CC2_Prelims_Lec: Specific Enzyme part 1 (AI)

Specific enzymes

Enzymes whose measurement can help detect tissue injury or identify abnormalities/absence of enzymes associated with inborn errors of metabolism.

Phosphatases

Enzymes that hydrolyze organic phosphate esters, producing an alcohol and inorganic phosphate.

Phosphatases enzyme class

Class 3 – Hydrolases.

Hydrolysis

Splitting of chemical bonds through addition of water.

Dephosphorylation

Removal of a phosphate group from a molecule.

Group specificity

Phosphatases are specific for the phosphate group rather than one particular substrate.

Major phosphatases

Alkaline phosphatase (ALP) and acid phosphatase (ACP).

Alkaline phosphatase (ALP)

Enzyme that cleaves phosphate-containing compounds at an alkaline pH.

ALP EC number

EC 3.1.3.1.

Former name of ALP

Alkaline Orthophosphoric Phosphohydrolase.

ALP reference value

30–90 U/L; reference ranges depend on the laboratory.

Major sources of ALP in healthy serum

Bone and liver.

Preferred specimen for ALP determination

Serum, because ALP is inactivated by EDTA.

ALP activator

Magnesium is the most important activator; zinc and other cations also activate ALP.

Biological roles of ALP

Dephosphorylation, bone mineralization, and digestive function.

ALP and bone mineralization

ALP provides inorganic phosphate needed for hydroxyapatite crystal formation.

Hydroxyapatite

Main mineral component of bone.

ALP isoenzymes

Placental, intestinal, liver, and bone isoenzymes.

Placental ALP

ALP isoenzyme that reaches its highest level during the 16th–20th week of gestation.

Placental ALP clinical use

Can help determine abnormalities involving the placenta.

Intestinal ALP

ALP isoenzyme whose serum presence depends on blood group and secretor status.

Intestinal ALP and blood group

B and O blood groups have higher intestinal ALP levels.

Intestinal ALP after eating

In B and O secretors, intestinal ALP can increase and peak about 2–3 hours after eating.

Liver ALP

ALP released by liver cells lining the canaliculi; significant in detecting post-hepatic/obstructive liver disease.

Bone ALP

ALP released by osteocytes; levels vary with age.

Bone ALP in children

Higher because osteocytes are actively replicating during growth.

Bone ALP in geriatrics

Higher due to damage to bone cells and release of ALP.

ALP heat fractionation

Technique using 56°C for 10–15 minutes to differentiate ALP isoenzymes based on heat stability.

Most heat-stable ALP isoenzyme

Placental ALP.

Heat stability order of ALP

Placental > Intestinal > Liver > Bone.

Most heat-labile ALP isoenzyme

Bone ALP.

Liver ALP electrophoretic migration

Fastest ALP isoenzyme toward the anode.

Intestinal ALP electrophoretic migration

Slowest ALP isoenzyme toward the anode.

Chemical inhibition of liver ALP

Levamisole inhibits liver ALP.

Chemical inhibition of bone ALP

Urea and levamisole inhibit bone ALP.

Chemical inhibition of placental ALP

Phenylalanine inhibits placental ALP.

Carcino-placental ALP

Placental-type ALP associated with certain cancers and used as a tumor marker.

Regan ALP

Carcino-placental ALP associated with lung, breast, ovarian, and gynecological cancers.

Regan ALP heat stability

Most heat-stable; remains stable at 65°C for 30 minutes.

Regan ALP inhibition

Inhibited by phenylalanine reagent.

Regan ALP electrophoresis

Co-migrates with bone ALP.

Nagao ALP

ALP variant associated with adenocarcinoma of the pancreas and bile duct and pleural cancer.

Nagao ALP inhibition

Inhibited by L-leucine and phenylalanine.

Kasahara ALP

ALP variant associated with hepatoma/hepatocellular carcinoma.

Bowers and McComb method

Continuous-monitoring method considered the most specific and routine method for ALP.

ALP optimum pH

Approximately 8.6–10; the Bowers and McComb method uses pH 10.15.

ALP wavelength

405 nm in the described Bowers and McComb method.

ALP substrate in routine assay

p-Nitrophenyl phosphate (PNPP).

ALP PNPP reaction

p-Nitrophenyl phosphate → p-nitrophenol + phosphate ion.

ALP activators

Zinc, magnesium, and other cations.

Most important ALP activator

Magnesium.

ALP chelators

EDTA, citrate, and oxalate.

Effect of chelators on ALP

Chelators can falsely lower ALP activity by binding metal-ion activators.

ALP storage at 4°C

Relatively stable for up to one week.

ALP storage for longer periods

Store at −20°C.

Effect of storage on ALP activity

Activity may increase slightly because of loss of inhibitors.

ALP inhibitors

Inorganic phosphate and proteins.

Conditions associated with increased ALP

Paget disease, osteomalacia, rickets, osteoblastic bone tumors, bone cancer, sprue, hyperparathyroidism, obstructive jaundice, hepatitis, and cirrhosis.

Condition with particularly significant ALP elevation

Obstructive jaundice.

Paget disease

Osteitis deformans involving enlargement of long bones or deformation of flat bones.

Osteomalacia

Softening of bones associated with increased ALP.

Rickets

Bone disorder associated with increased ALP.

Hyperparathyroidism and ALP

Parathyroid hormone affects osteocyte enzyme activity and may increase ALP.

ALP decrease after transfusion/cardiopulmonary bypass

A transient decrease can occur.

Malnutrition and ALP

Can decrease ALP due to decreased sources of inorganic molecules used as cofactors.

Hypophosphatemia and ALP

Prolonged severe low phosphate levels can decrease ALP.

Zinc deficiency and ALP

Prolonged severe zinc deficiency can decrease ALP activity.

Acid phosphatase (ACP)

Hydrolytic enzyme that catalyzes a reaction similar to ALP but functions at acidic pH.

ACP EC number

EC 3.1.2.3 according to the lecture.

ACP optimum pH

Approximately pH 5.0.

Major ACP source in males

Prostate.

Other ACP sources

RBCs, platelets, and bone.

Total ACP male reference value

2.5–11.7 U/L.

Prostatic ACP reference value

0–3.5 ng/mL.

ACP diagnostic significance

Can assist in evaluating prostatic carcinoma but is not itself the screening/diagnostic test for prostate cancer.

Preferred prostate cancer screening marker

Prostate-specific antigen (PSA).

ACP isoenzymes

ACP can be fractionated into five electrophoretic bands.

ACP Band 1

Major source is the prostate gland; prostatic ACP is inhibited by tartrate.

ACP Bands 2 and 4

Derived from granulocytes.

ACP Band 3

Major plasma form; derived from platelets, erythrocytes, and monocytes.

ACP Band 5

Found mainly in osteoclasts and is resistant to tartrate inhibition.

Tartrate inhibitor

Used to differentiate prostatic from non-prostatic ACP.

Tartrate-inhibited ACP

Prostatic ACP.

Tartrate-resistant ACP

Bone ACP/osteoclast-associated ACP.

TRAP

Tartrate-resistant acid phosphatase.

Bowers and McComb ACP

Preferred method according to the lecture.

Roy and Hillman method

Highest-specificity ACP method.

Roy and Hillman substrate

Thymolphthalein monophosphate.

Roy and Hillman end product

Thymolphthalein, producing a distinctive blue color, plus inorganic phosphate.

Copper inhibition of ACP

Copper inhibits RBC ACP but not prostatic ACP.

Copper-resistant ACP

Prostatic ACP.

Copper-sensitive ACP

RBC ACP.

Conditions increasing tartrate-inhibited/prostatic ACP

Prostatic cancer, benign prostatic hyperplasia, prostatic infarction, urinary tract obstruction, rectal carcinoid tumors, and after prostatic massage.

Medico-legal significance of ACP

ACP can be detected in vaginal swabs when semen is present, according to the lecture.

Conditions increasing tartrate-resistant ACP

Active osteoclast-mediated bone resorption, Gaucher disease, hairy cell leukemia, and hematological disorders.

Gaucher disease

Lysosomal storage disorder caused by glucocerebrosidase deficiency, associated with increased TRAP.

Hairy cell leukemia

Rare chronic leukemia characterized by abnormal lymphocytes infiltrating bone marrow, spleen, and peripheral blood.

Icteric serum and TRAP

Icteric serum causes falsely decreased TRAP activity but not total ACP.

Aminotransferases

Enzymes that transfer an amino group from an amino acid to a hydrocarbon/ketoacid, producing a different amino acid.

Two major aminotransferases

ALT and AST.