J. Pure & Appl. Phys., Vol. 22, No. 4, Oct – Dec, 2010, pp. 645 – 649 645 ……………………………………………………………………………………………………………
Quantitation of Glucose in Urine by Fourier Transform Infrared Spectroscopy SYED ISMAIL AHMAD, P. RAVI PRASAD1 & ADEEL AHMAD. Biophysics Unit, Nizam College (Autonomous), Osmania University, Hyderabad – 500 001, India. 1 Indian Institute of Chemical Technology, Uppal Road, Hyderabad, India.
[email protected] Received
26
September 2010
Accepted
18 November 2010
Abstract - The measurement of concentration of glucose has been achived using FT-IR Spectroscopy which can provide the physician an objective aid in identification of Diabetic mellitus. The FT-IR spectra of urine samples are recorded using liquid cell in Mid IR region 4000-500 cm-1 and also a second scan between 1500 -700 cm-1 using Thermo Nicolet Nexus 670 at IICT, Hyderabad in absorption mode. The overall spectra of urine samples are dominated by urea. The intense peak at 3400 cm-1 and a secondary peak at 1640 cm-1 can be assigned to urea. The normal urine is treated with glucose at different concentrations viz 10, 5, 2.5 and 1.25 gm/dL and the FT-IR spectra is recorded, this further confirms the specific peak for glucose. A graph between concentration of glucose and intensity of absorption shows a linear relation, there is an increase in the intensity of absorption at wave number 1034 cm-1 with the concentration, which suggests that this is the most specific peak for glucose.
Key Words – FT - IR Spectroscopy, Glucose, Urine. __________________________________________________________________________________
1. Introduction The glomerulus filtrate from the Bowman’s capsule contains waste products like urea, electrolytes, amino acids and glucose. When the filtrate passes into the proximal convoluted tubule (PCT), the glucose is reabsorbed back into the blood stream. The PCT can only absorb a limited amount of glucose. As and when blood glucose level exceeds 165 to 185 mg/dl, the PCT becomes over loaded and begins to excrete glucose in the urine. This point is called renal threshold of glucose (RTG). This glucose is a simple
sugar called corn sugar or blood sugar, which is an important carbohydrate in biological systems. The cells in the human body use glucose as a source of energy, and metabolic intermediate. For solid samples the far IR is also equally important and requires special instruments & techniques. To record IR spectra Fourier Transform Technique is used, called FT-IR Spectra. IR spectroscopy has been used by Biophysicist and chemist as a powerful tool to characterize compounds. It has been applied in biology for studying the structure and
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646 Syed Ismail Ahmad, et. al. …………………………………………………………………………………………………………….
conformation of molecules like proteins, nucleic acids and lipids. The advances made in instrumentation have paved the way for its utilization in medicine. Besides the application of FT-IR for tissue diagnostics, the investigation of body fluids has been gaining importance. The mid –IR region is very useful in the identification of disease patterns using the FT-IR spectrum of human urine. Precise quantification of several components such as glucose, total protein, and urea can be achieved using FT-IR spectroscopy. 2. Material and Methods The urine samples were collected from a healthy male in the age group of 35 years, and treated with research grade, SD fine chem make glucose anhydrase at concentrations 1.25, 2.5, 5 and 10 gm/dL. The FT-IR spectra were recorded with Thermo Nicolet Nexus 670. The table top Thermo Nicolet Nexus 670 is calibrated and checked with polysterene film. The sample was filled in the liquid cell of 1mm thickness with a micro syringe. The liquid cell was placed in the sample compartment. The resolution was kept at 4 cm-1 and scanning time was fixed at 38 Sec. A total number of 32 scans were carried out on each sample. The scanning range fixed from 4000 – 400 cm-1 for each sample. And also the ranges 2000-1400cm-1, 1400 – 600cm-1 and 1200 – 1000cm-1 were carried out. 3. Results and Discussion Table 1 gives the wave numbers and assigned functional groups obtained from standard FT-IR spectral library. Table 2 presents the wave numbers and assigned functional groups for glucose. Table 3 shows
the Intensity of absorption at wave numbers for urine added with glucose at different concentrations. Table 4 gives intensity of absorption and concentration of glucose added with which a graph is plotted between intensity of absorbance of glucose and concentration (Fig. 1.). The IR spectrum is like a ‘finger print’ or ‘signature’ of the molecular species making up of the sample. The intensities of IR spectra provide quantitative information while the absorption positions reveal qualitative characteristics about the nature of chemical bonds, their structure and their molecular environment. One can study the pathology of urine or any other bio-liquid using the FT-IR spectra. The major absorption bands arise from NH, C=0, C-H and X-O bonds found in urea, carbohydrates, proteins, lipids and nucleic acids [1]. The frequency, intensity and width of the particular vibration IR spectral bands are extremely sensitive to the packing constraints, conformational changes and chemical vibrations in lipids, carbohydrates, urea and proteins present in urine[1,2]. Specifically vibrations that have been previously used in the study of lipids are the CH2 stretching (2850 and 2920 cm-1) bending or scissoring (1450-1480 cm-1) and wagging (1180-1350 cm-1) modes, the lipid finger print region containing phosphate and diester stretch modes (1000-1450 cm-1), and the C=0 stretching (1700-1750 cm-1) vibrations [1]. It can be seen that an overall appearance of urine spectrum is dominated by urea, not surprisingly as urea concentration is expected to be much higher than any other
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Quantitation of Glucose in Urine by Fourier Transform Infrared Spectroscopy 647 …………………………………………………………………………………………………………….
urinary constituent in normal urine. The most intense bands for urea are 3400 and peaks between 1640-1620cm-1 and most intense absorption band in proteins is the amide-I peak, which is observed at 1652 cm-1. Amide–I is mainly associated both the C=0 and C-N stretching vibration
and is also related to the backbone conformation [4-6]. The next major absorption band is the amide-II that derives largely from the in plane N-H bending, and the C-N and C-C stretching vibrations. The peak at 1034 cm-1 can be assigned to glucose [3-4].
Table 1- Wave numbers and assigned functional groups for normal urine WaveNumber (cm-1) 3461 1641 591
Compound or functional group assignment H2O or N-H (N-H) Amide I. the C=N stretching absorption for open chain compound, helical structure – NH2. Strong C-H deformation , alkines
1458
CH2, CH3 bending modes. (N=O) symmetrical deformation.
1084
Very Weak, Sugar ring vibration
1399
Lactate, carboxylic acids and derivatives.
Table 2 - Wave number and assigned functional group for FT-IR spectra of normal urine added with Glucose Wave number (cm-1) 3449 – 3437 2927 - 2928 1633 – 1641 1457 - 1460 1403 – 1404 1077 1034
Compound or functional group assignment H2O or N-H (N-H) -C-H symmetrical stretching of CH2 Amide I. the C=N stretching absorption for open chain compound, helical structure –NH2, due to urea. CH2, CH3 bending modes. (N=O) symmetrical deformation. CH2, CH3 bending modes of lipids. C=O symmetric stretching vibration of COO-. Mannose, ester C-O-C asymmetric stretching vibrations of phospholiopids. Glucose, C-O region
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648 Syed Ismail Ahmad, et. al. ……………………………………………………………………………………………………………. Table 3 - Intensity of absorption at wave numbers for urine added with glucose at different concentrations Normal UG4 UG3 UG2 UG1
0 1.25 gm/dl 2.5 gm/dl 5 gm/dl 10 gm/dl
3461 (0.323) 1641(0.133) 591(0.0854) 1458(0.0072) 1084 (0.0068) 1399 (0.0052) 3449 (0.089) 1633 (0.0472) 1460 (0.0191) 1077.65 (0.0149)3753 (0.0047) 1077.44 (0.0094) 1034(0.0090) 1457 (0.0046) 1403 (0.0044) 3446 (0.0617) 1641 (0.0326) 571 (0.0213) 1036 (0.019) 1460 (0.0123) 3760 (0.0079) 3443 (0.152) 1636 (0.131) 1078 (0.107) 1460 (0.0887) 640 (0.0261) 2972 (0.0148) 3437 (0.326) 1034 (0.126) 1076 (0.119) 1634 (0.107) 561 (0.0926)1404 (0.0388)2928 (0.0374) Table 4 - Intensity of absorption and concentration of glucose added Glucose concentration Normal urine 1.25 gm/dl 2.50 gm/dl 5.00 gm/dl 10.0 gm/dl
Intensity of absorption 0 0.0094 0.0190 0.0850 0.1180
Fig. 1. Graph between intensity of absorbance of glucose and Concentration
The IR spectrum is like a ‘finger print’ or ‘signature’ of the molecular species making up of the sample. The intensities of IR spectra provide quantitative information while the absorption positions reveal qualitative characteristics about the nature of chemical bonds, their structure and their
molecular environment. One can study the pathology of urine or any other bio-liquid using the FT-IR spectra. The major absorption bands arise from NH, C=0, C-H and X-O bonds found in urea, carbohydrates, proteins, lipids and nucleic acids [1]. The frequency, intensity and width of the
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Quantitation of Glucose in Urine by Fourier Transform Infrared Spectroscopy 649 …………………………………………………………………………………………………………….
particular vibration IR spectral bands are extremely sensitive to the packing constraints, conformational changes and chemical vibrations in lipids, carbohydrates, urea and proteins present in urine. Specifically vibrations that have been previously used in the study of lipids are the CH2 stretching (2850 and 2920 cm-1) bending or scissoring (1450-1480 cm-1) and wagging (1180-1350 cm-1) modes, the lipid finger print region containing phosphate and diester stretch modes (1000-1450 cm-1), and the C=0 stretching (1700-1750 cm-1) vibrations [1]. It can be seen that an overall appearance of urine spectrum is dominated by urea, not surprisingly as urea concentration is expected to be much higher than any other urinary constituent in normal urine. The most intense bands for urea are 3400 and peaks between 1640-1620cm-1 and most intense absorption band in proteins is the amide - I peak, which is observed at 1652 cm-1. Amide – I is mainly associated both the C=0 and C-N stretching vibration and is also related to the backbone conformation [4-6]. The next major absorption band is the amide-II that derives largely from the in plane N-H bending, and the C-N and C-C stretching vibrations. The peak at 1034 cm-1 can be assigned to glucose [3,4]. Acknowledgement One of the authors (SIA) is very thankful to
the Director IICT, Hyderabad for providing the FT-IR facility. References [1] Benjamin Bird, Melissa. J. Romeo, Max Diem and Stephen Naber, Cytology by Infrared Micro-Spectroscopy: Automatic Distinction of Cell Type, J. Vibrational Spectroscopy, Vol.18, No. 1(2008), pp. 101. [2] M. A. Moharram, A. Higazi and A. A. Moharram, Infrared Spectra of Urine from Cancerous Bladder, J. Infrared Millimeter and Terahertz Waves, Vol. 17, No.6(1996), pp. 1103 – 1114. [3] Cyril Petibois,Vincent Rigalleau, AnneMarie Merlin, Annie Perromat, George Cazorla, Henrigin and Gerard Deleris, Determination of glucose in dried samples by FT-IR Spectroscopy, J. Clinical Chem, Vol. 45, (1999), pp. 1530-1535. [4] J. T. Grismer, L. T. Rozelle and R. B. Koch, Infrared Spectroscopy and Osmolality Analysis of Urine, Two simple sensitive methods for early detection of postoperative Anuria after thoracotomy, J. Chemistry, Vol. 49, No. 5(1996), pp. 467 – 478. [5] Do-Hyun Kim Ilev, Using Mid-Infrared glucose absorption peak changes for high precession glucose detection, J. Laser and Electro - Optics Society, 2007. [6] R. A. Shaw, S. Kotowich, H. H. Mantsch and M. Leroux – Quantitation of Protien, Creatinine and Urea in Urine by Near-Infrared Spectroscopy. J. Clin Biochem., Vol. 29, No.1(1996), pp. 11 – 19.
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