FTIR-ATR SPECTRAL AND BIOCHEMICAL VARIATIONS IN DIABETES MELLITUS INDUCED BY ALLOXAN IN WISTAR MALE RAT

Saira khatheeja 1* , Prabhakaran, A.R. 2 , Krishna Mohan, S 3 , Selvanathan, P 4 and Safiullah, A 5 . 1. Asst.Professor in Physics Department of Physics,JBAS College for Women,Chennai 600 018. 2. Assoc.Professor in Physics Department of Physics, Pachaiyappa’s College, Chennai-600 030. 3. Vice Principal, Saveetha Medical College and Hospital, Thandalam, Chennai 602. 4. Asst.Professor in Saveetha Dental College and Hospital, Chennai-600 077. 5. Lab Director S.S.Diagnostic Centre,Royapettah,Chennai-600 014. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Diabetes Mellitus
(DM) is a destructive disease of carbohydrate, fat and protein metabolism affecting a great amount of world's population. The experiment have been made on the White Wistar rats and recipient rats are injected with alloxan ( 40 mg/kg body weight ) subcutaneously. The experiment have developed DM with Clinical symptoms of haematuria, hyperglycemia etc.,. Blood glucose values were 489 ± 8.48 mg/dl recorded to assess the DM in experimental animals induced with alloxan. Current study focus on FTIR spectroscopy together with routine techniques as a diagnostic tool in the discrimination of diabetic samples from healthy ones . A variety of alterations in the spectral parameters, such as frequency and signal intensity/area was observed in diabetic rat compared to the control samples. Based on these spectral variations and routine analysis successful differentiation between diabetic and control groups was obtained in different spectral regions. The results of this current study further revealed the power and sensitivity of FTIR spectroscopy in precise and automated early diagnosis of diabetes. FTIR-ATR spectral variations and quantification of bio molecules among control and experimental animals observed are statistically more significant . The importance of FTIR -ATR Spectroscopy technique in evaluation of qualitative and quantitative nature of biochemical components is discussed.

…………………………………………………………………………………………………….... Introduction:-
Diabetes Mellitus (DM) is a metabolic disorder which includes a heterogeneous group of damages that have a different etiology, which have common hyperglycemia associated with lipid and protein damages. According to International Diabetic Federation the estimated diabetic prevalence in 2010 has raised to 285 million, representing 6.4 % of the world's adult population , with a prediction that by 2030, the number of people with diabetes will have raised to 438 million . With this alarming concern ,India has been declared as the " Diabetic Capital of World". 87 Currently 40.9 million people in India suffering from diabetes alone. It is also estimated that by the year 2030 , diabetes likely to be the seventh leading cause of death ,accounting 3.3% of the total deaths in the world. DM is characterized by abnormally high blood glucose levels due to decreased secretion or effectiveness in function of insulin . This property of DM causes some metabolic disturbances that lead to chronic, irreversible damage to vital organs and systems ( Stapleton, S. R, 2000). DM is classified into two types ( Kanazawa, Y 1999, Puavilai,G, 1985 Type I diabetes, also known as insulin-dependent diabetes mellitus (IDDM), is characterized by the autoimmune destruction of pancreatic β cells ( Thomas, W.H.K.,2000) Type II diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM), is a complex disease characterized by target organs, such as liver, muscle, and adipocytes. Complications of DM affect nearly all of the organs and systems in the body and a persistent DM can give rise to severe damage to these organs It has been reported that diabetes affects the nervous system, especially brain ( Brands, A. M et al., 2000, Chu, P.C et.al, 1986, Wahba, Z.Z, et al., 1988 the sensory system, especially eye ( Cunha-Vaz, J.G e t a l., 1975, Enea, N.A., 1989) causing diabetic retinopathy Klein, R 1995 ), the cardiovascular system ( Grossman, E. 1996), stomach, intestine the gastrointestinal and the respiratory system ( Thomson, A.B, 1983 , Schiller, R and Feldman, M, 1990), gall bladder ( Atkinson, M and Hosking, D.J, 1983 , Goyal, R. K and Spiro, H.M, 1983) and the reproductive system ( Hassan A.A et al., 1993). Liver is the central organ in the body that takes role in the control of metabolism in DM. A number of studies have been primarily focused on the quantitation of clinically relevant biomarkers present in blood, plasma and/or urine (glucose, electrolytes, proteins, lipids, hormones etc., ( Ollesch, J et al., 2013, Barman, I 2012 or tissue/organ imaging ( Olsztyńsk Janus S. 2 0 1 2 ) .
Although the functional and pathological abnormalities seen in diabetes are both clinically and experimentally defined, studies continued to understand the exact molecular mechanism of diabetes ( Feride Severcan , 2013) . Since DM is affecting all vital organs and it is necessary to rule out the early status of pathological condition by adopting simple method to support the existing techniques for the disease management. Earlier literature shows that Raman and infrared (IR) spectroscopy have been widely tested as powerful tools for medical diagnostics offering a great potential for the real time analysis of large sample number in the clinical setting ( Lasch , P and Kneipp. J , 2008 , Mitchell, A. L 2014 and Petrich, W. 2001). In recent years , FTIR together with chemometric methods , has had an increasingly important role to play in the field of pathology and diagnosis of disease status . Pathological conditions induces changes in content , structure and function of bio molecules in biological systems and these changes can be rapidly and sensitively monitored by FTIR spectroscopy even at very early stage.
To achieve this, author has chosen an animal model to induce diabetes Mellitus and study the biochemical variations using routine and FTIR -ATR spectroscopic study for control and management of disease. Alloxan has been demonstrated to be non-toxic to the human β cells, even in very high doses, because humans have different glucose uptake mechanisms as compared to rodents ( Eizirik, D.L, 1994 andTyrberg, B ,2001). It has been widely used to induce experimental diabetes in animals such as rabbits, rats, mice and dogs with different grades of disease severity by varying the dose of alloxan used ( Iranloye B.O, 2011 ). Moreover, Alloxan is most prominent chemical compound used in diabetogenic research. In research it is used for induction of Type 1 diabetes. Alloxan is a urea derivative which causes selective necrosis of the βcells of pancreatic islets ( Etuk , E.U.N.J , 2010 ). The chemical name of alloxan is 2,4,5,6 tetra oxypyrimidine; 2, 4, 5, 6-pyrimidinetetrone, which is an oxygenated pyrimidine derivative which is present as alloxan hydrate in aqueous solution . So, the current study focused on inducing DM in male wistar rat with alloxan and analyse the bio molecules variations adapting routine and FTIR-ATR spectroscopic method.

Materials and Methods:-
Male wistar rats were housed in the animal house of Research and Development , Saveetha Medical College and Hospital, Thandalam Chennai , India. All experiments were carried out according to the guidelines for care and use of experimental animals, and are approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Study proposal was approved by the Institutional Animal Ethical Committee. Six male wistar rats per cage were housed in polypropylene cages (32.5 ×21×14) cm lined with raw husk which was renewed every 48 hours. The animal house was maintained at an average temperature (24.0 0 C± 2 0 C) and 30-70 % RH, with 12 hr. light-dark cycle (lights on from 8.00 a.m. to 8.00 p.m.). Animals received human care and were fed with commercial pellet diet and the animals were acclimatized for one week 88 before the start of the experiment.

Induction of Diabetes Mellitus:-
Alloxan was prepared by the oxidation of uric acid by nitric acid and the monohydrate form is simultaneously prepared by oxidation of barbituric acid by chromium trioxide. The drug has been noted to its diabetogenic action and the dose of alloxan required for inducing diabetes depends on the animal species and route of administration ( Federiuk, I.F, 2 0 0 4 ) . Alloxan was obtained from Sigma Chemicals Co., St. Louis, MO, USA. Alloxan was dissolved in normal saline and always prepared freshly for immediate. The male wistar rats were divided into two groups: animals from the first group were made subcutaneous injection of Alloxan (40 mg/kg body weight ) for 0,5, 11 and 12 th da ys. and animals in the other group were untreated control . On day 14 th of the experiment animals were sacrificed by decapitation The blood glucose concentration was measured every day from the day of Alloxan induced and blood samples were collected from the tail vein once a day and checked for hyperglycemic condition by accu check method. Animals with a blood glucose concentration 200 mg/dL were considered to be diabetic (  Sample preparations for Biochemical studies:-At the end of experiment (14 th day ) the male wistar rats were fasted overnight . Blood samples of the male wistar rats were withdrawn on from the heart. under mild anaesthesia before killing and collected in plain and EDTA tubes for further analysis . Plasma and serum were separated by centrifugation at 3000 rpm for 15 minutes. The blood serum properly stored for estimation of biochemical parameters including, Glucose, Total cholestreol, triglycerides, LDL, urea, creatinine , uric acid, total protein, SGOT, SGPT e t c . , by CLIA and Total serum T 4 ,T 3 and TSH concentration were determined by ELISA (detection kits provided by Transasia , Zemun, SCG) in a reputed clinical laboratory in Chennai . FTIR-ATR Spectral Analysis the serum samples were properly preserved in ice bags and immediately transported to the wet laboratory.

FTIR-ATR spectral Measurements:-
The FTIR-ATR spectroscopy is based on the phenomenon known as Total Internal Reflection (TIR) . This radiation strikes the interface between the IRE and the serum sample composed of a lower refractive index. This internal reflectance creates an evanescent wave that extends beyond the surface of the crystal into the serum sample held in contact with the crystal. It can be easier to think of this evanescent wave as a bubble of infrared that sits on the surface of the crystal. This evanescent wave protrudes only a few microns (0.5µ-5µ) beyond the crystal surface and into the sample. The depth of penetration of infrared radiation from denser IRE into the test material depends on refractive indices of the materials to be investigated and the wave number of the infrared radiation. As the sample absorbs IR radiation at certain frequencies, the resultant totally reflected radiation (or) evanescent wave will be attenuated (altered) in regions of the infrared spectrum where the sample absorbs energy ( Katon JE andMicron 1996, Baulsir CF andSimler RJ 1996 ). This attenuated IR radiation of evanescent wave is passed back to the IR beam, which then exits the opposite end of the crystal and it is detected by the detector in IR spectrometer. The system generates an infrared spectrum.
FTIR-ATR spectral measurements of serum samples of male wistar rat induced diabetes were carried out at Sophisticated Analytical Instrumentation facility (SAIF-SPU), St. Peter's University, Avadi, and Chennai-600 054, using PerkinElmer Spectrum-Two FTIR Spectrophotometer with attenuated Total Reflectance accessory having highly reliable and single bounce diamond as its Internal Reflectance Element (IRE). Experimental serum samples were analyzed immediately for spectral recordings in the Mid IR region of 4000 -450 cm-¹. As water is a good absorbent of infrared radiation, it affects the actual spectral response of the test material and dominated in the FTIR spectrum of serum sample and therefore it was placed on the IRE crystal and water content on the serum sample is removed by air drier. FTIR spectral measurements were carried at room temperature and each measurement was repeated to ensure the reproducibility of the spectra. These spectra were subtracted against the background of air spectrum. After every scan, the crystal is cleaned with isopropyl alcohol or methanol soaked tissue and a background of new reference air was taken to ensure the crystal cleanliness.
Statistical Analysis:-All statistical analysis were performed using Statistical Package for Social Science (SPSS, version 17) for Microsoft windows. The data were not normally distributed. 89 and therefore Non -parametric tests were performed. Descriptive statistics were presented as numbers and percentages. The data were expressed as Mean and SD. A one way analysis of variance ( ANOVA ) /Kruskal-Wallis test with a post hoc Tukey HSD was used. Independent sample student t test / Mann-Whitney test were used to compare continuous variables between two groups. A two sided p value < 0.05 was considered statistically significant.

Results:-
Diabetes mellitus is group of metabolic disorders characterised by hyperglycemia, glycosuria and hyper lipidemia. DM accompanied not only by a permanently elevated level of blood glucose and altered levels of other biomarkers, but also by changes in the conformation of blood plasma proteins and other bio molecules associated with the pathogenesis of diabetes. The variations in biochemical composition of experimental rats induced with alloxan is given in table 1. The results shows that levels of glucose highly significant (P<0.001) among control and experimental animals. Further lipid profile found to be moderately significant between control and diabetic rat. But statistical significant values of thyroid hormones were obtained on control and rat induced alloxan . But other biochemical parameters shown in the table 1 are is not significant.   Internal Standard ratio Parameters calculation:-These spectra were used in Internal ratio Parameter calculation and analysis requires spectra with change in sensitive peaks and no change in sensitive peaks for control and experimental animals. Internal ratio parameter is calculated to fortify the results obtained from the FTIR intensity of absorptions. Internal ratio Parameter ignores the difference in the amount of sample analyzed, it nullifies the contradiction in the quantity of the sample and gives measured out exact deviations in the male wistar ratio (I 3282 / 1453, I 2961 / 1743, I 1537 / 1238 and I 1075 / 516 ). The internal ratio parameter of basic protein, lipid , Nucleic acid and glycogen of control and diabetic rats given in Table 3. The results shows that peak ratio for NH (protein and Urea) to Lipoproteins-(CH3) aym.bending among diabetic status of rat induced by alloxan (0.9152 ) and control male wistar rats ( 0.8707 ) is negligible . Significant elevation in the absorption peak ratio was documented in (Protein ) asym and Sym to TGL /HDL-cholesterol ester in diabetic induction on rat by alloxan and control healthy rat. Further , slight variation peak absorption ratio for Amide II to Amide III and Nucleic acid-PO 2 as well as Glucose Stretching to S-S stretch in cystic acid were noticed in control and experimental rats.

Discussion:-
The current diagnostic tools are insufficient for the early detection of many diseases, including type 1 diabetes mellitus. . However, the observation of these structural changes by infrared spectroscopy is limited. Therefore, we used FTIR-ATR Spectroscopy which is inherently sensitive to the structure of bio molecules and able to detect stretching of atoms in the functional group of molecules.. We investigated the blood plasma samples of diabetic rat and healthy rats using FTIR -ATR spectroscopic method. The results w e r e combined with conventional methods of molecules quantification i.e. ELISA and Spectrophotometer. The obtained data sets were statistically evaluated and focusing on the spectral ranges that correspond to the structure and conformation of proteins and other biomolecules. Our results suggest that FTIR -ATR spectroscopic method gives more detailed information about the structure of bio molecules; and therefore, might be a promising complement to conventional in diagnostic methods. The results obtained shows that elevated fatty acid types and its derivatives in addition to blood glucose level (Table 1 ) suggest that Diabetes is not a single disease it's group of heterogeneous syndromes and support the study of Patel, D 2011 where the author stated the clinical features such as heart attack, stroke and peripheral vascular disease in addition to diabetic condition.. Further the results obtained in this study also support earlier literature shows that diabetes, and higher-than normal blood glucose levels are well-known risk factors for development of type 2 diabetes recent prediction models have incorporated these with other readily measurable features of metabolic syndrome 92 (elevated blood pressure, low HDL cholesterol, and elevated triglycerides) to generate validated prediction rules (Stern M.P .2002;Wilson, P.W,2007, Schmidt ,M.I,2003). In the average FTIR-ATR spectra of diabetic and healthy controls (Fig. 1), we can recognize that the absorbance's for corresponding wave length to diabetes induced rat is significantly higher intense bands than control shows elevated levels of diabetic biomarker which support other methodology adopted in this study. Apart from this , the spectrum obtained for control and experimental model might be the additional supporting evidences to evaluate the bio molecule qualitatively and quantitatively as diagnostic tool in clinical study. The small changes in the absorption is also appreciable in the FTIR-ATR spectra as it depends on the short existing, effective evanescent wave with 0.5 µ-5 µ depth of penetrations. The trends observed on absorptions of internal peak ratio of experimental and control male wistar rat in this current study support earlier studies on different diseases like thyroid, Renal ,atherosclerosis, cancer, Hepatitis ( Khatheeja Saira et

Conclusion:-
Diabetes mellitus has been induced by administration of Alloxan in dose of 40 mg/kg, subcutaneous administration ( 0,5, 11 and 12 th days) for four dose. .
Biochemical blood exams showed an increase of Total cholesterol triglyceride , TSH etc., The results obtained suggested that to rat, subcutaneous administrated alloxan induces diabetes mellitus with clinical and complementary signs, this experiment being useful in experimental studies regarding diabetes mellitus. We have analyzed real clinical blood serum samples by FTIR-ATR spectroscopy and identified spectral regions that are most likely revels the qualitative and quantitative evaluation of bio molecules. The subsequent multivariate analysis of spectral data proved that the FTIR-ATR spectroscopic methods are able to detect more complex signal of serum bio molecules than conventional methods. The results obtained suggested that FTIR -ATR spectral evaluation might be an additional tool in clinical diagnosis , prognosis and disease management. The best predicting model included adiponectin, C-reactive protein (CRP), ferritin, interleukin-2 receptor A (IL2RA), glucose, and insulin, with area under the a receiver operator characteristic curve may be suggested for future study.