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enzyme methods 2


Enzyme Methods Part II
Bill McConnell
Scientist-in-Charge Biochemistry Clinipath Pathology

Enzyme Kinetics Refresher
Enzyme catalysed reaction over time.

? From Kaplan and Pesce 1989.

Variations on a Theme
Several different examples of Rate curves showing the possible variations that can occur.

From: The use of laboratory computers in monitoring kinetic enzyme assays G. Phillip Hicks, R.A. Ziesemer, and Norbert W. Tietz Clin Chem, Vol 19, No. 1, 1973.

Pick the linear bit
Autoanalyser must be able to pick the linear part of the assay to accurately calculate reaction rate and therefore enzyme activity.

From: The use of laboratory computers in monitoring kinetic enzyme assays G. Phillip Hicks, R.A. Ziesemer, and Norbert W. Tietz Clin Chem, Vol 19, No. 1, 1973.

More Kinetics

? Michaelis-Menton curve shows the relationship between substrate concentration and reaction velocity.

The Quest for Zero order
?In the first order part of the MM curve the reaction rate will change with substrate concentration. ?In the Zero order part of the curve the reaction rate is independent of Substrate concentration. ?Therefore the Zero order part of the curve is best for measuring enzyme activity.

Substrate Depletion
?When the substrate becomes depleted the reaction will move into First Order kinetics ?Reaction rate will no longer reflect enzyme activity. ?Substrate depletion is a risk where significantly higher levels of enzyme activity are present.

Substrate Depletion
?Automated assays are vulnerable to substrate depletion. ?Operators do not routinely review reaction curves and some analysers may not allow easy access to view this data if required. ?Operators rely on analysers to flag nonlinear results that may indicate Substrate Depletion.

Case:

35 y/o Female with acute Hep B.

21/01/2003 22/01/2003 23/01/2003 23/01/2003 Total Protein 69 70 70 71 Albumin 35 35 36 37 Bilirubin 171 207 223 218 Alk Phos 248 267 255 250 ALT 3490 3820 21 3970

Analyte

Units g/L g/L umol/L U/L U/L

Ref Interval 63-80 35-50 <20 35-105 <41

Doctor contacted lab concerned with results from 23/1. No error message on analyser (?).

Case:
?Repeat ALT was 23. No error message. ?Repeat on paired analyser was 3134 U/L with error (K) message alerting that curve was non-linear. ?After dilution result 3950 U/L. Consistent with clinical picture.

ALT: The Assay
? Alanine Amine Transferase. Serum Glutamate Pyruvate Transaminase (SGPT). L-alanine:2-oxoglutarate Aminotransferase

alpha-ketoglutarate + L-Alanine =ALT= L-Glutamate + Pyruvate This reaction is coupled to the following reporter reaction Pyruvate + NADH + H+ =LDH= L-Lactate + NAD+ The rate of change in absorbance at 340nm is used to calculate ALT activity.

Reaction Profile: Original

Result with no error message

Reaction Profile: Repeat

Result with error message

Other Enzymes to be wary of: ?Amylase and LDH; especially in fluid samples. ?AST in hepatitic patients ?CK in rhabdomyolysis ?Be alert and be suspicious of results out of character. Delta checks are very useful.

Simple Checks
?Sometimes all that is required is a simple dilution to check if the reaction curve is non-linear. ?Dilute in saline preferably unless water is shown to be okay for your method. ?Discrepant results on dilution could indicate substrate depletion.

More Recently:
?Request for Salivary Amylase level on Saliva sample. ??? ?Result low?

Analyser Reaction Curve

Know your analyser
?If your analyser is able to show you reaction curves then make sure you know how to find them and how to read them. ?Read the Kit insert information and note the linear range of the assay and any relevant warnings about substrate depletion or interferences.

AST
?AST. Aspartate Amino Transferase. Serum Glutamate Oxaloacetate Transaminase (SGOT). ?L-aspartate:2-oxoglutarate Aminotransferase

?MW. AST ~110 000 daltons

ALT
?ALT. Alanine Amine Transferase. Serum Glutamate Pyruvate Transaminase (SGPT). L-alanine:2-oxoglutarate Aminotransferase ?MW. ALT ~101 000 daltons

AST/ALT
?Aminotransferases constitute a group of enzymes that catalyse the interconversion of amino acids and 2-oxo-acids. ?2-oxoglutarate/L-glutamate couple serve as one amino group acceptor and donor pair in all amino-transfer reactions.

AST/ALT

AST ISOENZYMES
?Distinct isoenzymes of AST are present in the cytoplasm and mitochondria of cells. ?In conditions associated with a mild degree of tissue injury the predominant form in serum is from the cytoplasm. Severe tissue damage results in the release of significant mitochondrial enzyme as well

AST ISOENZYMES

AST/ALT
?AST and ALT catalyse reversible reactions but the formation of Aspartate and Alanine is favoured. ?AST ALT activity measurements use coupled reactions with MD/LD as indicator enzyme.

AST/ALT

AST/ALT
?Addition of P-5'-P is recommended in IFCC methods to ensure all transaminase activity in the sample is measured.

?Patients deficient in vitamin B6 may recover lower serum transaminase levels unless P-5'-P is added to the reaction mix.

AST/ALT
?P-5'-P functions as a coenzymes in the amino-transfer reactions. ?P-5'-P serves as a true prosthetic group and once bound to the apoenzyme accepts the amino acid from the first substrate and transfers it to the second substrate - regenerating P-5'-P

AST/ALT
?Holoaminotransferases = Enzymes with bound coenzyme ?Transaminases are widely distributed in animal tissues and normally present in human plasma, bile, csf and saliva. Not normally found in urine in absence of pathology.

Preferred Sample Types:
?Serum preferred but oxalate, heparin, EDTA and citrate do not inhibit the enzymes. ?Haemolysed samples must be avoidedRed cells contain transaminases. ?ALT and AST stable in serum for ~3 days at room temp and up to 1 week at 4oC.

AST/ALT
?Notes: Neonates and infants can have elevated AST up to twice adult levels. Declines to adult levels by 6 months of age.

ALP
?Alkaline phosphatase.
?Molecular weight: 70 000 - 120 000 Daltons

ALP
?Alkaline Phosphatase
?Catalyses the hydrolysis of a a large variety of naturally occurring and synthetic substrates. Actual substrates for the enzyme not fully understood.

ALP

?Predominant forms of enzyme in blood are liver and bone isoenzymes. Can also be Placental ALP in pregnant women.

ALP: Analytical Issues
?Require alkaline conditions pH ~10 for optimum activity ?Assay requires buffering to suit this pH ?Phosphate acceping buffers can increase activity of ALP by 2-6 fold

ALP: Analytical Issues
?Activating buffers include
2-amino-2-1-propanol (AMP) Diethanolamine (DEA) Tris hydroxy methyl aminomethane (TRIS) Ethylaminoethanol (EAE) N-methyl-D-glucamine

ALP: Analytical Issues
?These compounds are derivatives of aliphatic amines and act as buffers by binding protons at the nitrogen atom. Being hydroxyl compounds they can act as phosphate group acceptors. ?IFCC recommended method uses PNPP as the substrate and AMP as the phosphate accepting buffer.

ALP: Analytical Issues
?Divalent cations Mg2+ , Co2+ and Mn2+ are activators of the enzyme ?Zn is a constituent metal ion. The correct ratio of Mg/Zn ions is necessary to avoid displacement of Mg2+ and to obtain optimal activity.

ALP: Analytical Issues
?Mg2+ and Zn2+ are required for the reaction but excesses are inhibitory ?Mg2+ and Zn2+ are added to the reaction mix as well as the chelating agent HEDTA which acts as a metal ion buffer maintaining optimal concentration. ?Wavelength ~405 nm

ALP: Assay using PNPP

ALP Isoforms
?A single gene locus mapped to chromosome 1 determines the common primary structure of ALP from liver, bone and kidney. ?Same gene product - Differences in carbohydrate side chains. Isoenzymes may be differentiated by the following.

ALP Isoforms
?Electrophoretic mobility (with and without lectin addition) ?Heat/Urea denaturation ?Response to inhibitors ?Substrate preferences ?Immunochemical characteristics (antisera, wheat germ lectin)

ALP Isoforms
?Bone isoenzyme most susceptible to urea denaturation, liver has intermediate resistance with placental and intestinal forms showing greatest resistance to urea. ?Placental isoenzyme heat stable. Liver more stable than bone.

ALP Isoforms

Preferred Sample Types:
?ALP can be found in all body fluids and tissues. ?Samples should be drawn after a fast of at least 8 hours. ?Serum and heparin plasma give similar results. ?EDTA, oxalate and citrate complex Mg2+ and inhibit the enzyme.

ALP: Notes
?Mild haemolysis should not affect the assay but grossly haemolysed samples are not suitable. ?ALP activity increases slowly with storage at room temp or refrigeration. ?Age related reference ranges required to interpret data with confidence.

Gamma Glutamyl Transferase - GGT
?Peptidase that transfers the gamma glutamyl group from peptides and compounds to an acceptor.

?Acts as an amino acid transferase.

GGT
?GGT in serum is heterogeneous in molecular size and charge probably due to post translational modification of a single type of enzyme moloecule. Different forms not associated with different pathology.

GGT
?First identified in Kidney but found in serum and all cells except muscle cells.
?Enzyme found in cell cytosol but most in cell membrane

GGT: Assay

Preferred Sample Types:
?Serum or heparin sample preferred. ?EDTA acceptable but not ideal.

?Citrate, oxalate and fluoride preservatives can reduce GGT activity.

Bilirubin
?Molecular weight: 585 Daltons ?Bilirubin is the orange-yellow pigment formed from the metabolism of heme from dead red cells. Tetrapyrrole.

Bilirubin
?Four bilirubin fractions may be isolated from serum by chromatographic separation.
? ? ? ? Alpha Bilirubin (Unconjugated) Beta Bilirubin (Monoconjugated) Gamma Bilirubin (Diconjugated) Delta Bilirubin (Bound irreversibly to albumin)

Bilirubin
?Unconjugated bilirubin is highly non-polar and insoluble in water. This property is due to internal hydrogen bonding shaping the molecule. Exposure to UV light (450 nm) can change bilirubin conformation from ZZ conformation (with internal hydrogen bonds)to EE conformation (no internal hydrogen bonds) which is water soluble and readily excreted in urine (note: treatment of neonatal jaundice).

Bilirubin
?Conformational changes confered by esterification to glucuronic acid (liver metabolism) is the reason why conjugated bilirubin is readily excreted in bile and urine

Bilirubin: Structure

Bilirubin: Methods
?Reference method HPLC ?Most common methods use diazo reagents ?Generally modifications of the method by Evelyn and Malloy.

Bilirubin: Methods
?Hydrogen bond breaking chemicals such as caffeine, methanol, ethanol, various surfactants/detergents or urea required for unconjugated bilirubin to react with diazo reagent. ?These compounds collectively reffered to as "accelerators". ?Delta bilirubin reacts directly (without accelerators).

Bilirubin: Methods
?Evelyn Malloy method uses a low pH (~1.2) where the azobilirubin complex is red-purple in colour with an absorption maxima of approx 560nm. Prone to interference from haemolysis.

Bilirubin: Methods
?Jendrassik-Grof modification carried out at pH nearer to 6.5 but absorption of the reaction is measured after alkalinization of the reaction solution to around pH 13. At this pH the azobilirubin is a more intense blue color and absorbs at around 600nm. Has a higher molar absorptivity so more sensitive and precise at lower bilirubin concentrations.

Bilirubin: Diazo Reaction

Preferred Sample
?Serum, plasma and urine samples.

Bilirubin other Methods
?Urine dipsticks. ?Bilirubinometer/transcutaneous monitoring (neonates) ?Other methods include those using bilirubin oxidase and spectral shift (Ortho).

Bilirubin other Methods
?Bilirubin oxidase- Enzymatic oxidation of bilirubin to biliverdin. Reaction monitored at 405-460nm. ?Spectral shift- Bilirubin binds to hydrophobic cationic polymer causes shift in absorption spectrum of bilirubin. Magnitude of change, as monitored by reflectance photometry, proportional to bilirubin concentration.

Bilirubin: Final Note
?Make sure you can describe Bilirubin Metabolism. ?Understand Gilbert’s Disease and what we can measure to investigate.

References:
?Kaplan and Pesce Clinical Chemistry: Theory, analysis and Correlation. Second edition Chapter 52. Enzymes. ?Tietz 3rd Edition. Chapter 22 on Clinical Enzymology. ?Your analyser manual and kit inserts.

Historical Interest
?The use of laboratory computers in monitoring kinetic enzyme assays
G. Phillip Hicks, R.A. Ziesemer, and Norbert W. Tietz Clin Chem, Vol 19, No. 1, 1973.

Some Questions.
?Do you know how to look up the reaction curves on your Autoanalyser? ?Do you know how your analyser handles substrate depletion?

Biochemistry Humour

Biochemistry Humour

Ferrous Wheel.


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