Measurements of enzymes in serum within a tissue enzymes as markers of disease Injury to tissue releases cellular substances that can be used as plasma markers of tissue damage enzyme release is highly specific for cell death in some settings ID: 918494
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Slide1
Enzymes
Clinical significance & Methods
Slide2Measurements of enzymes
in serum
within a tissue
enzymes as markers of disease,
Injury to tissue releases cellular substances that can be used as plasma markers of tissue damage.
enzyme release is highly specific for cell death in some settings.
Slide3Some enzymes are predominantly found in the specialized tissue
while others, more widely distributed, have tissue specific
isoenzymes
or isoforms
The timing of the enzyme's diagnostic window
early indicators
late indicators
Several enzymes have diagnostic utility
Slide4Overlap occurs for some enzymes
may be used
for investigating disease
in several organs.
Slide5Enzymes of clinical
interest
MUSCLE ENZYMES
More commonly measured
CK, LD
CK
; adenosine
triphosphate
:
creatine
N-
phosphotransferase
Slide6Slide7Inhibitors
excess Mg
2+
Many metal ions, such as Mn
2+
, Ca
2+
, Zn
2+
,Cu
2+
sulfhydryl
-binding reagents
Iodoacetate
CK is a
dimer
(B and M)
the products of loci on chromosomes 14 and 19, respectively.
BB (or CK-1), MB (or CK-2), and MM (or CK-3).
numbered on the basis of their
electrophoretic
mobility, with the most anodal form receiving the lowest number.
The distribution of these
isoenzymes
in the various tissues
Slide9Slide10Serum CK activity is subject to a number of physiological variations.
Sex, age, muscle mass, physical activity, and race all interact to affect serum activities
Slide11Clinical Significance
Serum CK activity is greatly elevated in all types of muscular dystrophy.
may be increased long before the disease is clinically apparent.
Serum CK activity characteristically falls as patients get older and as the mass of functioning muscle diminishes with the progression of the disease.
Slide12Long time Physical inactivity reduce serum CK
Skeletal muscle that is diseased or damaged
(fetal reversion)
Renal failure , increase CK
Serum CK activity demonstrates an inverse relationship with thyroid activity.
Following a
myocardial
infarction
MB
isoenzyme
The assay of CK activity
Coupled enzyme methods
NADP+ to NADPH, monitored
spectrophotometrically
.
Slide15CK activity in serum is relatively unstable and is rapidly lost during storage.
Average stabilities are less than 8 hours at room temperature, 48 hours at 4°C, and 1 month at -20°C.
Methods for the Separation & Quantification of CK
Isoenzymes
Electrophoresis & various immunological methods.
Slide16Enzymes of clinical interest
Widely
used
enzymes
Aspartate aminotransferases (AST)
Alanine aminotransferases (ALT)
ALP
Slide17The aminotransferases
The aminotransferases
catalyze the
interconversion
of amino acids to 2-oxo-acids
L-aspartate:2-oxoglutarate
aminotransferase;AST
L-alanine:2-oxoglutarate aminotransferase; ALT
Slide18Slide19Slide20Pyridoxal-5'-phosphate ↔ pyridoxamine-5' –phosphate
Both the coenzyme-deficient
apoenzymes
and the
holoenzymes
may be present in serum.
All factors affecting the rate of reaction must be optimized and controlled
Slide21Transaminases
are widely distributed throughout the body.
Slide22Clinical Significance
Liver disease is the most important cause of increased
transaminase
activity in serum.
In most types of liver disease, ALT activity is higher than that of AST;
exceptions
alcoholic hepatitis, hepatic cirrhosis, and liver
neoplasia
.
elevated even before the clinical signs and symptoms of disease
Slide23Peak values
of
transaminase
activity occur between the 7th and 12th days
Medications
nonsteroidal
antiinflammatory
drugs, antibiotics.
ALT is the more liver-specific enzyme.
elevations of ALT activity persist longer than do those of AST activity.
Slide24After AMI, increased AST activity appears in serum
AST activity also is increased in some types of muscle diseases
Also serum CK
Mitochondrial AST (m-AST) activity
extensive liver cell degeneration and necrosis.
the ratio between m-AST and total AST activities
typical of alcoholic hepatitis
Slide25The increased AST activity might reflect decreased clearance
Macro-AST
Slide26Measurement of
Transaminase
Activity
coupling the
transaminase
reactions to specific
dehydrogenase
reactions.
Pyruvate
formed in the ALT reaction is reduced to lactate by LD.
Slide27The change in absorbance per minute
(
Δ
Almin
) is proportional to the
micromoles of NADH oxidized and in turn to micromoles of substrate transformed per minute.
AST activity in serum is stable for up to 48 hours at 4°C.
ALT stability is better maintained at -70°C.
Slide28Reference Intervals
AST
; 31 U/L for women and 35 U/L for men
Methods for the Separation & Quantification of AST
Isoenzymes
electrophoresis, selective inhibition, and immunoassays.
anionic (
cytoplasmic
AST) and a cationic band (m-AST)
Slide29m-AST (healthy individuals )
About 5% to I0% of the activity of total AST in serum
3.0 U/L.
Slide30ALKALINE PHOSPHATASE
ALP
Orthophosphoric
monoester
phospho
hydrolase
alkaline optimum
hydrolysis of a large variety of naturally occurring and synthetic substrates
ALP activity is present in most organs of the body
especially associated with membranes and cell surfaces
In the mucosa of the small intestine and proximal convoluted tubules of the kidney, in bone (osteoblasts), liver, & placenta
Slide31Metabolic function
Lipid transport in the intestine
Calcification process in bone
ALP exists in multiple forms
Slide32Slide33activators of the enzyme,
divalent ions, Zn
2+
Inhibitors
Phosphate, cyanide ions,…
The ALP activity in the sera of healthy adults
Liver
Skeleton
Slide34Clinical Significance
Common causes of elevation
Liver
Hepatobiliary
disease
bone
Bone disease associated with increased
osteoblastic
activity
Carcinoplacental
isoenzymes
derepression
of the placental ALP gene
non placental
isoenzymes
Modified forms of ALP
Slide35Determination of Alkaline
Phosphatase
Activity
The rate of formation of 4-NP at 405 nm is monitored
Slide36Determination of Alkaline
Phosphatase
Activity
Serum or
heparinized
plasma, free of
hemolysis
, should be used.
Freshly collected serum or up to 4 hours RT
*
Frozen specimens; ALP activity increases
kept at room temperature for 18 to 24 before assay
*Room temperature
Slide37Reference Intervals
ALP activities in serum vary with age.
Slide38Methods for the Separation and Quantification of
Alkaline
Phosphatase
Isoenzymes
Assays for ALP
isoenzymes
are needed when:
the source of an elevated ALP in serum is not obvious
to monitor the disease activity and the effect of appropriate therapies
Electrophoretic
mobility
Stability to
denaturation
by heat or chemicals
Response to the presence of selected Inhibitors
Slide39anodal mobility,
The liver, most rapidly
Bone ALP, slightly lower
Intestinal ALP
Placental
isoenzyme
Discrete band overlying the diffuse bone fraction
Improvement of
electrophoretic
separation
treated briefly with neuraminidase
Electrophoresis in the presence of wheat germ
lectin
Slide40Placental ALP is heat stable
65°C for 30 min
Other evidences
eg
;measurement of GGT
Immunological methods
Intestinal or placental ALPs.
Slide41Acid Phosphatase (ACP)
Include all phosphatases
that hydrolyze
phosphate esters with an optimum pH of less
than 7.0.
Produced
by
Primarily, prostate
gland,
also
found
in
erythrocytes, platelets
, leukocytes, bone marrow, bone, liver, spleen
, kidney
, and intestine.
Slide42Acid Phosphatase (ACP)
ACP is present
in
Lysosomes,
Extra
lysosomal
The
lysosomal
and
prostatic
enzymes
strongly
inhibited by
D-tartrate
ions
,
the
erythrocyte and
bone
isoenzymes
are not inhibited.
Slide43Acid Phosphatase (ACP)
Normal serum ACP
The
majority of the normally low
ACP activity
of (
unhemolyzed
) serum is of
a
Tartrate-resistant type
(TR-ACP)
Probably originates
mainly in osteoclasts
.
Increased
Physiologically in
growing children
Pathologically in conditions of increased
osteolysis
and bone remodeling.
High concentrations of TR-ACP
in
serum
Reflect increased
osteoclastic
activity
, whether appropriate as in normal
bone growth
, or
damaging
Slide44The only
nonbone
condition
Gaucher's
disease
of
spleen,
a
lysosomal
storage
disorder,
elevated activities of TR-ACP
are found in
serum,
abnormal macrophages
in spleen and other tissues,
overexpress ACP
Slide45Acid Phosphatase (ACP)
ACP-determining genes
At
least four
have
been
identified and
mapped
.
ACPs are labile
more
than 30% of
the ACP
activity may be lost in 3 hours at room temperature.
Acidification of the
serum
specimen
to a pH below 6.5
aids in
stabilizing the enzyme activity.
Slide46Acid Phosphatase
Prostatic acid phosphatase (PAP
),
an
optimum
pH of 5
to 6,
very
labile at a pH of greater than
7.0
very labile at
temperature greater
than 37°C
.
Distinguished from other
acid phosphatases
by
Using tartrate
,
Strongly inhibits
the prostatic form.
Select substrates
that are more specific for
PAP
thymolphthalein
monophosphate
and
β
-
naphthol
phosphate
.
Hydrolyzed by
PAP much more quickly
Slide47Acid Phosphatase
The clinical use of PAP
as
a screening tool for
prostate cancer.
to
help stage prostate cancer
,
to correlate with
the prognosis of the disease
,
to monitor therapy.
Elevated serum PAP may be seen in
malignant conditions,
osteogenic
sarcoma, multiple myeloma,
and bone
metastases of other cancers.
in some
benign
conditions,
Osteoporosis, benign prostatic hyperplasia
and hyperparathyroidism
.
Slide48Acid Phosphatase
The clinical use of
PAP
has
been replaced by
PSA
PAP
is not
as sensitive as PSA for screening or for detection of
early cancer.
restricted to
confirmation of
metastatic prostate cancer and staging of
prostate cancer.
Currently the method of choice for PAP
Measurement of enzymatic
activity.
Slide49Acid Phosphatase
Measurement of ACP
Methods
Continuous-monitoring
methods of ACP activity
Immunological
methods
Principle:
Thymolphthalein
monophosphate is hydrolyzed by prostatic ACP at pH 5.4
and
37°C.
The reaction stopped after 30 min by addition of NaOH-Na
2
CO
3
solution
This develops the alkaline color of the liberated
Thymolphthalein
Measured at 595 nm
Slide50Acid Phosphatase
Specimens:
Serum
should be immediately separated from
erythrocytes and
stabilized by the addition of
acetic
acid
to
lower the pH to
5.4
Under these conditions,
ACP activity
is maintained at room temperature for several hours,
for up to a week if the serum is refrigerated, and for 4
months if
stored at
-20°C
.
Slide51Acid Phosphatase
Specimens
:
Hemolyzed
serum specimens are contaminated with considerable amounts of the erythrocyte tartrate-resistant
isoenzyme
and should be rejected
.
Chylous
sera should be avoided
Interference with measurement due to turbidity
Slide52Acid Phosphatase