Transcript Metabol Nutri-ClinEnz Med 2_6 Nov 2012

Metabolism & Nutrition 2012
Clinical Enzymology
(Prof. Dr. Jerapan Krungkrai)
Objectives & Contents:
• Clinical correlations of enzymes
• Enzymes as markers for diagnosis
• Enzymes used in therapy
• Enzymes as target of drug
• Enzyme abnormalities in metabolisms
Enzymes: properties and measurements (I)
Nature: catalytic proteins / denaturation property
enzyme
Substrate
Product
cofactor/coenzyme
 3-dimensional structure: monomer
oligomer
 Variants:1) isozymes (different genes)- tissue-specific forms
2) allozymes (different alleles at single genetic locus)
3) post-translational modifications- cell and tissue
specific forms,
E.g., liver- and bone-specific alkaline phosphatase (ALP) differ
only in carbohydrate contents attached to the ALP proteins.
Enzymes: properties and measurements (II)
Measurements:
1) kinetic properties and activity of enzyme
[S], [P], Km, v, Vmax
2) expression of activity measurements
1 unit of enzyme activity (U) = 1 micromol per min.
Then, U/l of sample or U/ml of sample (e.g.,plasma,
serum, blood, urine etc.) are widely used.
3) direct measurement of enzyme protein concentration.
This is limited due to very low concentrations of
proteins.
I. Clinical Correlations of Enzymes:
Principles of Diagnostic Enzymology
1. Introduction:
A human cell contains 28,602 different proteins, 2,709 proteins of
which are enzymes. They are assigned roles in ~ 135 metabolic pathways
(2,645 metabolites)
They are distributed in: cytosol, nucleus, rough and smooth ER, Golgi
bodies, mitochondria, lysosomes, plasma and organellar membranes.
The enzymes are mainly synthesized in cytosol or ER (stay, or target to
other organelles and export to extracellular environment, or extracellular
enzymes).
A relatively small numbers are synthesized in the mitochondria and
these enzymes stay within this space.
The activity of an intracellular enzyme is determined by the
rates of synthesis, inactivation and degradation (= turnover).
Figure 1. Turnover of intracellular enzymes
Nobel Prize Chemistry 2004: Rose, Hershko, Ciechanover
Figure 2. Mechanism of enzyme release from damaged cell
Table 1. Half-lives of clinically important enzymes in plasma
Enzyme
Range (hours)
Lactate dehydrogenase (LD)
LD-1 (H4)
50-70
LD-5 (M4)
8-14
Alanine transaminase (ALT, GPT)
40-50
Aspartate transaminase (AST, GOT)
mitochondrial AST
6-7
cytosolic AST
12-17
Creatine kinase (CK)
CK-MM
10-20
CK-MB
7-17
CK-BB
3
Alkaline phosphatase (ALP)
liver ALP
190-230
bone ALP
30-50
Most enzymes are present in cells at much higher
concentrations than in plasma. Some occur predominantly in
cells of certain tissues.
Normal plasma enzyme level (normal or reference range,
e.g., 5’-nucleotidase, ALP, amylase =2-15, 30-95, 95-290 U/L).
The normal levels reflect the balance between the rate of
synthesis and release into plasma during cell turnover, and the
rate of clearance from the circulation.
The enzyme level in plasma may be:
•increased due to proliferation of cells, an increase in rate of
cell turnover or damage or in enzyme synthesis, or to reduced
clearance from plasma;
•lower than normal, due to reduced synthesis, congenital
deficiency.
Table 2. Serum normal (reference) ranges of clinical enzymes
Enzyme
Abbreviation
Range
(male> female)
Acid phosphatase
ACP, AP
Alkaline phosphatase
ALP
Alanine transaminase
ALT, G PT
Aspartate transaminase
AST, GOT
Alpha-amylase
AMS
Aldolase
ALS
Creatine kinase
CK, CPK
Gamma-glutamyl transferase
GGT
Glucose-6-phosphate dehydrogenase G-6-PD
Lactate dehydrogenase
LD, LDH
Lipase
LPS
Leucine aminopeptidase
LAP
5’-Nucleotidase
5’NT
Pseudocholineesterase
PChE
Ceruloplasmin (Copper-oxidase)
0.2-5.0 U/L
30-95 U/L
6-37 U/L
5-30 U/L
95-290 U/L
1.5-8.0 U/L
15-160 U/L
6-45 U/L
0-0.2 U/L
100-225 U/L
0-2 U/ml
11-30 U/L
2-15 U/L
5-12 U/ml
0.2-0.6 g/L
Stability
+
+++
++++
+++
++++
++++
-++++
+++
+
++++
+++
+++
++++
2. Assessment of cell damage and proliferation:
•Changes in plasma enzyme levels may help to detect and
localize tissue cell damage or proliferation, or to monitor
treatment and progress of disease.
•Plasma enzyme levels depend on:
a) the rate of release from damaged cells which, in turn, depends on the
rate at which damage is occurring;
b) the extent of cell damage.
•In the absence of cell damage, the rate of release depends on:
a) the rate of cell proliferation;
b) the degree of induction of enzyme synthesis.
•These factors are balanced by :
a) the rate of enzyme clearance from the circulation (only partly known);
b) half-life of enzyme.
II. Enzymes as markers for diagnosis
Introduction:
Isozymes are enzymes catalyzing the same catalytic
reaction, but they are synthesized from different genes/loci, and
most contain subunits (quaternary structure).
This will result in cell and tissue-specific forms of isozymes.
Examples of some clinically significant isozymes are as follows:
1. LDH
M4
M3H M2H2
MH3
(muscle) LD-5 LD-4 LD-3
LD-2
- ve ------- electrophoresis ------->
H4 subunit
LD-1 (heart)
+ ve
2. ALP
kidney (isozyme I) <intestine <placenta, kidney (isozyme II) <bone <liver
-ve --------- electrophoresis -------->
+ve
3. CK
CK-3, MM (heart, muscle) CK-2, MB (heart >> muscle) CK-1, BB (brain)
-ve --------- electrophoresis -------->
+ve
4. ACP
Prostate ACP >>> liver, red cells & bone ACPs
- ve
L-tartrate (prostatic acid phosphatase, PAP)
5. Prostatic specific antigen (PSA), a serine protease
(chymotrypsin type), functions in clot liquefaction of semen
coagulation.
Table 3. Enzyme markers of clinical significance
Enzyme (abbreviation)
Clinical significance
Acid phosphatase (ACP)
Prostatic carcinoma
Alkaline phosphatase (ALP)
Obstructive liver diseases, bone disorders
Alanine transaminase (ALT,GPT)
Hepatic disorders, viral hepatitis
Aspartate transaminase (AST,GOT) Myocardial infarction, hepatic disorders
Alpha-amylase (AMS)
Acute pancreatitis
Aldolase (ALS)
Skeletal muscle disorders
Creatine kinase (CK)
Myocardial infarction, muscle disorders
Gamma-glutamyl transferase (GGT) Hepatic disorders
G-6-PD
Drug-induced hemolytic anemia
Lactate dehydrogenase (LD)
Myocardial infarction, hepatic disorders,
carcionomas
Lipase (LPS)
Acute pancreatitis
Leucine aminopeptidase (LAP)
Hepatobiliary disorders
5’-Nucleotidase (5’NT)
Hepatobiliary disorders
Pseudocholineesterase (PChE)
(butyrylcholine as substrate)
Ceruloplasmin (Copper-oxidase)
Organophosphate poisoning
Wilson’s disease (abnormal Cu metabolism)
Plasma enzyme patterns in disease: diagnosis & monitor
Table 4. Time sequence of changes in plasma enzymes after myocardial
infarction (hours, h; days, d)
Enzyme
Onset of
Peak activity Degree of
elevation (h)
(h)
elevation
Duration of
elevation (d)
CK (total)
4-8
12-24
5-10 x normal
3-4
CK-MB
4-8
24-36
5-15 x normal
2-3
8-12
24-48
2-3 x normal
4-6
LD
12-24
48-72
2-3 x normal
10
LD-1>LD-2
12-24
AST (GOT)
Remarks: Precision of diagnosis can be improved by
• estimations of more than one enzyme
• isozyme determinations
• serial enzyme estimations
5
Figure 3. Time course of enzymes release into plasma during infarction
III. Enzymes as therapeutic agents, drugs
Table 5. Enzymes used in therapy are genetically engineered proteins.
Enzyme
Disease/therapy
Protease, e.g., Streptokinase,
Clot lysis in myocardial
Activase(plasminogen activator) infarction, trauma, bleedings
Aspariginase,
Acute lymphocytic leukemia
e g., Oncospar (pepasparagase)
Adenosine deaminase,
e.g., Adagen
Superoxide dismutase,
Severe combined immunodeficiency syndrome (SCID)
Head injury (clinical trial phase)
e.g., Dismutec peg
Nanoenzyme/nanozyme (2007)-catalase
Parkinson’s disease(attenuate neuroinflammatory process)
IV. Enzymes as drug targets
Table 6. Enzyme targets for drugs in clinical use.
Enzyme targeting
Drug
Dihydrofolate reductase
Antifolates: methrotrexate (cancer),
(Folate metabolism)
pyrimethamine (protozoa, malaria)
trimethoprim (bacteria)
Xanthine oxidase
Allopurinol (hyperuricemia, gout)
(Purine metabolism)
Thymidylate synthase
5-Fluorouracil &
(Pyrimidine metabolism)
5-fluorodeoxyuridine (cancer)
Glycopeptide transpeptidase
Antibiotics, penicillin
HIV-Reverse transcriptase
3’-azido-2’,3’-dideoxythymidine (AZT)
HIV & SARS proteases
Ritonavir, saquinavir (clinical trial phase)
Rational drug design, based of known targeting enzyme
A). Acquired immunodeficiency syndrome (AIDS) & Bird Flu
A.1. HIV-1 Protease: HIV-1 has an aspartate protease, which is essential for
delivery of structural and functional processing of the gag and gag-pol viral
gene products.
Figure 4.
Protease inhibtor design, with known
3-D structure at active site complexed
with the drug with a Ki of 0.25 nM
A.2. HIV-1 Reverse Transcriptase (RT):
RT is a viral polymerase responsible for synthesis of viral DNA strand.
The RT inhibitors are majority for drugs in clinical use.
K103L mutation -> HIV-1 resist to drugs1-3
Figure 5.
Crystal of drug 4
A.3. Avian flu, H5N1 virus: Neuraminidase (NA)
Enzyme NA is responsible for release of virion from the host epithelial
cells by cleaving the receptor sialic acid. NA inhibitor is the drug in
clinical use, e.g., zanamivir (Relenza) and oseltamivir (Tamiflu)
Tamiflu
Figure 6. Rational design of Tamiflu on viral NA
Crystal of NATamiflu complex
B). Alzheimer’s disease (AD)
B.1. AD involves mutation of gene APP (chromosome 21) coding for
amyloid beta protein, this APP deposits in neurons of brain as
amyloid fibrils -->and leads to cell damage.
Loss of cholinergic cell is accompanied by reduced :
- ChAT(choline acetyltransferase), ACh (acetylcholine),
- AChE (acetylcholine estease).
This is compensated by
increased BuChE
(butyrylcholinesterase)
from Glial cell.
Figure 7.
-ve
-ve
Rational design is to
inhibit the BuChE,
ACh is not destroyed.
Fig 8.
Abnormal or mutant APP (amyloid beta protein) deposits in
neurons as amyloid fibrils --> cell damage.
B.2. Design of drug for treatment of Alzheimer’s disease :
Cholinesterase as enzyme target, phenothiazine derivatives as drug.
Figure 9.
V.Enzymes abnormalities in metabolisms
1. Excess enzyme activity
Gout is characterized by elevated uric acid
levels in blood and urine, due to
overproduction of de novo purine
nucleotides.
E.g., Excess PRPP synthase activity (Xlinked recessive inheritance pattern)
+ ve
[PRPP]
purine nucleotides
then leads to increase degradation of purines
to uric acid through xanthine oxidase.
Figure 10.
2. Enzyme deficiency
Identification and treatment of enzyme deficiency.
Enzyme deficiencies usually lead to increased accumulation of
specific metabolites in plasma and hence in urine. This is useful in
pinpointing enzyme defects.
E.g., De novo pyrimidine pathway: defects of OPRT and OMPDC leads
to accumulation of orotate ----> Hereditary orotic aciduria
(Gene mapping, 3q13; inheritance pattern, autosomal recessive).
Figure 11.
Uridine
3. Enzyme defects found in all human metabolisms.
Table 7. Examples of enzyme defects.
Enzyme defect
Disease
Metabolism/molecule involved
Pyruvate kinase
Deficiency/Anemia Glycolysis
Pyruvate dehydrogenase
Pyruvate/Krebs cycle
Chronic lactic acidosis
G-6-PD
Deficiency
Pentose phosphate pathway
Glycogen debranching enzyme
Cori (type III )
Gylcogen storage
Iduronate sulfatase Hunter
Mucopolysaccharides
Acyl-CoA dehydrogenase
Deficiency
Fatty acid oxidation
Hexoaminidase A Tay-sachs
Lipid/sphingolipid storage
Acid lipase
Deficiency
Cholesterol/Triacylglycerol (TAG)
HGPRT
Lesch-Nyhan
Purine
OPRT/OMPDC
Orotic aciduria
Pyrimidine
Phenylalanine hydroxylase
Amino acids/Phe
Phenylketonuria
Arginase
Deficiency
Amino acids/Arg /Urea cycle
Lysyl hydroxylase Ehlers-Danlos
Collagen
List of References:
• Moss, D.W., and Rosalki, S.B. (1996) Enzyme Test in
Diagnosis, Arnold Group, London.
• Mayne, P.H. (1996) Clinical Chemistry in Diagnosis and
Treatment, 6th edition, Arnold Group, London.
• Cohn, R.M., and Roth, K.S. (1996) Biochemistry and
Diseases, chapters: 11, 12, 15-17 & 25, Williams& Wilkins,
Baltimore.
• Devlin, T.M. (2002) Textbook of Biochemistry with Clinical
Correlations, pp. 166-174, 5th edition, Wiley & Sons, New
York.
• Murray, R.K., et al. (2002) Harper's Biochemistry, pp. 8183, 26th edition, Appleton & Lange, Stamford.
Evaluation: MCQs, 4 questions/5 choices
Good Luck
Role of telomerase in telomere replication in eukaryotes
Shampay J, Szostak JW, Blackburn EH in Nature. 1984 Jul 12-18;310(5973):154-157.
DNA sequences of telomeres maintained in yeast.
Role of telomerase in telomere replication in eukaryotes
1. Enzyme telomerase activity is repressed in somatic cells of
multi-cellular organisms, resulting in chromosome shortening with each
cell generation. This may be important in cell aging.
2. In many cancer cells, there is reactivated telomerase activity.
3. DE Shippen (1990) found that older (70-year old) human chromosomes
are shorter than from 20 years old ones, due to reduced telomerase activity.
4. Recently, the shortening of telomere is associated with atherosclerosis
and diabetes mellitus type II.
[Ref: KD Salpea & SE Humphries (2010), Atherosclerosis 209, 35-38]