Assessment of Microcirculatory Hemoglobin Levels in Normal and Diabetic Subjects using Diffuse Reflectance Spectroscopy in the Visible Region — a Pilot Studyby N. Sujatha, B. S. Suresh Anand, K. Bala Nivetha, V. B. Narayanamurthy, V. Seshadri, R. Poddar

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Journal of Applied Spectroscopy, Vol. 82, No. 3, July, 2015 (Russian Original Vol. 82, No. 3, May–June, 2015)




N. Sujatha,a,* B. S. Suresh Anand,a K. Bala Nivetha,a UDC 616-073.584:547.963.4

V. B. Narayanamurthy,b V. Seshadri,b and R. Poddarb

Light-based diagnostic techniques provide a minimally invasive way for selective biomarker estimation when tissues transform from a normal to a malignant state. Spectroscopic techniques based on diffuse refl ectance characterize the changes in tissue hemoglobin/oxygenation levels during the tissue transformation process. Recent clinical investigations have shown that changes in tissue oxygenation and microcirculation are observed in diabetic subjects in the initial and progressive stages. In this pilot study, we discuss the potential of diffuse refl ectance spectroscopy (DRS) in the visible (Vis) range to differentiate the skin microcirculatory hemoglobin levels between normal and advanced diabetic subjects with and without neuropathy. Average concentration of hemoglobin as well as hemoglobin oxygen saturation within the probed tissue volume is estimated for a total of four different sites in the foot sole.

The results indicate a statistically signifi cant decrease in average total hemoglobin and increase in hemoglobin oxygen saturation levels for diabetic foot compared with a normal foot. The present study demonstrates the ability of refl ectance spectroscopy in the Vis range to determine and differentiate the changes in tissue hemoglobin and hemoglobin oxygen saturation levels in normal and diabetic subjects.

Keywords: diffuse refl ectance spectroscopy, tissue hemoglobin, hemoglobin oxygen saturation, diabetic foot.

Introduction. Diabetes mellitus (DM) is defi ned as a complex disease with diversifi ed etiology. With the risk of foot ulceration, diabetes affects people worldwide with estimations of 334 million by 2025 [1]. DM has common complications associated with lower extremities of the body. Understanding the tissue pathology associated with progressing diabetes is essentially complex and hence challenging. Later complications of diabetes lead to nerve fi ber damage resulting in loss of sensation in the foot, known as diabetic neuropathy. This in turn reduces blood supply causing tissue hypoxia and altered metabolism. Changes in blood fl ow along with loss of sensation in foot lead to the pathogenesis of diabetic neuropathy [2] succeeded by diabetic foot ulcer. Also, the synthesis of vasoactive agents such as nitrous oxide is observed to contribute to the changes in diameter of blood vessels associated with diabetes [3].

Early identifi cation of indicators related to complications of diabetes enables one to select the proper choice of treatment and reduces the chances of amputation in the later stages. Several research groups have studied the changes in microcirculation in the diabetic foot using different methods. The majority of the studies focused on determining blood fl ow and hemoglobin oxygen saturation for normal and diabetic feet using different techniques. Basal skin blood fl ow investigations were reported for normal and diabetic subjects using laser Doppler blood fl owmeter measurements. A reduction in blood fl ow was observed in diabetic subjects with neuropathy compared to normal subjects. This can be attributed to changes in microangiopathy [4]. In another report, cutaneous microcirculation in feet and forearms were studied to compare normal and diabetic patients [5]. Variations related to skin blood fl ow in the dorsal region of the diabetic foot was also studied [6]. Reduced microcirculation was observed in diabetic subjects compared to the controls. Transcutaneous oxygen pressure (TcPO2) was reduced in the skin of diabetic subjects because of the impaired microcirculation. It was also observed that changes in skin aBiophotonics Lab, Biomedical Engineering Group, Department of Applied Mechanics, Indian Institute of

Technology Madras, Chennai-600 036, India; e-mail:; bDiabetic Foot Clinic, Sundaram Medical

Foundation, Chennai-600 040, India. Published in Zhurnal Prikladnoi Spektroskopii, Vol. 82, No. 3, pp. 423–428, May–June, 2015. Original article submitted July 24, 2014. _____________________ *To whom correspondence should be addressed. 432 0021-9037/15/8203-0432 ©2015 Springer Science+Business Media New York

DOI 10.1007/s10812-015-0125-9 433 oxygenation levels may occur before developing diabetic foot ulcer. In another study, microlightguide spectrophotometry was used to measure the hemoglobin oxygen saturation and blood fl ow in human sural nerve. Hemoglobin oxygen saturation in subjects with diabetic neuropathy was reduced when compared with the control subjects. In addition, fl uorescein rise time was prolonged in the subjects with neuropathy, which may be due to impaired blood fl ow and nerve perfusion [7].

In this paper, we explore the potential of diffuse refl ectance spectroscopy (DRS) in the Vis range for assessing the difference in the tissue hemoglobin and hemoglobin oxygenation saturation levels between normal and diabetic subjects.

Vis spectroscopy instrumentation is found to be cost effective compared with their near infrared counterparts. The potential of UV-Vis spectroscopy has been previously explored to assess the tissue hemoglobin concentration qualitatively based on a ratiometric approach [8]. Trials to quantify tissue hemoglobin using spectral methods in the UV-Vis range were also tried out using inverse Monte Carlo [9] as well as polarized light studies [10]. The present paper extends this application of UVVis spectroscopy for possible quantifi cation studies on tissue hemoglobin based on the apparent absorbance method and its related application in the case of diabetic patients.

Experimental. Diffuse refl ectance spectroscopy. The spectral signatures obtained from a diffuse refl ectance spectrum depend on the absorption and scattering properties of a tissue. DRS techniques have been widely used previously to characterize normal and malignant states of different types of tissues [11–13]. DRS can provide information on tissue morphology and total hemoglobin concentration (THb) [11]. The information regarding hemoglobin concentration can be extracted from DR spectra by converting to apparent absorbance using the formula λ − λλ = λ − λ ( ) ( ) ( ) log ( ) ( ) ,r b t b