Phosphatases and Alkaline Phosphatases

• Phosphatases can hydrolyze a wide range of organic phosphate esters, resulting in alcohol and a phosphate ion.
• Dephosphorylation involves the removal of phosphate groups from molecules.
• Phosphatases are specific because they recognize the unique structures of their target molecules.
• Phosphatases are classified as hydrolases, belonging to Class 3.

Alkaline Phosphatases (EC 3.1.3.1)

• Alkaline Orthophosphoric Monoester Phosphohydrolase - non-specific enzyme
• Reference value is between 30-90 U/L

Function

• ALP cleaves phosphate-containing compounds in alkaline pH.
• Catalyzes hydrolysis of phosphate groups from organic molecules.
• ALP functions optimally at between pH 8-10 or 9-10.
• Facilitates the movement of substances across cell membranes.

Biological Roles

• Dephosphorylation of molecules
• Bone demineralization where ALP provides inorganic phosphate from hydroxyapatite crystals
• Digestive function involves breaking down dietary phosphates for absorption.
• The liver primarily produces ALP in healthy serum.

Types of ALP Isoenzymes

• Placental isoenzyme is seen in pregnant women, peaking between the 16th-20th week, and is used to assess placental health.
• Lower levels of placental isoenzyme are seen in women with blood types A and AB.
• Intestinal isoenzyme depends on blood group and secretor status, increasing in individuals with blood types B and O after fatty meals.
• Liver isoenzyme is used for detecting post-hepatic or obstructive liver disease.
• Bone isoenzyme increases with osteoblastic activity, during growth in children, and in adults over 50.
• Bone destruction releases ALP.

Carcinoplacental ALP

• Present only in individuals with cancer, serving as a tumor marker.
• Regan ALP is a bone ALP co-migrator found in lung, breast, ovarian, and gynecological cancers.
• Regan ALP is heat stable at 65°C for 30 minutes.
• Phenylalanine reagent can inhibit Regan ALP.
• Nagao ALP is a variant of Regan seen in adenocarcinoma of the pancreas and bile duct and pleural cancer.
• L-leucine and phenylalanine also inhibit Nagao ALP.
• Kasahara ALP occurs in hepatoma/hepatocellular carcinoma.

Methods of Determination

• Assays use p-nitrophenyl phosphate substrate at an alkaline pH to measure ALP activity.
• Activators like zinc and magnesium facilitate phosphate bond hydrolysis and stabilize enzymes in substrate binding.
• Magnesium is the most important activator.
• Chelators like EDTA, citrate, and oxalate can falsely lower activity by binding to essential metal ions.
• Enzyme activity may increase slightly on storage.
• ALP is relatively stable at 4°C for up to one week and optimal at between pH 8.6-10.

Bowers and Mc Comb Method

• The most used and specific method
• Continuous-monitoring technique with a pH of 10.15, 405 nm.
• p-nitrophenylphosphate converts to p-nitrophenol + phosphate ion.

Bowers and Mc Comb Method

• Most specific method
• Continuous-monitoring technique
• pH 10.15; 405 nm
• p-nitrophenylphosphate converts to p-nitrophenol + phosphate ion

Increased ALP

• Osteomalacia or softening of bones, rickets, and osteitis deformans/Paget's disease
• Osteoblastic bone tumors of the bone, bone cancer.
• Hyperparathyroidism where overproliferation of cells
• Obstructive jaundice.
• Hepatitis and cirrhosis, sprue, and celiac disease.

Decreased ALP

• After blood transfusions or cardiopulmonary bypass
• ALP is diluted from donor's blood.
• Malnutrition.
• Hypophosphatemia or prolonged severely low levels and zinc deficiency
• Liver and bone diseases are the most common causes
• Zinc, a necessary cofactor, is needed for ALP activity

ALP Elevations

• Cholestasis is connected to post-hepatic type of liver disease
• Osteosarcoma and bone tumor metastases increase bone and ALP activity
• Diabetes or renal failure elevate due to secondary complications like bone disorders and liver dysfunction

Acid Phosphatase

• Catalyzes the same reaction as ALP.
• Optimum pH: 5.0

Diagnostic significance

• Used in detection of prostatic carcinoma
• ACP is not a sensitive marker

Tissue sources

• Prostate as major source
• RBC, platelets and bone

Reference values

• Total ACP male: 2.5-11.7
• Prostatic ACP: 0-3.5 ng/mL

Isoenzymes

• Have tissue specificity and can be fractionated into five bands.
• Band 1’s major source is prostate gland
• Prostatic ACP activity is inhibited by tartrate

Tartrate-Inhibited ACP (Prostatic Isoenzyme)

• Prostatic Cancer: over proliferation of cells
• Benign prostatic hyperplasia
• Prostatic infarction: disruption of blood flow to prostatic gland
• Urinary tract obstruction

Medicolegal Cases

• ACP has implications in suspected rape cases.
• Positive ACP is evident in vaginal swab if semen is present for 12 hours up to four days from the incident.
• Bands 2 and 4 are from granulocytes.

Band 3 in Plasma

• Derived from platelets, erythrocytes, and monocytes.
• Band 5 is found mainly in osteoclasts.
• Resistant to tartrate inhibition
• TRACP involves active osteoclast-mediated bone resorption.
• Lysosomal storage disease

Gaucher's Cells

• Deficiency in enzyme glucocerebrosidase
• Glucocerebrosidase within the macrophages accumulate

Hairy Cell Leukemia

• Rare type of chronic B-cell leukemia
• Infiltrate the bone marrow, spleen, and peripheral blood

Roy and Hillman

• Uses thymolphthalein monoPO4
• Has the highest specificity
• Operates under optimal conditions to minimize interferences
• Thymolphthalein yields a blue color in alkaline pH

ACP Elevations

• Female Breast CA with Moderate elevation of Total ACP
• Hyperparathyroidism and Paget's disease
• Non-prostatic ACP elevations:

Aminotransferases

• Catalyzes the transfer of an amino group of one amino acid to a hydrocarbon for amino acid metabolism
• Optimum pH: 7.4.

Types

• Aspartate Aminotransferase (AST): L-aspartate + 2-oxoglutarate yielding oxaloacetate + glutamate
• Alanine Aminotransferase (ALT): L-alanine + alpha ketoglutarate yielding pyruvate + glutamate
• Needs cofactor P-5'-P, an active form of vitamin B6, as a coenzyme

ALT & AST

• Amino acid metabolism- Transamination, decarboxylation, and deamination rxn
• Neurotransmitter or Hemoglobin synthesis
• Energy production aids in the breaking down of amino acids

Specimen stability

• AST’s half-life: 17. Range: 12-
• ALT’s half-life: 47. Range: 37-

AST

• Stable for serum at room temp (2-8*C) for up to three weeks, indefinitely if frozen.
• Rises quickly and falls faster due to shorter half-life
• Factors affecting half-life include, Liver function, Overall health & Tissue damage severity

Methods of Determination

• AST & ALT specimens are stable in whole blood for up to 12-24 hours.
• Levels increase with time.
• Kidney helps clear AST and ALT in blood

A. Aspartate Aminotransferase (AST)

• Formerly known as Serum Glutamate Oxaloacetate Transaminase
• L-aspartate + 2-oxoglutarate leads to oxaloacetate + glutamate
• Involved in amino group transfer
• 2 isoenzyme fractions: Cytoplasm (ASTc)- abundant in healthy serum & Mitochondrial (ASTm)- predominant in people with necrosis
• Major tissue source: cardiac tissue, liver and skeletal muscles

Normal values

• Value lies at (5-37 U/L)

Factors that Affect Results

• Increase: AMI, diagnosis of chronic alcohol abuse and drug hepatotoxicity & pulmonary infarction
• Decrease: Pregnancy, hemolysis & hemolyzed and icteric samples

B. Alanine Aminotransferase (ALT)

• Also known as Serum Glutamate Pyruvate Transferase and subtrate is alpha-ketoglutarate
• Elevates during alanine conversion to pyruvate
• Evaluation: Hepatocyte Injury, Liver and kidney damage, Hepatic disorders & ALT
• Used to screen donors and exposure cases

Diagnostic significance

• ALT>AST (except in alcohol-related liver damage).
• Liver injury: High AST and Higher ALT

Creatinine Kinase

• Involved in the reversible phosphorylation of creatine by ATP
• Energy tissues and active muscle can lead to used up and CK-dependent
• ATP allows muscle contractions, and facilitates continual CK conversation by facilitating magnesium usage
• M: 15-160 U/L and F: 15-130 U/L (LD value)
• CK-1 or CK-BB found brain smooth muscles
• CK2 is CK-MB: ✓ Oligomer releases into the mitochondrial membrane ✓ Occurs when there is damage to the tissue

Measurements

• Electrophoresis is the way to indicate all isoenzymes because tech issues.
• Immunoassays use the measuring capacity residual antibodies
• Immunoassays the measurement of antibodies

Assay Considerations

• Assays performed carefully to see if they will be in the patients liver
• Fluoride and oxalates (anticoagulants) are used to counter CK
• Rapid lost storage of heat and exercise lead to elevation

Diagnostic Significance

• Acute Myocardial Infaction & Duchenne's Muscular dystrophy
• CNS issues may suggest shock, degeneration- CK testing may be helpful to detect, and discover
• CK/progesterone testing for ectopic
• CK/Vibrio vulnificus testing for cardiac and stroke events

Gammal-Glutamyl transferase (GGT)

• GGC catalyzes the transfer of y-glutamyl from amino acids
• Cells containing have a high affinity
• Half life for normal persons is from 7 to 10 days but can be extended w/ hepatic functions

Normal testing ranges

• Male: 6-45 u/L
• Female: 5-30 u/L
• Measuring steps: By spectrometric processes at 405 mm wave lengths
• GGT Elevations: damage, smoking, medications.

Lactate Dehydrogenase (LD)

• Zinc containing enzyme
• Allows glycolycitic pathways
• H transffering enzy
• Heart and Liver have presence
• Isomers: LD occurs as a form of tetra peptides due to tetral chains

Ld values

• Found in tissues, the myocardium, and red cells
• Has many uses to test tissues
• Evaluation and abnormalities
• Evaluation of Liver injuries

Diagnostic Significance

• Liver disease elevated testing
• Alcholol abuses
• Clinical, and chemical, inhibitory, and spectrophotometerical ways exists to test the effects of high/low counts

Amylase/Diastase

• amylases, enzymes that are part of of hydrolase groups, used to break down starch
• calcium based enzymes of the body
• body can take the enzyme in at great temp
• calcium and chloride are activators
• value: 60-180 SU/dL testing range

ISO ENZYMES

• Salivary fast moving that can be secreted by the body
• Pancreatic slow activity secretion for digestion and measurement

Method of determination

• saccharogenic measure
• Amyloclastic evaluation for enzyme
• The method can be measured in any amount

Diagnostic Significant

• Elevated by many factors
• Salivary Gland for Mumps
• The significance occurs when the patient becomes hyperthyroid. Alpha is the main function
• SPE is elevated to normal standards

Lipase

• Enzymes breaking down fatty acids
• The range is between 0-1.0 U/ML
• In order and effect ways.

Determination

• The process can be performed by Cherry standard methods for 24 hours with Hcl and testing.

G6PD

• Catalyzed to a new form and reaction can tested with light testing
• The value comes as 250 to 5000
• Diagnostic testing: for rbc counts and hemoglobin protection
• Can indicate MI for future diagnosis

LAP

• exhibits naphithylamidase activity to test peptides
• Acyrl B is a sign

ALDOLASE

• enzyme works with ALD conversion
• muscles damage with elevation
• measures ALDs reactions

Enyme markers and diseases

• acute liver: Increased AST and ALT counts as well as the other measurements

ASTR/ALT

• time markers that signal events
• Testing and indicators that work for testing events

ACE

• measured to find molecules
• most found through the lower test and functions to lower high tests

OCT

• Excellent for marker for liver disease use to rarely Cholinesterase and Pseudocholinesterase = break down molecules The levels of enzyme markers from the liver and heart are altered in characteristic patterns in different types of disorders.

Phosphatases hydrolyze organic phosphate esters into alcohol and a phosphate ion through dephosphorylation. Alkaline phosphatases (ALP) cleave phosphate-containing compounds in alkaline conditions, with optimal function between pH 8-10. They play roles in bone demineralization and digestive processes.