PAMANTASAN NG LUNGSOD NG MAYNILA

COLLEGE OF MEDICINE
DEPARTMENT OF BIOCHEMISTRY AND NUTRITION
Intramuros, Manila, Philippines

CASE DISCUSSION ON ENZYME:

MUSCLE INJURY

Section 1C Group 2

Dimaano, Allen Karlo 2017-70123 ________________

Dimatatac, Mary Joy 2017-70003 ________________
Dioneda, Sjerlive Clare 2017-70079 ________________
Frayre, Alyssa Marie 2017-70107 ________________
Galang, Dianne 2017-70039 ________________
Garcia, Ericka Mae 2017-70023 ________________

Date Submitted:
August 30, 2017
I. INTRODUCTION

Enzymes are proteins that primarily work as an accelerator of chemical reactions involved
in biological systems. They accelerate chemical reactions by lowering the activation energy, which
is the amount of energy needed for the reaction to occur. Without the aid of these catalysts,
several various reactions necessary for the maintenance of a living cell would not be possibly
carried out at a sufficient pace. Thus, in order to achieve the high reaction rates observed in living
organisms, catalysts are required to facilitate the speed of the process.
All enzymes are proteins. They are high molecular weight compounds principally made up
of chains of amino acids linked together by peptide bonds. Like other proteins, their molecular
weights range from about 12,000 to more than 1 million. Additionally, their catalytic activity
depends on the integrity of their native protein conformation. Enzymes consist of a protein portion
(called the apoenzyme) and a non-protein portion (cofactor). This complex of apoenzyme and
cofactor constitute a haloenzyme. Cofactors may be organic groups permanently bound to the
enzyme (prosthetic groups), positively-charged metal ions (activators), or organic molecules,
usually derived from vitamins, but are not perminantly bound to the enzyme molecule and only
combine with the enzyme-substrate complex temporarily (coenzymes).
The distinguishing feature of an enzyme-catalyzed reaction is that it takes place within the
confines of a pocket on the enzyme called the active site. The molecule that is bound in the active
site and acted upon by the enzyme is called the substrate. The surface of the active site is lined
with amino acid residues with substituent groups that bind to the substrate molecule and hold it
in position while the reaction takes place.
There are two models that illustrate how enzymes work. In the lock and key model, the
substrate (“key”) fits into the active site (“lock”) to form a reaction intermediate. On the other hand,
in the induced-fit model, the substrate induces a conformational change in the enzyme, allowing
for a better fit between the two.
Two important characteristics of enzyme catalysts are that (a) the enzyme is not changed
as a result of catalysis and (b) the enzyme does not change the equilibrium constant but simply
increases the rate at which the reaction approaches equilibrium. It lowers the energy of activation.
Therefore a catalyst increases the rate but does not change the thermodynamic properties of the
system with which it is interacting.
Enzymes are also central in making diagnoses of certain pathological conditions or
diseases. An ideal enzyme for diagnostic enzymology should have the following characteristics:
(a) specific for the tissue or organ under study, (b) appears in the plasma or other fluid during the
“diagnostic window”, and (c) can be used for an automated assay.
Many enzymes are functional constituents of the blood, including lipoprotein lipase,
pseudocholinesterase, and enzymes associated with the clotting cascade. Other enzymes are
not normally found in blood plasma but can be released into it as a result of cell death or injury.
These particular enzymes serve no physiological function in the plasma but they act as
biomarkers. These are molecules whose levels or appearance in the plasma can assist in
diagnosis and prognosis of diseases and injuries in specific tissues.
It is important to note that these enzymes are not absolutely specific for just a single kind
of tissue or indicators of a particular disease only. As a consequence, enzyme assay data must
be used in conjunction with a comprehensive clinical examination. Other factors such as patient
age, sex, drug history, family history, as well as the sensitivity of the particular enzyme test must
be considered when interpreting enzyme assay data (Rodwell et al., 2015).

At the end of the activity, the students should be able to:

• Describe briefly the biological and chemical structures of enzyme
• Discuss how and why enzymes/specific isozymes are used as diagnostic tools
II. CASE
Muscle Injury

A 29 year old laborer developed chest pain while operating a pneumatic hammer on a construction
project. The pain was of moderate intensity, but he felt well enough to continue to work. As he
continued, he became more apprehensive, particularly since he experienced sharp pain on
respiration and tight feeling across the anterior chest wall. He was rushed to ER of a local hospital
and was admitted following a brief examination. ECG and chest xray examination were negative,
as were all the other blood tests that were performed except for elevated level of plasma lactate
dehydrogenase (LDH) of 6.9 mkat/L (400 IU/L). The elevation in plasma LDH persisted during the
next 4 days of hospitalization; no other laboratory or physical abnormalities appeared; and chest
pain gradually improved with bed rest.

Signs and Symptoms

ü chest pain of moderate intensity
ü sharp pain on respiration
ü tight feeling across the anterior chest wall
ü elevated level of plasma lactate dehydrogenase (LDH) of 6.9 mkat/L (400 IU/L)

III. CONCEPT MAP

 Initial presentation of the patient showed chest pain of moderate intensity, which was due
to the strenuous activity caused by operating the pneumatic hammer (jackhammer). Strenuous
work increased the oxygen demand of the skeletal muscles, which resulted in increased heart
rate to increase blood flow to the skeletal muscles. Eventually, oxygen could no longer meet the
demand, which results in a shift to anaerobic respiration. Lactate dehydrogenase (LDH) catalyzes
the reduction of pyruvate to lactate which regenerates NAD+ from NADH. NAD+ then enters
glycolysis to produce a net amount of 2 ATPs. Lactate then accumulated, which should have
provided feedback inhibition (to create more pyruvate from lactate and resume aerobic
respiration), but the patient continued his activity without resting. As a result, lactate threshold
was reached, causing lactic acidosis. This lactic acid buildup caused chest pain.
As the patient continued working, more strain was placed on his muscles due to the
vibration and heavy weight of the pneumatic hammer. This resulted in sarcomeric degeneration
due to Z-disk fragmentation (the Z-disk is the junction between sarcomeres, and it anchors actin
filaments to the Z line). The Z-line contains many cytoskeletal proteins which lessen the stress on
the plasmalemma by transmitting forces from the extracellular matrix to the cytoskeleton itself.
Due to the disruption of the Z-disk, there was an increase in the mechanical stress placed on the
sarcolemma, resulting in membrane failure. Consequently, there was a loss of cytoplasmic
components, including LDH, and an influx of extracellular ions, including Ca2+. This increase in
Ca2+ triggered degradative pathways, including calpain which is a calcium-sensitive cysteine
protease. Calpain then cleaved proteins on their cysteine residues. Denatured proteins were
tagged with ubiquitin, to mark them for degradation. These ubiquitylated proteins were then
identified and pulled into proteasomes, which contains many proteases for cleaving proteins.
Extensive local tissue damage to the muscles of the anterior chest wall then manifested as sharp
pain upon respiration (anterior chest wall contains primary and accessory muscles for respiration,
such as the intercostals, serratus anterior, and pectoral muscles) and tight feeling across anterior
chest wall.

IV. BIOCHEMICAL QUESTIONS

1. What reaction does lactate dehydrogenase (LDH) catalyze? Is an elevation in plasma of

LDH specific for damage to a given organ or tissue in the body?
Lactate dehydrogenase is an enzyme which facilitates the oxidation of lactate to pyruvate
and reduction of NAD+ to NADH and vice versa. In an anaerobic environment, with the decrease
in oxygen, pyruvate and the resulting NADH will go into lactic acid fermentation. The purpose of
lactic acid fermentation is to re-use the NADH by oxidizing it into NAD+ in order to start glycolysis
again. The byproduct in this reaction is pyruvate which is turned into lactate (or lactic acid).
Increase in lactic acid in turn can cause lactic acidosis which is a subtype of metabolic acidosis.
Normally, it shouldn’t occur unless concentration of NADH exceeds the capacity of the cell to
maintain redox homeostasis.
In this case, damage on the muscle tissue can cause cell lysis which results to intracellular
lactate dehydrogenase to be released into blood. In cases of myocardial infarction, LDH and
creatine kinase are used as markers to determine the size or degree of the infarction. However,
LDH can be found in almost all cells including the brain, the skeletal muscles and the liver making
them unspecific markers. However, by differentiating LDH isozymes present in the plasma it is
possible to discern or narrow down which organ(s) sustained damage. In plasma, LDH-2 is
predominant. If perchance, the amount of LDH-5 increases more than LDH-2 in plasma, it might
indicate a damage of the skeletal muscle or the liver. While an increase in LDH-1 might indicate
myocardial infarction. Although, an increase in LDH-1 can also be caused by in vitro or in vivo
hemolysis.
2. What are isozymes? How many isozymes of LDH normally occur in the serum? How
might LDH isozyme assay help to determine the cause of this patient’s condition? Is
there a similar proportional increase in the individual isozymes of LDH in all disease?
Explain.
Isozymes or isoenzymes are proteins with different structure which catalyze the same
reaction. The term had come into use when multiple forms of an enzyme, having the same or
almost the same biological activity, being derived from the same cells of the same tissues, could
be separated.
There are, in man, five principal isozymes of lactate dehydrogenase. These are the L1,
L2, L3, L4, and L5. Studies of these isoenzyme variants state that they differ with respect to their
amino acid composition, immunological and physical properties, and in their contribution to the
catalytic activity of the enzyme molecule. L1 is found mainly in the heart, L2 is chiefly in
association with the system in the body that functions as a defense against infection, L3 could be
seen in the lungs and other tissues, L4 in the kidney, placenta, and pancreas, and L5 particularly
in liver and striated (skeletal) muscles. The normal Lactate Dehydrogenase value usually ranges
from 2.34 mkat/L to 4.68 mkat/L.
Alterations in the levels or appearance of these molecules, in this case, the LDH, in the
plasma can aid in diagnosis of the said injury. An LDH isozyme assay may assist in the
identification of the cause of the patient’s condition since it is stated that a high-level of LDH is
indicative of tissue damage or injury.
No similar proportional increase in the individual isozymes of LDH would be observed
since certain diseases have their own patterns of elevated LDH isoenzyme levels depending
particularly on which area of the body is affected.

3. In setting up a valid enzymatic assay for LDH, the laboratory technician added lactate
and NAD+ to plasma specimen. Why were these substances added? Should lactate and
NAD+ have been added in very small or in excessive amounts? Could NADH or FAD
have been substituted for NAD+ in the assay?
Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to lactate, which is a
reversible reaction (Nelson & Cox, 2004).
Pyruvate + NADH + H+ <--> Lactate + NAD+
LDH can be detected by a special chromogenic reaction (Hegyi et al., 2013). In the
reaction, LDH binds to NAD+ to catalyze the reaction. Since this reaction is reversible, the addition
of NAD+ (coenzyme) and lactate (substrate) will disturb the equilibrium which in turn, will lead to
the onset of the oxidation of lactate to pyruvate. Thus, in the enzymatic assay for LDH, lactate
and NAD+ were added. In most clinical enzyme procedures, the reaction is started by the addition
of excess substrate. However, as the substrate is consumed, it becomes inadequate to saturate
the enzyme which then leads to substrate inhibition (Wilkinson, 1971). In contrast to the substrate,
the coenzyme (NAD+) should be added in excess to effectively convert all lactate to pyruvate.
This is done with the help of hydrazine which traps the formed pyruvate (Sigma-Aldrich, 2010).
While FAD cannot be used as a substitute for NAD+, NADH can be used as a substitute in the
assay along with the condition that pyruvate should also be used instead of lactate.

4. Depending on the specific assay method employed, which suitable substrate in an LDH
assay could be used, pyruvate or lactate? Why? Explain also how would assay
condition differ with use of pyruvate or lactate as the substrate?
Lactate Dehydrogenase (LDH), also termed as lactic acid dehydrogenase, is a tetrameric
enzyme abundant in major tissues and blood levels of the enzymes are normally low. However
when tissues are damaged either by injury or disease, the LDH enzymes inside the cells are
released into the bloodstream causing increased blood levels of LDH. Although elevation in LDH
levels is a non-specific finding, it is still useful in diagnosing tissue damage.
LDH plays a role in energy production as it catalyzes the interconversion of lactate and
pyruvate that involves the use of the coenzyme NAD+. Since the reaction is a reversible oxidation-
reduction reaction, it can either proceed in a forward (lactate) or reverse (pyruvate) direction. In
this reaction, lactate loses two electrons, becomes oxidized, and is converted to pyruvate. NAD+
gains two electrons, is reduced, and is converted to NADH. The coupling of oxidation-reduction
reactions is often depicted in the following manner.
Both lactate and NAD+ bind to the active site of the LDH enzyme and both lactate and
NAD+ participate in the catalysis reaction. In fact, catalysis could not occur unless the coenzyme
NAD+ bound to the active site.
Under normal physiological conditions, pyruvate is generated from glucose by glycolysis
and enters the citric acid cycle in the mitochondria, where it is oxidatively decarboxylated to form
acetyl-CoA, which is used to fuel oxidative phosphorylation, theoretically generating 36 net
adenosine triphosphate (ATP) per molecule of glucose.
However, when oxygen becomes scarce, as in the case of over-exertion of muscles, cells
are unable to use oxidative phosphorylation to efficiently generate ATP. In this hypoxic condition,
anaerobic glycolysis takes its place, wherein glycolysis becomes the main generator of ATP,
producing 2 net ATP per molecule of glucose. As part of glycolysis, hydrogen from glucose is
placed on NAD+ to form NADH. Normally, these hydrogen atoms are then transferred to oxygen
to form water. If oxygen isn't available, the NADH builds up and there isn't enough NAD+ to
continue using glycolysis to make [Link], NAD+ is required to enable the sixth step of glycolysis
as glyceraldehyde phosphate dehydrogenase (GAPDH) uses NAD+ to convert glyceraldehyde 3-
phosphate (GADP) to D-1,3-biphosphoglycerate (1,3 BPG). NAD+ is usually regenerated through
oxidative phosphorylation by the electron transport chain, so when the oxygen supply is restricted,
NAD+ is regenerated from NADH by LDH in order to maintain glycolysis, generating lactate as a
by-product. The NAD+ can then be recycled to do another round of glycolysis, quickly producing
more ATP.
There are 2 enzymatic assays involved in LDH measurement, namely Wacker method and
the Wroblewski-LaDue method. Wroblewski and LaDue published the first UV kinetic method for
the determination of LDH activity in serum in 1955. Their method utilizes the reverse reaction from
pyruvate to lactate. In 1956, Wacker et al created a procedure that followed the forward reaction
from lactate to pyruvate. The rate of reverse reaction is approximately three times faster, allowing
smaller sample volumes and shorter reaction times. However, the reverse reaction is more
susceptible to substrate exhaustion and loss of linearity. Furthermore, the forward reaction does
not require pre-incubation to exhaust endogenous a-keto acids and displays linearity over a wider
range of activity in patient samples. Thus, the most commonly used method is the Wacker Method,
which follows the forward reaction.
The principle behind the enzymatic assay is based on the simultaneous reduction of NAD+
to NADH when LDH catalyzes the oxidation of lactate to pyruvate. NADH strongly absorbs light
at 340nm, whereas NAD+ does not. The rate of NAD+ reduction can be measured as the rate of
increase in absorbance at 340nm. The rate of increase in absorbance is also directly proportional
to the LDH activity in the serum.

5. What other enzyme assay might be useful in diagnosing and following up this case?
What enzyme assays/markers can be used to completely rule out myocardial
infarction?
LDH is a tetrameric enzyme that exists in five isoenzymes which differs structurally in their
subunits – H for heart (myocardium) and M for muscle. These isoenzymes are tissue specific
hence they can serve as markers that can be used diagnostically. LDH assay following
electrophoresis can be used in the qualitative and quantitative determination of the LDH
isoenzymes by electrophoresis on agarose gel and specific enzymatic detection. There are five
different LDH isoenzymes that can be detected in serum. In standard LDH isoenzymes
electrophoretic patterns five bands are observed, identified according to their electrophoretic
mobility from anode to cathode as LDH 1, LDH 2, LDH 3, LDH 4, and LDH 5. Quantitation of these
fractions provides useful information on the identification of specific tissue or organ damage. In
the case of skeletal muscle injury, high level of LDH 5 found on skeletal muscle would be
observed.
In MI, the lack of oxygen supply (Ischemia) leads to the necrosis of cardiac muscle tissue.
Upon tissue damage, the contents of the cell is released in the bloodstream increasing its
concentration in the blood relative to the normal levels. Hence, enzymes found specifically in
cardiac muscle (Myocardium) that leak in the blood may serve as markers indicating tissue
damage. Isoenzymes of Troponins (Tn-T and Tn-I) and Creatinine kinase (CK-MB) serves as
biological markers that can be used to detect myocardial infarction. Hence, if laboratory results
would indicate normal levels of these enzymes in the serum, myocardial infarction can be ruled
out. Enzyme-linked Immunosorbent Assay are used in the detection of this markers.

MYOCARDIAL NECROSIS MARKERS

Aspartate Aminotransferase was first used as biomarkers for myocardial infarction
however, due to its lack of specificity to the cardiac tissue, it is no longer used for the diagnosis
of MI.
Specific isoenzyme of lactate dehydrogenase can also be an indicator of MI. Normally, the
activity of the LDH 2 isozyme is higher than that of LDH 1, however, in the case of infarction the
activity LDH1 becomes greater than LDH2. The change in dominance known “LDH flip”.
Troponin is a regulatory proteins in the skeletal and cardiac muscle with a complex of three
protein subunits-- Toponin C (Tn-C), Toponin T (Tn-T), and Toponin I (Tn-I). Tn-C is the Ca2+
binding protein that is identical to both skeletal and cardiac muscles. Hence, they are not specific
for myocardial injury. Tn-T (tropomyosin-binding) and Tn-I (inhibitory) are specific for cardiac
necrosis. These proteins are released to the blood once tissue damage occurs.
High levels of creatinine kinase (CK) may also indicate myocardial infarction. There are
three isoenzymes of CK—MM, BB, and MB. Similar to troponin, each isoenzymes is tissue
specific. CK-MM is the skeletal muscle fraction, CK-MB is the cardiac muscle fraction and CK-BB
is the brain fraction of the total CK. Since the primary source of CK-MB is the myocardium, the
level of this isozyme increase with myocardial damage. Unlike Toponin I and T, detection of this
enzyme allows correlation with infarction size, detection of progression and reinfarction. ELISAs
assays are used in the determination of the CK-MB, Tn-T, and Tn-I.

Type Abundance Composition Location

LDH1 14-26% HHHH Myocardium and RBC

LDH2 29-39% HHHM Myocardium and RBC

LDH3 20-26% HHMM Lung, lymphocytes, spleen, pancreas, brain and

kidney

LDH4 8-16% HMMM Liver

LDH5 6-16% MMMM Skeletal muscle

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