Erythrocyte Protoporphyrin Fluorescence as a Potential Marker of Diabetes

Abstract

Protoporphyrin (PpIX), a porphyrin derivative, is the intermediate metabolic precursor of the heme molecule. Abnormal metabolism of total erythrocyte PpIX has been observed in diseases such as cancer, lead poisoning, psoriasis, iron deficiency anemia and acute porphyries. Diabetes mellitus (DM) is a complex metabolic syndrome in which hyperglycemia is the primary clinical manifestation and contributes to the diabetic complications. The aim of this study was to evaluate the utility of fluorescence spectroscopy of erythrocyte PpIX for monitoring the early stages of diabetes. A total of 14 male C 57BL mice, 6 weeks old, were divided into two groups: diabetic and non-diabetic. Diabetes was induced by intraperitoneal injection of streptozotocin (SZT). Blood cells were cultured with standard and 50 mM supplemented RPMI medium. Blood smears were prepared and stained for qualitative morphology analysis under optical microscopy. Blood porphyrin autofluorescence was analyzed by fluorescence spectroscopy. Characteristic PpIX emission spectra were obtained by exciting the samples at 405 nm. Average blood glucose was lower in the control group than in the diabetic group (156.50 +/- 8.11 mg/dL vs. 371.10 +/- 14.43 mg/dL, P < 0.05). Both diabetic and glucose-cultured erythroblasts showed a significant decrease (around 30.5% and 40%, respectively) in the emission band intensity at 635 nm. Our results indicate that the erythrocyte PpIX profile could be used as a biological monitor for diabetes.

Dual‐wavelength excitation for fluorescence‐based quantification of zinc protoporphyrin IX and protoporphyrin IX in whole blood

“…Lead and other heavy metal intoxication [15][16][17] and a variety of sideroblastic [18] and inherited microcytic anemias [19] may also be responsible for alterations in erythrocyte PPIX and ZnPP. A recent study found decreased PPIX fluorescence in erythrocytes from diabetic compared to non-diabetic mice, suggesting the possible use of PPIX as a diagnostic marker for monitoring of diabetes [20]. In addition to their diagnostic utility, measurements of PPIX and ZnPP may be useful in following the effects of therapy on iron deficiency, the course of chronic disorders [9], and some forms of porphyria.…”

Autofluorescence of blood and its application in biomedical and clinical research

“…It was determined that this fluorescence is actually due to the formation of a hemoglobin photoproduct (Shirshin et al, 2018;Wu et al, 2020), as native hemoglobin alone is nonfluorescent at single photon excitation due to ultrafast electron transfer to heme's iron atom (Qiqi et al, 2015;Tripathy & Steer, 2007). However, porphyrins like heme precursors and other derivatives do possess inherent fluorescence when lacking the quenching iron atoms, typically exhibiting emission peaks in the 620-635 nm range when excited at 405 nm (Fauaz et al, 2010) (Figure 2c,d). This porphyrin fluorescence is especially notable when the Wetlaufer, 1963), (b) Emission spectra (redrawn from Albani, 2004) of aromatic amino acids tryptophan, tyrosine, and phenylaniline.…”

“…(c) Excitation spectra and (d) emission spectra of collagen (black), elastin (blue), porphyrins (red), lipo-pigments (cyan) and pyridoxine (green) (redrawn from Wagnieres et al, 1998). (e) Absorption (dotted line) and emission (solid line) spectra of NADH (red) and FAD (blue) (redrawn from Islam et al, 2013) molecules have been extracted from RBCs with a solvent like acetone (Fauaz et al, 2010;Masilamani et al, 2004). Thus, while hemoglobin is a dominant contributor due to heme in autofluorescence of RBC and can be excited at ~438 nm, porphyrin is another source which can be excited at ~405 nm.…”

“…However, emission spectra and lifetime measurement using two photon excitation fluorescence (TPEF) showed that Hb is the dominant contributor in autofluorescence of RBC as well as their washed membrane among NADH, riboflavin, hemoglobin, bilirubin, biliverdin (Saytashev et al, 2016). The presence of hemoglobin and porphyrins (Fauaz et al, 2010) like heme gives RBCs their autofluorescent properties (Yakimov et al, 2019), and depending upon the selection of excitation wavelength, the corresponding emission spectra will vary. This is due to different variants of hemoglobin and their subunits (Alpert et al, 1980; Hirsch et al, 1980; Zhurova et al, 2014).…”

“…However, porphyrins like heme precursors and other derivatives do possess inherent fluorescence when lacking the quenching iron atoms, typically exhibiting emission peaks in the 620–635 nm range when excited at 405 nm (Fauaz et al, 2010) (Figure 2c,d). This porphyrin fluorescence is especially notable when the molecules have been extracted from RBCs with a solvent like acetone (Fauaz et al, 2010; Masilamani et al, 2004). Thus, while hemoglobin is a dominant contributor due to heme in autofluorescence of RBC and can be excited at ~438 nm, porphyrin is another source which can be excited at ~405 nm.…”