
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39237619
70573
10.1038/s41598-024-70573-6
Article
Bio-spectroscopic investigation linking changes of retinal structure with short-term administration of Amiodarone and revealing the ameliorative effect of vitamin E supplementation
Mahmoud Sherif S. sherif.siddick@rio.sci.eg

1
Morsy Sahar A. 2
Aly Eman M. 1
Mohalhal Islam A. 3
1 https://ror.org/01h0ca774 grid.419139.7 0000 0001 0529 3322 Biophysics and Laser Science Unit, Research Institute of Ophthalmology, Giza, Egypt
2 https://ror.org/05fnp1145 grid.411303.4 0000 0001 2155 6022 Physics Department, Faculty of Science, Al-Azhar University (Girls Branch), Cairo, Egypt
3 https://ror.org/01h0ca774 grid.419139.7 0000 0001 0529 3322 Retina Department, Research Institute of Ophthalmology, Giza, Egypt
5 9 2024
5 9 2024
2024
14 2074612 3 2024
19 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Long term use of Amiodarone (AMIO) is associated with the development of ocular adverse effects. This study investigates the short term effects, and the ameliorative consequence of vitamin E on retinal changes that were associated with administration of AMIO. This is accomplished by investigating both retinal structural and conformational characteristics using Fourier transform infrared spectroscopy (FTIR) and Fundus examination. Three groups of healthy rabbits of both sexes were used; the first group served as control. The second group was orally treated with AMIO (160 mg /kg body weight) in a daily basis for two weeks. The last group orally received AMIO as the second group for two weeks then, oral administration of vitamin E (100 mg/kg body weight) for another two weeks as well. FTIR results revealed significant structural and conformational changes in retinal tissue constituents that include lipids and proteins due to AMIO administration. AMIO treatment was associated with fluctuated changes (increased/decreased) in the band position and bandwidth of NH, OH, and CH bonds. This was concomitant with changes in the percentage of retinal protein constituents in particularly α-helix and Turns. AMIO facilitates the formation of intra-molecular hydrogen bonding and turned retinal lipids to be more disordered structure. In conclusion, the obtained FTIR data together with principal component analysis provide evidence that administration of vitamin E following the treatment with AMIO can ameliorate these retinal changes and, these biophysical changes are too early to be detected by Fundus examination.

Keywords

Retina
Fundus
FTIR
Amiodarone
Vitamin E
Subject terms

Biophysics
Medical research
Research Institute of OphthalmologyOpen access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Since its introduction in 1960 as a powerful coronary vasodilator for treating anginal symptoms, Amiodarone (AMIO) is still the most effective drug for the treatment of arrhythmia. In one hand, it was labeled by the US-FDA for the treatment of life-threatening ventricular arrhythmias in 19851–3. On the other hand, it is used off-label to treat atrial fibrillation as well as for the prevention of ventricular tachyarrhythmia in high-risk patients2. The mechanism of action of AMIO comes from the alteration of the function of many membrane protein ion channels, ion exchangers, and adrenergic receptors, which lead to prolongation of the action potential duration of atrial and ventricular muscles without altering the resting membrane potential; therefore, this contributes to complex therapeutic and toxicity profiles3. Because of the long elimination half-life of AMIO and its effect on multiple ion channels and receptors, the toxicity profiles include pulmonary4, renal5 genital6, liver7, thyroid dysfunction8 and peripheral neuropathy9.

The dominance of Amiodarone therapy-adverse effects reaches 15% at the first year and 50% for long-term2. Ocular side effects associated with AMIO were firstly reported in 196910 and include corneal microdeposits11 in at least 90% of patients12,13, lens opacities without visual impairment11, Optic neuropathy and retinopathy14, abnormal phospholipid accumulation in the retinal pigment epithelial cells and induced apoptosis15. Although Amiodarone-induced retinopathy is rare and the main cause of visual loss is due to optic neuropathy during long-term AMIO therapy16, histopathological studies indicate that intra-cytoplasmic deposits of AMIO were detected in retinal pigment epithelial cells and ganglion cells as well; in addition to cornea, lens and optic nerve17. These deposits in particularly that located in the retina was reported to act as photosensitizing agent and results in retinal phototoxicity. Joshi and Gill (2017) reported the first case with Amiodarone induced retinal phototoxicity following vitrectomy surgery. A 66-year-old male was on oral Amiodarone and developed retinal phototoxicity from intraoperative light exposure.

This study reports for the first time, to the best of knowledge, the effects of short term oral administration (two weeks) of Amiodarone on vibrational characteristics of retina and the potential effect of Vitamin E (Vit. E) post-administration. After ophthalmic examination, retinal characteristics were investigated by Fourier transform infrared spectroscopy (FTIR) and FTIR-data were statistically evaluated by principal component analysis as well.

Results

Ophthalmic examination

In Fig. 1.a, examination of the control rabbit retina showed optic nerve head, choroid and retinal blood vessels. The optic nerve head is orange-red in color and oval in shape. Retinal blood vessels and myelinated nerve fibers were crossing the retina in a horizontal plane from the optic disc. Anatomically, and by morphology retinal arteries were thinner than retinal veins18. Choroid was visible under the retina, but there was no distinguishable macular like structures. The optic disc is situated above the horizontal midline of the eye so during examination of the retina we have to look upwards. There were no clinical changes in the retinae that were treated with Amiodarone for two weeks and those post-administered with Vit. E (Fig. 1b,c).Fig. 1 Fundus examination of retinae; (a) Control, (b) AMIO group, and (c) Vit. E group.

Infrared spectroscopy

Detailed analysis of the FTIR spectra was carried out for the following three ranges: 4000 -3000 cm-1 (NH-OH region), 3050—2800 cm−1 (CH stretching region), and 1800—900 cm-1 (fingerprint region) with special consideration of the bands at 1800—1595 cm-1 (lipid carbonyl and protein Amide I absorption bands).

NH-OH region

The stretching NH-OH region of the control and the treated animals in the absorption range 4000–3000 cm−1 is shown in Fig. 2. After normalizing the existing data (panel a), the second derivative spectra are displayed in panel (b). The overlaid spectra in panel (a) clearly show that the pattern of AMIO group is different than the pattern of both control and Vit. E groups and, there are some similarities between the last two groups. The differentiated spectra in panel (b) indicate that AMIO treatments increase the absorption intensity not only that, but also the discrepancy in results is more pronounced. In AMIO group, the non-hydrogen bonded OH groups in the range 4000 – 3650 cm−1 show five absorption bands (arrows) with two strong absorptions at 3702 and 3588 cm−1 and, another absorption band at 3563 cm-1 that belongs to OH-hydrogen bonded group. A strong absorption due to OHasym is also obvious in AMIO group.Fig. 2 Normalized-FTIR spectra of the NH-OH region of the control retinae and the other groups that received AMIO either alone or with Vit. E (a), and their second derivative spectra (b).

Comparing the control pattern with that of Vit. E group; the main absorption in the non-hydrogen bonded-OH group is noticed at 3625 cm−1 for the control while; it located at 3756 cm−1 in Vit. E group. The absorption pattern of both groups is similar in the frequency range 3580–3400 cm−1 which comprises both hydrogen bonded OH group and asymmetric OH vibrations. On the other hand, OHsym mode was characterized by higher intensity in Vit. E group relative to the control one. NH symmetric stretching mode was found to be associated with Vit. E group only.

CH region

The band fitting of the CH stretching vibrations in the range 3000 – 2800 cm−1 is displayed in Fig. 3. These vibrations are associated with retinal lipids (νCH2) and proteins (symCH3, if any). The control pattern revealed the presence of three underlying bands at 2920 cm−1 (asymCH2), 2854 cm-1 (symCH2) and 2800 cm-1 (CH). The absorption pattern of AMIO group show more underlying bands where; unsaturated CH band (olefinic = CH, 2970 cm−1) and asymCH3 (2951 cm-1) vibrations were detected. Asymmetric CH2 mode of vibration also influenced by AMIO administration where; its band position is increased concomitant with reduced band width. In addition, the unidentified CH mode of vibration was characterized by increased band position associated with increased bandwidth. Although asymCH3 vibrational mode is detected in Vit. E group, the rest of vibrational bands matches their control values.Fig. 3 FTIR spectra related to the CH stretching region of the control retinae and the treated groups. The constituting bands are displayed in gray lines.

Table 1 displays these variations and their significance as well. The band area ratio of asymCH2/symCH2 was calculated and found to be increased from 1.3 in the control to 2.8 in both AMIO and Vit. E groups.Table 1 Band fitting analysis of CH stretching region of control and treated groups.

	 = CH	asymCH3	asymCH2	symCH2	CH	
Control			2920 ± 2

49 ± 4

	2854 ± 2

42 ± 6

	2800 ± 3

28 ± 5

	
AMIO group	2970 ± 2

23 ± 5

	2951 ± 2

33 ± 4

	†2925 ± 1

†33 ± 4

	2855 ± 2

33 ± 3

	†2827 ± 2

†49 ± 3

	
Vit. E group		2957 ± 3

36 ± 5

	2921 ± 1

41 ± 4

	2854 ± 2

35 ± 4

	2801 ± 3

33 ± 4

	
First line in each cell displays the band position (cm-1) and, second line displays bandwidth (cm-1).

†Statistically significant.

Fingerprint region

The fingerprint region (1800—900 cm−1) results from the absorption of functional groups relate to all retinal constituents. In Fig. 4, and as compared to the control pattern, the absorption pattern of AMIO group is characterized by increased absorption intensity, and administration of Vit. E reduces this absorption intensity.Fig. 4 Fingerprint region of the control retinae and the treated groups. Underlying bands are displayed in grey lines.

The band position of both Amide I and Amide II was upshifted due to AMIO treatment. The bending mode of CH2 group was found to be affected by AMIO treatment; upshifted band position associated with decreased band position. These two observations are quite similar to Vit. E group. Regarding the phosphate group vibrations; the changes were noticed only for asymPO2 where, the band width was reduced in AMIO as well as Vit. E groups. More vibrational bands were detected in AMIO and Vit. E groups namely; symCOOC, wagging CH2 and (CH3)3N. Comparing the last mentioned vibrational band of both treated groups; it is noticed that Vit. E administration reduces the band characteristics; position and width. Table 2 summarizes all these values and their statistical significance as well.Table 2 Fingerprint region band characteristics of control and treated groups.

	Control	AMIO	Vit. E	
Amide I	1644 ± 2

111 ± 10

	†1651 ± 2

91 ± 8

	†1652 ± 1

92 ± 11

	
Amide II	1534 ± 1

49 ± 6

	†1544 ± 3

54 ± 5

	†1542 ± 2

58 ± 6

	
bendCH2	1436 ± 2

80 ± 4

	†1455 ± 3

†37 ± 3

	†1453 ± 3

†31 ± 5

	
symCOOC		1401 ± 2

49 ± 3

	1399 ± 1

53 ± 3

	
wagCH2		1303 ± 3

51 ± 4

	1304 ± 3

54 ± 6

	
asymPO2	1234 ± 3

116 ± 8

	1234 ± 2

†59 ± 5

	1232 ± 2

†74 ± 7

	
symPO2	1074 ± 3

70 ± 5

	1077 ± 4

68 ± 4

	1074 ± 4

78 ± 6

	
(CH3)3N		977 ± 1

52 ± 2

	972 ± 2

31 ± 4

	
First line in each cell displays the band position (cm-1) and, second line displays bandwidth (cm-1).

†Statistically significant.

Ratiometric analysis of Amide I/II absorption intensity revealed difference in its value for all groups; in the control, this value was calculated and found to be 8.7 while; it reduced to 2.7 and 0.2 for AMIO and Vit. E groups respectively. The intensity ratio of PO2 sym/asym bands was also calculated and its value for the control is 0.7 while in AMIO and Vit. E groups it was 1.3 and 1.2 respectively.

Carbonyl bands

Absorptions due carbonyl bands were carefully investigated by differentiated spectra. Second derivative spectroscopy revealed interesting information that was masked in the original data due to variation in the absorption intensities. In Fig. 5, the two bands that discernible at 1797 and 1744 cm-1 are associated with esterC = O of the lipid moiety of retina. The higher frequency band in both AMIO and Vit. E groups were characterized by reduced band position and increased band width. The position of the second band is reduced in AMIO group while; in Vit. E treated group, it mimics the control value.Fig. 5 Second derivative of carbonyl region of both lipids and proteins from the control group and the other treated groups showing the underlying bands.

On the other hand, retinal proteins secondary structure is obviously influenced by short term administration of Amiodarone. Detection of Turns constituent and reduced contents of α-helix, β-sheet and β-turns represent the major findings as given in table 3. Vitamin E administered group pattern indicates an increased content of β-turns only while, both α-helix and β-sheet contents are mimicking the control values.Table 3 Second derivative spectra of carbonyl bands of retinal lipids and proteins.

Control	Ester C = O of lipids	Amide I of protein	
β-turns	Turns	α-helix	β-sheet	
1797 ± 2
15 ± 5 (w)	1744 ± 2
25 ± 4 (w)	1683 ± 2
10.3 ± 4 (a)		1646 ± 1
57 ± 3 (a)	1623 ± 2
28.7 ± 5 (a)	
AMIO group	†1784 ± 3

†38 ± 3 (w)

	†1736 ± 1

30 ± 6 (w)

	1680 ± 3

†4.1 ± 1 (a)

	1660 ± 4

38.5 ± 4 (a)

	1641 ± 2

†46 ± 4 (a)

	1625 ± 2

†11.4 ± 3 (a)

	
Vit. E group	†1780 ± 3

†43 ± 3 (w)

	1742 ± 1

29 ± 4 (w)

	1674 ± 1

†20.6 ± 3 (a)

		1649 ± 2

57 ± 3 (a)

	1626 ± 4

22.4 ± 3 (a)

	
(w) Indicate the width in cm−1, and (a) indicates the area percentage.

†Statistically significant relative to the control.

Principal component analysis

Principal component (PC) analysis was applied to full range of FTIR data for all groups, and the results are displayed in Fig. 6. The first two PCs cover 100% of the data where; the percentage of variance of PC1 is 94.89% and that of PC2 is 5.11%. This loading plot reveals the relationships between the studied groups. It is clear that AMIO group and Vit. E group has similar loading to PC1 but the angle between them is right angle, and both groups have different directions i.e. reverse relationship according to PC2. Comparing the two vectors of the control and Vit. E groups; it is noteworthy that they have similar loading to PC1 and the angle between them is acute angle.Fig. 6 Loading plot of the principal component analysis of FTIR data for AMIO group and Vitamin E Co-administered group compared with the control group.

Discussion

FTIR investigation is based on the absorption of infrared light by vibrational transitions in the covalent bonds of the analyzed sample. Therefore, this analysis gives detailed information about the structure and conformation of the examined specimen19. Numerous studies have evidenced the accuracy of this approach in diagnostic diseases such as diabetes, cancer, and Alzheimer's disease20.

Full clinical effects of oral Amiodarone can be achieved after 6 weeks; and upon discontinuation of AMIO therapy, the pharmacological effects could continue for 1 to 3 months2. In addition, histopathological studies reveal that during AMIO therapy, intracytoplasmic deposits of Amiodarone can be found in the cornea, lens, optic nerve, and retina21. Other studies have shown that the prevalence of Amiodarone usage is increasing globally, especially among the elderly population22, and ocular changes induced by Amiodarone have also been reported. The incidence of visual disturbances in patients taking Amiodarone ranges from 1.4% to 40%. Corneal epithelial deposits are very common, occurring in 70% to 100% of patients receiving Amiodarone11,23.

The retinal blood vessel network is unique in that it is the only blood vessel network in the body visible through non-invasive imaging methods. Retinal fundus color imaging is a common technique used for evaluation of this vascular structure. Analyzing the structure of the retinal vessel network serves as a reliable tool for the early detection of retinopathies24. In this short term study, ophthalmic examinations by Fundus camera reveal no detectable clinical changes in the retinae while; the FTIR spectra demonstrated various retinal structure changes due to AMIO treatment that were noted through the absorption frequencies and bandwidth changes of many retinal functional groups. The changes observed in the NH-OH region (strO-H, O-Hasym, and O-Hsym vibrational bands) are related to hydrogen bonding. AMIO treatments affect retinal constituents with OH groups, and alter the molecular structure through induction of hydrogen bonding. Vitamin E was found to reduce these effects where the FTIR-pattern was different than that of AMIO group and have similarities to control pattern. In addition, Vit. E increases the order of retinal membranes through affecting the symmetric vibrational mode of both NH and OH groups. Detection of NHsym band in Vit. E group is related to molecular alteration in retinal proteins25.

Protein misfolding is a detrimental effect that can lead to several structural/functional consequences that involving the inactivation of enzymes and protein aggregation. Typically, ocular-protein aggregation begins with forming insoluble protein fragments. Protein insolubility is dependent on the contents of β-sheet structure and α-helix as well. The association between the decreased α-helix content and increased Turns content noticed in AMIO group is indicative of a protein structure with different compositional characteristics. Taking this into consideration with the decreased content of β-turns and β-sheets, gives the impetus that the helical structure of retinal proteins becomes more folded while; the polypeptide chains were lost the ability to abruptly changing the direction due to decreased β-turns26. This situation is totally differed when Vit. E administered; Vit. E can buffer the undesired changes due to AMIO treatment as eluded by resuming the content of α-helix and β-sheets to their control value which is directly related to enhancing protein solubility. Not only that, but also the ability of the polypeptide chains to abruptly changing their directions is greatly enhanced as well. Moreover, the difference in Amide I/II ratio is attributed to difference in secondary structure and indicates protein rearrangement27. The large variation in the value of this ratio between AMIO and Vit. E groups indicate that this rearrangement is different.

Oral administration of AMIO for two weeks induce changes in the molecular structure of retinal lipids. It increases the disorder of retinal membranes; the detection of asymCH3 vibrational mode- which is mainly due to lipids with little contribution from proteins, carbohydrates and nucleic acids- at higher frequency20,28 and the increased concentration of double bonds of lipids within the retinal tissue; olefinic = CH band, can be used as index of unsaturation level20,28,29. In the same context, the increased band position and the increased vibrational motion (band narrowing) of asymCH2 also support the increased disorder as well as the formation of hydrogen bond. Administration of Vit. E for two weeks after two weeks of AMIO treatment provides protection to these alterations.

Note that, the increase in the band area ratio of asymCH2/symCH2 in both AMIO and Vit. E reflect an increase in the lipid content30,31 therefore, Vit. E has no effect in this regard. This increased ratio also indicates that lipid chain length and branching is different than the control32 and, again no ameliorative effect of Vit. E.

There are certain requirements for biological membranes to be functioning. These requirements include barrier properties, physical characteristics and certain mobility for proteins. All these requirements are achieved by the lipid disorder which can be characterized by the vibrational frequency of symCH2 band where; lipid disorder can be induced by structural disorder due to protein-lipid interactions or by dynamic disorder that associated with bulk lipid interactions33. Our results clearly indicate that retinal lipid disorder can be correlated to structural disorder as the frequency of symCH2 band is the same in all groups.

In another context, the absorption characteristic of PO2 bands is a measure of the cell activities. Phosphorylation processes of retinal tissue i.e., cellular activities, is increased due to AMIO treatment and, administration of Vit. E did not alter this increase. This conclusion was derived from the intensity ratio of PO2 sym/asym34. The band narrowing observed for asymPO2 in AMIO group indicate increased motional freedom around the phosphate group in other words; increased disorder and, Vit. E has no effect in this regard.

The acyl chain packing of lipid bilayer is monitored by the bending CH2 vibrational band. The upshift in its frequency after AMIO treatment concomitant with reduced band width indicate changes in the lateral packing of phospholipid hydrocarbon chains and, these changes could not be contained by post administration of vitamin E.

Finally, with all these changes and discrepancies in the results, principal component analysis comes to clarify the reality about these discrepancies, that is the structural and conformational changes of the retinal tissue as a result of AMIO treatment are completely different from their control counterparts, and that vitamin E returned these changes to their control state, as the acute angle between the vectors of the control and Vit. E groups indicate a positive correlation, while the right angle between AMIO and Vit. E vectors indicate no correlation35.

Vitamin E supplementation provides several benefits for eye health, particularly in preventing the progression of ocular diseases such as age-related macular degeneration (AMD) and diabetic retinopathy. Studies have shown that vitamin E, especially α-tocopherol, acts as a powerful antioxidant, protecting the retina from oxidative damage and reducing the risk of developing AMD. Research indicates that antioxidant vitamins like vitamin E can slow the progression of AMD, with those at higher risk benefiting the most from these supplements36,37. Additionally, tocotrienol-rich vitamin E has been found to play a crucial role in preventing the progression of retinal microhaemorrhages and diabetic macular edema in patients with diabetic retinopathy, highlighting its importance in the early treatment and prevention of this condition38. Additionally, vitamin E levels in the retina and retinal pigment epithelium increase with age, with older individuals showing higher concentrations of vitamin E compared to younger age groups39. The exact optimal dose may vary based on individual health needs and age-related requirements.

In conclusion, retinal changes associated with short-term administration of AMIO were mainly due to the formation of hydrogen bonding and deterioration of retinal proteins as well. These observations were conquered and contained by vitamin E administration, and this is supported by the results of principal component analysis. Moreover, these biophysical changes at the vibrational level of the retinal tissue could not be clinically detected by Fundus examination therefore; ophthalmic examination of patients should be a prerequisite for continuation of AMIO treatment, and vitamin E supplementation is recommended. The calculated human equivalent dose40 relative to the animal applied dose is 32.4 mg/kg.

Materials and methods

Materials

Drug Cordarone®, 200 mg with active ingredient AMIO hydrochloride, was purchased from Global Napi Pharmaceuticals Company, Cairo, Egypt. Vitamin E (alpha-tocopherol acetate, 400 mg) was purchased from Pharco Pharmaceuticals Company, Cairo, Egypt. Potassium bromide powder (KBr-IR grade) was purchased from Sigma Aldrich (St. Louis, MO, USA).

Animals and experimental design

Three groups of healthy colored rabbits (Chinchilla, 2–2.5 kg) of both sexes (male/female, 5/5) were used in this study, where each group comprises ten rabbits (20 eyes). Animals were obtained from the animal house facility at Research Institute Ophthalmology, Giza, Egypt, and were kept separately in stainless steel cages under good ventilation and 12 h light/dark cycle during the experimental period. They have free access to an adequate standard diet and water ad-libitum, and the ambient temperature was set at 25 ± 2 °C. Animals were treated according to the guidelines of using animals in ophthalmic research established by the Association of Research in Vision and Ophthalmology (ARVO), and the protocol was approved by the Research Institute of Ophthalmology ethical committee. The study is reported in accordance with ARRIVE guidelines.

The experimental design was performed as follows; rabbits were treated orally for two weeks with Amiodarone through polypropylene orogastric tube attached to 20 ml syringe. The dose was 160 mg Kg−1 (AMIO group). The second group was on oral administration as the AMIO group for two weeks then, received oral dose of vitamin E (100 mg Kg−1) for another two weeks (Vit. E group). The last group was served as the control and received 5 ml of water by gavage tube. Rabbits were sedated as a prerequisite for administration by intramuscular injection of a mixture of ketamine (80 mg/kg) and xylazine (20 mg/kg)41.

Ophthalmic examination

High resolution Fundus images were taken using Topcon TRC-50EX (Japan) which is a mydriatic retinal camera incorporating digital ready features to provide complete retinal imaging including color, red-free and fluorescein angiography, with SONY DXC-950P (Japan) which is a 3CCD color video camera. The angle of coverage is 50°, 35°, and 20°.

Sample preparation and FTIR measurement

At the end of the administration period and after the ophthalmic examination, rabbits were killed by intravenous administration of sodium pentabarbitone (30 mg Kg−1)42, and their eyes were enucleated then opened by the corneal section through the ora Serrata where the anterior segment constituents can be removed so that the retina is exposed, and can easily be obtained. Each retina was kept in a sterilized dark glass vial, flushed with dry nitrogen gas, and immediately processed for FTIR investigation (Supplementary information).

For recording the mid-infrared absorption spectrum (4000–900 cm−1), 10 mg of retinal tissue were mixed with 90 mg KBr powder and pressed to form a transparent KBr disk using the pressing kit provided by the manufacture. Measurements were done using an infrared spectrophotometer model Nicolet-iS5 (Thermo Fisher Scientific Inc, USA) with an effective resolution of 2 cm-1. The spectra were recorded under a continuous nitrogen gas flow to prevent the effect of environmental vapor (CO2 and H2O) where; one hundred and fifty interferograms were co-added. These spectra were then baseline corrected and smoothed with Savitsky–Golay function (9 points). The individual spectrum that was recorded from each studied group was averaged to obtain the group spectrum using OriginPro 2015 (64-bit) software package (Origin Lab Corporation, Northampton, MA 01,060, USA). This group spectrum is displayed in the study. Bands were carefully examined by spectral resolution techniques that comprise derivative spectroscopy or band fitting.

Statistical analysis

Results were expressed as the mean ± standard deviation (SD). The comparison between groups was performed using one-way ANOVA (OriginPro, 2015) where the significance level was set at p < 0.05. Multivariate analysis was applied to FTIR raw data for further discrimination between the studied groups. Principal component analysis was performed using the statistical tools provided by OriginPro (2015) software.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-70573-6.

Author contributions

S.S.M.: Conceptualization, Methodology, Resources, Investigation, Statistical analyses. S.A.M. and E.M.A.; Methodology, Investigation, Resources. I.A.M.: Conceptualization, Ophthalmic Investigation, Resources. All authors wrote the main manuscript text, prepared figures, and review the manuscript.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability

All data generated or analyzed during this study are included in this published article and/or its supplementary information files.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Niimi N Takaku S Yako H Sango K Drug-Induced Demyelinating Neuropathies Adv. Exp. Med. Biol. 2019 1190 357 369 10.1007/978-981-32-9636-7_23 31760656
Niimi, N., Takaku, S., Yako, H. & Sango, K. Drug-Induced Demyelinating Neuropathies. Adv. Exp. Med. Biol. 1190, 357–369 (2019).31760656 10.1007/978-981-32-9636-7_23
2. Florek JB Girzadas D Amiodarone 2023 StatPearls
Florek, J. B. & Girzadas, D. Amiodarone (StatPearls, 2023).
3. Rusinova R Koeppe RE Andersen OS A general mechanism for drug promiscuity: Studies with amiodarone and other antiarrhythmics J. Gen. Physiol. 2015 146 463 475 10.1085/jgp.201511470 26573624
Rusinova, R., Koeppe, R. E. & Andersen, O. S. A general mechanism for drug promiscuity: Studies with amiodarone and other antiarrhythmics. J. Gen. Physiol. 146, 463–475 (2015).26573624 10.1085/jgp.201511470
4. Zidan RA Effect of long-term administration of amiodarone on rat lung and the possible protective role of vitamin E Egypt. J. Histol. 2011 34 117 128 10.1097/01.EHX.0000395190.66459.04
Zidan, R. A. Effect of long-term administration of amiodarone on rat lung and the possible protective role of vitamin E. Egypt. J. Histol. 34, 117–128 (2011).10.1097/01.EHX.0000395190.66459.04
5. Sakr SA El-Gamal EM Effect of grapefruit juice on amiodarone induced nephrotoxicity in albino rats Toxicol. Ind. Health 2016 32 68 75 10.1177/0748233713498443 24021428
Sakr, S. A. & El-Gamal, E. M. Effect of grapefruit juice on amiodarone induced nephrotoxicity in albino rats. Toxicol. Ind. Health 32, 68–75 (2016).24021428 10.1177/0748233713498443
6. Özkaya AK Effects of chronic amiodarone treatment on rat testis Acta Histochem. 2016 118 271 277 10.1016/j.acthis.2016.02.003 26947592
Özkaya, A. K. et al. Effects of chronic amiodarone treatment on rat testis. Acta Histochem. 118, 271–277 (2016).26947592 10.1016/j.acthis.2016.02.003
7. Kim G Increased hepatic acylcarnitines after oral administration of amiodarone in rats J. Appl. Toxicol. 2020 40 1004 1013 10.1002/jat.3960 32084307
Kim, G. et al. Increased hepatic acylcarnitines after oral administration of amiodarone in rats. J. Appl. Toxicol. 40, 1004–1013 (2020).32084307 10.1002/jat.3960
8. Jiang L-QQ Dronedarone and Amiodarone induce dysliiidemia and thyroid dysfunction in rats Cell. Physiol. Biochem. 2016 38 2311 2322 10.1159/000445585 27197836
Jiang, L.-Q.Q. et al. Dronedarone and Amiodarone induce dysliiidemia and thyroid dysfunction in rats. Cell. Physiol. Biochem. 38, 2311–2322 (2016).27197836 10.1159/000445585
9. Pulipaka U Lacomis D Omalu B Amiodarone-induced neuromyopathy: three cases and a review of the literature J. Clin. Neuromuscul. Dis. 2002 3 97 105 10.1097/00131402-200203000-00001 19078662
Pulipaka, U., Lacomis, D. & Omalu, B. Amiodarone-induced neuromyopathy: three cases and a review of the literature. J. Clin. Neuromuscul. Dis. 3, 97–105 (2002).19078662 10.1097/00131402-200203000-00001
10. Watillon M Lavergne G Weekers JF Corneal lesions during treatment with cordarone (amiodarone hydrochloride) Bull. Soc. Belge Ophtalmol. 1968 150 715 726 5757945
Watillon, M., Lavergne, G. & Weekers, J. F. Corneal lesions during treatment with cordarone (amiodarone hydrochloride). Bull. Soc. Belge Ophtalmol. 150, 715–726 (1968).5757945
11. Mäntyjärvi M Tuppurainen K Ikäheimo K Ocular side effects of amiodarone Surv. Ophthalmol. 1998 42 360 366 10.1016/S0039-6257(97)00118-5 9493278
Mäntyjärvi, M., Tuppurainen, K. & Ikäheimo, K. Ocular side effects of amiodarone. Surv. Ophthalmol. 42, 360–366 (1998).9493278 10.1016/S0039-6257(97)00118-5
12. Tauveron I Batisse-Lignier M Maqdasy S Challenges in the management of amiodarone-induced thyrotoxicosis Presse Med. 2018 47 746 756 10.1016/j.lpm.2018.09.001 30274916
Tauveron, I., Batisse-Lignier, M. & Maqdasy, S. Challenges in the management of amiodarone-induced thyrotoxicosis. Presse Med. 47, 746–756 (2018).30274916 10.1016/j.lpm.2018.09.001
13. Moore BM Cordina RL McGuire MA Celermajer DS Adverse effects of amiodarone therapy in adults with congenital heart disease Congenit. Heart Dis. 2018 13 944 951 10.1111/chd.12657 30239160
Moore, B. M., Cordina, R. L., McGuire, M. A. & Celermajer, D. S. Adverse effects of amiodarone therapy in adults with congenital heart disease. Congenit. Heart Dis. 13, 944–951 (2018).30239160 10.1111/chd.12657
14. Wang A-GG Cheng H-CC Amiodarone-associated optic neuropathy: Clinical review Neuro-Ophthalmology 2017 41 55 58 10.1080/01658107.2016.1247461 28348626
Wang, A.-G.G. & Cheng, H.-C.C. Amiodarone-associated optic neuropathy: Clinical review. Neuro-Ophthalmology 41, 55–58 (2017).28348626 10.1080/01658107.2016.1247461
15. Liao R Amiodarone-Induced Retinal Neuronal Cell Apoptosis Attenuated by IGF-1 via Counter Regulation of the PI3k/Akt/FoxO3a Pathway Mol. Neurobiol. 2017 54 6931 6943 10.1007/s12035-016-0211-x 27774572
Liao, R. et al. Amiodarone-Induced Retinal Neuronal Cell Apoptosis Attenuated by IGF-1 via Counter Regulation of the PI3k/Akt/FoxO3a Pathway. Mol. Neurobiol. 54, 6931–6943 (2017).27774572 10.1007/s12035-016-0211-x
16. Shaikh S Retinal evaluation of patients on chronic Amiodarone therapy Retina 2003 23 354 359 10.1097/00006982-200306000-00011 12824836
Shaikh, S. et al. Retinal evaluation of patients on chronic Amiodarone therapy. Retina 23, 354–359 (2003).12824836 10.1097/00006982-200306000-00011
17. Ingram DV Jaggarao NSV Chamberlain DA Ocular changes resulting from therapy with amiodarone Br. J. Ophthalmol. 1982 66 676 679 10.1136/bjo.66.10.676 7115651
Ingram, D. V., Jaggarao, N. S. V. & Chamberlain, D. A. Ocular changes resulting from therapy with amiodarone. Br. J. Ophthalmol. 66, 676–679 (1982).7115651 10.1136/bjo.66.10.676
18. Iwase T Differences of retinal blood flow between arteries and veins determined by laser speckle flowgraphy in healthy subjects Medicine (Baltimore) 2015 94 e1256 10.1097/MD.0000000000001256 26287409
Iwase, T. et al. Differences of retinal blood flow between arteries and veins determined by laser speckle flowgraphy in healthy subjects. Medicine (Baltimore) 94, e1256 (2015).26287409 10.1097/MD.0000000000001256
19. Derenne A Van Hemelryck V Lamoral-Theys D Kiss R Goormaghtigh E FTIR spectroscopy: A new valuable tool to classify the effects of polyphenolic compounds on cancer cells Biochim. Biophys. Acta Mol. Basis Dis. 2013 1832 46 56 10.1016/j.bbadis.2012.10.010
Derenne, A., Van Hemelryck, V., Lamoral-Theys, D., Kiss, R. & Goormaghtigh, E. FTIR spectroscopy: A new valuable tool to classify the effects of polyphenolic compounds on cancer cells. Biochim. Biophys. Acta Mol. Basis Dis. 1832, 46–56 (2013).10.1016/j.bbadis.2012.10.010
20. Bozkurt O Severcan M Severcan F Diabetes induces compositional, structural and functional alterations on rat skeletal soleus muscle revealed by FTIR spectroscopy: a comparative study with EDL muscle Analyst 2010 135 3110 10.1039/c0an00542h 20967384
Bozkurt, O., Severcan, M. & Severcan, F. Diabetes induces compositional, structural and functional alterations on rat skeletal soleus muscle revealed by FTIR spectroscopy: a comparative study with EDL muscle. Analyst 135, 3110 (2010).20967384 10.1039/c0an00542h
21. Ebeid WM New insights into amiodarone induced retinal and optic nerve toxicity: functional and structural changes Ther. Adv. Ophthalmol. 2023 10.1177/25158414231194159 37701727
Ebeid, W. M. et al. New insights into amiodarone induced retinal and optic nerve toxicity: functional and structural changes. Ther. Adv. Ophthalmol.10.1177/25158414231194159 (2023).37701727 10.1177/25158414231194159
22. Kervinen M Falck A Hurskainen M Hautala N Bilateral optic neuropathy and permanent loss of vision after treatment with amiodarone J. Cardiovasc. Pharmacol. 2013 62 394 396 10.1097/FJC.0b013e31829f9e40 23921312
Kervinen, M., Falck, A., Hurskainen, M. & Hautala, N. Bilateral optic neuropathy and permanent loss of vision after treatment with amiodarone. J. Cardiovasc. Pharmacol. 62, 394–396 (2013).23921312 10.1097/FJC.0b013e31829f9e40
23. Kim H-L The incidence and predictors of overall adverse effects caused by low dose amiodarone in real-world clinical practice Korean J. Intern. Med. 2014 29 588 10.3904/kjim.2014.29.5.588 25228834
Kim, H.-L. et al. The incidence and predictors of overall adverse effects caused by low dose amiodarone in real-world clinical practice. Korean J. Intern. Med. 29, 588 (2014).25228834 10.3904/kjim.2014.29.5.588
24. Miri M Amini Z Rabbani H Kafieh R A comprehensive study of retinal vessel classification methods in fundus images J. Med. Signals Sens. 2017 7 59 70 10.4103/2228-7477.205505 28553578
Miri, M., Amini, Z., Rabbani, H. & Kafieh, R. A comprehensive study of retinal vessel classification methods in fundus images. J. Med. Signals Sens. 7, 59–70 (2017).28553578 10.4103/2228-7477.205505
25. Gamal EM Aly EM Mahmoud SS Talaat MS Sallam ASM FTIR assessment of the effect of Ginkgo biloba leave extract (EGb 761) on mammalian retina Cell Biochem. Biophys. 2011 61 169 177 10.1007/s12013-011-9173-9 21369798
Gamal, E. M., Aly, E. M., Mahmoud, S. S., Talaat, M. S. & Sallam, A. S. M. FTIR assessment of the effect of Ginkgo biloba leave extract (EGb 761) on mammalian retina. Cell Biochem. Biophys. 61, 169–177 (2011).21369798 10.1007/s12013-011-9173-9
26. Usoltsev D Systematic FTIR spectroscopy study of the secondary structure changes in human serum albumin under various denaturation conditions Biomolecules 2019 9 359 10.3390/biom9080359 31409012
Usoltsev, D. et al. Systematic FTIR spectroscopy study of the secondary structure changes in human serum albumin under various denaturation conditions. Biomolecules 9, 359 (2019).31409012 10.3390/biom9080359
27. Ricciardi V Portaccio M Manti L Lepore M An FTIR microspectroscopy ratiometric approach for monitoring X-ray irradiation effects on SH-SY5Y human neuroblastoma cells Appl. Sci. 2020 10 2974 10.3390/app10082974
Ricciardi, V., Portaccio, M., Manti, L. & Lepore, M. An FTIR microspectroscopy ratiometric approach for monitoring X-ray irradiation effects on SH-SY5Y human neuroblastoma cells. Appl. Sci. 10, 2974 (2020).10.3390/app10082974
28. Kneipp J Lasch P Baldauf E Beekes M Naumann D Detection of pathological molecular alterations in scrapie-infected hamster brain by Fourier transform infrared (FT-IR) spectroscopy Biochim. Biophys. Acta Mol. Basis Dis. 2000 1501 189 199 10.1016/S0925-4439(00)00021-1
Kneipp, J., Lasch, P., Baldauf, E., Beekes, M. & Naumann, D. Detection of pathological molecular alterations in scrapie-infected hamster brain by Fourier transform infrared (FT-IR) spectroscopy. Biochim. Biophys. Acta Mol. Basis Dis. 1501, 189–199 (2000).10.1016/S0925-4439(00)00021-1
29. Cakmak G Zorlu F Severcan M Severcan F Screening of protective effect of amifostine on radiation-induced structural and functional variations in rat liver microsomal membranes by FT-IR spectroscopy Anal. Chem. 2011 83 2438 2444 10.1021/ac102043p 21410135
Cakmak, G., Zorlu, F., Severcan, M. & Severcan, F. Screening of protective effect of amifostine on radiation-induced structural and functional variations in rat liver microsomal membranes by FT-IR spectroscopy. Anal. Chem. 83, 2438–2444 (2011).21410135 10.1021/ac102043p
30. Mahmoud SS The impact of elevated blood glycemic level of patients with type 2 diabetes mellitus on the erythrocyte membrane: FTIR study Cell Biochem. Biophys. 2010 58 45 51 10.1007/s12013-010-9092-1 20652761
Mahmoud, S. S. The impact of elevated blood glycemic level of patients with type 2 diabetes mellitus on the erythrocyte membrane: FTIR study. Cell Biochem. Biophys. 58, 45–51 (2010).20652761 10.1007/s12013-010-9092-1
31. Guldag D Bozkurt O Sreeparna Banerjee FS Guldag D Screening and Diagnosis of Diabetes by Vibrational Spectroscopy Vibrational Spectroscopy in Diagnosis and Screening 2012 IOS Press
Guldag, D., Bozkurt, O. & Sreeparna Banerjee, F. S. Screening and Diagnosis of Diabetes by Vibrational Spectroscopy. In Vibrational Spectroscopy in Diagnosis and Screening (ed. Guldag, D.) (IOS Press, 2012).
32. Yu P Christensen CR Christensen DA McKinnon JJ Ultrastructural-chemical makeup of yellow-seeded (Brassica rapa) and brown-seeded (Brassica napus) canola within cellular dimensions, explored with synchrotron reflection FTIR microspectroscopy Can J. Plant Sci. 2005 85 533 541
Yu, P., Christensen, C. R., Christensen, D. A. & McKinnon, J. J. Ultrastructural-chemical makeup of yellow-seeded (Brassica rapa) and brown-seeded (Brassica napus) canola within cellular dimensions, explored with synchrotron reflection FTIR microspectroscopy Can. J. Plant Sci. 85(533), 541 (2005).
33. Szalontai B Kóta Z Nonaka H Murata N Structural Consequences of Genetically Engineered Saturation of the Fatty Acids of Phosphatidylglycerol in Tobacco Thylakoid Membranes. An FTIR Study † Biochemistry 2003 42 4292 4299 10.1021/bi026894c 12680783
Szalontai, B., Kóta, Z., Nonaka, H. & Murata, N. Structural Consequences of Genetically Engineered Saturation of the Fatty Acids of Phosphatidylglycerol in Tobacco Thylakoid Membranes. An FTIR Study †. Biochemistry 42, 4292–4299 (2003).12680783 10.1021/bi026894c
34. Sabbatini S Infrared microspectroscopy of oral squamous cell carcinoma: Spectral signatures of cancer grading Vib. Spectrosc. 2013 68 196 203 10.1016/j.vibspec.2013.07.002
Sabbatini, S. et al. Infrared microspectroscopy of oral squamous cell carcinoma: Spectral signatures of cancer grading. Vib. Spectrosc. 68, 196–203 (2013).10.1016/j.vibspec.2013.07.002
35. Yan W Tinker NA Biplot analysis of multi-environment trial data: Principles and applications Can. J. Plant Sci. 2006 86 623 645 10.4141/P05-169
Yan, W. & Tinker, N. A. Biplot analysis of multi-environment trial data: Principles and applications. Can. J. Plant Sci. 86, 623–645 (2006).10.4141/P05-169
36. Chandrinos A Tzamouranis D Kakoura S Vitamin E and supplements offer eye neuroprotection – myth or reality? Ophthalmol. Res. An Int. J. 2023 18 16 24 10.9734/or/2023/v18i6404
Chandrinos, A., Tzamouranis, D. & Kakoura, S. Vitamin E and supplements offer eye neuroprotection – myth or reality?. Ophthalmol. Res. An Int. J. 18, 16–24 (2023).10.9734/or/2023/v18i6404
37. Edwards G Olson CG Euritt CP Koulen P Molecular mechanisms underlying the therapeutic role of vitamin E in age-related macular degeneration Front. Neurosci. 2022 10.3389/fnins.2022.890021 36699526
Edwards, G., Olson, C. G., Euritt, C. P. & Koulen, P. Molecular mechanisms underlying the therapeutic role of vitamin E in age-related macular degeneration. Front. Neurosci.10.3389/fnins.2022.890021 (2022).36699526 10.3389/fnins.2022.890021
38. Ho J-I The effects of vitamin E on non-proliferative diabetic retinopathy in type 2 diabetes mellitus: Are they sustainable with 12 months of therapy SAGE Open Med. 2022 10 205031212210953 10.1177/20503121221095324
Ho, J.-I. et al. The effects of vitamin E on non-proliferative diabetic retinopathy in type 2 diabetes mellitus: Are they sustainable with 12 months of therapy. SAGE Open Med. 10, 205031212210953 (2022).10.1177/20503121221095324
39. Organisciak DT Berman ER Wang H-M Feeney-Burns L Vitamin E in human neural retina and retinal pigment epithelium: Effect of age Curr. Eye Res. 1987 6 1051 1055 10.3109/02713688709034876 3665559
Organisciak, D. T., Berman, E. R., Wang, H.-M. & Feeney-Burns, L. Vitamin E in human neural retina and retinal pigment epithelium: Effect of age. Curr. Eye Res. 6, 1051–1055 (1987).3665559 10.3109/02713688709034876
40. Nair A Jacob S A simple practice guide for dose conversion between animals and human J. Basic Clin. Pharm. 2016 7 27 10.4103/0976-0105.177703 27057123
Nair, A. & Jacob, S. A simple practice guide for dose conversion between animals and human. J. Basic Clin. Pharm. 7, 27 (2016).27057123 10.4103/0976-0105.177703
41. Green CJ Knight J Precious S Simpkin S Ketamine alone and combined with diazepam or xylazine in laboratory animals: A 10 year experience Lab. Anim. 1981 10.1258/002367781780959107 7043078
Green, C. J., Knight, J., Precious, S. & Simpkin, S. Ketamine alone and combined with diazepam or xylazine in laboratory animals: A 10 year experience. Lab. Anim.10.1258/002367781780959107 (1981).7043078 10.1258/002367781780959107
42. Kodama I Suzuki R Kamiya K Iwata H Toyama J Effects of long-term oral administration of amiodarone on the electromechanical performance of rabbit ventricular muscle Br. J. Pharmacol. 1992 107 502 509 10.1111/j.1476-5381.1992.tb12774.x 1422596
Kodama, I., Suzuki, R., Kamiya, K., Iwata, H. & Toyama, J. Effects of long-term oral administration of amiodarone on the electromechanical performance of rabbit ventricular muscle. Br. J. Pharmacol. 107, 502–509 (1992).1422596 10.1111/j.1476-5381.1992.tb12774.x
