Clinical Enzymology 2 PDF

Clinical Enzymology2 Saeedeh Salimi Professor of clinical biochemistry Saeedeh Salimi 1  Alkaline phosphatase ALP ALPs are a type...

Clinical Enzymology2 Saeedeh Salimi Professor of clinical biochemistry Saeedeh Salimi 1  Alkaline phosphatase ALP ALPs are a type of hydrolase ALPs represent a family of enzymes coded for by different genes. Their physiologic role is not completely understood. The most abundant plasma ALP isoforms are the tissue-nonspecific isozyme found in the kidney, liver, and bone. However, in different tissues, this parent isozyme is subjected to different posttranslational modifications, resulting in differences in their carbohydrate side chains. Two other genes on chromosome 2 code for ALP of placental and intestinal origin; another gene on chromosome 2 codes for the so-called germ cell or placental-like isoenzyme, which has some antigenic and physical similarities to the placental isozyme Saeedeh Salimi 2 Saeedeh Salimi 3  Alkaline phosphatase ALP In cells, ALP is primarily bound to cell membranes, where it appears to be involved in the cleavage of phosphate-containing compounds and may facilitate the movement of substances across cell membranes. Hepatocytes produce ALP in the liver, where it is found attached to the canalicular surface of the cells Osteoblasts produce bone ALP, which appears to be involved in the cleavage of pyrophosphate, an inhibitor of bone mineralization. Intestinal epithelial cells produce intestinal ALP, which is released into the intestine following ingestion of fatty foods. Saeedeh Salimi 4  Some divalent ions, such as Mg2+, Co2+, and Mn2+, are activators of the enzyme, and Zn2+ is a constituent metal ion. Phosphate, borate, oxalate, and cyanide ions are inhibitors of ALP activity. Saeedeh Salimi 5  Alkaline phosphatase ALP There appear to be different mechanisms for the release of ALP from cells, leading to varying forms of ALP in plasma. With liver injury, ALP synthesis increases, but bile acids dissolve fragments of canalicular cell membranes with attached enzymes (including ALP, GGT, leucine aminopeptidase, and 5′-NT) In normal serum, a single form (of liver or bone origin) of ALP is typically seen. However, with hepatobiliary disease, both the normal product and the membrane-attachment form (high molecular weight) bound to lipoproteins can be found. The intestinal isoenzyme of ALP is released in large amounts into duodenal fluid, and large amounts enter lymphatic fluid, draining the intestinal tract following a meal. Saeedeh Salimi 6  Alkaline phosphatase ALP The half-lives of isoenzymes of ALP differ significantly. Thus, it is necessary to know the isoenzyme that is elevated before the rate of clearance can be evaluated: intestine, minutes; bone, 1 day; liver, 3 days; and placenta, 7 days. Day-to-day variation in total ALP is 5% to 10%, although the bone isozyme shows 20% day-to-day variability. Saeedeh Salimi 7 Saeedeh Salimi 8  Alkaline phosphatase ALP Clinical Significance Increases in serum ALP activity commonly originate from one or both of two sources: liver and bone. hepatobiliary disease and bone disease The response of the liver to any form of biliary tree obstruction induces the synthesis of ALP by hepatocytes. Higher ALP activity in extrahepatic obstruction (eg, by stone, by cancer of the head of the pancreas) than in intrahepatic obstruction. Saeedeh Salimi 9  Alkaline phosphatase ALP Serum enzyme activities may reach 10 to 12 times and usually return to baseline on surgical removal of the obstruction. A similar increase is seen in patients with advanced primary liver cancer or widespread secondary hepatic metastases. ALP increase in patients with primary biliary cirrhosis. Liver diseases that principally affect parenchymal cells, such as infectious hepatitis, typically show only moderately (less than threefold) increased or even normal serum ALP activities. Saeedeh Salimi 10 Saeedeh Salimi 11  Alkaline phosphatase ALP An increase of up to two to three times URL is observed in women in the third trimester of pregnancy, with the additional enzyme being of placental origin. Transient, benign increases in serum ALP may be observed in infants and children Saeedeh Salimi 12  Alkaline phosphatase ALP These carcinoplacental isoenzymes (eg, Regan isoenzyme) appear to result from de- repression of the placental ALP gene. Tumors have also been found to produce ALPs that appear to be modified forms of nonplacental isoenzymes (Kasahara isoenzyme). Saeedeh Salimi 13  Alkaline phosphatase ALP Increased osteoblastic activity in Paget disease, osteosarcoma, tumor metastatic to bone, and metabolic bone disease are the most common causes of elevated bone isoenzyme. Occasionally, patients will have elevations of both bone and liver isoenzymes, especially in metastatic carcinoma. Placental alkaline phosphatase (PLAP) is a useful tumor marker in serum and cerebrospinal fluid (CSF) for most germ cell tumors. Saeedeh Salimi 14 Saeedeh Salimi 15 Saeedeh Salimi 16 Saeedeh Salimi 17 Saeedeh Salimi 18 Saeedeh Salimi 19 Saeedeh Salimi 20 Saeedeh Salimi 21 Saeedeh Salimi 22 Saeedeh Salimi 23  Alkaline phosphatase ALP Reference Ranges and Preanalytic Variation Reference ranges for ALP are highly dependent on age and gender. During childhood, levels gradually rise higher in boys than in girls. The higher values in children are due to the bone isoenzyme. After menopause, the bone isoenzyme increases slightly in women Pregnancy causes a two- to threefold increase in ALP, mainly due to the placental isoenzyme, but also because of an increase in bone isoenzyme. Saeedeh Salimi 24  Alkaline phosphatase ALP Reference Ranges and Preanalytic Variation A number of other factors affect ALP levels as well. High body mass index is associated with a 10% average increase in ALP Oral contraceptives decrease ALP fibric acid derivatives decrease total ALP by 25% and the liver isoenzyme by 40%. Antiepileptic agents commonly cause increased total ALP, mainly because of increases in the liver isoenzyme. However, in some cases, the bone isozyme may also be elevated Smoking causes an average 10% increase in total ALP as the result of pulmonary production of placental-like ALP Blood transfusion and cardiopulmonary bypass decrease alkaline phosphatase, often causing low levels this may be due to chelation of necessary cations by citrate. Saeedeh Salimi 25  Alkaline phosphatase ALP Saeedeh Salimi 26  IsoEnzymes Electrophoresis Thermal Resistance Selective Inhibition Saeedeh Salimi 27  IsoEnzymes Electrophoresis HPLC six different isoforms These are bone/intestinal (B/I), two bone isoforms, called B1 and B2, and three liver isoforms termed L1, L2, and L3. Thermal Resistance The most heat-stable isoenzyme is placental (and germ cell) ALP; the liver isoenzyme is moderately stable, and the bone isoenzyme is the most heat labile. Selective Inhibition phenylalanine reduces reactivity of intestinal and placental isoenzymes, and levamisole inhibits bone and liver isoenzymes. Immunoassays for bone and placental isoenzymes of ALP are available commercially Affinity for specific lectins Saeedeh Salimi 28 Saeedeh Salimi 29  Tissue non specific ALP(TNALP) Germ cell ALP(GCALP) Placental ALP(PALP) Intestinal Saeedeh Salimi 30  Sample collection Serum Plasma (Heparine) Fasting Hemolysis Saeedeh Salimi 31  5′-Nucleotidase 5NT 5′-Nucleotidase (EC 3.1.3.5; 5′-ribonucleotide phosphohydrolase; NTP) is a phosphatase that acts only on nucleoside-5′-phosphates, such as adenosine-5′-phosphate (AMP) and adenylic acid, releasing inorganic phosphate. 5′-Nucleotidase is a glycoprotein that is widely distributed throughout the tissues of the body and is principally localized in the cytoplasmic membrane of the cells in which it occurs (“ecto-5’-nucleotidase”). Its pH optimum is between 6.6 and 7.0 Saeedeh Salimi 32 Saeedeh Salimi 33  5′-Nucleotidase 5NT Clinical Significance Despite its ubiquitous distribution, serum NTP activities appear to reflect hepatobiliary disease with considerable specificity. NTP is increased three- to sixfold in those hepatobiliary diseases in which there is interference with the secretion of bile. This may be due to extrahepatic causes (a stone or tumor occluding the bile duct), or it may arise from intrahepatic conditions, such as cholestasis caused by chlorpromazine, malignant infiltration of the liver, or biliary cirrhosis. But, when parenchymal cell damage is predominant, as in infectious hepatitis, serum NTP activity is only moderately increased. Saeedeh Salimi 34  5′-Nucleotidase 5NT The assay of NTP activity has been considered of value as an addition to measurement of nonspecific total ALP in patients with suspected hepatobiliary disease, and increased NTP activity is routinely interpreted as evidence of a hepatic origin of increased ALP activity in serum. However, approximately half of the individuals in whom liver ALP activity is increased in serum may simultaneously show a normal NTP. On the other hand, increased NTP in the serum of patients with normal liver ALP is very often associated with the presence of liver disease. Thus, the frequent dissociation of the two enzyme activities supports the usefulness of determining both (liver) ALP and NTP to enhance diagnostic efficiency in patients with suspected liver disease. Saeedeh Salimi 35  5′-Nucleotidase 5NT Reference Interval The reference interval for NTP activity at 37°C is from 3 to 9 U/L, with no sex-related differences. Saeedeh Salimi 36  γ-Glutamyltransferase GGT. γ-Glutamyltransferase catalyzes the transfer of the γ-glutamyl group from peptides and compounds to an acceptor. The enzyme acts only on peptides or peptide-like compounds containing a terminal glutamate residue joined to the remainder of the compound through the terminal (-γ-) carboxyl. Glycylglycine is five times more effective as an acceptor than glycine or the tripeptide (gly-gly-gly), but little is known about the optimal properties of the acceptor cosubstrate. Saeedeh Salimi 37  γ-Glutamyltransferase GGT Saeedeh Salimi 38  γ-Glutamyltransferase GGT GGT is present (in decreasing order of abundance) in proximal renal tubule, liver, pancreas, and intestine. The enzyme is present in cytoplasm (microsomes), but the larger fraction is located in the cell membrane and may transport amino acids and peptides into the cell across the cell membrane in the form of γ-glutamyl peptides. GGT is critical for the maintenance of adequate intracellular concentrations of reduced glutathione, a major antioxidant agent. Saeedeh Salimi 39 Saeedeh Salimi 40 Saeedeh Salimi 41  γ-Glutamyltransferase GGT GGT activity in serum comes primarily from the liver, where it is predominantly found in the biliary pole of the hepatocyte. However, it is also found in both the cytosol and the smooth endoplasmic reticulum (where it is susceptible to induction). The enzyme in serum is heterogeneous with respect to both net molecular charge (eg, shown by electrophoresis) and size. These forms appear to derive from posttranslational modifications of a single type of enzyme molecule rather than resulting from the existence of true isoenzymes. For example, high molecular weight forms may represent the release of cell membrane fragments into the circulation. Despite numerous investigations, clear correlations between patterns of multiple forms and particular diseases cannot be discerned. Saeedeh Salimi 42  γ-Glutamyltransferase GGT Clinical Significance Even though renal tissue has the highest concentration of GGT, the enzyme present in serum appears to originate primarily from the hepatobiliary system. GGT is a sensitive indicator of the presence of hepatobiliary disease, being increased in most subjects with liver disease regardless of cause, but its usefulness is limited by lack of specificity. Similar to ALP, it is highest in cases of intrahepatic or posthepatic biliary obstruction, reaching activities some 5 to 30 times the URL. High increases of GGT are also observed in patients with primary or secondary (metastatic) liver neoplasm and other hepatic space-occupying lesions, presumably caused by intrahepatic obstruction. Saeedeh Salimi 43  γ-Glutamyltransferase GGT Moderate increases (two to five times the URL) occur in infectious hepatitis. Small increases in GGT activity are observed in more than 50% of patients with NAFLD, and similar but transient increases are noted in cases of drug intoxication. In acute and chronic pancreatitis and in some pancreatic malignancies (especially if associated with hepatobiliary obstruction), enzyme activity may be 5 to 15 times the URL. Saeedeh Salimi 44  γ-Glutamyltransferase GGT Increased activities of GGT are found in the sera of patients with alcoholic hepatitis and in the majority of sera from people who are heavy drinkers. GGT is also increased with increased body weight and obesity, and the effect of alcohol is more marked in these groups. Increased concentrations of the enzyme are also found in the serum of subjects receiving anticonvulsant drugs such as phenytoin and phenobarbital. Such an increase in GGT activity in serum may reflect induction of new enzyme activity by the action of the alcohol and drugs or their toxic effects on microsomal structures in liver cells. In acute MI, GGT activity is usually normal. If there is a rise, it occurs at about the fourth day, reaches a maximum value in another 4 days, and probably implies liver damage secondary to cardiac insufficiency Saeedeh Salimi 45  γ-Glutamyltransferase GGT Unlike ALP, serum GGT is not increased in conditions in which osteoblastic activity is increased, so the enzyme measurement can be useful in differentiating the source of ALP activity increase in serum, whether it is of bone or liver origin. Epidemiologic evidence has shown that serum GGT activity possesses an independent prognostic value for cardiovascular morbidity and mortality.53 Indeed, experimental work has documented that active enzyme is present in atherosclerotic plaques, and this appears related to the ability of GGT to mediate redox/pro-oxidant reactions at a cellular level. Saeedeh Salimi 46 Saeedeh Salimi 47  γ-Glutamyltransferase GGT Reference Intervals In adults, the URL for GGT activity in serum is 40 U/L for females and 70 U/L for males, when measured with an assay traceable to the IFCC reference procedure. Reference limits are approximately twofold higher in people of African ancestry. In normal full-term neonates, GGT activity at birth is approximately six to seven times the adult reference interval. The activity then declines, reaching adult values by the age of 5 to 7 months Saeedeh Salimi 48  Glutathione S-Transferase GST Cytosolic glutathione S-transferases (EC 2.5.1.18; GST) are dimeric enzymes that catalyze the nucleophilic addition of glutathione to the electrophilic centers of a wide variety of chemical structures, accomplishing detoxification reactions. In addition, GSTs exert part of the glutathione peroxidase activity and have an important function in intracellular binding and transport of a wide variety of both endogenous and exogenous compounds. Saeedeh Salimi 49  Glutathione S-Transferase GST Clinical Significance Unlike aminotransferases, which are found predominantly in the periportal hepatocytes, α-GST is evenly distributed across the liver acinus and therefore is released in all types of hepatocyte injury. In liver transplant recipients, α-GST was found to be more valuable than AST in detecting early rejection episodes postoperatively and less susceptible to the confounding effects of infection. Saeedeh Salimi 50 Saeedeh Salimi 51  Glutathione S-Transferase GST Methods for Determination of Glutathione S-Transferase Several problems have been associated with GST activity measurements. First, normal plasma activity is low and difficult to measure. Second, GST binds a number of anions, such as bile salts and bilirubin, that inhibit enzyme activity. Immunoassays have been described that allow the precise and specific measurement of α-GST concentrations. The only methodologic problem relates to the assay turnaround time, which takes several hours. Reference Interval Using a commercially available enzyme immunoassay for α-GST, the URL was 11.4 µg/L. Saeedeh Salimi 52  Acid Phosphatase ACP ACPs belong to the hydrolase class of enzymes and occur as several isoenzymes with a common enzymatic function (the hydrolytic breakdown of phosphate monoesters). ACP is present in lysosomes They all show optimal enzyme activity below a pH of 7.0. They possess some tissue specificity (greatest concentrations occur in prostate, liver, spleen, erythrocytes, and bone). The major forms are coded for by different genes and possess different molecular weights and structures, as well as differences in sensitivity to tartrate inhibition. Lysosomal, prostatic, erythrocyte, macrophage, and osteoclastic ACPs are five important types found in humans. Saeedeh Salimi 53 Saeedeh Salimi 54 Saeedeh Salimi 55 Saeedeh Salimi 56  Acid Phosphatase ACP Normally, concentrations in serum are low The activity of erythrocyte ACP can be distinguished from that of the other ACP isoenzymes in that it is inhibited by 2% formaldehyde solution and 1-mM cupric sulfate solution. In addition, a particular isoform of TRAP, called TRAP-5b, occurs predominantly in osteoclasts in bone marrow and is used as a marker for bone remodeling. Saeedeh Salimi 57  Tartrate-resistant acid phosphatase (TRAP or TRAPase) Glycosylated monomeric metalloprotein enzyme A basic isoelectric point (7.6–9.5), and optimal activity in acidic conditions. TRAP is highly expressed by osteoclasts, activated macrophages, neurons, and the porcine endometrium during pregnancy. leukemic reticuloendotheliosis (hairy cell leukemia), Gaucher’s disease, HIV-induced encephalopathy, osteoclastoma and osteoporosis, and metabolic bone diseases. Saeedeh Salimi 58  Acid Phosphatase ACP Reference Ranges and Preanalytic Variation Reference values depend on age, gender, and hormonal status (in women). Total and tartrate-resistant ACP values are high in children In women, total and tartrate-resistant ACPs increase after menopause. The half-life of prostatic ACP is about 1 to 3 hours Saeedeh Salimi 59  Acid Phosphatase ACP Measurement Assays for all phosphatases utilize the strategy that phosphate esters, which have no visible light absorbance, are hydrolyzed to inorganic phosphate and a strongly visible light-absorbing alcohol or alcohol anion. Total ACP is typically measured by its ability to cleave phosphate groups at an acid pH High bilirubin causes falsely low values for TRAP activity but not for total ACP. Isozymes of ACP can be separated by electrophoresis; however, there is usually little interest in isoenzymes other than prostate and bone. Immunoassays for both prostatic and bone isoenzymes of ACP have been developed; the former are widely available. The reference range for serum ACP is ≤ 2 ng/mL Saeedeh Salimi 60  Acid Phosphatase ACP Causes of Abnormal Results The main cause of increased ACP is prostate disease; with the development of PSA as the major serum test for prostate, ACP has become less popular for use in prostate cancer although, with the availability of newer immunochemical methods, its use in the diagnosis of prostate cancer has been maintained. In early prostate cancer, the sensitivity of ACP is inferior to that of PSA although ACP, similar to PSA, is elevated in a significant percentage of patients with benign prostatic hyperplasia or prostatic infarction, making ACP of little use for prostate cancer screening. Urinary tract obstruction and acute urinary retention may cause elevated ACP. Saeedeh Salimi 61  Acid Phosphatase ACP Extensive prostatic massage, prostatic inflammation, infarction/ischemia, and prostatic manipulations such as needle biopsy and cystoscopy may also cause a transient increase in serum ACP; testing should be done before any procedures are performed. Another important use of ACP is in the differential diagnosis of acute lymphoblastic leukemia (ALL). Saeedeh Salimi 62  Acid Phosphatase ACP ACP has been used for many years in cases of suspected rape. Fluid collected from the vagina on a cotton swab will give a positive test for ACP if semen is present, provided that a stabilizing fluid with an acidic pH is used Peak values are generally present in the first 12 hours, and values remain elevated for up to 4 days. Saeedeh Salimi 63  Isoenzymes Specific inhibition Tartrate prostatic and lysosomal Formaldehyde RBC Immunochemistry Saeedeh Salimi 64  ACP Prostate Cancer Prostate Hyperplasia Breast Cancer Saeedeh Salimi 65 Saeedeh Salimi 66