ACP TO ALT

• Major Clinical Enzymes


  • Phosphatases
  • Transferases / Transaminases
  • Amylase
  • Lipase
  • Lactate Dehydrogenase
  • Creatine Kinase
  • Aldolase
• Phosphatases


  • Alkaline Phosphatase (ALP)
  • Acid Phosphatase (ACP)
• Transferases / Transaminases


  • Aspartate Aminotransferase (AST)
  • Alanine Aminotransferase (ALT)
• Amylase


  Alpha-1-4 Glucan-4-Glucohydrolase (AMS)
• Lipase


  Triacylglycerol Acylhydrolase (LPS)
• Lactate Dehydrogenase


  LD
• Creatine Kinase


  ATP-Creatine-N-Phosphotransferase (CK)
• Aldolase


  Fructose 1,6-Diphosphate Aldolase
• Alkaline Phosphatase (ALP)


  • Nonspecific enzyme capable of reacting with many different substrates
  • Functions to liberate Inorganic phosphate from an organic phosphate ester with the concomitant production of an alcohol
• Tissue sources of ALP

  Liver, bone, placenta and intestinal
• Reference value for ALP: 30-90 U/L
• Major Isoenzymes of ALP

  • Liver ALP
  • Bone ALP
  • Placental ALP
  • Intestinal ALP
• Acid phosphatase


  An enzyme that catalyzes the hydrolysis of phosphate esters at acidic pH
• Diagnostic Significance of ALP

  • When total ALP levels are increased, it is the major liver fraction that is most frequently elevated, especially in obstructive jaundice
  • For bone disorders, highest elevations occur in Paget's disease (osteitis deformans)
  • Bone ALP isoform, B1x, was detected in the serum of dialysis patients and is used to study low bone mineral disease (BMD) in patients with chronic kidney disease
  • B1x isoform is also increased in the serum of individuals with BMD of the hip, which is predominantly made up of trabecular bone
• Acid phosphatase assay method


  1. 1-naphthyl phosphate is hydrolyzed by acid phosphatase to phosphate and 1-napththol

  2. 1-napththol is converted with FRTR-salt to an azo dye

  3. Increase of absorbance at 405nm is proportional to the total acid phosphatase activity in the sample
• Carcinoplacental ALP


  • Regan ALP - found in lung, breast, ovarian and gynecological cancers; bone ALP co-migrator; mostly heat stable ALP (65°C for 30 minutes); Inhibited by phenylalanine reagent
  • Nagao ALP - found in adenocarcinoma of the pancreas and bile duct, pleural cancer; variant of Regan ALP; Inhibited by L-leucine and phenylalanine
• Methods for ALP analysis

  • Electrophoresis
  • Heat Fractionation/Stability Test
  • Chemical Inhibition Test
  • Bowers and Mc. Comb (Szasz modification)
• Assay materials


  • Buffer: Citrate Buffer (pH5.2) 100mmol/l
  • Substrate: 1-naphthyl phosphate 19umol/l
  • FR TR-salt 2umol/l
  • Stabiliser: Acetic acid 0.7mul
  • Calibrator: AUTOCAL
  • Controls: Huma Trol N, Huma Trol P, SERODUS, SERODUS plus
• Summary of ALP Methods

  • Bodonsky
  • Shinowara
  • Jones
  • Reinhart
  • King and Armstrong
  • Bessy, Lowry & Brock
  • Bowers and McComb
  • Huggins and Talalay
  • Moss
  • Klein, Babson & Read
• Reagent preparation


  Dissolve the contents of 1 bottle Substrate with exactly 2 ml of Buffer
• Zinc is a component of ALP, and magnesium is the enzyme activator
• Specimen


  Serum (No plasma)
• Ingestion of food leads to release of intestinal ALP into lymphatic fluid, and may transiently increase plasma levels of ALP
• Sample considerations


  • Separate serum from RBC after centrifugation
  • Analyze immediately (within 1-2 hours)
• Hemolysis and diet (fatty meals) are sources of analytical errors that can lead to elevated serum ALP
• Sample preparation


  1. Stabilise samples, AUTOCAL, and Controls by addition of one drop of stabiliser to 1 ml sample immediately after separation/reconstitution

  2. Samples will remain stable for 3 days at 2-8°C, 24 hours at 15-25°C
• ALP is sensitive if stored at low temperature (4°C) - leads to increased serum level
• ALP is inhibited by phosphorus - the addition of 2-amino-2-methyl-1-propanol (AMP) buffer binds phosphorus under Bowers-McComb method
• Assay conditions


  • Wavelength: Hg 405nm
  • Optical path: 1 cm
  • Temperature: 37°C
  • Measurement: against air (increasing absorbance)
• Decreased ALP is seen in zinc deficiency
• Assay procedure


  1. Warm working reagent and cuvettes up to 37°C

  2. Pipette 100ul sample and 1000ul working reagent into cuvette

  3. Mix, read absorbance A1 after 5 minutes and start stopwatch

  4. Read absorbance A2 exactly after 3 minutes at 37°C

  5. Calculate A = A2 - A1
• Transient low serum ALP may occur after blood transfusion or cardiopulmonary bypass
• Prolonged low levels of ALP occur in hypophosphatasia
• Calculation


  Total Acid phosphatase activity (U/l) = A x 248
• Placental alkaline phosphatase (PLAP) is a useful tumor marker in serum and cerebrospinal fluid (CSF) for most germ cell tumors - CSF levels of PLAP are of diagnostic value in differentiating whether a tumor in the pineal body is a pinealoma or a germ cell tumor
• Conversion: 1U/l = 16.67 x 10-3 ukat/l, 1ukat/l = 60U/l
• Conditions with increased ALP

  • Osteitis deformans
  • Obstructive jaundice
  • Osteomalacia
  • Rickets
  • Osteoblastic bone tumors - osteosarcoma
  • Sprue
  • Hyperparathyroidism
  • Hepatitis and cirrhosis (slight increased)
• Reference values for total acid phosphatase

  • Men up to 6.6 U/l
  • Women up to 5.5 U/l
• Acid Phosphatase (ACP)


  • Catalyzes the same reaction made by ALP, except that it is active at pH 5.0
  • ACP activity > 50 IU/L indicates the presence of seminal fluid in the sample
• Aspartate aminotransferase (AST)


  An enzyme that catalyzes the transfer of an amino group from aspartate to 2-oxoglutarate, producing oxaloacetate and glutamate