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Vibrational Spectroscopy 53 (2010) 181–188

Contents lists available at ScienceDirect

Vibrational Spectroscopy
journal homepage: www.elsevier.com/locate/vibspec

Neural network algorithm for the early detection of Parkinson’s disease from
blood plasma by FTIR micro-spectroscopy
Shiek S.S.J. Ahmed a, Winkins Santosh a,*, Suresh Kumar b,*, T. Hema Thanka Christlet c
a
Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu 603203, India
b
Department of Neurology, SRM Medical College Hospital and Research, SRM University, Kattankulathur, Tamil Nadu 603203, India
c
Department of Physics, Arignar Anna Government Arts College, Cheyyar, Thiruvannamalai -604 407, India

A R T I C L E I N F O A B S T R A C T

Article history: Parkinson’s disease (PD) is a progressive bradykinetic disorder. Diagnosis of PD is entirely clinical, no
Received 31 October 2009 biochemical parameters exist to diagnose PD. The diagnostic problem associated with the early detection
Received in revised form 25 January 2010 has led to an interest in the development of spectroscopic based diagnostic techniques. Fourier-
Accepted 25 January 2010
transform infrared micro-spectroscopy analysis (FTIR) was applied to analyze human plasma samples of
Available online 2 February 2010
69 healthy and 61 drug-naive PD patients in order to detect spectral parameters, which serve as
biomarkers for monitoring and identification of PD. The analysis showed the bands at 1078, 1169 and
Keywords:
1244 cm 1 corresponding to carbohydrates, significantly increased in all tested patient samples
Parkinson’s disease
(p  0.01). Several other spectral regions that attribute to amino acids, lipids and proteins indicate the
FTIR
Blood plasma unique detection of disease stages. Cluster analysis of variable regions provided excellent grouping of the
Spectral diagnosis healthy and the patient samples, which correlate completely with the clinical diagnosis. For automatic
Neural network detection, artificial neural network (ANN) was implemented on the variable regions showed 96.29%
accuracy in the detection of disease progression. These parameters could be used, as a basis for
developing a spectral method for detecting PD. Execution of neural network will be useful in clinical
screening and rapid detection of PD.
ß 2010 Elsevier B.V. All rights reserved.

1. Introduction Many fundamental diagnostic decisions in medical practices


were made with the help of biomarkers. Biomarkers serve as
Parkinson’s disease (PD) is a chronic neurodegenerative disorder diagnostic tools for diseases that are usually performed on readily
characterized by progressive loss of substantia nigra pars compacta accessible body fluids, such as saliva, urine, serum or cerebrospinal
(SNc) neurons of unknown etiology [1,2]. The diagnosis of PD is fluid (CSF). Several studies have suggested the diagnosis of PD from
entirely clinical, no biochemical tests are currently available to the serum. Fourier-transform infrared (FTIR) micro-spectroscopy is
diagnose PD. At present, the diagnosis is done by standard a powerful technique applied in biochemistry for studying the
neuropathological examination and medical history [3]. The severity secondary structure of protein molecules [7], nucleic acids [8],
of disease is categorized as stages based on the overall motor carbohydrates [9] and lipids [10]. FTIR micro-spectroscopy has
function evaluation using Unified Parkinson’s Disease Rating Scale been used effectively to diagnose and characterize various types of
(UPDRS) or on an equivalent rating scale such as Hoehn and Yahr disorders. Encouraging results were obtained in an attempt to
scale and Schwab and England Activities of Daily Living Scale. [4]. detect cancer [11], gastric inflammation [12], Alzheimer’s disease
Major clinical symptoms like tremor, rigidity and motor dysfunction [13], kidney stones [14], leukemia [15], cervical cancer [16],
significantly help in detection of PD. However, the clinical diagnosis melanoma [16], urinary crystals [17], changes in the biochemistry
fails to identify PD before any significant loss of dopamine neurons of oral cavity tissues [18] and congenital hypothyroidism [19]. In
[5]. There is immense need for early and rapid detection and more microbiology, FTIR is used to classify and differentiate various
effective drugs for cure or to stop the ongoing nigral degeneration strains and species of microorganisms [20], in the detection of
[6]. The understanding of molecular mechanism and the changes virus infected cells [21] and fungi in wood [22].
acquired during disease progression are ultimately important for In this study, FTIR analysis of plasma samples of 69 normal and
developing biomarkers in the early detection of the disease, which 61 drug-naive patients were carried out to investigate the
subsequently provides high value therapeutics. presence of spectral markers for PD. The aim is to implement
the band intensity values of spectral markers in neural network
for rapid detection of PD. The results presented contribute to
* Corresponding authors. understanding FTIR spectral differentiation patterns in various
E-mail address: parkinresearch@gmail.com (S. Kumar). stages of PD.

0924-2031/$ – see front matter ß 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.vibspec.2010.01.019
182 S.S.S.J. Ahmed et al. / Vibrational Spectroscopy 53 (2010) 181–188

2. Materials and methods Baseline correction was done by rubber band method and
normalization to amide II was performed by vector method. Five
2.1. Clinical sample biological replicate spots were measured for each sample and there
are no significant differences in the spectra of replicates each
Blood samples of 61 drug-naive PD patients of South Indian sample were noticed. The multiple spectra of each population were
origin were obtained from the outpatients of the Department of normalized prior to average of biological and experimental
Neurology at SRM Hospital, Tamil Nadu, India and compared with replicates. The band positions were determined using second
69 healthy controls of similar origin. The control samples were derivate of Savitzky-Golay algorithm by panorama software.
grouped into three classes, namely ‘‘Stage 1 control’’, ‘‘Stage 2
control’’ and ‘‘Stage 3 control’’ based on age and gender, in order to 2.4. Statistical analysis
compare with PD stages, respectively (Table 1). All individuals
chosen for this study had no history of smoking, alcoholism, drug Statistical analyses were performed using GeneSpring GX
abuse and anti-oxidant exposure for a period of three months software version 7.3 (Agilent Technologies Inc., Santa Clara, CA).
before sample collection. A written consent was obtained from The significance of spectral variation between the PD stages and
each participant during an in-person interview and blood their control groups were calculated by analysis of variance
donation. For participants with PD, date of the symptom onset, (ANOVA). The cluster analysis was executed to determine the
date of diagnosis and family history of PD were recorded. The grouping ability of identified variable regions into stages. The
patients were shown to have symptom onset for the average of analysis was performed on the absorption intensities of each peak
three years before the diagnosis and the samples were collected using similarity measurement of Pearson correlation and average
immediately after diagnosis. Two patients of stage 3 PD and four linkage algorithm.
patients of stage 2 PD had reported a family history of PD. All the
patients were evaluated by one or more qualified neurologists and 2.5. Artificial neural network (ANN) detection
the pathological status was classified by UPDRS. The ethical
committee of SRM Medical College and Hospital, India reviewed The neural network design consisted of a three-layer network:
and approved the protocol of this study. an input layer, with 13 units containing the information on the
diagnostic criteria; a hidden layer, with six units; and an output
2.2. Preparation of samples layer. A NeuNet Pro software (CorMac Technologies Inc., Canada),
back prop algorithm was used for the prediction of disease stages.
Blood samples (4 ml) were collected in EDTA vacutainer tube Back prop algorithm of NeuNet Pro software accepts the clinical
(Becton-Dickinson, Franklin Lakes, NJ) from individuals and data as numerical inputs.
immediately centrifuged for 5 min at 14,000 rpm to separate The training variables include age, gender, intensities of 10 FTIR
plasma from other cellular material. Subsequently, the plasma was peaks (689, 1078, 1169, 1244, 1542, 2953, 2930, 3060, 3293 and
transferred to fresh tube and kept frozen at 20 8C before 3296 cm 1) and disease status (normal: coded 0, stage 1: coded 1,
processing. Thallium bromide iodide crystal was used as the slide stage 2: coded 2, stage 3: coded 3). The input variables associated
materials. 1 ml of the plasma sample was placed on the slide, air- with diagnoses were trained with randomly selected 103
dried at room temperature for 30 min. The size of each dried spots individuals, which include 53 normal and 50 PD patients (16
was 0.5 cm in radius and the examination was further carried out samples of stage 1, 16 samples of stage 2 and 18 samples of stage
using FTIR micro-spectroscopy. 3). The training process continued until the differences between
the network classifications and the clinical diagnoses diminished.
2.3. FTIR data analysis Once the network was trained, the remaining 27 individuals were
‘‘tested,’’ by the trained network. The neural network classifica-
All experimental samples were subjected to FTIR micro- tions were then compared with the known clinical data, to see the
spectroscopy Bruker IFS 66 V (Bruker Biospin, Inc., Milton, Canada) classification ability network.
operating at 450–4000 cm 1 coupled with panorama (LabCogni-
tion Inc., Germany) software. The FTIR measurements were 3. Results and discussion
performed in transmission mode with a liquid nitrogen-cooled
detector. The spectrum was obtained in the wavenumber range of The present study is focused on identification of the spectral
450–4000 cm 1. A spectrum was taken as an average of 137 scans markers for the detection of PD from blood plasma. Few successful
to increase the signal to noise ratio, and the spectral resolution was breakthroughs have shown metabolite variations in serum/plasma
at 1.0 cm 1. The baseline correction, smoothing and normalization of patients with PD. For instance, glycine, aspartate and glutamate
were performed prior to peak finding using panorama software. were found to be increased in parkinsonian plasma in comparison
with control subjects [23]. Increase in valine and reduction in
arginine and methionine concentrations in serum were noticed in
Table 1
Statistics of research participants involved in the study. PD [24]. Additionally, a significant increase of 8-OHdG in serum
and urine was also observed in PD [25]. Our previous study also
Sample information No. of samples Gender Mean age
showed the feasibility of potential diagnosis of PD from the
Male Female detection of 22 differentially regulating metabolites in plasma of
Stage 1 control 23 14 9 57.60 PD [26]. The recent study of early PD plasma using near infrared
Stage 2 control 22 10 12 62.80 spectroscopy (NIR) showed significant variations in oxidative
Stage 3 control 24 14 10 71.46 stress sensitivity bands in comparison with control [27]. And, it
PD-stage 1 20 12 8 57.84
was suggested that reactive oxidative species (ROS) modify the
PD-stage 2 20 11 9 62.68
PD-stage 3 21 12 9 71.00 proteins, lipids and other cellular substrates in plasma [27]. From
the inferences of these studies, FTIR micro-spectroscopy analysis
The samples were divided into four categories. The normal groups of 69 samples
were categorized into three sub-groups as ‘‘stage 1 control’’, ‘‘stage 2 control’’ and
was executed to scan entire plasma metabolites via functional
‘‘stage 3 control’’. Each sub-group was compared with corresponding PD stages. group between normal and PD. The typical analysis of FTIR spectra
Stage 1 (20 samples) stage 2 (20 samples) and stage 3 (21 samples), respectively. is aimed for early three stages of disease in order to determine the
S.S.S.J. Ahmed et al. / Vibrational Spectroscopy 53 (2010) 181–188 183

Fig. 1. Variations in stage 1 Parkinson’s disease in comparison with their control group. Spectra representing the region 450–4000 cm 1 of stage 1 control and stage 1 PD. The
presented spectra are an average of 20 patient samples and 23 healthy samples. (A) FTIR spectra of healthy individuals and PD patients in the region 1078–1244 cm 1. (B) Peak
at 3060 cm 1 area of healthy persons and patients with PD.

early markers for PD. Moreover, this study was made with and the variations in spectral regions at 689, 1078, 1169 and
unmedicated individuals to avoid the confounding effects of 1244 cm 1 were identified in stage 2 PD (Figs. 3 and 4). Similarly,
medication including antioxidant that could potentially interfere the difference in band intensities were noticed at 1078, 1169, 1244,
with the spectrum [27]. The results show that the FTIR spectra of 1542, 2930, 2953, 3293 and 3296 cm 1 for stage 3 PD in
both healthy individuals and PD patients were similar although comparison with their control (Figs. 5 and 6). The presented
there were variations in certain regions of patient samples Figs. 1, 3 and 5 are the average spectra of stage 1, stage 2 and stage
compared to normal samples. The identified variations confirm 3 PD with their respective control groups. The results show a
the statistical significance at all stages of PD (p  0.01). significant and detectable increase of bands at 1078, 1169 and
The bands at 1078, 1169, 1244 and 3060 cm 1 (Figs. 1 and 2) 1244 cm 1 in all tested plasma of PD patients compared to the
were altered in stage 1 PD in comparison with their control group control groups. The overlap of Figs. 1, 3 and 5 at 1078, 1169 and

Fig. 2. Band intensities difference between the variable regions of stage 1 controls and stage 1 patients. The band intensities at variable regions of 23 examined stage 1 control
and 20 stage 1 PD samples were shown. Each point represents the average of five measurements of biological replicates of each sample.
184 S.S.S.J. Ahmed et al. / Vibrational Spectroscopy 53 (2010) 181–188

1
Fig. 3. Spectral variations in stage 2 Parkinson’s disease. The average spectra of 22 ‘‘stage 2 control’’ and 20 stage 2 PD samples in the region 450–4000 cm . (A) FTIR spectra of
healthy persons and PD patients in the region 1078–1244 cm 1. (B) Peak at 689 cm 1 area of healthy individuals and patients with PD.

1244 cm 1 shows a systematic increase in absorption intensities as disease and spinocerebellar degeneration [30]. Also, variation in
the disease progress from stage 1 to 3 (Fig. 7). These spectral serum glucose was previously reported in early PD [31] and the
regions were attributed to C–O bending mode of carbohydrates validation of plasma glucose level in these samples was shown to
bands, suggesting variations in plasma carbohydrates of PD be abnormal (unpublished result). With these carbohydrate
samples [16]. This result supports the previous report of cerebral abnormalities in FTIR spectra could be used as a basis for the
hypometabolism of glucose [28] and dysfunction of the glycolysis detection of PD irrespective of stages. And, these common variable
pathway in PD [29]. Furthermore, a study suggests the role of regions can be used to determine the disease progression in PD
carbohydrate metabolisms in the pathogenesis of motor neuron (Fig. 7).

Fig. 4. Band intensities difference between the variable regions of stage 2 controls and stage 2 patients. The band intensities at variable regions of 22 examined stage 2 control
and 20 stage 2 patients samples were shown. Each point represents the average of five measurements of biological replicates of each sample.
S.S.S.J. Ahmed et al. / Vibrational Spectroscopy 53 (2010) 181–188 185

Fig. 5. Variations in stage 3 Parkinson’s disease in comparison with their control group. FTIR spectra in the region of 450–4000 cm 1 of stage 3 control and stage 3 of PD. The
presented spectra are an average of 21 stage 3 patient samples and 24 healthy person samples. (A) FTIR spectra of healthy individuals and PD patients in the region 1078–
1244 cm 1. (B) Peak at 1542 cm 1 area of healthy individuals and patients with PD. (C) Indicating the variations at 2930 and 2953 cm 1. (D) Spectra representing the variable
regions at 3293 and 3296 cm 1 between normal and stage 3 PD.

3.1. Identification of unique markers described in PD patients compared to control [35]. Furthermore,
the result from a recent meta-analysis showed an increased
In order to identify the stage specific spectral markers, the risk of PD in carriers of the APOE2 allele [36], which is associated
results were compared. In stage 1 PD, absorbance at 3060 cm 1 with lower plasma levels of cholesterol. In addition, in vitro
was reduced when compared with the control and other two stages study of alpha-synuclein protein was shown to have close
of PD samples. This band corresponds to unsaturated fatty acid association with cholesterol-enriched lipid rafts in the
[32], which indicates abnormality in unsaturated fatty acid cell membrane [37]. The aggregation of alpha-synuclein
derivatives in early PD. This unique variation can serve as spectral might be regulated by fatty acids [35]. The increase in alpha-
marker for early detection of PD. synuclein protein is the pathological structures, termed Lewy
In stage 2 PD, the spectra show an increased intensity at bodies, in the brain of patient with PD. Thus, variation in protein
689 cm 1 compared to the control and other disease groups. This region occurs at stage 3 PD due to abnormal regulation of
region assigned to C–C twist, C–C stretch and C–S stretch mode fatty acids.
of amino acids [32], show their variation in stage 2 PD samples. The present spectral variations correlate with molecular
Furthermore, unique spectral variations were observed at 1542, abnormalities of previous studies of PD [25]. In other words, the
2930, 2953, 3293 and 3296 cm 1 of stage 3 PD samples. The underling molecular variations in plasma have been detected by
band at 1542 cm 1 shows a significant increase in intensity in FTIR micro-spectroscopy. And, these supporting spectral changes
stage 3 samples compared to the control and other stages of PD. can be used as spectral marker for detection of PD. This is the first
This region is attributed to stretching C5 5C vibration of lipids study using FTIR micro-spectroscopy for the detection of PD stages
[32]. In addition, the bands at 2930 and 2953 cm 1 were from plasma samples. Based on these findings, it seems that
significantly increased contributing to stretching vibrations of plasma is an easy and rapid target for detection and identification
C–H of saturated lipids [32]. Also, an increase in absorbance is of PD.
observed at 3293 and 3296 cm 1 in stage 3 PD samples. These
regions were attributed to amide I band of proteins [32]. 3.2. Cluster analysis
Variations in these regions suggest the abnormality of proteins
in stage 3 PD samples. Hence, the cumulative analysis shows The cluster analysis was carried out in order to determine the
that these regions are relatively important for stage 3 diagnosis classification ability of variable regions into PD and normal. The
of disease. absorption intensities of all samples for the regions at 689, 1078,
The observed changes in these spectral regions indicate the 1169, 1244, 1542, 2953, 2930, 3060, 3293 and 3296 cm 1 were
metabolite abnormalities in plasma of patients with PD. used as input for the cluster analysis. Two major clusters are
The majority of unique changes in PD stages contribute to formed (Fig. 8). Among these, one of the clusters contains the
amino acids, lipids and proteins. The variations in plasma normal, whereas the second cluster is composed of stage 1, stage 2
amino acids were previously reported. The variation of amino and stage 3 of PD. The normal cluster was highly deviated from the
acid region is may be due to the abnormal concentration of disease stages suggesting an excellent grouping of three stages
arginine, aspartate, glutamate, glycine and methionine in PD from the absorption intensity values of above spectral regions.
plasma [23,24]. Also, this study shows alterations in spectral This confirms that these regions can be potentially used as
regions of lipids. Several studies suggest the alterations in markers for detection of PD by stages. Hence, the intensities of
fat metabolism are involved in the pathogenesis of PD these regions were considered for neural-network for automatic
[33,34]. Lower levels of total cholesterol have been previously detection.
186 S.S.S.J. Ahmed et al. / Vibrational Spectroscopy 53 (2010) 181–188

Fig. 6. Band intensities difference between the variable regions of stage 3 controls and stage 3 patients. The band intensities at variable regions of 24 examined stage 3 control
and 21 stage 3 patients samples were shown. Each point represents the average of five measurements of biological replicates of each sample.

3.3. Artificial neural network (ANN) for rapid detection

The network was tested with 27 test samples. The success rate
in classification of the test set is 96.29% (26/27) accuracy, the
sensitivity is 91% (10/11, one case of stage 2 was misdiagnosed as
stage 3 PD), the specificity is 100% (16/16). Moreover, the values
generated by the neural network were ranged from 0 to 3. Based on
these values, individuals were assigned into normal or diseased
group. Individuals whose final predicted values x  0 are assigned
as normal and the values x > 0 are assigned to the PD groups
(Table 2). For instance, the predicted range from x = 0.51–0.99 was
assigned as stage 1, x = 1.51–1.94 as stage 2 and the values
x = 2.10–3 as stage 3 (Table 2). The utility of this prediction
algorithm is questionable since the analysis was made with a
limited number of samples and comparison with other neurode-
generative disorders was not made. However, the spectral
differentiation between PD and other neurodegenerative disorders
Fig. 7. Comparative analysis of control groups with PD stages. FTIR spectra of is possible with the understanding of early study of NIR in
control groups and PD stages in the region 1055–1245 cm 1, indicating the comparison with Alzheimer’s and Parkinson’s disease [38]. To
increased spectral intensities as the disease progress. confirm this possibility, further work needs to be done to
S.S.S.J. Ahmed et al. / Vibrational Spectroscopy 53 (2010) 181–188 187

Table 2
Neural network prediction.

S. no. ANN Neural network Clinical


predicted value classification designation

1 0.845 Normal Normal


2 0.7931 Normal Normal
3 0.676 Normal Normal
4 0.951 Normal Normal
5 0.735 Normal Normal
6 0.9837 Normal Normal
7 0.539 Normal Normal
8 0.898 Normal Normal
9 0.497 Normal Normal
10 0.635 Normal Normal
11 0.978 Normal Normal
12 0.559 Normal Normal
13 0.631 Normal Normal
14 0.809 Normal Normal
15 0.997 Normal Normal
16 0.851 Normal Normal
17 0.174 PD-stage 1 PD-stage 1
18 0.5125 PD-stage 1 PD-stage 1
19 0.767 PD-stage 1 PD-stage 1
20 0.974 PD-stage 1 PD-stage 1
21 1.864 PD-stage 2 PD-stage 2
22 1.945 PD-stage 2 PD-stage 2
23 1.51 PD-stage 2 PD-stage 2
24 2.992 PD-stage 2* PD-stage 3
25 2.133 PD-stage 3 PD-stage 3
26 2.576 PD-stage 3 PD-stage 3
27 2.738 PD-stage 3 PD-stage 3

Neural network classification values, neural network designation and disease


designation, for 36 Individuals who have known clinical information. Individuals
denoted by asterisk.
*
Indicate the misclassification of PD-stage 3 as stage 2 by ANN.

differentiate the PD spectral markers from other neurodegenera-


tive disorders.

4. Conclusions

FTIR micro-spectroscopy technique in detection of PD is simple


and more feasible. It requires a small amount of plasma (1 ml),
which can be easily obtained from patients, and the results can be
obtained within a short interval of time (30 min). ANN can be
further implemented as a tool for the rapid detection of PD. The
spectral markers obtained in this study are crucial and may be
considered as a promising basis for future studies by including
other neurodegenerative disorder samples. At present, it is
suggested to execute the FTIR technique along with the clinical
diagnosis, which additionally improves the accuracy of PD
detection.

Acknowledgments

We thank Dr. K. Ramasamy for his intellectual input and Indian


Institute of Technology, Madras for the instrument facility. This
work was supported by SRM University, Tamil Nadu, India. We
specially thank all the patients and healthy volunteers for their
selfless donation of blood. SSSJA thanks to Mahmoud Huleihel,
Faculty of Health Sciences, Ben-Gurion University of the Negev,
Israel for his support throughout the data analysis.

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