World Journal of Pharmaceutical Sciences

ISSN (Print): 2321-3310; ISSN (Online): 2321-3086

Available online at: http://www.wjpsonline.org/
Original Article

Role of enzyme alkaline phosphatase in parkinsons disease patients

Sujitha M1* and Durai Rajan P2*

1
Tutor, 2Professor & HOD, Department of Pharmacology, Sri Muthukumaran Medical
College Hospital & Research Institute, Chikkarayapuram (Near Mangadu), Chennai -600069
[Affiliated to the Tamil Nadu Dr. M.G.R Medical University, Chennai, India]
Received: 19-06-2018 / Revised Accepted: 01-07-2018 / Published: 02-07-2018

ABSTRACT

Parkinson’s Disease (PD) is a debilitating illness associated with considerable impairment of

quality of life and substantial cost of Health care systems. Our aim of this present study was
to find the levels of alkaline phosphatase enzyme in Parkinson’s disease patients for
correlation. Methodology: Blood samples were collected from patient of parkinson’s disease
and also from Healthy volunteers in a government hospital by venipuncture and the serum
was separated. The serum was then used for the estimation of Total protein by Lowry’s
method. The Results indicates the Alkaline phosphatase levels were found to be increased in
the view of this present study correlates well with the finding of study. Hence we suggest for
the cure of Neurodegenerative Disorder need further Research to confirm the cause of the
elevated alkaline phosphatase level in Parkinson’s Disease and to develop a newer approach
as a novel therapy for Parkinson’s Disease.

Keywords: Parkinson’s Disease, Alkaline Phosphatase, Neurodegenerative Disease, Lowry’s

Method, L-DOPA, Spectrophotometer

Address for Correspondence: Mrs. M. Sujitha, M.Sc, Tutor, Department of Pharmacology, Sri Muthukumaran
Medical College, Tamil Nadu, India; E-mail: jei.communication2016@gmail.com

How to Cite this Article: Sujitha M and Durai Rajan P. Role of enzyme alkaline phosphatase in
parkinsons disease patients. World J Pharm Sci 2018; 6(7): 58-64.

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 Sujatha and Durai Rajan, World J Pharm Sci 2018; 6(7): 58-64

INTRODUCTION The basis for aggregation varies across diseases.

For example, it may be directly related to an
The central nervous system consists of the brain intrinsic feature of a mutated protein (e.g.,expanded
and spinal cord. The brain is situated within the polyglutamine repeat in Huntington disease), a
cranium and comprises a forebrain feature of a peptide derived from a larger precursor
(prosencephalon), a midbrain (mesencephalon), and protein (e.g., Aβ in Alzheimer disease), or an
a hindbrain (rombencephalon).The forebrain is unexplained alteration of a normal cellular protein
subdivided into a telecephalon consisting of the (e.g., α-synuclein in sporadic Parkinson disease).
two cerebral hemispheres laterally and a central The aggregated proteins are generally cytotoxic,
part (the diencephalon) composed mainly of two but the mechanisms by which protein aggregation
thalami and the hypothalamus . The midbrain is linked to cell death may be different in these
(mesencephalon) made up of crus cerebri, various diseases.
substantia nigra, tegmentum, tectum. The hindbrain
is subdivided into the Pons, medulla and DEGENERATIVE DISEASES OF BASAL
cerebellum. The midbrain Pons and medulla are GANGLIA AND BRAINSTEM
collectively known as the brain stem.(1)There are Diseases affecting these regions of the brain are
many central nervous system diseases, frequently associated with movement disorders,
including infections of the central nervous system including rigidity, abnormal posturing, and chorea.
such as encephalitis and poliomyelitis, In general, they can be categorized as manifesting
neurodegenerative such as Alzheimer's disease, either a reduction of voluntary movement or an
Parkinsonism, amyotrophic, autoimmune and abundance of involuntary movement. The basal
inflammatory diseases such as multiple or acute ganglia and especially the nigrostriatal pathway
disseminated encephalomyelitis, and genetic play an important role in the system of positive and
disorders such as Krabbe's disease, Huntington's negative regulatory synaptic pathways that serve to
disease, oradrenoleukodystrophy. modulate feedback from the thalamus to the motor
cortex. The most important disorders in this group
DEGENERATIVE DISEASE are those associated with Parkinsonism and
These are diseases of gray matter characterized Huntington chorea. (2 )
principally by the progressive loss of neurons with
associated secondary changes in white matter ALKALINE PHOSPHATASE
tracts. Two other general characteristics bring them (ALP, ALKP) (EC 3.1.3.1) is
together as a group. First, the pattern of neuronal a hydrolase enzyme responsible for
loss is selective, affecting one or more groups of removing phosphate groups from many types of
neurons while leaving others intact. Second, the molecules, including nucleotides, proteins,
diseases arise without any clear inciting event in andalkaloids. The process of removing the
patients without previous neurologic deficits. The phosphate group is called dephosphorylation. As
neuropathology findings observed in the the name suggests, alkaline phosphatases are most
degenerative diseases differ greatly; in some, there effective in an alkaline environment. It is
are intracellular abnormalities with some degree of sometimes used synonymously as basic
specificity (e.g., Lewy bodies, neurofibrillary phosphatase.
tangles), while in others, there is only loss of the
affected neurons. It is convenient to group the PHYSIOLOGY
degenerative diseases according to the anatomic In humans, alkaline phosphatase is present in all
regions of the CNS that are primarily affected. tissues throughout the entire body, but is
Some degenerative diseases have prominent particularly concentrated in liver, bile duct,
involvement of the cerebral cortex, such as kidney, bone, and the placenta. Humans and most
Alzheimer disease; others are more restricted to sub other mammals contain the following alkaline
cortical areas and may present with movement phosphatase isozymes:
disorders such as tremors and dyskinesias. As ▪ ALPI – intestinal
genetic and molecular studies of these diseases ▪ ALPL – tissue non-specific
have progressed, there has been recognition of (liver/bone/kidney)
shared features across many of the disorders. ▪ ALPP – placental (Regan isozyme)

A common theme among the neurodegenerative INTESTINAL ALKALINE PHOSPHATASE

disorders is the development of protein aggregates Intestinal alkaline phosphatase is secreted by
that are resistant to normal cellular mechanisms of enterocytes and seems to play a pivotal role in
degradation through the ubiquitin-proteasome intestinal homeostasis and protection as well as in
system. These aggregates can be recognized mediation of inflammation via repression of the
histological as inclusions, which often form the downstream toll-like receptor (TLR)-4-dependent
diagnostic hallmarks of these different diseases. and MyD88-dependent inflammatory cascade. It

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dephosphorylates toxic/inflammatory microbial In addition, the following drugs have been

ligands like lipopolysaccharides, demonstrated to reduce alkaline phosphatase:
unmethylatedcystosine –guanine dinucleotides, ▪ Oral contraceptives
flagellin and extracellular nucleotides such as
uridine diphosphate or ATP. Thus,altered IAP Leukocyte alkaline phosphatase:
expression has been implicated in chronic Leukocyte alkaline phosphatase (LAP) is found
inflammatory diseases such as IBD. It also seems within white blood cells. White blood cell levels of
to regulate lipid absorption and bicarbonate LAP can help in the diagnosis of certain conditions.
secretion in the duodenal mucosa, which regulates ▪ Higher levels are seen in polycythemia
the surface pH. vera (PV), essential
thrombocytosis (ET), primary
DIAGNOSTIC USE myelofibrosis (PM), and the leukemoid
The normal range is 20 to 140 IU/L. High ALP reaction.
levels indicate blocked bile ducts. Levels are ▪ Lower levels are found in chronic
significantly higher in children and pregnant myelogenous
women. Also, elevated ALP indicates that there leukemia (CML), paroxysmal nocturnal
could be active bone formation occurring as ALP is hemoglobinuria (PNH) and acute
a byproduct of osteoblastactivity (such as the case myelogenous leukaemia (AML).(4)
in Paget's disease of bone).
Lowered levels of ALP are less common than PARKINSONISM
elevated levels. Typical use in the lab for alkaline Parkinsonism is a clinical syndrome known to
phosphatases includes removing phosphate occur in elderly people and form 1% of
monoester to prevent self ligation. neurodegenerative disease. It is characterized by
diminished facial expression, stooped posture,
ELEVATED LEVELS slowness of voluntary movement, festinating gait
If it is unclear why alkaline phosphatase is (progressively shortened, accelerated steps),
elevated, isoenzyme studies rigidity, and a "pill-rolling" tremor. This type of
using electrophoresis can confirm the source of the motor disturbance is seen in a number of conditions
ALP. Heat stability also distinguishes bone and that have in common damage to the nigrostriatal
liver isoenzymes ("bone burns, liver lasts"). dopaminergic system. Parkinsonism may also be
Placental alkaline phosphatase is elevated induced by drugs that affect this system,
in seminoma and active form of Rickets, as well as particularly dopamine antagonists and toxins. The
in the following diseases and conditions. principal diseases to be discussed here that involve
Biliary obstruction the nigrostriatal system are as follows:
Bone conditions
Osteoblastic bone tumors PARKINSON DISEASE (PD)
Osteomalacia Multiple system atrophy, a disorder that may have
Osteoporosis Parkinsonism as a prominent symptom (clinical
Hepatitis presentation as striatonigral degeneration) as well
Cirrhosis as other symptoms (cerebellar ataxia and
Hyperthyroidism autonomic dysfunction). Post encephalitic
Myocardial Infarction Parkinsonism, which was observed in the wake of
Pregnancy the influenza pandemic that occurred between 1914
and 1918 and is now vanishingly rare. Progressive
LOWERED LEVELS supranuclear palsy (PSP) and corticobasal
The following conditions or diseases may lead to degeneration (CBD), which are movement
reduced levels of alkaline phosphatase: disorders that may also exhibit cognitive
▪ Hypophosphatasia, an autosomal impairment; they share some pathologic and
recessive disease genetic features with each other and with other
▪ Postmenopausal women receiving estrogen tauopathies (see the discussion above in the section
therapy because of osteoporosis on front temporal dementias).
▪ Men with recent heart
surgery, malnutrition, magnesium This diagnosis is made in patients with progressive
deficiency, hypothyroidism, or severe anemia parkinsonism in the absence of a toxic or other
▪ Children with achondroplasia and cretinism known underlying etiology. Familial forms with
▪ Children after a severe episode of enteritis autosomal-dominant or autosomal-recessive
▪ Pernicious anemia inheritance exist. Although these make up a limited
▪ Aplastic anemia number of cases, they have contributed to our
▪ Chronic myelogenous leukemia understanding of the pathogenesis of the disease. In
▪ Wilson's disease addition to the movement disorder, there are other,

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less well-characterized changes in mental function, all tissue but in lower concentration .its is also seen
which may include dementia, in a subset of to be lower in neurodegeration disorder Wilson and
individuals with Parkinson’s disease. Symptoms of anemia, plastic anemia, bone disorder and Gastric
Parkinson’s disease include manifestation of intestinal tract disorder .the wide range of
Dopamine in the substania nigra in the tremor, distribution of this enzyme prompts in it
rigidity, Bradykinesia, postural instability, investigation in Parkinson’s Disease. (2 )
Difficulty in swallowing and speech, urinary and
other bowl problem leads to other cognitive MATERIALS AND METHOD
abnormal problem fatigue is also. The hallmark of
Parkinson’s disease is neurodegenerative of Sample Collection:
dopamine in substania nigra lead to dopamine and The sample 1ml were collected from patient of
cholinergic imbalance.(3) Parkinson’s disease (5 patients) in a government
hospital by venipuncture and kept in a slanting
Pathological the ethiopathogenesis of Parkinson’s position for one hour for the separation of serum.
disease characterized by deposition of abnormal α- This serum was then centrifuged at 2000 r.p.m for
synuclein in the neuron of substania nigra which 5 minutes and then was used for the estimation of
have under neurodegenerative. The pathology of total protein by Lowry’s method. The samples were
the condition has suggested the use of dopamine in collected from healthy volunteers for control (5
the form of L-DOPA as standard treatment in these samples).
patients. Other amylolting drug are also being
consider. However treatment of Parkinson’s INSTRUMENTS USED:
disease with respect to rapid alleviation of Various bio-instruments are used in the lab which
symptom remains a difficult. A similar challenge is facilitates the biochemistry test. The bio-
found is faced in treating patients of younger age instruments used in the laboratory as follows,
group[less than 40 years] ,which have been 1. Centrifuge
increasingly observed although free radical changes 2. Spectrophotometer
and metal toxicity have implicated along with the 3.PH meter
genetic role in which protein implicated are α-
synuclein and parkin. CENTRIFUGE:
A laboratory centrifuge is a piece of laboratory
Other treatment modalities include neurosurgical equipment, driven by a motor, which spins liquid
approach of where lesion in the extrapyrimidal samples at high speed. There are various types of
system has compensively nigrostive function by centrifuges, depending on the size and the sample
provide motor symptom in Parkinson’s disease. capacity.

Symptomatic response to l-DOPA therapy is one of Centrifuge tubes or centrifuge tips are tapered tubes
the features, in addition to clinical signs and of various sizes made of glass or plastic. They may
symptoms, which support a diagnosis of PD. While vary in capacity from tens of milliliters, too much
l-DOPA therapy is often extremely effective in smaller capacities used in micro centrifuges used
symptomatic treatment, it does not significantly extensively in molecular biology laboratories. The
alter the intrinsically progressive nature of the most commonly encountered tubes are of about the
disease. Over time, l-DOPA becomes less able to size and shape of a normal test tube (~ 10 cm long).
help the patient through symptomatic relief and Micro centrifuges typically accommodate micro
begins to lead to fluctuations in motor function on centrifuge tubes with capacities from 250 all to
its own. As a result, there has been a search for 2.0ml. These are exclusively made of plastic.
alternative therapies that might alter the disease
course. Given the well-characterized biochemical Glass centrifuge tubes can be used with most
defect in PD, therapy through neural solvents, but tend to be more expensive. They can
transplantation has been attempted. Clinical be cleaned like other laboratory glassware, and can
improvement has been reported in patients with PD be sterilized by autoclaving. Plastic centrifuge
or MPTP-induced Parkinson disease treated with tubes, especially micro centrifuge tubes tend to be
stereotactic implants of fetal mesencephalic tissue less expensive. Water is preferred when plastic
into the striatum. Other current treatment modality centrifuge tubes are used. They are more difficult
includes neurosurgical approach where lesion in to clean thoroughly, and are usually inexpensive
the extrapyramidal system has compensively enough to be considered disposable.
nigrostive function by placement of simulative
electrode which could provide motor symptoms in The load in a laboratory centrifuge must be
Parkinson disease. Alkaline phosphates to occur carefully balanced. This is achieved by using a
ubiquitously: its elevated level are more common combination of samples and balance tubes which
than lower level. Liver, bile duct, kidney, Bone and all have the same weight or by using various

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balancing patterns without balance tubes. Small does not cover time-resolved spectroscopic
differences in mass of the load can result in a large techniques.
force imbalance when the rotor is at high speed.
This force imbalance strains the spindle and may Spectrophotometry involves the use of a
result in damage to the centrifuge or personal spectrophotometer. A spectrophotometer is a
injury. Some centrifuges have an automatic rotor photometer that can measure intensity as a function
imbalance detection feature that immediately of the light source wavelength. Important features
discontinues the run when an imbalance is of spectrophotometers are spectral bandwidth and
detected. linear range of absorption or reflectance
measurement.
Before starting a centrifuge, an accurate check of
the rotor and lid locking mechanisms is mandatory. A spectrophotometer is commonly used for the
Centrifuge rotors should never be touched while measurement of transmittance or reflectance of
moving, because a spinning rotor can cause serious solutions, transparent or opaque solids, such as
injury. Modern centrifuges generally have features polished glass, or gases. However they can also be
that prevent accidental contact with a moving rotor designed to measure the diffusivity on any of the
as the main lid is locked during the run. listed light ranges that usually cover around 200nm
- 2500nm using different controls and calibrations.
Centrifuge rotors have tremendous kinetic energy Within these ranges of light, calibrations are
during high speed rotation. Rotor failure, caused by needed on the machine using standards that vary in
mechanical stress from the high forces imparted by type depending on the wavelength of the
the motor, can occur due to manufacturing defects, photometric determination. An example of an
routine wear and tear, or improper use and experiment in which spectrophotometer is used is
maintenance. Such a failure can be catastrophic the determination of the equilibrium constant of a
failure, especially with larger centrifuges, and solution. A certain chemical reaction within a
generally results in total destruction of the solution may occur in a forward and reverse
centrifuge. While centrifuges generally have safety direction where reactants form products and
shielding to contain these failures, such shielding products break down into reactants. At some point,
may be inadequate, especially in older models, or this chemical reaction will reach a point of balance
the entire centrifuge unit may be propelled from its called an equilibrium point. In order to determine
position, resulting in damage to nearby personnel the respective concentrations of reactants and
and equipment. Uncontained rotor failures have products at this point, the light transmittance of the
shattered laboratory windows and destroyed solution can be tested using spectrophotometry.
refrigerators and cabinetry. To reduce the risk of The amount of light that passes through the
rotor failures, centrifuge manufactures specify solution is indicative of the concentration of certain
operating and maintenance procedures to ensure chemicals that do not allow light to pass through.
that rotors are regularly inspected and removed
from service or derated (only operated at lower The use of spectrophotometers spans various
speeds) when they are past their expected lifetime. scientific fields, such as physics, materials science,
chemistry, biochemistry, and molecular biology.
UV- VISIBLE SPECTROPHOTOMETER: They are widely used in many industries including
PRINCIPLE: semiconductors, laser and optical manufacturing,
The Beer-Lambert law states that the absorbance of printing and forensic examination, and as well in
a solution is directly proportional to the laboratories for the study of chemical substances.
concentration of the absorbing species in the Ultimately, a spectrophotometer is able to
solution and the path length. Thus, for a fixed path determine, depending on the control or calibration,
length, UV/Vis spectroscopy can be used to what substances are present in a target and exactly
determine the concentration of the absorber in a how much through calculations of observed
solution. It is necessary to know how quickly the wavelengths UV-visible spectrophotometry
absorbance changes with concentration. This can
be taken from references (tables of molar extinction The most common spectrophotometers are used in
coefficients), or more accurately, determined from the UV and visible regions of the spectrum, and
a calibration curve. some of these instruments also operate into the near
infrared region as well.Visible region 400–700 nm
Spectrophotometry is the quantitative measurement spectrophotometry is used extensively in
of the reflection or transmission properties of a colorimetry science. Ink manufacturers, printing
material as a function of wavelength. It is more companies, textiles vendors, and many more, need
specific than the general term electromagnetic the data provided through colorimetry. They take
spectroscopy in that spectrophotometry deals with readings in the region of every 5–20 nanometers
visible light, near-ultraviolet, and near-infrared, but along the visible region, and produce a spectral

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reflectance curve or a data stream for alternative carbonate was added and added and left for 20min
presentations. These curves can be used to test a the intensity of blue color developed was measured
new batch of colorant to check if it makes a match at 640nm againt the blank in spectronic
to specifications, e.g., ISO printing standards. 20,spectrophotometer and the serum enzme activity
expressed as IU/L.(6)
pH METER:
A pH meter is an electronic instrument used RESULT
for measuring the pH (acidity or alkalinity) of a
liquid (though special probes are sometimes used to Statistical analysis of the study is shown in Table 1,
measure the pH of semi-solid substances). A which showed the response of highly elevated
typical pH meter consists of a special measuring Alkaline Phosphate level in the Test sample group
probe (a glass electrode) connected to an electronic when compared to the Control sample group.
meter that measures and displays the pH reading. whereas the response is P > 0.001 with the degree
of freedom 4 (Hence the null hypothesis is
ALKALINE PHOSPHATASE accepted)
(ALP, ALKP) (EC 3.1.3.1) which is
a hydrolase enzyme responsible for DISCUSSION
removing phosphate groups from many types of
molecules, including nucleotides, proteins, and In the present study five serum sample from known
alkaloids. It was estimated on the bases of the Parkinson’s disease patients where estimated for
below principle. alkaline phosphatase. There is no current evidence
PRINCIPLE: suggesting this enzyme alteration in Parkinson’s
Phenol released by enzymatic hydrolysis disease. In addition the classical ethiopathogenic
at alkaline cause like α-synuclein, lewy body, metal, toxin,
(pH -10)disodium phenyl phosphatase is measured genetic causes are also implicated(9) .It remains to
in spectrophotometer. investigate the role of phosphatases in view of
REAGENTS: genetic abnormality
1. Sodium carbonate sodium bicarbonate buffer
0.1 (pH 10): 3.18 anhydrous Sodium carbonate and The study showed the highly elevated alkaline
1.68g sodium bicarbonate in 500ml of phosphatase level in the sample group when
2. Disodium phenyl phosphate: 0.001 In distilled compared with normal serum levels alkaline
water (used as substrate (prepare before use). phosphatase which is distributed widely and
3. Folin and ciocalteus reagents: Folin and elevated in number of disorder like liver, bone
ciocalteus reagents (2N phenol solutin SRL disease and tumor as per previous studies.(10)
bamney) distilled water (prepare before use).
4. Sodium carbonate: 20% (w/v) in distilled In one study (11) the bone density was measured
water. using computerized radiography (CCT) in patients
5. Magnesium chloride: Magnesium chloride with Parkinson’s disease with no other disorder and
0.1M in distilled water. no other drugs administered .Alkaline phosphatase
6. phenol standard: 100 mg of phenol in 100ml levels where found to be increased in view of this
distilled water kept in refrigerated. present study correlates well with the finding of
Reference standard: study. It remains to confirm the cause of the
Before use the stock standard diluted to yield 10 elevated alkaline phosphatase levels in parkinson’s
to the power-6-moles/ml with distilled water and disease.
standard graph constructed.
CONCLUSION
PROCEDURE:
To 0.05ml of serum and standard phenol The current study estimated alkaline phosphatase in
(0.05-0.2 micromole) in 15x 150 mm test tubes ,1.5 a sample group of long standing Parkinson’s
ml of Sodium carbonate sodium bicarbonate buffer disease patients with symptom and on treatment
0.1 (pH 10),1ml of Disodium phenyl phosphate .However estimated showed elevated serum
and 0.1 Magnesium chloride were vortex well. The alkaline phosphatase level when compared to
final volume was adjusted to 3ml with distilled normal control. The increased alkaline phosphatase
water ,after incubation at 37*c for 15min ,1ml of value was found to be more than the normal range
folin and ciocalteus reagent was added to arrest the of alkaline phosphatase in human serum ;20 to 140
reaction blank was processed similarly but the IU/L. This suggest gross increase in the enzyme
serum added after arresting the reaction .The level and therefore requires consideration in view
precipitate formed after addition of folins reagents of the abnormality detected by alkaline
was separated by centrifugation at 1000xg for 10 phosphatase estimation and in relation to bone
mins to the supernant 1.0 ml of 20% sodium pathology in Parkinson’s disease patients.

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This study includes small population, in absence of patients is doubtful but the altered levels (increase)
other abnormality (includes drugs that could alter suggests its estimation in the disorder in
the serum alkaline phosphatase levels). Relevance consideration with statistical correlation.
of alkaline phosphatase in parkinson’s disease

Table:1 Showing the Concentration Of Alkaline Phosphatase(IU/L) in control sample

S.NO SAMPLE Conc of alkaline phosphatase in IU/l
1. Control-1 61.7647 IU/L
2. Control-2 76.190 IU/L
3. Control-3 71.428 IU/L
4. Control-4 63.80 IU/L
5. Control-5 60.952 IU/L

Table:2 Showing the Concentration Of Alkaline Phosphatase(IU/L) in test sample

S.NO Sample ID Conc of alkaline phosphatase in IU/L
1. OPNO 121106 240 IU/L
2. OPNO 25293 235.55 IU/L
3. OPNO 17298 200 IU/L
4. OPNO 3792 177.77 IU/L
5. CDM NO72/11 248.88 IU/L

250

200

150

Control
100
Test

50

0
Sample

X axis- Number of patients ,Y axis – level of Alkaline phosphatase

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