International Journal of Experimental Spectroscopic Techniques
Volume 7, Issue 1
Oxidative effect of alcohol on human hemoglobin
Eleazar Chukwuemeka Anorue1*, Henry Amaechi Onwubiko1 and Grace Nneka Onwubiko2
Table of Content
- Baan RS, Straif K, Grosse T, Secretan B, elGhissassi F, Bouvard V, et al. (2007) Carcinogenicity of alcohol beverages. Lancet Oncol 8: 292-293.
- Balinus D, Rehm J, Irving H, Shuper P (2010) Alcohol consumption and risk of incident human immunodeficiency virus infection: A metal analysis. Int J Public Health 55: 159-166.
- Puukka K, Hietala J, Koivisto H, Anttila P, Bloigu R, et al. (2006) Age-related changes on serum GGT activity and the assessment of ethanol intake . Alcohol Alcohol 41: 522-527.
- Borges G, Loera CR (2010) Alcohol and drug use in suicidal behavior. Curr Opin Psychiatry 23: 195-204.
- Stahre M, Roeber J, Kanny D, Brewer RD, Zhang X (2014) Contribution of excessive alcohol consumption to death and years of potential life lost in the United States. Prev Chronic Dis 11: E109.
- Agarwal S, Fulgoni VL, Lieberman HR (2015) Assessing alcohol intake and its dose-dependent effects on liver enzymes by 24-h recall and questionnaire using NHANES 2001-2010 data. Nutr J 15: 62.
- Sharpe PC (2001) Biochemical detection and monitoring of alcohol abuse and abstinence. Ann Clin Biochem 38: 652-664.
- Rehm J, Room R, Monteiro M, Gmel G, Graham K, et al. (2003) Alcohol as a risk factor for global burden of disease. Eur Addict Res 9: 157-164.
- Rusyn I, Bataller R (2013) Alcohol and toxicity. J Hepatol 59: 387-388.
- Chenet L, Britton A, Kalediene R, Petrauskiene J (2001) Daily variations in deaths in Lithuania: the possible contribution of binge drinking. Int J Epidemiol 30: 743-748.
- Bouchery EE, Harwood HJ, Sacks JJ, Simon CJ, Brewer RD (2006) Economic costs of excessive alcohol consumption in the United States. Am J Prev Med 41: 516-524.
- Seitz HK, Pehicchi C, Barnardi V, La Vecchia C (2012) Epidemiology and pathophysiology of alcohol and breast cancer. Alcohol Alcohol 47: 204-212.
- Parry CD, Patra J, Rehm J (2011) Alcohol consumption and non-communicable diseases: Epidemiology and policy implication. Addiction 106: 1718-1724.
- Hills AG (1973) pH and the Henderson-Hasselbalch equation. Am J Med 55: 131-133.
- Robert WR, Mauri D, Lisa PN (1994) Discovering the Beer-Lambert law. J Chem Educ 71: 983.
- Maclntyre NR (2014) Tissue hypoxia: Implications for the respiratory clinician. Respir Care 59: 1590-1596.
- Mostofsky E, Mukamai KJ, Giovannucci EL, Stampfer MJ, Rimm EB (2016) Key findings on alcohol consumption and a variety of health outcomes from the nurses’ health study. Am J Public Health 106: 1586-1591.
- Chopra K, Tiwari V (2012) Alcohol neuropathy: Possible mechanisms and future treatment possibilities. Br J Clin Pharmacol 73: 348-362.
- Guo R, Ren J (2010) Alcohol and acetaldehyde in public health; from marvel to menace. Int J Environ Res Public Health 7: 1285-1301.
- Manzo-Avalos S, Saavedra-Molina A (2010) Cellular and mitochondrial effects of alcohol consumption. Int J Environ Res Public Health 7: 4281-4304.
- Lachenmeier DW, Kanteres F, Rehm J (2009) Carcinogenicity of acetaldehyde in alcoholic beverages: Risk assessment outside ethanol metabolism. Addiction 104: 533-550.
- Meng F, Alayash AI (2017) Determination of extinction coefficients of human hemoglobin in various redox states. Anal Biochem 521: 11-19.
- Carey KB, Hustad JTP (2005) Methods for determining blood alcohol concentration: Current and retrospective. In Comprehensive Handbook of Alcohol Related Pathology. Elsevier Inc: London, Uk, 1429-1444.
- Bhogal AS, Mani AR (2017) Pattern analysis of oxygen saturation variability in healthy individuals: Entropy of pulse oximetry signals carries information about mean oxygen saturation. Front Physiol 8: 555.
- Clark DJ, Byrne PO, Casssells SA (1992) Measurement technique for the determination of blood oxygen saturation. J Biomed Eng 14: 168-172.
- Nitzan M, Barchenko A, Khanokh B, Taitelbaum H (2000) Measurement of oxygen saturation in venous blood by dynamic near IR spectroscopy. J Biomed Opt 5: 155-162.
- Anorue EC, Onwubiko GN, Onwubiko HA, Asogwa CN (2021) Oxidative effects of cyanogenic glycosides residuals in cassava products on human haemoglobin. Food Biosci 41: 100846.
- Anorue EC, Ekpo DE (2020) Non-oxidative effects of the flavonoid-rich fraction of Lasiantheraafricana leaveson human haemoglobin. All Life 13: 658-667.
- Iwu MM, Igboko AO, Onwubiko HA, Ndu JE (1988) Effects of Cajanus cajan on gelation and oxygen affinity of sickle cell hemoglobin. J Ethnopharmacol 23: 99-104.
- Honnamurthy JB, Shivashankara AR, Mathai PJ, Malathi M (2016) Biochemical and hematological profile in patients with alcohol dependence syndrome (ADS) co-morbid with nicotine dependence syndrome (NDS). Int J Biochem Res 13: 1-10.
- Jain R, George AB, Narnoli S (2020) Haematological changes in alcohol and substance use disorders- an overview. Int Arch Subst Abuse Rehabil 2: 006.
- Taasan VC, Block AJ, Boysen PG, Wynne JW (1981) Alcohol increases sleep apnea and oxygen desaturation in asymptomatic men. Am J Med 71: 240-245.
- Parry C, Rehm J, Poznyak V, Room R (2009) Alcohol and infectious diseases: An overlooked causal linkage? Addiction 104: 331-332.
- Borne PV, Mark AL, Montano N, Mion D, Somers VK (1997) Effects of alcohol on sympathetic activity, hemodynamics and chemoreflex sensitivity. Hypertension 29: 1278-1283.
- Hu TM, Lee RP, Lee CJ, Subeq YM, Lin NT, et al. (2013) Heavy ethanol intoxication increases proinflammatory cytokines and aggravates hemorrhagic shock-induced organ damage in rats. Mediators Inflamm 2013: 121786.
- Ferreira A, Balla J, Jeney V, Balla G, Soares MP (2008) A central role for free heme in the pathogenesis of severe malaria: The missing link? J Mol Med 86: 1097-1111.
- Alayash AI (2009) Oxidative mechanisms of hemoglobin-based blood substitutes. Artif Cells Blood Substit Immobil Biotechnol 29: 415-425.
- Ioannou GN, Dominitz JA, Weiss NS, Heagerty PJ, Kowdley KV (2004) The effect of alcohol consumption on the prevalence of iron overload, iron deficiency, and iron deficiency anemia. Gastroenterology 126: 1293-12301.
- Faivre B, Menu P, Labrude P, Vigneron C (1998) Hemoglobin autoxidation/oxidation mechanisms and methemoglobin prevention or reduction processes in the bloodstream. Artif Cells Blood Substit Immobil Biotechnol 26: 17-26.
- Perutz MF (1979) Regulation of oxygen affinity of hemoglobin: Influence of the structure of globin on the heme iron. Annu Rev Biochem 48: 327-386.
- Marden MC, Griffon N, Poyart C (1995) Oxygen delivery and autoxidation of hemoglobin. Transfus Clin Biol 2: 473-480.
- Winterbourn CC (1985) Free-radical production and oxidative reactions of hemoglobin. Environ Health Perspect 64: 321-330.
- Chen PS, Chiu WT, Hsu PL, Lin SC, Peng IC, et al. (2020) Pathophysiological implication of hypoxia in human diseases. J Biomed Sci 27: 63.
- Brahimi-Horn MC, Pouyssegu J (2007) Oxygen, a source of life and stress. FEBS Lett 581: 3582-3591.
- Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, et al. (1998) Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci U S A 95: 11715-11720.
- Abe H, Semba H, Takeda N (2017) The roles of hypoxia signaling in the pathogenesis of cardiovascular diseases. J Atheroscler Thromb 24: 884-894.
- Toescu EC (2004) Hypoxia sensing and pathways of cytosolic Ca 2+ Cell Calcium 36: 187-199.
- Loor G, Schumacker PT (2008) Role of hypoxia-inducible factor in cell survival during myocardial ischemia reperfusion. Cell Death Differ 15: 686-690.
- Greijer AE, van der WE (2004) The role of hypoxia indicible factor 1 (HIF-1) in hypoxia induced apoptosis. J Clin Pathol 57: 1009-1014.
- Kristian T (2004) Metabolic stages, mitochondria and calcium in hypoxia/ischemic brain damage. Cell Calcium 36: 221-233.
- Chandel NS, Budinger GR (2007) The cellular basis for diverse responses to oxygen. Free Radic Biol Med 42: 165-174.
- Pierce GN, Czubryt MP (1995) The contribution of ionic imbalance to ischemia/reperfusion-induced injury. J Mol Cell Cardiol 27: 53-63.
- Butler J, Vijayakumar S, Pitt B (2018) Revisiting hyperkalaemia guidelines: Rebutall. Eur J Heart Fail 20: 1255.
- Hunter RW, Bailey MA (2019) Hyperkalemia: Pathophysiology, risk factors and consequences. Nephrol Dial Transplant 34: iii2-iii11.
- Adrogue HJ (2005) Consequences of inadequate management of hyponatremia. Am J Nephrol 25: 240-249.
- Tracz MJ, Alam J, Nath KA (2007) Physiology and pathophysiology of heme: Implications for kidney disease. J Am Soc Nephrol 18: 414-420.
- Nagy E, Eaton JW, Jeney V, Soares MP, Varga Z, et al. (2010) Red cells, haemoglobin, heme, iron, and atherogenesis. Arterioscler Thromb Vasc Biol 30: 1347-1353.
- Chen Q, Vasquez EJ, Moghddas S, Hoppel CL, Lesnefsky EJ (2003) Production of reactive oxygen species by mitochondria : Central role of complex III. J Biol Chem 278: 36027-36031.
- Fruehauf JP, Meysken FL (2007) Reactive oxygen species: A breath of life or death? Clin Cancer Res 13: 789-794.
- Rifkind JM, Nagababu E, Ramasamy S, Ravi LB (2003) Hemoglobin redox reactions and oxidative stress. Redox Rep 8: 234-237.
- Sauer H, Wartenberg M, Hescheler J (2001) Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 11: 173-186.
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
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.
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.
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.
Alcohol is a psychoactive substance use as an active ingredient in drinks such as beers, wine, distilled spirits (hard liquor) and in some beverages . 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 .
Most of the times, alcoholic beverages and drinks are abused, leading to alcoholic intoxication, also known as alcoholic poisoning . 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 . 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 . Furthermore, abuse of alcohol contributes to over 3 million deaths each year globally . 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 .
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 . Unlike food which can take hours to digest, alcohol is absorbed more rapidly than it is eliminated .
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 . 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).
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 .
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 . 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.
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 .
Measurement of oxy-Hb and met-Hb concentration
Oxy-Hb and met-Hb concentrations were determined using the Beer-Lambert equation . 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 . To calculate the concentrations of oxy-Hb, the following equation was used:
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 . 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.
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).
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).
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 . How much the analyte absorbs light is directly proportional to the concentration of that analyte in the solution . 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 . 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 . 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% .
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 . Each polypeptide subunit of haemoglobin contains a haem group and each haem group contains an iron atom . 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 . 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 . 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 . 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 .
First, the inability of the ferric iron of the heme protein to bind oxygen leads to hypoxemia and subsequently to hypoxia . 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 . 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 . During hypoxia, oxidative phosphorylation which accounts for the major generation of ATP is inhibited, thereby leading to reduced cell viability in living organism . 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 . But during hypoxia there is an enhanced influx of 3 Na+ inside the intracellular fluid and efflux of 2 K+ outside the extracellular fluid . This leads to a disturbance in membrane potential causing depolarization of the cell membrane and activation of the voltage-gated Ca2+ channels . 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 .
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 . Hyperkalemia (an increase in intracellular K+) and hyponatremia (a decrease in extracellular Na+) has been suggested to be harmful to cells . 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 .
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 .
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 . 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 . 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].
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.
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.
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.
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.