Disparity of selenourea and selenocystine on methaemoglobinemia in non-diabetics and diabetics

Abstract

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Human haemoglobin A (HbA), the highest abundant protein in erythrocytes, is made of two α and two β subunits with 141 and 146 amino acid residues, respectively, to form an active tetramer (1, 2). Each subunit of tetrameric HbA has a pocket like structure which contains a haem group that reversibly bind to oxygen. Haem is a heterocyclic organometallic compound containing a Fe²⁺ encapsulated inside a porphyrin ring which is composed of four pyrrole groups that are joined by methane bridges. The oxidation of Fe²⁺ atom of haem group causes the formation of methaemoglobin (metHb) which is less capable of binding and transporting oxygen leading to low oxygen concentration in tissues (3, 4). Usually methaemoglobin levels are kept below 1% by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent methaemoglobin reductase enzyme (5). But methaemoglobin concentration is increased due to the exposure of various dietary foods, chemicals, drugs, etc., or in the patients who have pathological disorders like deficiency of G-6-PD (glucose-6-phosphate-dehydrogenase), methaemoglobin reductase, etc., or diseases like diabetes, chronic artery disease, etc. (6, 7). Methaemoglobinemia, a hematological disorder where the ratio of metHb to HbA is high, can cause cyanosis, hypoxia and sometimes death (8). Administration of nitrate containing drugs for prolonged period of time to the patients suffering from coronary artery disease develops chronic methaemoglobinemia (9). Again, in diabetic condition due to high oxidative stress the rate of oxidation of HbA is higher than that of non-diabetic one (10). Clinically methylene blue is used which function as electron acceptor and accelerates the enzymatic reduction of metHb by NADPH-metHb reductase while ascorbic acid and reduced glutathione reduce metHb in non-enzymatic pathway (11, 12). Several reports showed that natural dietary antioxidants like curcumin, resveratrol, caffeine, etc. are capable of inhibiting nitrite-induced oxidation of HbA, though they fail to reverse the oxidation reaction if metHb is already formed from HbA (13–15).

Selenium, an essential micronutrient, is found to be present in several proteins at their active sites and play vital roles in several biological functions like cellular redox balance, cancer prevention and anti-inflammatory activity (16–19). Recently, organoselenium compounds have drawn major attraction due to their higher bioavailability and lesser toxicity as compared to inorganic selenium compounds and due to several biological activities like anticancer, antitumor, antioxidant and enzyme inhibitors (20, 21).

Selenourea (SeU) and its derivatives are found to inhibit several enzymes like inducible nitric oxide synthase, which is overexpressed in cancer cells as compared to normal cells (22). Hence, few SeU derivatives are shown to act as anticancer agent against different human cancer cell lines like melanoma cell lines (WM115), ovarian carcinoma (OVCAR-3), fibrosarcoma (HT-1080), adenocarcinoma (MDA-MB-231), prostate adenocarcinoma (PC-3) and lung adenocarcinoma (A549) (22). SeU derivatives also increase apoptosis and inhibit the uncontrolled proliferation of colon cells significantly by inhibiting phosphoinositide 3-kinase and phosphorylated Akt. SeU derivatives are also found to bind DNA with partial intercalation in the range 10⁴ M⁻¹.

Another organoselenium compound, selenocystine (SeC) is a redox active diselenide, which is a product of oxidation of amino acid selenocysteine. It has been shown that SeC acts as an anticancer agent and treatment of melanoma cells with SeC follows apoptotic cell death via extrinsic/death-receptor pathway (23). SeC was found to exhibit higher anticancer activity with IC₅₀ values ranging from 3.6 to 37.0 μM than other organoselenium compounds like selenomethionine, Se-methyl-selenocysteine, etc., and inorganic selenium containing drugs like selenite and selenite (24–26).

Selenium nanoparticles (SeNPs) is reported to be a promising candidate with reduced risk of selenium toxicity and better bioavailability and biological activities like antioxidant and antitumor activity, higher cellular uptake and high degree of biodegradability, which are significantly better than noble metals such as silver, gold and platinum (27). The LD₅₀ value was found 113 mg Se/kg for SeNPs while 8–12 mg Se/kg for sodium selenite and 25.6 mg Se/kg for SeMet (27, 28). A large number of reports suggest unique biomedical applications of SeNPs with varied roles of antioxidant, antimicrobial, antiparasitic, antifungal, antiviral activities, neuroprotection, anticancer effects and redox modulatory property (29).

In the present study, our aim is to find out the functional implication of HbA binding of SeC and SeU on the oxidative damage of HbA. Herein, we have observed that SeU was found to generate nanoselenium in presence of HbA and nitrite. This generated SeNPs tend to complex with haemoglobin which may affect other cellular activities. An earlier report showed that SeC catalyzes the reduction of metHb by 2 mM reduced glutathione at pH 7.4 (30). We found that SeC itself is capable of reducing metHb even in absence of reduced glutathione, suggesting a novel property of SeC. The effect of these organoselenium drugs has been studied on both non-diabetic and diabetic samples in presence and absence of nitrite. The results are compared with similar experiment on pristine metHb for validation of the process. We also compared the efficiency of dose-dependent metHb reduction by SeC both in non-diabetic and diabetic samples and found the significance of the results using paired t-test.

Materials and methods

Materials

L-SeC, SeU, haemin chloride and methaemoglobin from bovine (pristine metHb) were purchased from Sigma, India. Ammonium iron (II) sulphate hexahydrate GR, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate, potassium cyanide, potassium ferricyanide and sodium nitrite were purchased from Merck.

Methods

Preparation of blood sample and pristine metHb solution

The haemolysate was processed, as described elaborately in our previous study according to a published protocol, from the blood samples collected from the non-diabetic and diabetic patients following proper ethical committee guideline (31). The packed red blood cell (RBC), isolated from plasma and buffy coat of white blood cell by centrifugation at 3,000 g for 10 min at 4°C, was dissolved in 20 volumes of 1 mM phosphate buffer, pH 7.4 at 4°C for overnight followed by centrifugation at 12,000 g for 1 h at 4°C to obtain membrane free haemolysate. The concentration of hemolysate was considered that of HbA as it occupies 96% weight of whole RBC. Concentration of HbA was estimated using a Cary 50 UV–visible spectrophotometer assuming the molar extinction coefficient values of HbA as 125,000 M⁻¹cm⁻¹ at 415 nm and 13,500 M⁻¹cm⁻¹ at 541 nm (32).

Pristine metHb was prepared by adding pinch of metHb to 1 mM phosphate buffer, pH 7.4 and MetHb concentration was calculated using extinction coefficient as 3.78 mM⁻¹cm⁻¹ at 630 nm (33).

Blood glucose estimation

The estimation of free glucose in hemolysate of non-diabetic and diabetic samples was also carried out using the same protocol as mentioned above.

Absorption measurements

Spectral changes of HbA from 350 to 700 nm in presence of nitrite and two organoselenium compounds were determined using UV–visible spectroscopy. First, UV–visible spectra of both untreated and 400 µM nitrite-treated HbA (200 µM) were recorded. Then HbA was subjected to pretreatment with 400 µM of SeU and SeC individually for 30 min at 37°C followed by incubation with 400 µM of nitrite for another 30 min at 37°C and corresponding differences in spectra were determined. Spectra of 400 µM of both SeC and SeU-treated HbA were recorded to find out the effect of these two organoselenium compounds on HbA. Additionally, the effect of SeU and SeC on nitrite-treated HbA was also estimated by incubating HbA (pre-incubated with 400 µM of nitrite for 30 min at 37°C) with SeC and SeU for 30 min at 37°C. The spectra of SeU and SeC were also recorded as blank.

Kinetics of nitrite-induced metHb in non-diabetic and diabetic hemolysates as well as pristine metHb in presence of organoselenium compounds

First, the formation metHb was induced in vitro by incubating 200 μM hemolysate of non-diabetic and diabetic hemolysates with addition of sodium nitrite (final concentration 400 μM) and the changes in the concentration of metHb were monitored at 630 nm at 37°C for 30 min. Then to investigate the role of pretreatment of hemolysate with SeU and SeC, the hemolysates were pre-incubated with these compounds followed by the exposure to nitrite. This was done to estimate the protective role (if any) of these compounds towards metHb formation in both non-diabetic and diabetic subjects. The corresponding kinetics of the reactions were recorded at 630 nm as a function of time for 30 min at 37°C. All the experiments were repeated several times.

To find out the reducing behaviour of these compounds (SeU and SeC) towards pristine metHb, they were added to pure metHb also and the corresponding kinetics of the reactions were recorded for 30 min at 37°C at a λ_max of 630 nm.

Preparation and characterization of nanoselenium

Red particles of nanoselenium were obtained upon mixing equimolar SeU and hydrogen peroxide (400 µM each). The red nanoparticles were centrifuged at 5,000 rpm for 30 min and supernatant was discarded to isolate the particles from the reaction mixture and washed vigorously with water to separate the unreacted reagents followed by air-drying for further characterization using TEM analysis.

Interaction of the prepared nanoselenium with haemoglobin by UV–visible spectroscopy

The absorption spectra of haemoglobin both in absence and presence of SeNPs have been recorded on a Cary absorption spectrophotometer using a 1 cm path length cuvette over a wavelength range from 500 to 700 nm at 37°C. In this study, concentration of HbA was always kept 4 μM followed by addition of successive aliquots of an aqueous suspension of SeNPs. Dilution factors were subtracted for each binding experiment to find out the actual effect of complexation on the absorbance of HbA in presence of nanoselenium.

Fluorescence analysis of interaction of nanoselenium with haemoglobin variants

The interaction of haemoglobin with nanoselenium was studied by steady-state fluorescence experiments using a Cary Eclipse Spectro-fluorometer with a 1 cm path length quartz cuvette. Small aliquots of SeNPs were added successively to 4 μM HbA for tryptophan fluorescence measurements using excitation wavelength at 295 nm and keeping slits with band passes at 5 nm for both excitation and emission channels at 37°C. In all fluorescence measurements, baseline correction was done with buffer containing different concentrations of SeNPs in absence of protein.

CD spectroscopic analysis for nanoselenium binding of HbA

Dose-dependent in-vitro study of SeC on nitrite-induced metHb in non-diabetic and diabetic hemolysates as well as pristine metHb

Two hundred micromolars of hemolysate of both non-diabetic and diabetic blood samples was subjected to nitrite (final concentration 400 µM)-induced oxidation. Then various concentrations of SeC ranging from 10 µM to 1 mM were added to the above reaction mixtures as well as to 50 µM pristine metHb individually. After 30 min of incubation at 37°C, formation of metHb was monitored by analyzing its concentration at wavelength 630 nm for each sample. To check the effect of glucose on metHb reduction by SeC, pristine metHb solution was pretreated with 10 mM glucose for 30 min before its exposure to SeC. Similar experiment was then done by adding different concentrations of SeC to pristine metHb treated with10 mM glucose. Additionally, to find the effect of SeC in case of fever condition, a similar experiment was also performed at 40°C.

Methaemoglobin assay

Statistical analysis of the in-vitro clinical study of metHb reduction by SeC

A further in-vitro clinical study of SeC-mediated metHb reduction was done to justify the underlying reasons behind the phenomenon exhibited by SeC. Blood samples were collected from patients on nitrate containing drugs, both non-diabetic and diabetic, keeping non-diabetic and diabetic patients not on nitrate drugs as control. Total 20 clinical samples (age ranging from 55 to 75 years) were collected based on four groups as described in Table I, keeping five samples in each group. Fasting serum glucose level was measured by GOD–POD method to divide the samples into four groups. Usually metHb level in group A is found to be very less. Therefore, if analysis is done as soon as the collection of blood sample is made, metHb-related studies are not possible to perform. However, metHb level is found to increase with a detectable amount after 3 weeks of storage at 4°C due to spontaneous autoxidation of HbA when it is taken out from blood mainstream of non-diabetic non-nitrates. Hence to check the effect of SeC on group A, and keep a parity with all other types of samples, all the samples of each group were allowed to undergo air oxidation keeping for 3 weeks at 4°C. MetHb level was checked for each sample by monitoring the changes in absorbance at 630 nm before and after the addition of specific amount of SeC at 37°C.

Paired t-test was performed to find out the significance of difference between non-diabetic and diabetic samples using GraphPad Quickcalcs software which suggests that P < 0.05 is considered as significant value.

Results and discussion

Effect of organoselenium drugs on non-diabetic and diabetic hemolysates as well as pristine metHb

The characteristic peak for metHb at 630 nm was found (Fig. 1A) when HbA was incubated with nitrite and both the peaks at 540 and 575 nm, characteristic for oxy HbA was almost negligible as well indicating the formation of metHb. Figure 1A also shows that SeC and SeU alone has no such effect on normal hemolysate. There were no changes in absorption peak at 630 nm when SeU was added after the addition of nitrite to HbA although the peak intensity at 630 nm was found to decrease slightly when SeU was added before the addition of nitrite, suggesting SeU cannot prevent metHb formation. On the contrary, HbA pretreated with SeC did not show any peak at 630 nm in presence of nitrite and the absorption peak at 630 nm of HbA treated with nitrite (metHb) was found to disappear when incubated with SeC. These findings suggest that SeC not only inhibits metHb formation but also reduces already formed metHb. Figure 1B shows changes in Soret peaks due to the treatment with SeU and SeC both in absence and presence of nitrite. This also supports the earlier observations. Soret peak of HbA at 415 nm was unaltered when HbA was treated with SeU, again suggesting that SeU alone has no effect on HbA. The intensity of Soret peak at 415 nm was found to increase slightly in presence of SeC as it reduces the naturally occurring metHb found in hemolysate, although Soret peak remains unchanged when HbA was treated with SeC both before and after the addition of nitrite as expected. On the contrary, Soret peak of HbA was shifted to 406 nm in both SeU-treated and SeU-untreated HbA in presence of nitrite.

Fig. 1.

(A) UV–visible spectra (500–700 nm) of untreated HbA (200 μM) (a); HbA (200 μM) in presence of nitrite (400 μM) (b); HbA (200 μM) in presence of SeU (400 μM) (c); 400 μM of SeU-treated HbA (200 μM) followed by addition of nitrite (400 μM) (d); treatment with 400 μM of SeU to HbA (200 μM) pre-incubated with 400 μM nitrite (e); 400 μM of SeU. (B) UV–visible spectra (500–700 nm) of untreated HbA (200 μM) (a); HbA (200 μM) in presence of nitrite (400 μM) (b); HbA (200 μM) in presence of SeC (400 μM) (c); 400 μM of SeC-treated HbA (200 μM) followed by addition of nitrite (400 μM) (d); treatment with 400 μM of SeC to HbA (200 μM) pre-incubated with 400 μM nitrite (e); 400 μM of SeC. (C) UV–visible spectra (350–500 nm) of untreated HbA (200 μM) (a); HbA (200 μM) in presence of nitrite (400 μM) (b); HbA (200 μM) in presence of SeU (400 μM) (c); 400 μM of SeU-treated HbA (200 μM) followed by addition of nitrite (400 μM) (d); treatment with 400 μM of SeU to HbA (200 μM) pre-incubated with 400 μM nitrite (e). (D) UV–visible spectra (350–500 nm) of untreated HbA (200 μM) (a); HbA (200 μM) in presence of nitrite (400 μM) (b); HbA (200 μM) in presence of SeC (400 μM) (c); 400 μM of SeC-treated HbA (200 μM) followed by addition of nitrite (400 μM) (d); treatment with 400 μM of SeC to HbA (200 μM) pre-incubated with 400 μM nitrite (e).

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Analysis of kinetics data

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The initial slow oxidation of HbO₂ produces nitrogen dioxide and peroxide. In-situ generated nitrogen dioxide leads to the fast autocatalytic formation of reactive peroxynitrite ion. Another study showed that in presence of biological oxidizing agents like peroxide, peroxynitrite, etc., SeU gets converted to nanoselenium according to the following reactions (38):

Hence, the increase in the rate of reaction between nitrite and HbA in presence of SeU can be explained by the fact that SeU is converted to nanosized elemental selenium in presence of reactive oxygen species (ROS) which is generated during the reactions occurring in presence of nitrite. This explanation can be further validated by the kinetic (absorption time scan) profile for the reaction of both the untreated and pretreated HbA with nitrite (Fig. 2). The initial patterns in both the cases look similar until ∼10 min, while after ∼10 min of the initiation of the reaction (the inset of Fig. 2), the slope increases at a much higher rate for the reaction between HbA pretreated with SeU and nitrite. This initial time lag accounts for the in-situ generation of superoxide radicals. It is also observed that ROS generation is higher in presence of higher nitrite concentration leading to higher rate of increase in absorption spectral intensity at 630 nm.

Fig. 2.

Time scan of reaction of 200 μM of non-diabetic hemolysate at 630 nm for 30 min in presence of (a) nitrite (400 μM), (b) SeU (400 μM) and nitrite (400 μM) and (c) SeU (400 μM).

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The effect of SeU was also studied on a diabetic hemolysate. The rate of the reaction between the diabetic hemolysate and nitrite both in absence and presence of SeU is summarized in Table III. The rate of metHb formation was found higher in the diabetic hemolysate due to higher oxidative stress associated with diabetes. Moreover, the effect of SeU on diabetic Hb (gly Hb) in presence of nitrite was found to be more prominent, probably because of the combined effect of both higher metHb (formed by the action of nitrite on HbA) and nanoselenium formation due to the presence of higher ROS concentration in diabetic hemolysate. The increase in absorbance at 630 nm in all the samples in presence of SeU can be explained on the basis of interaction of Hb with nanoselenium which is formed in presence of ROS. However, further formation of metHb, i.e. direct oxidation of Fe²⁺ to Fe³⁺ owing solely to nanoselenium is not evidenced in any of the cases. These results are further validated with direct reactions between nanoselenium (as prepared in the lab) and HbA in the subsequent sections.

On the contrary, 400 µM SeC when pretreated with non-diabetic hemolysate was found to slow down the rate of the reaction between the hemolysate and nitrite significantly, as reflected in Table II, suggesting inhibition of metHb formation, as validated in Fig. 1. To further validate whether SeC only protects HbA from nitrite-induced oxidation or it is also capable of reducing metHb already formed, 200 µM of SeC was added to 50 µM of pristine metHb and the absorbance value was found to decrease with respect to time yielding a negative slope, which indicates reducing behaviour of SeC towards metHb (Supplementary Fig. S1). This phenomenon of SeC is explained later elaborately.

Characterization of SeNP and its interaction with HbA

SeNPs were formed in the reaction mixture containing equimolar SeU and H₂O₂ and was found to reduce the oxidized ABTS (2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as described in an earlier study (38). TEM image of isolated Se nanoparticles in Fig. 3A shows particle size of <10 nm and indicates a porous nature. Further analysis of characteristic selected area electron diffraction (SAED) pattern (Fig. 3B) shows bright diffraction points suggesting nanocrystallinity (39).

Fig. 3.

(A) TEM image of SeNP. (B) SAED pattern of SeNP.

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SeNPs are studied extensively as anticancer and anti-inflammatory drugs due to their lesser toxicity as compared to other selenium containing drugs (29). However, the role of SeNPs on major blood protein, HbA has not been studied so far. The present study shows the interaction of SeNPs with HbA using different spectroscopic techniques like UV–visible, fluorescence and circular dichroism (Fig. 4). It was found that the absorption peak intensity at 630 nm increases (Fig. 4A) with the increase in concentration of nanoselenium upon interaction with a fixed concentration of HbA suggesting complex formation. This observation is also in agreement with the earlier observation of the increase in rate of absorbance at 630 nm due to reaction between nitrite and HbA in presence of SeU (Figs 1 and 2). Again, the binding behaviour of SeNPs with HbA was further investigated by fluorescence spectroscopy (Fig. 4B) where the emission intensity of tryptophan fluorescence was found to increase in presence of increasing concentration of SeNPs. It suggests the partial unfolding and surface exposure of the microenvironment of tryptophan of HbA, due to the adsorption on surface of SeNP, which is also validated by CD spectroscopy (Fig. 4C). The characteristic peaks of native HbA at 208 and 222 nm were found to be altered in presence of SeNPs. Further quantitative analysis of CD spectra showed that % α-helicity of untreated HbA was decreased from 75% to 71% in presence of SeNPs, suggesting moderate changes in secondary structure of HbA.

Fig. 4.

(A) Absorption spectra, changes in absorbance value of HbA at 630 nm, downward arrow suggests decrease in absorption intensities at both 540 and 580 nm while upward arrow shows increase in absorbance values at 630 nm with increasing concentration of SeNP (successive addition of 10, 20, 35, 50, 70, 90, 120, 150, 200 μl of SeNP). (B) Variation in fluorescence emission intensity of HbA tryptophan at 332 nm while excited at 295 nm, black upward arrow shows increase in fluorescence with intensities increasing concentration of SeNP (successive addition of 5, 15, 35, 50, 100, 200 μl of SeNP). (C) CD spectra of HbA (a), changes in ellipticity at both 208 and 222 nm in presence of 100 μl SeNP (b).

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Effect of SeC on non-diabetic and diabetic hemolysates as well as pristine metHb

As mentioned earlier, SeC shows a different effect on the hemolysates as Table II gave clear indication of decrease in rate of nitrite-induced metHb formation in presence of 400 µM of SeC. Additionally, Supplementary Fig. S2 shows that higher the concentration of SeC lower is the rate of metHb formation suggesting that the reaction rate depends on the concentration of SeC. To find out if SeC is capable of reducing metHb, two sets of experiments were done with (i) 50 µM pristine metHb and (ii) 200 µM hemolysate of both non-diabetic and diabetic hemolysates. They were pretreated with 400 µM nitrite and incubated for 30 min followed by treatment with increasing concentration of SeC ranging from 10 µM to 1 mM. Figure 5A shows that increasing the concentration of SeC, lowered concentration of pristine metHb. These results were also validated by the cyanohaemoglobin method (Fig. 5B). Similar trend was also found in case of nitrite-induced metHb in both non-diabetic and diabetic samples. Supplementary Figure S3 shows absorbance versus concentration of SeC, where nitrite-induced metHb in hemolysates of both non-diabetic and diabetic blood samples were treated with SeC. The results of percent reduction of metHb calculated using these absorption spectral data and are tabulated in Table IV. It was found that in case of nitrite-induced metHb of diabetic blood sample (fasting blood sugar >126), percent reduction of metHb was lower than that of non-diabetic blood samples. For pristine metHb, reduction of metHb by SeC was found to reach saturation beyond 100 µM of SeC as metHb concentration did not undergo further decrease even at higher concentration of SeC. However, no such saturation was observed for the hemolysates. The results, as validated using cyanohaemoglobin method, indicate direct reduction of Fe³⁺ to Fe²⁺ by SeC. The reducing property of SeC can be justified by the presence of two electron donating –NH₂ groups present in the amino acid structure. Moreover, it can also be explained on the basis of available XPS data that the outer electron (3d_{3/2, 5/2}) binding energy of selenium (55.5 ± 0.1 eV) is much lower than outer electron (2p_3/2) binding energy of iron (711 ± 4 eV), suggesting that selenium has a higher tendency to get oxidized (40, 41). As the electron rich SeC, species encounters the electron deficient Fe³⁺ in metHb a subsequent reduction of metHb takes place. Figure 5B shows the % change in metHb due to the treatment of pristine metHb with SeC, which is in good agreement with the data calculated by monitoring the changes in absorbance at 630 nm using extinction coefficient of metHb mentioned above.

Fig. 5.

(A) Changes in absorption intensity at 630 nm with the concentration variation of SeC (0–1,000 μM). (B) cyanohaemoglobin assay with 50 μM of pristine metHb pre-incubated with 400 μM of SeC. (C) Effect of fever (40°C) on efficiency of 400 μM of SeC towards reduction of 50 μM of pristine metHb as compared to physiological temperature (37°C).

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At a higher temperature (40°C) metHb formation is found to be higher than that of normal body temperature (37°C), leading to deterioration of the pathological condition of the patients consuming nitrate/nitrite containing drugs regularly (15). Hence, the effect of high temperature on reducing capability of SeC towards pure metHb was also studied to check whether this drug can be used to administer to the patients during fever (Fig. 5C). At 40°C, the reducing activity of SeC was found even higher suggesting higher efficacy, which may be due to the temperature-dependent behaviour of the interaction between SeC and HbA.

Effect of glucose on metHb reduction by SeC

The differential activity of SeC towards metHb reduction in non-diabetic and diabetic samples can arise out of any of the three possible reasons (Table IV). First, it is well established that in diabetic samples oxidative stress is high due to high concentration of ROS which can affect the reducing behaviour of SeC (42). Second, some of the SeC may be engaged in binding with the free glucose which is high in concentration inside erythrocytes in case of diabetes (43). If SeC interacts with free glucose, its reducing activity may be decreased due to mutual interactions. Finally, due to non-enzymatic glycation of haemoglobin, glycated Hb percentage is found to be higher in case of diabetes (44). Reducing potential of SeC towards Hb and glycated Hb may not be similar which may thereby result in the lesser reducing action of SeC.

To find and establish the actual reason, SeC was allowed to interact with free glucose which revealed that there was no change in absorption spectra of SeC in presence of varying concentrations of glucose (Supplementary Fig. S4). Again, Fig. 6A shows that when free glucose was added together with SeC to pristine metHb, there was no change in absorbance value as compared to glucose-untreated metHb. This shows that the second possibility is not valid. However, when SeC was added to pristine metHb with a prior incubation with free glucose for 30 min, appreciable changes in absorbance value was observed which corresponds to metHb level as shown in Fig. 6B. This suggests that pretreatment of pristine metHb with free glucose generates glycated metHb (gly metHb) which may be lesser susceptible towards reduction by SeC. Hence, it can be inferred that either higher ROS concentration or formation of glycated Hb or both are responsible for this differential behaviour of SeC.

Fig. 6.

(A) Variation of the absorbance value at 630 nm when 10 mM of dextrose was added together with 400 μM of SeC to 50 μM pristine metHb. (B) Variation of the absorbance value at 630 nm when 400 μM of SeC was added to 50 μM of pristine metHb pre-incubated with 10 mM of dextrose for 30 min at 37°C.

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Comparison of reduction activity of SeC towards metHb in non-diabetic and diabetic samples for statistical analysis

To further investigate whether ROS is responsible for the differential activity of SeC in non-diabetic and diabetic hemolysates, a prospective clinical study was done with four different groups of blood samples classified based on different health conditions (Table I).

Table V shows the comparative data set for the reduction of metHb by SeC in samples with different health conditions. For similar concentration ratio of metHb:SeC (metHb:SeC = 1:8) % reduction of metHb were 27.46 ± 0.82 and 16.1 ± 2.4 for groups A and C, respectively. The comparative effect of SeC for non-diabetic and diabetic samples was found to be highly significant with two tailed P-value of 0.0040, which was in good agreement with our earlier data. Similar trend was found for group B and group D with P-value of 0.0015. Again, it was found that for similar metHb reduction activity of SeC, ratio of concentration of metHb:SeC was 1:8 and 1:20 for group A and group C, respectively. Earlier reports established that nitrate causes the generation of ROS in higher concentration which also agrees with the data presented in Table V (45). This maybe explain why the samples collected from the non-diabetic patients regularly taking nitrate containing drugs (group B) needed higher concentration of SeC than control (group A).

Conclusion

The effect of two selenium containing anticancer drugs SeU and SeC on haemoglobin was studied using different biophysical and biochemical techniques. There was a profound disparity in the mode of action of the two drugs. SeU has no effect towards cause or remediation of nitrite-induced metHb formation. However, it gets converted to nanoselenium in presence of nitrite which generates ROS during an autocatalytic stage of reaction and interacts with HbA. Hb upon adsorbing on the surface of nanoparticle undergoes conformational change, suggesting that use of SeNPs as anticancer drug could induce adverse effect on stability and function of haemoglobin. This effect is even higher in case of a diabetic hemolysate where there is further increase in ROS production.

On the other hand, another organoselenium drug SeC was found to function as novel agent towards the reduction of metHb. The ability of metHb reduction is even higher in fever condition which is due to temperature dependence of the ensuing chemical interaction. However, in the metHb reduction capacity is poor in glycated condition of Hb as in case of diabetes. A statistical analysis of both non-diabetic and diabetic blood samples yields quite significant results in this direction. Hence, this study opens a new avenue to show that the anticancer drug SeC can be used to treat chronic methaemoglobinemia as well.

Supplementary Data

Supplementary Data are available at JB Online.

Acknowledgements

The authors gratefully acknowledge Dr. Shantanu Bhakta, KPC Medical College & Hospital for providing blood samples from selected OPD patients.

Funding

Author Debashree Das acknowledges the award and funding of DS Kothari postdoctoral fellowship (CH/17-18/0150) from University Grant Commission, India.

Conflict of Interest

None declared.

References

Abbreviations

 
• CD

  circular dichroism

 
• GOD–POD

  glucose oxidase and peroxidase

 
• HbA

  human haemoglobin A

 
• IC₅₀

  half maximal inhibitory concentration

 
• MetHb

  methaemoglobin

 
• MRE

  mean residual ellipticity

 
• NADPH

  nicotinamide adenine dinucleotide phosphate

 
• RBC

  red blood cell

 
• ROS

  reactive oxygen species

 
• SAED

  selected area electron diffraction

 
• SeC

  selenocystine

 
• SeNPs

  selenium nanoparticles

 
• SeU

  selenourea

 
• TEM

  transmission electron microscopy.