
International Journal of Experimental Spectroscopic Techniques
(ISSN: 2631-505X)
Volume 7, Issue 1
Research Article
DOI: 10.35840/2631-505X/8530
Oxidative effect of alcohol on human hemoglobin
Eleazar Chukwuemeka Anorue1*, Henry Amaechi Onwubiko1 and Grace Nneka Onwubiko2
Table of Content
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Author Details
Eleazar Chukwuemeka Anorue1*, Henry Amaechi Onwubiko1 and Grace Nneka Onwubiko2
1Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria
2Natural Science Unit, School of General Studies, University of Nigeria, Nsukka, Enugu State, Nigeria
Corresponding author
Eleazar Chukwuemeka Anorue, Medical Biochemical and Pharmacognosy Research Unit, Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, 410001, Nsukka, Enugu State, Nigeria, Tel: +234 7065284325.
Accepted: July 08, 2022 | Published Online: July 11, 2022
Citation: Anorue EC, Onwubiko HA, Onwubiko GN (2022) Oxidative effect of alcohol on human hemoglobin. Int J Exp Spectroscopic Tech 7:030
Copyright: © 2022 Anorue EC, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Some studies have suggested that people who drink too much alcohol suffer from hypoxia and other hematological damages due to oxidation of the hemoglobin. Therefore, increasing concentration of alcohol is likely to oxidize human hemoglobin. This study was designed to investigate the effect of increasing concentration of alcohol on oxidation of human hemoglobin. The study was carried out using UV-visible spectroscopy and was divided into 5 groups comprising of the control (normal oxy-Hb) and test groups (0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL and 1 mg/mL alcohol reacted with1000 μL oxy-Hb). It was found that alcoholled to concentration and time dependent oxidation of hemoglobin and the mechanism of the oxidation was also discussed.
Keywords
Abuse, Alcohol, Metabolism, Oxidation, Oxy-Hemoglobin
List of Abbreviation
Oxy-Hb: Oxy-Hemoglobin; Met-Hb: Met-Hemoglobin; EDTA: Ethylene Diaminetetraacetic Acid
Significance of Study
Abuse of alcohol is becoming an increasing problem in preteens and teenagers. As a result, it has been reported that abuse of alcohol contributes to over 3 million deaths each year globally and account for 7.1% and 2.2% of the global burden of disease for males and females respectively. Since during metabolism, alcohol is mostly absorbed in the blood; this study investigated the effect of alcohol on the oxidation of human hemoglobin. It was found that increasing concentration of alcoholled to oxidation of human hemoglobin which could lead to pathophysiological changes in the red blood cell, and some organs of the body. Therefore, this study aims at discouraging abuse of alcohol.
Introduction
Alcohol is a psychoactive substance use as an active ingredient in drinks such as beers, wine, distilled spirits (hard liquor) and in some beverages [1]. Although ethanol is only one of the several types of alcohol, alcohol is often referred to as ethanol because it is the only type of alcohol found in alcoholic beverages and drinks used for recreational purposes [2,3]. It is also the only alcohol produced by fermentation of grains, fruits and other sources of sugar [4].
Most of the times, alcoholic beverages and drinks are abused, leading to alcoholic intoxication, also known as alcoholic poisoning [5]. Abuse of alcohol is becoming an increasing problem in preteens and teenagers; binge drinking defined as consuming greater than 5 drinks per occasion for men and greater than 4 drinks per occasion for ladies is a particular problem among younger people [6,7]. As a result, abuse of alcohol accounts for 7.1% and 2.2% of the global burden of disease for males and females respectively [8]. Consequently, this has been suggested to be the major cause of death and impairment among young people aged 15-49 years, accounting for 10% of mortality in this age group [9]. Furthermore, abuse of alcohol contributes to over 3 million deaths each year globally [10]. Only in the USA, acute intoxication directly results in about 2,200 deaths per year and indirectly about 30,000 deaths per year [11,12]. Also, according to Centers for Disease Control (CDC) data released January 2013, an average of 6 people died of alcohol poisoning each day in the USA from 2010 to 2012 [13].
Apart from death, respiratory depression, aspiration, hypotension, cardiovascular collapse, drowsiness, stupor, coma, disinhibition, confusion, memory difficulty, anterograde amnesic, slowed speech, in coordination, dysmetria, ataxia, diminished reflexes, unsteady gait, hypoglycemia and hypothermia has been reported to result from alcoholic intoxication [14-17]. During metabolism, alcohol is majorly absorbed into the blood than the stomach [18]. Unlike food which can take hours to digest, alcohol is absorbed more rapidly than it is eliminated [18].
Some studies have suggested that since alcohol is majorly absorbed into the blood, people who drink too much alcohol will suffer from hypoxia and other hematological damages due to oxidation of the hemoglobin [18]. Therefore, increasing concentration of alcohol is likely to oxidize human hemoglobin. Base on this, this study was designed into 5 groups comprising of the control (normal oxy-Hb) and test groups (0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL and 1 mg/mL alcohol reacted with 1000 μL oxy-Hb) to investigate the effect of increasing concentration of alcohol on the oxidation of human hemoglobin.
Materials and Method
Procurement of ethanol
The ethanol used for this study was of analytical grade and was purchased from Sigma-Aldrich Co.,St. Louis, Missouri, USA.
Chemicals and reagents
The chemical and reagents used for this study were of analytical grade: Sephadex G-150 (Thomas Scientific, Swedesboro, New Jersey, USA), 39.4% sodium chloride, 95% methylated spirit, 97% potassium phosphate salts, 92.5% sodium hydroxide, 35% concentrated hydrochloric acid (AllPanhongChemical Co., Shenzhen, Guangdong, China) and 100% distilled water (Energy Centre University of Nigeria, Nsukka, Enugu, Nigeria).
Equipment, apparatus and instruments
V-750 ultraviolet-visible (UV visible) spectrophotometer (Jenway Co., Stone, Staffordshire, UK), Allegra X-14R centrifuge, (Beckman Coulter Co., Indianapolis, Indiana, USA), Spectrum Lab 23A spectrophotometer (Spectrum Laboratories, Stamford, Connecticut, USA), cotton wool (Jinniu District Hengnuan Cotton Plant, Chengdu, Sichuan, China) 2 by 30 cm glass column (Sigma-Aldrich Co.,St. Louis, Missouri, USA), glass wool (Gupta Glass Wool Industries, Nangloi, Delhi, India), hypodermic syringes (Troge Medical GmbH, Hamburg, Germany), micropipettes (Globe Scientific Inc., Mahwah, New Jersey, USA), Pasteur pipettes(Globe Scientific Inc.), pH meter (Hanna Instruments, Woonsocket, Rhode Island, USA), refrigerator (Thermo-coolPublic Ltd Co., Ilupeju, Lagos, Nigeria), water bath (Qinhuangdao YuanchenHardwares, Qinhuangdao, Hebei, China), dialysis membrane (Thermo Fisher Scientific, Waltham, Massachusetts, USA).
Ethical approval
The study was conducted in accordance to the regulations and approval of the Ethics and Biosafety Committee of the Faculty of Biological Sciences, University of Nigeria, Nsukka with the approval number UNN/FBS/EC/2019/1008.
Preparation of potassium phosphate buffer
The buffer was prepared using the Henderson-Hasselbalch equation and 10 mM potassium phosphate buffer (pH 7.6, pK 7.2) was used for this study [19].
Collection of blood sample
Whole blood sample was collected from a human volunteer (a 28 yr old non-smoking male student of the University of Nigeria, Nsukka), confirmed to be of genotype Hb AA at Renascent Hospital, Nsukka, Nigeria. The whole blood (5 mL) was collected using venepuncture into a Venoject tube containing ethylene diaminetetraacetic acid (EDTA) as an anticoagulant and kept at 4 ℃ for a maximum of 1 day.
Preparation of oxy-Hb
Oxy-Hb was prepared according to the method of Meng and Alayash [20]. Briefly, 5 mL of the whole blood sample was washed 4 times using normal saline and centrifuged at 2600 × g (3000 rpm) at 25 ℃ for 45 min. The supernatant was aspirated and the pellet was diluted 3 folds using distilled water, rocked gently at 25 ℃ for 10 min and kept in an ice bath for 1 hr (for lysis). The lysate was then centrifuged at 7100 × g (5000 rpm) at 25 ℃ for 45 min. The lysate was loaded onto a Sephadex G-150 column (bed dimension 3 × 40 cm, 300 mL), which was equilibrated using 3 column volumes 10 mM potassium phosphate buffer of pH 7.6. Oxy-Hb was eluted at 4 ℃ using a linear gradient of 25-100% of the buffer in 2 column volumes. The column was eluted at a flow rate of 2 mL/min, and the effluent was observed at 541, 576 and 630 nm. Oxy-Hb was collected and stored at -18 ℃ for a maximum of 1 day.
For spectrophotometry, stock solutions of the oxy-Hb and buffer were mixed in the ratio 10:1.
Experimental design
This study was divided into 5 groups as follows:
Group 1: 1000 μL of 0.081 mM oxy-Hb (normal control). See "Figure 1".
Group 2: 100 μL of 0.2 mg/mL ethanol + 1000 μL of 0.081 mM oxy-Hb
Group 3: 100 μL of 0.4 mg/mL ethanol + 1000 μL of 0.081 mM oxy-Hb
Group 4: 100 μL of 0.6 mg/mL ethanol + 1000 μL of 0.081 mM oxy-Hb
Group 5: 100 μL of 0.8 mg/mL ethanol + 1000 μL of 0.081 mM oxy-Hb
Group 6: 100 μL of 1 mg/mL ethanol + 1000 μL of 0.081 mM oxy-Hb
The alcoholic concentration used were simulated using different range of blood alcoholic concentrations [21].
Measurement of oxy-Hb and met-Hb concentration
Oxy-Hb and met-Hb concentrations were determined using the Beer-Lambert equation [22]. The concentrations of oxy-Hb were expressed as mM and the absorption spectra were scanned between 400 to 700 nm. The concentrations (mM) for oxy-Hb species were determined from the extinction coefficients derived by Meng and Alayash [20]. To calculate the concentrations of oxy-Hb, the following equation was used:
Where:
C = Concentration
A = Absorbance at 541 and 576 nm
e = Extinction coefficient (15.5 and 16.6 for 541 and 576 nm, respectively).
l = Length of cuvette
Determination of haemoglobin bound oxygen
Hemoglobin bound oxygen and partial pressure of oxygen was determined according to the method of Hafen and Sharma; Clark, Byrne, and Casssells; and Nitzan, Barchenko, Khanokh, and Taitelbaum [23-25]. To calculate the hemoglobin bound oxygen, the following equation was used:
Total hemoglobin bound oxygen = HbO2/g × Hemoglobin concentration × %O2 Saturation
Where %O2 saturation is ~89%
HbO2/g is hemoglobin bound oxygen per gram (1.4)
Determination of oxidation of oxy-Hb
Oxidation of oxy-Hb was determined according to the method of Ibrahim, EL-Gohary, Saleh, and Elashry [26]. Briefly, the degree of the reduction in the magnitude of the absorbance maxima characteristic of oxy-Hb (541 and 576 nm) is proportional to the amount of oxidation of oxy-Hb.
Statistical analyses
Data on oxy-Hb concentration were analyzed using the Statistical Package for the Social Sciences, SPSS version 22 (International Business Machine Corp., Armonk, New York, USA). Specific tests between means were carried out using Duncan Multiple Range Test (DMRT). Generally, statistical significance was carried out at p < 0.05. Results were shown as mean ± standard deviation (SD).
Results
Effects of different concentration of alcohol on the oxidation of oxy-hemoglobin
Spectra characterization of unreacted oxy-Hb showed two distinct absorbance maxima (namely β and α band) at 541 and 576 nm, respectively, (Figure 1). Having this as a baseline, the effects of alcohol on the oxidation of hemoglobin was determined. The results showed that there was a concentration and time dependent reduction in absorbance maxima of oxy-Hb reacted with 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL and 1 mg/mL of ethanol. It was observed that the higher the concentration of ethanol, the higher the reduction in the absorbance maxima of the reacted oxy-Hb. Worthy of note is that1 mg/mL ethanol had the highest reduction in the absorbance maxima of the reacted oxy-Hb (Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6).
Effects of different concentration of alcohol on the oxy-hemoglobin concentration, met-hemoglobin concentration and ferrous-ferric iron ratio
The result also revealed that the different concentration of ethanol caused concentration dependent significant (p < 0.05) decrease in oxy-Hb concentrations and ferrous-ferric iron ratio of the reacted oxy-Hb, especially 1 mg/mL ethanol had the highest reduction in oxy-Hb concentration and ferrous-ferric iron ratio. On the other hand, there was a significant (p < 0.05) increase in met-hemoglobin concentration of the reacted oxy-hemoglobin (Table 1).
Effects of different concentration of ethanol on hemoglobin bound oxygen
The result showed significant decrease (p < 0.05) in hemoglobin bound oxygen. The highest decrease was seen at 1 mg/mL alcoholic content (Table 1).
Discussion
The result of this study showed that increasing concentration of alcohol led to oxidation of the human hemoglobin. Thus, this corroborate with the hypothesis of this research and confirm that people who drink too much alcohol suffer from hypoxia due to oxidation of the human hemoglobin. This study was conducted using UV-visible spectroscopy. UV-visible spectroscopy is a quantitative technique used to measure how much an analyte substance absorbs light. This is done by measuring the intensity of light that passes through a sample with respect to the intensity of light through a reference sample or blank [22]. How much the analyte absorbs light is directly proportional to the concentration of that analyte in the solution [20]. The Fe2+-O-O single bond of Oxy-hemoglobin absorbs light at 576 and 541 nm wavelength of the UV-visible spectrum representing α and β subunits of the polypeptide subunits respectively [27]. Decrease in the absorbance maxima at 576 and 541 nm bands shows reduction in the concentration of oxygen molecule bound to the iron moiety of the hemoglobin and oxidation of the ferrous iron of the hemoglobin to ferric iron [27-29].
Findings from the study showed that alcohol led to time and concentration dependent reduction in the concentration of oxy-hemoglobin and also led to the oxidation of the ferrous iron moiety of the hemoglobin to ferric iron. This is clearly shown by the reduction in the absorbance maxima of the oxy-Hb reacted with different concentration of ethanol. The reduced oxy-Hb concentration proves that there is a decrease availability of oxygen in the cell due to gradual knocking off of the oxygen bound to the iron moiety of the hemoglobin [30,31]. Interestingly, the decrease in oxy-Hb concentration was spectral-wise significant even at 0.2 mg/mL alcoholic concentration.
Several kinds of alcoholic beverages contain varying concentration of alcohol, some containing more alcohol than others and all these are exposed for public consumption without consideration of the possible effect on the public [4]. For example, gin, a spirit made from juniper berries contain ~35% to 55% alcohol, brandy a distilled wine contains ~35% to 60% alcohol, whisky a spirit made from fermented grain contains ~40% to 50% alcohol, rum a distilled drink made from fermented sugarcane and molasses contain ~40% to 75.5% alcohol, tequila a type of liquor contain 40% alcohol, vodka a liquor usually made from fermented grain and potatoes has a standard alcoholic concentration of ~40%, absinthe a spirit made from a variety of leaves and herbs contain between ~40% to 90% alcohol while everclear, a grain-based spirit contains a minimum alcoholic concentration of 60% and a maximum of ~95% [4].
Therefore, this study suggests that increasing intake of a highly concentrated alcoholic beverage can have deleterious impact on the human hemoglobin by oxidizing the ferrous iron moiety of the hemoglobin to ferric iron and reducing the oxy-Hb concentrations [31,32]. This study is consistent with the result of Borne, Mark, Montano, Mion, and Somers who reported decreased concentration of hemoglobin when treated with ethanol. Also, this study is consistent with the result of Hu, Lee, Lee, Subeq, Lin, and Hsu; and Ballard who reported decreased hemoglobin concentrations and characteristic damages in the blood cell of drunkards [30,33,34].
The chemistry/mechanism underlying the oxidation of hemoglobin in the presence of increasing concentration of ethanol can be explained by understanding the structure of hemoglobin [35,36]. Haemoglobin is the oxygen-carrying pigment found in red blood cells, and is a globular protein made up of four polypeptide subunits [37]. Each polypeptide subunit of haemoglobin contains a haem group and each haem group contains an iron atom [36]. This iron atom has six coordination valences, four with pyrole nitrogen atoms of the haem group, one with the nitrogen of the F8 proximal histidine of the globin protein, and one that binds one molecule of oxygen reversibly in response to the partial oxygen pressure [38-40]. In the presence of alcohol, the OH- group of alcohol acts as an electron donor, it provides an electron and reduces the bound dioxygen to peroxide [41]. Subsequently, the peroxide may be displaced by water and the haemoglobin is left in the aquomet (oxidized, HbFe3+) form and peroxide appears in the solution [41]. The reaction is thus;
HbFe2+O2+ C2H5OH-HbFe3++ C2H5OH+ + O2˙− (1)
O2˙− + 2H+↔ H2O2 (2)
Where HbFe2+O2 is the oxy-hemoglobin, C2H5OH- is the electron donor, HbFe3+ is oxidized hemoglobin, C2H5OH+ is the reduced electron donor, O2˙− is superoxide, H2O2 is hydrogen peroxide [38]. In all, two electrons are transferred to bound 02 to yield H2O2, one electron from HbFe2+ and one electron from C2H5OH. Thus, the H2O2 generated can react with a new oxy-hemoglobin molecule, and so on. Clearly the hemoglobin inside red blood cells or in plasma would rapidly become unable to carry oxygen since there are no enzymatic or chemical systems to block these reactions [41].
First, the inability of the ferric iron of the heme protein to bind oxygen leads to hypoxemia and subsequently to hypoxia [42]. Hypoxemia is decrease oxygen content of the blood. This decrease is shown by the reduction in the hemoglobin bound oxygen shown in Table 1. Normally, when the blood oxygen concentration is high, the absorbance maxima at the 541 and 576 nm band will be high, but when the blood oxygen concentration starts reducing, the absorbance maxima at the 541 and 576 nm band of oxy-Hb spectrum will decrease steadily and as a result hemoglobin will not have enough oxygen molecules to transport to different tissues of the body for their cellular function, a term called hypoxia. Thus, hypoxia is a state of decreased oxygen supply to a level insufficient for cellular functions [43]. The importance of oxygen to aerobic organisms cannot be overemphasized; oxygen plays a vital role in aerobic organism because it is the final acceptor of electrons in the mitochondrial respiratory chain, thereby allowing the process of oxidative phosphorylation [44]. During hypoxia, oxidative phosphorylation which accounts for the major generation of ATP is inhibited, thereby leading to reduced cell viability in living organism [45]. For instance, under normal viable cell conditions, there is an influx of 2 K+ inside the intracellular fluid and efflux of 3 Na+ outside the intracellular fluid, this helps to maintain a high and constant ratio of ATP/ADP, signifying high dependence of the cell on oxygen [46]. But during hypoxia there is an enhanced influx of 3 Na+ inside the intracellular fluid and efflux of 2 K+ outside the extracellular fluid [43]. This leads to a disturbance in membrane potential causing depolarization of the cell membrane and activation of the voltage-gated Ca2+ channels [47]. Consequently, the overload of the intracellular Ca2+ resulting from the activation of the voltage-gated Ca2+ channels causes harmful changes in the mitochondrial metabolisms, activation of lipases and proteases leading to reduced cell viability [48,49].
Reduced oxygen concentration of the hemoglobin could also lead to stroke and other pathological responses such as ataxia, confusion, disorientation, hallucination, severe headache, reduced level of consciousness, papilloedema, breathlessness, pallor, tachycardia and pulmonary hypertension eventually leading to late signs cyanosis, slow heart rate, low blood pressure followed by heart failure eventually death [50].
Two conditions known as hyperkalemia and hyponatremia might occur following the disturbance of the heomostasis of the membrane caused by the reduced oxygen concentration of hemoglobin [51]. Hyperkalemia (an increase in intracellular K+) and hyponatremia (a decrease in extracellular Na+) has been suggested to be harmful to cells [52]. Hyperkalemia is associated with an increased risk of death and could also contribute to peripheral neuropathy, renal tubular acidosis, muscle weakness, numbness or tingling, sudden collapse, chest pain, shortness of breath, weak pulse and irregular heartbeat [53,54]. While hyponatremia could contribute to neurological dysfunction, decreased mental function, cerebral edema, gait disturbance, osteoporosis, fractures, seizure, coma and even death, this might account for myriad of neurological dysfunctions seen in drunkards [55].
The further reaction of the H2O2 generated with new oxy-hemoglobin molecule can lead to deleterious effects in the hemoglobin and degenerative changes in the erythrocyte such as discoloration of hemoglobin from red to brown and to green, formation of insoluble precipitates called Heinz bodies consisting of various combination of denatured globin and membrane proteins, formation of hemichromes (in which the heme iron is attached not only to the proximal histidine, but to the distal histidine or in an irreversible bond to the side chain of another amino acid), oxidative haemolysis, oxidation of membrane lipids, release of free haem, cell deformity and cell rigidity [47,56]. The free haem released has been implicated in other damages in the body such as denaturation of DNA and increased damage to proteins, depletion of cytosolic enzymes including glucose-6-phosphate dehydrogenase and glutathione reductase, activation of cell-damaging enzymes such as caspases and cathepsins, cytoskeleton damage [57].
Finally, continuous reaction of hydrogen peroxide with new oxy-hemoglobin molecule will yield the met-Hb (Hb-Fe3+) product in the circulation, which will readily react with an additional molecule of H2O2 to release its heme group, a hydrophobic molecule that has recently been designated as a major erythrocytic danger-associated molecular pattern (DAMP) that activates and amplifies thrombotic and inflammatory mechanism [58]. Free heme and excess free radicals released during the reaction of peroxides and oxy-hemoglobin is implicated in endothelia damage by activation of cell-damaging enzymes such as caspases and cathepsins [59]. Endothelial cell (EC) injury, as a consequence of free-heme induces abnormal surface expression/mobilization of tissue factor (TF), P-selectin, and von Willebrand Factor (vWF), via binding of the heme to toll-like receptor (TLR)-4 or via the transfer of heme from heme- and hemoglobin-laden microparticles derived from erythrocytes; a major phenomenon that drive the thrombotic events, which is a precursor to venous thromboembolism (VTE), stroke, pulmonary hypertension, cardiovascular disease and chronic organ failure [60,61].
Conclusion
This study investigated the effect of different concentration of alcohol on human hemoglobin. It was found that reaction with increasing concentration of alcoholled to concentration and time dependent oxidation of oxy-hemoglobin which have been shown to cause hypoxia and other hematological damages. However, in vivo studies were not carried out, therefore in vivo studies need to be carried out to ascertain the findings of this study.
Acknowledgement
Thanks to Department of Biochemistry, University of Nigeria, Nsukka for providing the chemical reagents and equipment for this study.
Sources of Support
No funding was received for this work.
Author Contributions
All authors made significant contributions to the conceptualization and design of the study, read and approved the final manuscript.
Eleazar Chukwuemeka Anorue: Conceptualization; Investigation; Data curation; Formal analysis; Methodology; Resources; Writing original draft; Validation.
Grace Ngozi Adama: Conceptualization; Investigation; Data curation; Formal analysis; Methodology; Resources; Writing original draft; Validation.
Nnamdi Lawrence Obasi: Conceptualization; Investigation; Data curation; Formal analysis; Methodology; Resources; Writing original draft; Validation; Supervision.
Author Declarations
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.