Enzymes – Part 2 – Diagnostic Values of various enzymes

These enzymes have their clinical significance for the diagnosis of diseases.

Acid phosphatase (ACP)

Sample

• Blood collected to prepare the serum.
• Acidification of the serum to pH below 6.5 will stabilize the enzyme.
  • Avoid lipemic serum which interferes with the test.

Definition:

1. Acid phosphatase has an optimal activity below a pH of 7.
2. Prostatic ACP has optimal activity at the range of pH 5 to 6.
3. The ACPs are unstable at the temperature above 37 °C  and at a pH level above 7.0.

Biochemical features:

1. Acid phosphatase (ACP) is present in the lysosomes, except the RBCs. Extralysosomal ACP is also present in many cells.

2. The greatest concentration of ACP activity occurs in the liver, spleen, milk, RBCs, bone marrow, platelets, and prostate glands.
   1. The prostate is the richest source of ACP.
   2. The osteoclast is the source of raised ACP in growing children compared with adults.

Clinical significance:

1. 1. Osteoclasts are the source of ACP in growing children compared to adults.
   2. This is used to diagnose or monitor the prostatic carcinoma.
      1. Now, this is not used for the detection of prostate carcinoma.
   3. A slight or moderate increase in the ACP is seen in Paget’s disease.
   4. In hyperparathyroidism with skeletal involvement, ACP is raised.
   5. It is raised in the case of tumor infiltration of the bone, such as breast cancer.
   6. ACP is raised in:
      1. Osteoclastoma (Giant-cell tumor).
      2. Osteoclastic tumors.
      3. Osteopetrosis (marble bone disease).
      4. Hairy – cell leukemia also shows osteoclastic type ACP.

Medicolegal importance:

1. There is a very high concentration of ACP in the semen, so used in case of a rape investigation.
2. Method 1: Take a swab from the alleged case of a rape victim, and preserved it in 2.5 mL of broth.
   1. Broth contains in one liter of the solution:
      1.  Bovine albumin = 50 grams.
      2. Sodium azide = 0.2 gram.
      3. Phosphate buffer at pH at 7.4 (phosphate buffer 10 mmol + 9 gram NaCl).
   2. Store the sample in the broth at 4 °C  or room temperature.
   3. The specimen can retain ACP activity for up to one month.
3. Interpretation of vaginal sample:
   1. Vaginal ACP in noncoital women is <10 U/L (broth sample).
   2. Post-coital women ACP is >50 U/L.
   3. Post-coital vaginal ACP takes 4 days to become a non-coital level. So the sample can be taken during this time.
4. Method 2: Take a vaginal swab and keep in 1 mL of isotonic saline.
   1. Freeze the swab.
   2. Before the test, thaw the swab at 2 to 4 °C for 24 hours before the assay.

Diagnosis of prostate carcinoma:

1. ACP is now rarely used for the diagnosis of carcinoma of the prostate.
2. ACP is replaced by the following tests:
   1. Digital rectal examination.
   2. Transurethral ultrasonic images.
   3. Histopathological examination of the prostatic biopsy.
   4. Advise total body scan.
   5. Advise prostatic specific antigen (PSA).

Alkaline phosphatase (ALP)

Sample

1. It is done on the Serum of the patient.
2. A fasting sample is a better choice.
3. This test can be done on the random sample as well.
   • How to get good serum: Take 3 to 5 ml of blood in the disposable syringe or in vacutainer. Keep the syringe for 15 to 30 minutes at 37 C and then centrifuge for 2 to 4 minutes to get the clear serum.
4. Keep the sample refrigerated as soon as you separate the serum.
5. Serum at 0 to 4 °C is stable for 2 to 3 days and at -25 °C is for one month.
6. Perform the test as soon as possible because ALP activity increases 3 to 10% on standing at 25° C or 4 ° C for several hours.

Indications

1. Storage At room temperature increases the ALP activity.
2. Avoid EDTA and oxalate anticoagulants which decreases Alkaline phosphatase activity.
3. If serum left at room temperature:
   • Then there is a 1% increase in 6 hours.
   • 3 to 6% in 1 to 4 days.
   • Even may see an increase if refrigerated the serum which is 2%/day.
4. Recent intake of food may increase the value.
   1. Values may be 25% higher after taking the high-fat meal.
5. Drugs like allopurinol, antibiotics, colchicine, indomethacin, fluorides, isoniazid (INH), methotrexate, nicotinic acid, methyldopa, phenothiazine, and probenecid can increase the alkaline phosphatase level.
6. Drugs like arsenal, cyanides, nitrofurantoin, and zinc salts may decrease the alkaline phosphatase level.
7. Hemolysis may cause a slight increase in the ALP. ALP is 6 times more in the RBC than the serum.

Pathophysiology

1. ALP is found in many tissues, at or in the cell membrane.
   1. ALP is a nonspecific enzyme, capable of reacting with many different substrates.
   2. The highest concentration is found in the liver. Within the liver, ALP is present in the Kupffer cells and these cells line the biliary collecting system. From there this enzyme is excreted in the bile.
   3. ALP is mainly from the liver, biliary tree, nearly up to 50% from the skeleton (osteoblastic cells), a small amount from the intestinal epithelium, renal tubule cells, and lower concentration in leucocytes and placenta.
   4. ALP is also found in the intestinal epithelium and the placenta.

1. This ALP is age-dependent.
2. ALP (serum) is denatured at 56 °C and stable at lower temperatures.
   1. The liver isoenzyme is moderately heated stable at 55° C but bone isoenzyme is heat-labile.
   2. Placental isoenzyme is stable at 65 °C  but not others.
3. Alkaline phosphatase is called Alkaline because its function is seen between a pH of 9 to 10 and best at a pH of 9.0.
4. ALP enzymes require Magnesium for the reaction.
5. The functions of this enzyme in the alkaline medium are better.
6. This is raised in the growing children due to osteoblastic activity.
7. This enzyme helps to diagnose obstructive jaundice.

Alkaline phosphatase is raised in:

1. It is raised in bone associated diseases with increased osteoblastic activity.
2. In obstructive jaundice, it may be raised from 10 to 20 times the normal.
3. This will differentiate hepatocellular disease from the cholestatic disease.

Amylase

Sample

1. This test is done in the serum of the patient.
2. The serum is stable at room temp. for 7 days and at 4 °C for one month.
3. Take 3 to 5 ml of blood in the disposable syringe. Keep the syringe for 15 to 30 minutes and then centrifuge for 2 to 4 minutes. In this way can get a clear serum.

Precautions

1. Avoid alcohol intake before sampling because it gives rise to an increase in the amylase level.
2. Urine can be collected at 2 hours or 24 hours sample. Refrigerate the urine.
3. Avoid contamination with saliva.
4. Lipemia, anticoagulant EDTA, fluoride, and citrate decreased amylase levels.

Purpose of the test (Indications)

1. To diagnose acute pancreatitis and monitor the treatment.
2. To differentiate other abdominal pain, epigastric discomfort, nausea, and vomiting.

In the case of ascites, may be done to rule out pancreatitis.

Pathophysiology

1. Amylase is present in a number of organs and tissue.
2. Amylase molecular weight is 55,000 to 60,000, this small molecule can pass the glomeruli of the kidneys and normally found in the urine.
3. It is of two types:
   1. α-amylase present in human tissue.
   2. β-amylase present in the plants and the bacteria.
4. The greatest concentration is in the pancreas.
   1. This enzyme is synthesized by the acinar cells and then secreted into the intestinal tract via a pancreatic duct system.
   2. In the duodenum, a slightly alkaline medium favors the effective action of pancreatic and intestinal amylase.
   3. The salivary glands also produce amylase to initiate the hydrolysis of starch while the food is still in the oral cavity and esophagus.
   4. The amylase activity is destroyed by the trypsin activity.

5. Amylase activity is present in:
   1. Semen.
   2. Testes.
   3. Ovaries.
   4. Fallopian tubes.
   5. Milk.
   6. Colostrum.
   7. Tears.
   8. Striated muscles.
   9. Adipose tissue.
   10. Sometimes the tumors of the lung and ovary may contain amylase activity.
   11. Ascitic and pleural fluid may contain amylase as a result of pancreatitis or neoplasm.
   1. The amylase in the serum and urine are mainly from the pancreas and salivary glands origin.
6. Serum amylase has an optimum sharp pH at 6.9 to 7.0. It is tested in routine at 37 °C but is still active at 50. °C.
7. This enzyme in the serum and urine can diagnose acute pancreatitis.
   1. Initial rise is seen in 2 to 12 hours
   2. The peak level is 12 to 72 hours
   3. The normal level is seen after 3 to 14 days.

Angiotensin-converting enzyme

1. This enzyme is used to detect the clinical course of sarcoidosis.
   1. It differentiates active and dormant sarcoidosis.
2. It differentiates between sarcoidosis and other granulomatous diseases.
3. This is found in the epithelial cells of the lungs.
   1. It converts angiotensin-1 to angiotensin II which is a potent vasoconstrictor.
   2. It helps in controlling hypertension.
4. The raised level is seen:
   1. Sarcoidosis.
   2. Other diseases showing a raised level are:
      1. Gaucher’s disease.
      2. Leprosy.
      3. Tuberculosis.
      4. Alcoholic cirrhosis.
      5. Hodgkin’s lymphoma.
      6. Multiple myelomas.
      7. Scleroderma.
      8. Pulmonary embolism.
      9. Active histoplasmosis.

Creatine kinase (CK)

1. This is a dimeric enzyme that causes the reversible phosphorylation of creatine.
2. CK activity is greatest in striated muscles, brain, and heart tissues.
   1. While kidney has less activity.
   2. The liver and RBCs have no CK activity.
3. It has subunits:
   1. CK-1 is CK-BB.
   2. CK-2 is CK- MB.
   3. CK-3 is CK – MM.
4. CK enzymes activity is increased in the diseases of skeletal muscles, cardiovascular system,  brain and, thyroid.
5. CK-MM is increased in:
   1. Muscular atrophy, especially in Duchenne’s type.
   2. In progressive muscular dystrophy.
   3. The enzymes level is high in children between the age of 7 to 10 years.
      1. It falls in the older patient.
6. CK-MB is raised after the myocardial infection.

7. CK-BB is an increase in cerebrovascular diseases.
   1. It increases in the neurosurgical intervention.
   2. In cerebral ischemia.

Gamma-Glutamyltransferase (GGT)

1. This enzyme originates from the hepatobiliary system.
   1. It is raised in all forms of liver disease.
   2. It is raised in intrahepatic and posthepatic biliary obstruction.
   3. The level reaches 5 to 30 times the normal value.
   4. This is a more sensitive test for the diagnosis of obstructive jaundice, cholangitis, and cholecystitis.
   5. The rise is early than other enzymes and persists longer.
      1. Moderate elevation 2 to 5 times occurs in infectious hepatitis.
      2. A high level was seen in primary or metastatic malignancies.
      3. 2 to 5 times increase seen in fatty liver and drug intoxication.
      4. In acute or chronic pancreatitis when there is a hepatobiliary obstruction, the GGT level may be raised to 5 to 15 times the normal.
      5. It is normal in a skeletal disease like Paget’s disease and bone malignancy, while alkaline phosphatase is raised.
      6. GGT is raised in alcoholic cirrhosis.

Lactate dehydrogenase (LDH)

1. Lactate dehydrogenase is a hydrogen transfer enzyme, it catalyzes the oxidation of L-Lactate to pyruvate.

2. The optimum pH for LDH to pyruvate is 8.8 to 9.8.
3. It’s isoenzymes are:
   1. LD-1 is predominant in the heart, muscles, kidneys, and RBCs.
   2. LD-2 is like LD-1.
   3. LD-3.
   4. LD-4.is more in the liver and skeletal muscles.
   5. LD-5 is more in the liver and skeletal muscles.
   6. LD-6
4. LDH activity is present in all the cells and is found in the cytoplasm of the cells.
5. The enzymes level is variable in concentration in various tissues.
   1. The tissue level is 500 times higher than the serum, so the leakage from the cells increases the level in the serum.
6. LDH is raised in:
   1. Acute myocardial infarction.
   2. The moderate increase may be seen in myocarditis and cardiac failure.
   3. This is normal in angina and pericarditis.
   4. Severe shock and anoxia.
   5. Megaloblastic anemia.
   6. In the case of liver diseases.
   7. In 1/3 of the cases in renal diseases like tubular necrosis and pyelonephritis.
   8. In liver metastatic tumor infiltration.
7. LDH raised the value in the urine is seen in case glomerulonephritis, diabetic nephropathy, systemic lupus erythematosus, urinary bladder, and kidneys malignancies.
8. CSF infiltration by the granulocytes increases the LDH value.
   1. In bacterial meningitis, there is an increase in the LD-4 and LD-5.

Lipase

1. The pancreas is the main organ for the secretion of lipase which goes into pancreatic juice.
   1. Lipase may be found in the stomach and intestinal mucosa but don’t have a significant role in the processing of fats.
2. Lipase concentration in the pancreas is 100 times greater than the other tissue.
   1. The difference of amylase in the pancreas and serum is 20,000 times.
   2. A very small amount is found in the serum.
   3. Lipase estimation is very difficult and not reproducible, so not common in the labs.
   4. Lipase hydrolyzes glycerol esters of long-chain fatty acids.
   5. 

      Lipase function for hydrolysis of triglycerides

3. This enzyme can diagnose acute pancreatitis.
   1. Initial rise is seen in 4 to 8 hours.
   2. The peak level is 24 hours (another reference say peak level after 72 to 96 hours).
   3. The normal level is seen after 8 to 14 days.

SGOT, Aspartate Aminotransferase (AST)

1. This is present widely in the tissue, in plasma, bile, saliva, and cerebrospinal fluid.
   1. It is absent in the urine unless there is kidney disease.
2. In liver injury, SGOT appears before the patient will have signs and symptoms.

3. This enzyme is helpful to diagnose acute myocardial infarction
   1. Initial rise is seen in 6 to 8 hours
   2. The peak level is 18 to 24 hours
   3. The normal level is seen after 4 to 5 days.

2. For the diagnosis of Acute Viral hepatitis
   1. Initial rise before the onset of jaundice.
   2. The peak level is seen 7 to 12 days after the onset of jaundice.
   3. The normal level is seen after 3 to 4 weeks of the onset of jaundice when the patient is recovering from the disease.

 SGPT, Alanine Aminotransferase (ALT)

1. This is present widely in the tissue, in plasma, bile, saliva, and cerebrospinal fluid.
   1. It is absent in the urine unless there is kidney disease.
2. In liver injury, SGPT appears before the patient will have signs and symptoms.

3. This enzyme helps to diagnose Acute Viral Hepatitis.
   1. Initial rise is seen before the onset of jaundice.
   2. The peak level is seen 7 to 12 days after the onset of jaundice.
   3. The normal level is seen 3 to 4 weeks after the onset of jaundice and in case the patient is recovering from the disease.

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