Introduction
Magnitude
of type 2 diabetes mellitus (T2DM) is ever increasing in India and at present ~
69 million people are living with diabetes [1] and another ~ 77 million people are pre-diabetic
subjects, having high potential for the development of T2DM [2]. Uncontrolled
serum glucose levels for extended durations are associated with retinopathy,
nephropathy, neuropathy as well as cardiovascular, cerebrovascular and
peripheral vascular diseases. Recently cognitive dysfunction (CD) in T2DM is
gaining much attention due to their co-occurrence. Major cognitive dysfunctions associated with
T2DM are psychomotor speed, executive function, verbal memory and processing
speed, working memory, immediate and delayed recall, verbal fluency, visual
retention and attention [3].
More
than half of the brain is constituted by lipids. They play critical roles in maintaining
the brain’s structural and functional components [4,5,6].Sphingolipids are
class of lipids that are present in higher concentration in the brain compared
to that in the plasma. Sphingolipids constitutes ~22% dry weight of the human
brain white matter where as it constitutes only 5% dry weight of plasma
[7,8,9]. Certain sphingolipid species are enriched highly in brain while their
levels are relatively low abundance in all other tissues and some of their
levels are 20-90 fold higher in brain than in plasma [10].Presence of certain
specific classes of brain lipids in the serum makes it an ideal marker to study
the possible abnormalities occurring in its composition during disease
conditions.
Thus
performing a targeted lipidomics of the serum samples from T2DM patients with
cognitive dysfunction and its comparison to T2DM patients without CD can
provide us an insight into the role of these molecules in management of such
patients.
Literature survey
In a French population study with 59-71 age group diabetic
patients’ cognitive decline was higher compared to non-diabetic individuals [11].
A study in older than 60 years of Latinos indicated that diabetes was a
significant predictor for major cognitive decline [12] with 4.8% diabetic
patients showing severe CD known as dementia at this age group while prevalence
of CD was 31% in diabetic people aged more than 85 years [13].A
systematic review of the cognitive decline in diabetes showed 1.2 to 1.5 fold
increase in rate of decline in cognitive ability in diabetes compared to
non-diabetic population [14]. In a Japanese elderly population study, compared
to non-diabetic groups, diabetics had a significant cognitive decline, which
was well correlated with hippocampal atrophy but not whole brain atrophy [15]. In a Croatian study on adult population diabetics had
higher cognitive dysfunction compared to controls [16]. In an US study,
diabetes was associated with cognitive decline (1.39 fold) in elderly person
above 70 years of age [17]. In a Polish study, 31.5% diabetics had CD,
the age group was above 65 years [18]. In a Taiwanese
study with an age group of 65 or above, 11.5% of the diabetic population had CD
compared to control population [19]. In a Chinese large cross sectional study
13.5% of the diabetic population above 65 years of age had mild cognitive
dysfunction [20].In an Australian adult population study, cognitive decline was
associated with impaired cerebrovascular responsiveness in T2D [21]. In a
Greece population study of diabetic patients above 65 years of age, 2 fold
higher chances of CD was observed compared to control population [22].
The relationship
between T2DM and dysfunction of lipids have led to many lipidomics studies. Large number of studies
showed variations in lipidomics in T2DM patients which is not a surprise based
on the fact that alterations in lipid
metabolism is a well-known fact in T2DM. Additionally, both targeted and untargeted
lipidomics studies have been performed in cognitive dysfunction. Non-targeted lipidomics of
frontal cortex grey and white matter in control and mild cognitive impairment
subjects revealed that phosphatidylethanolamines were reduced in white matter
where as diacyl glycerol levels were elevated in grey matter [23]. Targeted fatty
acid lipidomics of plasma in mild cognitive impairment revealed higher levels
of arachidic, erucic, mead and vaccenic acid and
lower levels of cerotic and linoleic acid compared to healthy controls [24].
In another study, targeted lipidomics of plasma and frontal cortex
diacylglycerols in mild cognitive impairment revealed elevated levels of diacylglyerols with saturated, unsaturated,
and polyunsaturated fatty acid substituents compared to healthy controls [23].
Research gaps identified
We have magnitude data about cognitive dysfunction
in type 2 diabetes mellitus from several parts of the world although with much
variations in sample size and age groups. However, we do not have the detailed
data of magnitude of T2DM patients with CD from the Indian population which we
assume to be different due to significant dietary variations as well as life
style factors which are well associated with cognitive functions. Further, we did
not find any studies that were performed to link T2DM with CD through lipidomics
approach.
Objectives
·
To measure the magnitude of cognitive dysfunction
in T2DM patients
·
To compare serum targeted lipidomics in T2DM
patients with and without cognitive dysfunction
·
Identifying key lipid molecules altered in
cognitive dysfunction in type 2 diabetes mellitus for future studies
Detailed methodology
Following
approval from the IEC and obtaining informed consent from the previously
identified T2DM patients, digital symbol Substitution and Montreal cognitive
tests will be performed to measure the magnitude of cognitive dysfunction in
T2DM patients. Anticipating 8% of diabetes to report of cognitive dysfunction
with a 20% relative precision at 95% confidence level accounting for 20%
non-response minimum of 1278 T2DM patients will be recruited for this study. For
targeted lipidomic studies, 2ml of fasting blood will be collected from 20 T2DM
patients each with and without cognitive dysfunction. If the patient already
gives the serum sample for biochemical analysis the residual serum samples will
be collected from the Biochemistry lab, KMC Manipal. The serum samples will be
stored in -80 degree centigrade freezer. The samples will then be transported to
CCAMP- NCBS Mass spectrometry Facility in Bangalore in a box of liquid nitrogen
maintaining -80 degree centigrade temperature .Less than 100 μl serum will be
used for lipidomic studies. Lipidomics analysis will be done by Liquid
Chromatography – Mass Spectrometry (LC-MS) technology at CCAMP- NCBS Mass
spectrometry Facility, Bangalore.
Inclusion
and exclusion criteria
— Inclusion
criteria for the 1st objective: All T2DM patients above 20 years of age with informed consent visiting the hospital of various age groups
ready to volunteer
— Minimum of 5 years of formal education
— Patients having all the co-morbidities including
hypo and hyperthyroidism, hypertension, CAD, kidney and liver diseases, HIV,
Vitamin B12 deficiency and any other infectious diseases
·
Exclusion criteria for 1st
objective:T2DM patients
who are severely sick and un co-operative for performing the cognitive tests
·
Patients with
visual impairment, confusion and delirium
·
Inclusion criteria for the 2nd
objective- T2DM patients above 60 years of age with more
than 10 years of diabetes without any complications who are ready to volunteer
with informed consent
·
Exclusion criteria for the 2ndobjective - T2DM patients suffering from hypo and hyper thyroidism, hypertension,
HIV, Vitamin B12 deficiency, kidney, liver and other infectious diseases
Montreal
Cognitive Assessment (MoCA)
The instrument which can be used for screening mild
cognitive impairment is Montreal Cognitive Assessment (MoCA). The
screening instrument which was designed for detecting mild cognitive impairment
is Montreal Cognitive Assessment (MoCA). Ziad Nasreddine
from Montreal, Quebec developed the MoCA scoring system. This is a worldwide used screening assessment. The MoCA test is a paper and pencil test. It is
a single page 30-point test which approximately takes 10 minutes to be
administered. The availability of the full test and the instructions to be
followed in administering the MoCA are accessible in online for clinicians and educational purposes. The availability of the test is in 56 languages and
dialects. A validation study done by Nasreddine in
the year 2005 proved MoCA to be more useful tool than MMSE in detecting mild
cognitive impairment.
The MoCA is an instrument
which is used for screening cognitive dysfunction. This is basically a paper
and pencil test and the time administered for this test is 10 minutes. Various
cognitive domains such as language, memory, executive functions, visuospatial
skills, calculation, abstraction, attention, concentration and orientation are
assessed from this test. The validation of the test has been established in
detection of mild cognitive impairment suffering from Alzheimer’s disease and
other pathological conditions in subjects with mild cognitive impairment who
scored in the normal range of MMSE According to a study in 2015 the sensitivity
and specificity of the MoCA for detecting MCI were found to be 90% and 87%
respectively, compared with 18% and 100% respectively for the MMSE. Various
features in the MoCA designing explain its sensitivity to a superior level for
mild cognitive impairment detection. More number of words, fewer trials of
learning and a longer delay before recall are there in the MoCA test rather
than the MMSE. MoCA can also assess executive functions, language abilities,
processing of complex visuospatial abilities in a higher level than MMSE. The
general MoCA was basically normed for a highly educated population because it
was developed in a setting of memory clinic. Later a basic MoCA which is well
known as MoCA B was developed for the less educated and literate people to
fulfill the limitation of the general MoCA test. The scoring system of MoCA
ranges in between 0 and 30. A person is said to have proper cognitive
function when he/she scores 26 or above. A study in the past showed normal
people scoring an average of 27.4 and people with mild cognitive impairment (MCI)
scoring an average of 22.1 in the MoCA test.
Digital Symbol Substitution test
A frequent consequence of brain disorders
slowed mental processing and impaired ability for focused behavior (Duncan and
Mirsky 2004, Leclercq and Zimmermann 2002). Damage to the brain stem or diffuse
damage involving the cerebral hemispheres, especially the white matter
interconnections can produce a variety of attentional deficits. Attentional
deficits are very common in neuropsychiatric disorders. Most neuropsychological
deficits are very common in neuropsychiatric disorders. The Wechsler’s part of
intelligence test contains several such relevant tests among which Digit Symbol
substitution test is one of them. Digit Symbol Substitution test is an
excellent measure of focused attentional capacity. This test requires
concentration plus motor and mental speed for successful performance and
requires rapid processing of symbolic informations and coding of symbol number
pairs. The patient must accurately and rapidly code numbers into symbols.
Performance is determined by the number of correct numbers transcribed in 90
seconds. Copyright versions of Montreal Cognitive test will be used for this
study after taking permission from the respective website. The DSST test is a
part of Weschler’s Adult Intelligence Skill which assessed mental speed. It is
a test which is procured by the Clinical Psychology department, KMC Manipal.
This test can be administered from patients within age groups 16-85 years. The
time limit for the subtest is a maximum of 120 seconds [25].
Expected Outcome
There is a possibility of getting certain
class of sphingolipids which may be up-regulated and down-regulated in T2DM
patients suffering from CD. This information may be helpful for therapeutic
intervention studies in future.
Importance of the
proposed research
The data generated may give critical inputs for the role of sphingolipids
subclasses in the occurrence of CD in T2DM patients. |