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Near-infrared absorbance measurements of hemoglobin solutions incubated with glucose Olga S. Zhernovaya,1* Valery V. Tuchin,1 Igor Meglinski,2 Laurie Ritchie2 1 Saratov State University, Russia 2 Cranfield University, United Kingdom ABSTRACT It is known that glucose influences on spectral properties of blood and hemoglobin and interacts with plasma proteins and hemoglobin in erythrocytes. Changes of optical properties of blood and hemoglobin at glucose concentration within physiological level are important for diagnosis and monitoring of diabetes. The purpose of this study is to investigate the effect of presence of glucose and glycation of hemoglobin on absorbance of aqueous hemoglobin solutions with different glucose concentrations. Measurements were taken using spectrophotometer EQUINOX 55 (Bruker Optic GmbH) in a range 1000-1800 nm. Water has absorption bands in the near-infrared region which may be influenced by glucose presence. We have hypothesized that glucose and hemoglobin, especially glycated hemoglobin, may influence the absorption band of water in solution. The hemoglobin solutions with different amount of glucose (from 0 to 1000 mg/dl with a step 100 mg/dl) were incubated up to 28 days. Our measurements show that presence of glucose affects the spectra of aqueous hemoglobin solutions. The magnitude of absorbance depends on glucose concentration. At the beginning of incubation hemoglobin solution without glucose has the lowest absorbance magnitude, but after a rather long time of incubation (28 days) the absorbance of hemoglobin solutions with glucose become smaller compared to the absorbance of hemoglobin solution without glucose. This fact may be explained by assumption of hemoglobin glycation, when glucose molecules chemically bind to hemoglobin, and water binding to hemoglobin. In the case of water binding to hemoglobin molecules the amount of free water molecules in solution decreases, so the water aborbance is excepted to decrease. Keywords: near-infrared spectroscopy, glucose, glycated hemoglobin

1. INTRODUCTION Monitoring of blood glucose and glycated hemoglobin level is an urgent requirement for diabetic patients. The elevation of blood glucose levels for prolonged periods of time leads to a number of micro- and macrovascular complications, including retinopathy, nephropathy, neuropathy, and cardiovascular diseases. Diabetes complications are the dominant causes of morbidity and mortality in diabetic patients. The goal of diabetes management is the prevention of diabetesassociated complications and, therefore, diagnosis of the disease at an early stage. Plasma glucose level is not a suitable parameter for long-term glycemic control since it fluctuates widely during a day. Glycated hemoglobin is used both as an index of mean glycemia and as a measure of risk for the development of diabetes complications. HbA1c is the major component of glycated hemoglobin, which is formed by a non-enzymatic irreversible process of combination of aldehyde group of glucose with the amino-terminal valine of the β-chain of hemoglobin. This process comprises a sequence of non-enzymatic reactions (Fig. 1). The first is the rapid but reversible formation of an aldimine (or Schiff base). The next is the considerably slower formation of a stable ketoamine via a process known as the Amadori rearrangement. The ketoamine accumulates over the life of the erythrocyte and forms the main part of the glycated hemoglobin. The next stage of the reaction is formation of hemoglobin-advanced glycation end products (Hb-AGE).

*

e-mail: [email protected]; Phone: +7 (8452) 285722

Complex Dynamics and Fluctuations in Biomedical Photonics IV, edited by Valery V. Tuchin, Proc. of SPIE Vol. 6436, 64360S, (2007) · 1605-7422/07/$18 · doi: 10.1117/12.711053

Proc. of SPIE Vol. 6436 64360S-1

The level of glycated hemoglobin in a blood sample reflects the mean glycemia of about the previous 120 days. In a norm less than 7% of Hb is glycated and this value rises at the increasing of free glucose concentration.1 The rate of glycated hemoglobin formation is proportional to free blood glucose concentration, each 1% increase of HbA1c (the major component of glycated hemoglobin) amount corresponds to a 35-mg/dl (1.95-mmol/l) increase of mean plasma glucose.2

HbA-Va1-NH +

HCO

HC

HC I

HCOH

HbA-VarN

HbA-VarN

Amadori

I

HLDH rearmngement

Rb-AGE HOCH

HOQi

HOSH

HCOH

HCOH

HCOH

HCOH

HCOH

HCOH

CHOH fl-glucose

CHOH Aldireine, Shift base

CHOH Keteam'ne. HbAIC

Fig. 1. Nonenzymatic formation of HbA1c and Hb-AGE from hemoglobin and glucose (L. Maillard).

There are many (greater than 30) different glycated hemoglobin assay methods in current use.3 Most methods quantify hemoglobin A1c, defined as hemoglobin A with glucose attached to the NH2-terminus valine of one or both beta chains. Over the last years various optical methods for monitoring of free glucose and glycated hemoglobin levels have been investigated, such as near-infrared spectroscopy4-13, optical coherence tomography14, polarimetry15, Raman spectroscopy16-18 and fluorescence19,20. Refractive index is promising parameter for estimation the glycated hemoglobin amount in blood.14,21 For example, optical coherence tomography (OCT) may be applied for measurements of highly absorbing and scattering mediums, such as whole blood. The OCT system is based on fiber-optic Michelson interferometer, which is illuminated by lowcoherence light, usually superluminescent diode (SLD).22 Sample is placed in one interferometer arm, and sample reflections are combined with the reflection from the reference mirror. The amplitudes and delays of sample reflections are measured by scanning the reference mirror position and simultaneously recording the amplitude of the interferometric signal. The optical path length between two points of cuvette along the depth direction is proportional to the refractive index and geometrical path length. Recently OCT was applied for refractive index measurements of hemoglobin solutions with glucose.14 The obtained degree of refractive index increase, its saturation and subsequent fall down was explained using concept of interaction of glucose and hemoglobin molecules and glycated hemoglobin formation. The disadvantage of OCT-based refractive index measurements is relatively big coherence length of SLD. The coherence length of the light source determines the reproducibility of refractive index measurements. In case of SLD with the width of the power spectrum about 40 nm, the coherence length is not enough to obtain sufficient precise and accuracy of refractive index measurements. Vigneshwaran et.al.19,20 has investigated glycated hemoglobin fractions by fluorescence measurements. It was showed that in vitro glycated hemoglobin gave the maximum intensity of emitted light at 345 nm upon excitation at 308 nm. The hemoglobin solutions with glucose concentrations of 5, 10, 20, 30, 40 and 50 mmol/l were investigated. Initially, the fluorescence of all samples was close to zero for all samples. Small rise of fluorescence intensity was detected after 7 weeks of incubation, and at 11 weeks of incubation the increase was substantial, which finally reached threshold at 17 weeks for all samples. The threshold intensity varied linearly with the concentration of glucose. In addition, colour


change was noticed from red to dark brown after 2 months of incubation, which perhaps indicated the advanced stage of glycation. Among optical techniques for estimating of blood glucose and glycated hemoglobin level, non-invasive measurements offers several advantages, such as absence of pain and, hence, ability to increase frequency of testing. The possibility of non-invasive glucose testing using near-infrared spectroscopy was discussed in several studies.4,7-13 Near-infrared spectroscopy glucose measurement enables investigation of tissue depths in the range of 1 to 100 millimeters with a general decrease in penetration depth as the wavelength value is increased.7 Non-invasively determined blood glucose values may vary by body site, depending on the differences in tissue and vascular properties. Burmeister et.al.10 presented non-invasive near-infrared human spectra for several potential measurement sites, such as lip, cheek, webbing and tongue. Obtained results showed that the tongue is the most suitable site for non-invasive glucose measurements with near-infrared spectroscopy. The disadvantages of using the near-infrared measurements for glucose value determination are caused primarily by absorption of other blood and tissue component, especially by strong absorption of water in this region. Raman spectroscopy offers the possibility to obtain measurements of glucose because, in contrast to infrared spectroscopy, its spectral signature is not obscured by water.17 In addition, Raman spectral bands are considerably narrower than infrared spectra and Raman excitation in the near infrared region (700-1300 nm) encounters minimal fluorescence in aqueous media. The vibrational spectra produced as a result of Raman scattering reveals the state of the atomic nuclei and chemical bonding within a molecule, as well as the interactions between the molecule and its local chemical environment. The disadvantages of Raman spectroscopy include instability in the laser wavelength and intensity, errors due to presence in whole blood and tissue highly absorptive, containing many fluorescent and Ramanactive components, and long spectral acquisition time. An alternative approach is to use Raman spectroscopy for noninvasive measurement of glucose in the ocular aqueous humor.16 Benefits include increased resistance to interference from luminescence and fluorescence. Polarimetric measurement of glucose concentration is based on optical rotatory dispersion, when a solution containing a chiral molecule rotates the plane of polarization for linearly polarized light passing through it.15 The rotation is the result of a difference in refractive indices nL and nR for left and right circularly polarized light. Glucose in the body rotates light in the right-handed direction and has a specific rotation at the sodium D-line of 589 nm. Advantages of polarimetric methods for glucose sensing include the use of available visible source and ability to use substantial pathlengths in aqueous solutions. The anterior chamber of the eye was proposed as a non-invasive glucose sensing site for polarimetric measurements since it provides measurements with minimal scattering and a glucose concentration well correlated to that of the blood.23 In this study we investigate near-infrared absorbance of aqueous hemoglobin solutions incubated with glucose and changes of absorbance during the incubation. Measurements were taken using spectrophotometer EQUINOX 55 (Bruker Optic GmbH) in a range 1000-1800 nm.

2. MATERIALS AND METHODS Hemoglobin solutions (Biokont-GK, Agat-Med, Russia) with the initial concentration 120 g/l were dissolved in distilled water. The initial solution of D-glucose (Dia-M, Russia) in water was 2000 mg/dl. This solution was dissolved in distilled water to obtain lower concentrations of glucose (1800 mg/dl, 1600 mg/dl etc.). These glucose solutions were added to hemoglobin solution in the ratio 1:1 to obtain different concentrations of glucose in samples (from 1000 to 0 mg/dl with a step 100 mg/dl). Two groups of hemoglobin solutions were investigated: (1) hemoglobin concentration was 20 g/l in all samples; glucose concentration was 0-1000 mg/dl (with a step 100 mg/dl). Time of incubation was up to 7 days. (2) hemoglobin concentration was 15 g/l in all samples; glucose concentration was 0-1000 mg/dl (with a step 100 mg/dl). Time of incubation was 28 days.


Measurements were taken using spectrophotometer EQUINOX 55 (Bruker Optic GmbH) in a range 1000-1800 nm, the thickness of cuvette was ≈ 1 mm.

3. RESULTS AND DISCUSSION Figures 2 and 3 represent the absorbance of hemoglobin solutions with glucose at 1455 nm.

1,31

5 days, group (2) 7 days, group (2) 28 days, group (3)

1,30

Absorbance

1,29

1,28

1,27

1,26 0

200

400

600

800

1000

Glucose concentration, mg/dl Fig. 2. Absorbance of hemoglobin solutions with glucose at 1455 nm after 5, 7 and 28 days of incubation.


1,4

Hb Water

1,2

Absorbance

1,0 0,8 0,6 0,4 0,2 0,0 -0,2 1000

1200

1400

1600

1800

Wavelength, nm Fig. 3. Absorbance of aqueous hemoglobin solution and water in the range 1000-1800 nm, group (1). Hemoglobin concentration is 20 g/l.

Water has absorption bands in the near infrared region which may be influenced by the presence of glucose and other substances.9,11 Absorption band with the maximum at 1455 nm is typical for all spectra. The magnitude of absorbance depends on glucose and hemoglobin concentration. Figure 2 represents the absorbance changes of hemoglobin solutions with different glucose concentrations in a range 1000-1800 nm. It can be seen that the absorbance of hemoglobin solutions with glucose (group (1)) after 7 days of incubation decreased compared to the absorbance after 5 days of incubation, while the absorbance of hemoglobin solution without glucose did not change during this period of incubation. The absorbance behavior of hemoglobin solution after 7 days of incubation is the same for solutions after 28 days of incubation, where the absorbance of hemoglobin solutions with glucose is smaller than the absorbance of solution without glucose. The results can be possibly explained by assumption of hemoglobin glycation, which may influence the spectra of hemoglobin solutions incubated with glucose. As it can be seen from Figure 2, the decrease of the absorbance may be observed for hemoglobin solutions with glucose after rather long time of incubation (7 days and 28 days), and the difference between the absorbance of hemoglobin solutions with and without glucose in group (2) is more evident than in group (1). Water is the main absorber in near infrared region, so the measured spectra are dominated by the water spectrum. As it can be seen from the Figure 3, the presence of hemoglobin molecules leads to transmission increase and absorbance decrease of aqueous hemoglobin solution, compared to water spectra. The absorption band with the maximum at 1455 nm can be possibly determined only by absorption of water. The amount of water in hemoglobin solution with the concentration 15 g/l is bigger, than in solution from group (1) (hemoglobin concentration 20 g/l), so the latter has smaller absorbance value. In addition, hemoglobin solutions with glucose have lowest absorbance magnitudes, which also may be explained by decrease of water content in solution with glucose concentration. In tissues and blood, water can be found in two states: free and bound with bimolecules. Water molecules can combine


with proteins and form the water layer surrounding the protein molecule.24,25 The absorbance decrease after long time of incubation can be explained by assumption of water binding to hemoglobin molecules. In this case the amount of free water decrease during the incubation period and absorbance value of free water at 1455 nm is also decreases. Novskova et.al.26 presented dielectric spectra of hemoglobin solution. The imaginary part of dielectric permittivity has three peaks related to hemoglobin (1 MHz), bound water (316 MHz) and free water (1 GHz). The experimental value of the peak related to bound water was explained by assumption of considerable amount of water bound with hemoglobin, about 3000 water molecules per one hemoglobin molecule. Maleev et.al.27 reported that total hydration of DNA duplex in B-form take place when about 20 water molecules bind to one nucleotide, and total dehydration of DNA occurs at the temperature of 105°C. The temperature as an external condition impacts greatly to the near-infrared absorbance measurements. The quantitative effect of temperature to the absorbance of aqueous glucose was investigated by Cui et.al.28 The results showed that the absorption of aqueous glucose decreases with the increasing of temperature, also the absorbance decreases. In addition, only 1°C change in the temperature induces about -7x10-3 and -4.10-3 errors in the absorbance of the aqueous glucose at the wavelength of 1550 nm, 1610 nm respectively.

CONCLUSIONS This study shows that presence of glucose affects near-infrared spectra of aqueous hemoglobin solutions. Hemoglobin does not have absorption bands in a range 1000-1800 nm, so the absorbance maximum at 1455 nm is determined only by water. This explains less absorbance of aqueous hemoglobin at 1455 nm compared to water absorbance. The magnitude of absorbance depends on glucose concentration. The decrease of absorbance of hemoglobin solution with glucose concentration for longer time of incubation may be explained by hemoglobin glycation that may possibly cause the increase of amount of water molecules bound to hemoglobin. It can be seen than glycated hemoglobin has more ability to bind water, which is possibly due to water-glucose interactions, which leads to decrease of free water in a sample and, therefore, decrease of absorbance at 1455 nm.

AKNOWLEDGEMENTS The research was supported by grant of Royal Society, grant of Federal Agency of Education of RF № 1.4.06, RNP.2.1.1.4473 of the RF Program “The Development of Scientific Potential of the High School (2006-2008)” and by CRDF BRHE grant RUXO-006-SR-06.

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