA (BLOOD UREA NITROGEN) AND URIC ACIDAlthough serum creatinine and cystatin C are more commonly used to assess renal function, urea (blood urea nitrogen) and uric acid measurements also have some clinical values. Urea is the result of catabolism of proteins and amino acids. Measurement of urea levels is inferior when assessing renal function because serum or plasma concentration of urea may be increased in the following situations: dehydration, hypoperfusion of kidneys, high-protein diet, protein catabolism, and steroid administration. Uric acid is produced from transformation of xanthine and hypoxanthine by xanthine oxidase. Increased uric acid level in the blood can be associated in gout and can cause the formation of renal stones. However, the serum level of uric acid may also be elevated due to decrease function as observed in patients with renal failure. 11.7 PROTEINS IN URINE AND PROTEINURIATotal urinary protein is