Article

ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 8, pp. 1417-1431 © Pleiades Publishing, Ltd., 2026.

1417

Age-Related Changes in bis-Retinoids of Lipofuscin

Granules in Human Retinal Pigment Epithelium Cells

Marina A. Yakovleva

1,2

, Alexey A. Kostyukov

, Natalya L. Aronshtam

Patimat M. Shilkrot

, Sergei A. Borzenok

, Vladimir A. Kuzmin

Tatiana B. Feldman

1,2,a

*, Aleksandr E. Dontsov

, and Mikhail A. Ostrovsky

1,2

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia

Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

Dagestan Eye Microsurgery Center, 368305 Kaspiysk, Republic of Dagestan, Russia

Svyatoslav Fyodorov Eye Microsurgery Complex, Ministry of Health of the Russian Federation,

127486 Moscow, Russia

e-mail: feldmantb@mail.ru

Received March 29, 2026

Revised May 25, 2026

Accepted May 25, 2026

Abstract— Lipofuscin granules (LGs) in retinal pigment epithelium (RPE) cells contain bis-retinoids and

their oxidation and degradation products, rendering them photo- and cytotoxic to intracellular structures.

LGs are implicated in the pathogenesis of multiple visual pathologies, including age-related macular degen-

eration (AMD). They exhibit strong autofluorescence, which has led to the development of fundus autofluo-

rescence (FAF) imaging as a non-invasive diagnostic method in ophthalmology. Spectral analysis of autoflu-

orescence can expand the capabilities of this method, including for preclinical diagnostics, as pathological

conditions are often associated with increased proportions of oxidized bis-retinoid derivatives that alter LG

autofluorescence parameters. However, limited knowledge of age-dependent changes in LG bis-retinoid com-

position remains a key limitation. Inthis study, we combined fluorescence spectroscopy, confocal fluorescence

microscopy, and fluorescence lifetime imaging (time-correlated single-photon counting) to demonstrate that,

under physiological conditions, aging is accompanied by a progressive increase in the relative abundance

of oxidation and degradation products of bis-retinoids in LGs. These findings provide an age-dependent

baseline for distinguishing physiological and pathological states, thereby improving the potential of FAF

imaging for early (preclinical) diagnosis.

DOI: 10.1134/S0006297926600985

Keywords: eye, retinal pigment epithelium, lipofuscin granules, bis-retinoids, autofluorescence

* To whom correspondence should be addressed.

INTRODUCTION

Throughout life, retinal pigment epithelium(RPE)

cells accumulate lipofuscin granules (LGs) as products

of incomplete lysosomal degradation of phagocytosed

photoreceptor cell debris [1-3].

Until the early 1990s, LGs had been regarded as

relatively harmless cellular ballast, occupying up to

20% of the RPE cell volume by the age of 70 [4]. The

first studies on the phototoxicity of LGs demonstrated

their ability to generate reactive oxygen species(ROS)

upon light exposure [5-7]. It is now well established

that the photo- and cytotoxic properties of LGs con-

tribute to the development of many visual pathologies,

including age-related macular degeneration(AMD)[3,

8-10]. These detrimental effects are primarily associ-

ated with the accumulation of bis-retinoids and their

derivatives (Fig.  1), which are byproducts of the ret-

inoid cycle  [11].

The most toxic compounds are oxidation and

degradation products of bis-retinoids, which can dif-

fuse from LGs into the cytoplasm of RPE cells, where

they damage cellular structures even in the absence

of light [12-16]. Because all these compounds exhibit

YAKOVLEVA et al.1418

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Fig.  1. Structures of retinoid cycle byproducts: a)bis-retinoids: A2E (N-retinylidene-N-retinylethanolamine), A2E-DHP-E (dihy-

dropyridine derivative with A2E and ethanolamine), ATR (all-trans-retinal) dimer; b)  products of A2E oxidation: 5,6-epoxy,

7,8-epoxy, and 5,8-furan derivatives. c)  Degradation products of bis-retinoids: low-molecular-weight aldehydes and ketones

(glyoxal and methylglyoxal).

strong fluorescence, they have enabled the develop-

ment of a new noninvasive ophthalmic diagnostic

technique, fundus autofluorescence (FAF) imaging

[17-19], and subsequently an entire experimental

field focused on FAF spectral analysis [20-23]. Consid-

erable effort is currently directed toward improving

FAF spectral analysis because of its potential for the

preclinical detection of degenerative changes in the

retina and RPE.

We previously discovered and later confirmed

that in AMD, the relative content of bis-retinoid oxi-

dation and degradation products increases compared

to normal tissue, leading to a shift in the fluorescence

spectrum toward the short-wavelength (blue) region

relatively to fluorescence spectra of non-oxidized

bis-retinoids (particularly A2E)  [22,  23]. Based on

these findings, FAF spectral analysis was proposed as

a method for preclinical diagnostics of AMD  [24,  25].

Currently, another noninvasive diagnostic ap-

proach, fluorescence lifetime imaging ophthalmosco-

py (FLIO), is being actively developed[26]. Itis based

on a large set of data produced in the studies using

fluorescence lifetime imaging microscopy (FLIM) to

investigate the dynamics of fluorescence decay of LGs

in various model systems [21]. The principal quanti-

tative parameter used to assess retinal pathology is

the mean fluorescence lifetime (t

) [21, 26,27], which

has been shown to be prolonged in pathologies.

Our studies of the spectral and temporal fluores-

cence characteristics of individual groups of bis-ret-

inoids and their oxidation and degradation products

have shown that oxidized bis-retinoids exhibit lon-

ger fluorescence decay times than their non-oxidized

forms [28, 29]. These data explain the increase in t

observed under pathological conditions: the higher

the content of oxidized bis-retinoids in LGs, the lon-

ger the t

of LGs.

Although the relationship between the composi-

tion of bis-retinoids and pathological changes in the

retina has already been demonstrated  [22,  23], the

question of age-related alterations in the composition

of LG fluorophores remains largely unresolved. Sev-

eral studies have shown that, in healthy individuals,

FAF intensity increases progressively up to approxi-

mately 70 years of age [3,  30-32]. In addition, FLIO

studies in healthy elderly subjects revealed an in-

crease in fluorescence lifetime, suggesting age-related

changes in the retinoid composition of LGs, accumu-

lation of additional fluorophores, or alterations in the

intracellular environment surrounding LGs [33-35].

AGE-RELATED CHANGES IN BIS-RETINOIDS OF LIPOFUSCIN GRANULES 1419

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

While these clinical observations are highly valuable,

direct evidence regarding age-dependent qualitative

and quantitative changes in the fluorophore composi-

tion of LGs in human RPE cells is still lacking.

The aim of the present study was to compara-

tively evaluate the relative content of non-oxidized

bis-retinoids and their oxidation and degradation

products in LGs isolated from RPE cells of cadaveric

eyes from different age donors. The study employed

fluorescence spectroscopy, high-performance liquid

chromatography (HPLC), confocal fluorescence mi-

croscopy, and fluorescence lifetime measurements

using the time-correlated single-photon counting

(TCSPC) method.

MATERIALS AND METHODS

Reagents. Reagents and solvents used in the

work were from Sigma-Aldrich (USA), Fluka (USA),

and Komponent-Reaktiv (Russia).

Material. Human cadaveric eyes without signs of

fundus pathology were obtained from the Eye Tissue

Bank of the Svyatoslav Fyodorov Eye Microsurgery

Complex under a scientific cooperation agreement

between the Emanuel Institute of Biochemical Phys-

ics and the Eye Microsurgery Complex in accordance

with all ethical requirements described previous-

ly [36].

RPE was isolated immediately after receiving the

donor material and stored at −80°C in the dark un-

til use in order to minimize additional oxidation of

bis-retinoids. The experiments described in this arti-

cle had been conducted over several years, with all

sample preparation procedures performed according

to a standardized protocol in the identical time and

temperature regimes.

LG isolation from RPE cells and preparation of

chloroform extracts of bis-retinoids and their de-

rivatives. LGs were isolated from the RPE of cadav-

eric donor eyes of different age groups according to

the method described in [6] and suspended in 0.1  M

K-phosphate buffer (pH  7.3).

LG samples were irradiated with visible light us-

ing a KGM 24-150 lamp (390-700  nm; optical system

of a slide projector with a heat filter; irradiation in-

tensity, 80  mW/cm

) with continuous stirring.

Bis-retinoids and their derivatives were extract-

ed from LGs using the Folch method with a chloro-

form  :  methanol mixture (2  :  1, v/v)[37]. Fluorescence

spectra of chloroform extracts were recorded with a

Shimadzu RF-5301PC spectrofluorimeter (Shimadzu,

Japan).

HPLC analysis. Chromatographic separation of

bis-retinoids and their oxidation and degradation

products in LG chloroform extracts was performed

using a Knauer chromatograph (Germany) on a Dias-

fer 120 C18 column (4×250  mm, particle size, 5  µm;

BioKhimMak ST, Russia) in an 80-100% linear gradient

of acetonitrile in 0.05% aqueous solution of trifluoro-

acetic acid over 20  min at a flow rate of 1.5  mL/min.

The products of chromatographic separation were

detected with a Knauer K-2501 photometric detector.

Prior to chromatographic analysis, LG chloroform

extracts were evaporated to dryness under reduced

pressure using a Vacuubrand MZ 2C NT+AK+M+D vac-

uum pump system and then reconstituted in 200  µL

of methanol.

Because oxidized forms of bis-retinoids eluted

at short retention times, the solvent peak (front)

was removed from the chromatograms to exclude

its contribution. The data were processed using the

EuroChrom  5.05 software. Analytical reproducibili-

ty was assessed from three independently acquired

chromatograms for each sample. As the majority

of detected oxidation and degradation products of

bis-retinoids remained structurally unidentified,

their relative abundances were estimated without

correction for individual molar extinction coeffi-

cients.

For comparative analysis, all chromatographic

datasets were categorized into three groups according

to donor age: group  1, 17-30 years (n  =  21); group  2,

34-61 years (n  =  49); group  3, 65-78 years (n  =  22).

A2E synthesis. A2E (standard) was synthesized

from all-trans-retinal and ethanolamine and puri-

fied on a silica gel column according to the standard

method [38].

Photo-oxidation of the synthesized A2E was car-

ried out in a methanol  :  0.1  M K-phosphate buffer

mixture (1  :  1, v/v) by irradiation with a blue LED

lamp (emission maximum, 450  nm; irradiation inten-

sity, 4  J/cm

) for 150  min. Theextent of A2E oxidation

was determined spectrophotometrically by measuring

the decrease in the absorption maximum at 430  nm.

Irradiation for 150  min resulted in almost complete

disappearance of the absorption maximum, whereas

further irradiation produced no additional spectral

changes. Fully oxidized A2E samples were used to

prepare A2E mixtures containing different propor-

tions of native and oxidized A2E. The purity of A2E

was controlled by HPLC.

Preparation of model lipofuscin containing

different amounts of oxidized A2E. Model lipofus-

cin was prepared from photoreceptor outer segments

(POSs) [39]. POSs were isolated from bovine retinas

using the standard method described in [40], resus-

pended in 0.1  M K-phosphate buffer (pH  7.6), and

stored at −18°C.

POS modification was performed in the presence

of methylglyoxal at 37°C with constant stirring for

24  h  [41]. During this process, methylglyoxal reacted

YAKOVLEVA et al.1420

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

with amino groups of POS proteins, forming cross-

links characteristic of natural lipofuscins. The incu-

bation medium for POS modification contained 0.1  M

K-phosphate buffer (pH  7.6), 1.3-1.5  mg/mL POS pro-

tein, and 10  mM methylglyoxal. After incubation, the

mixture was dialyzed at 6°C for 24  h against 0.1  M

K-phosphate buffer to remove unreacted low-mo-

lecular-weight compounds using Float-A-Lyser cellu-

lose ester membranes (SPECTRUM Lab., USA) with

a 3.5-kDa cut-off.

To obtain model lipofuscin containing different

amounts of oxidized bis-retinoids, modified and pu-

rified POSs were incubated for 30  min with meth-

anol solutions of non-oxidized A2E, photo-oxidized

A2E, or their mixtures at volume ratios of 2  :  1, 1  :  1,

and 1  :  2 [41]. The samples were then centrifuged at

15,000g for 20  min in a Beckman Allegra 64R centri-

fuge, and the pellets containing model lipofuscin with

varying proportions of oxidized A2E were resuspend-

ed in 0.1  M K-phosphate buffer and used for fluores-

cence measurements.

Measurement of total fluorescence of model

lipofuscin. The fluorescence spectra of model lipo-

fuscin were recorded with a Shimadzu RF-5301PC

spectrofluorimeter at the excitation wavelengths

of 488  nm (commonly used in FAF imaging) and

440  nm. Fluorescence measurements of samples were

carried out in two modes with yellow (ZhS-18) and

orange (OS-14) emission filters, respectively, as de-

scribed in [20]. The total fluorescence intensity  (I)

was measured at 510-715  nm (yellow filter) and 575-

715  nm (orange filter). The fluorescence intensity in

the 510-575  nm range, corresponding to green-yellow

fluorescence, was determined by subtracting the flu-

orescence measured with the orange filter from that

measured with the yellow filter. For model lipofus-

cins containing different amounts of oxidized A2E

fluorophore, the ratio of yellow-green fluorescence to

orange-red fluorescence  (R) was calculated using the

formula (1): R = I

ZhS-18

OS-14

 − 1.

Isolation of RPE monolayer. RPE monolayers

were isolated separately from selected cadaveric

eyes without visible signs of pathology as described

in [42].

Cadaveric donor material was screened and

selected based on clinical and age-related criteria.

Following selection, each cadaveric eye underwent

a comprehensive examination by an ophthalmolo-

gist to evaluate the fundus condition. To isolate RPE

monolayer, the anterior segment of the enucleated

eyeball was circumferentially excised 2-3  mm pos-

terior to the limbus together with the iris–lens dia-

phragm. The vitreous body was carefully removed

as completely as possible up to the central retinal

region. The resulting vitrectomized eye was filled

with 3-4  mL of neutral 5% formalin solution and

left for 24  h at room temperature in a tightly sealed

container. After fixation, the formalin solution was

aspirated using an automated pipette, and the tissue

was rinsed three times with distilled water. Using a

stereomicroscope (×30 magnification), the superficial

retinal layers within the macular region (7-10  mm in

diameter) were carefully excised down to the RPE

level using microsurgical instruments (retinal spatu-

la, retinal scissors, and forceps). The RPE monolayer

was subsequently separated using a specialized mi-

crosurgical delaminator with gentle strictly horizon-

tal forward movements. The isolated monolayer was

transferred onto a microscope slide while simulta-

neously applying 700-1000  µL of antifade mounting

solution dropwise to the specimen surface to mini-

mize fluorophore degradation and photobleaching

under visible light exposure. The  antifade solution

contained 50%  glycerol and 2%  1,4-diazabicyclo[2.2.2]

octane in 0.02  M Tris-HCl buffer (pH  7.4). The pre-

pared RPE monolayer was covered with a coverslip

and stored horizontally in a tightly sealed container

containing a cotton ball moistened with 0.5% forma-

lin solution. Samples prepared in this manner were

stored at 4°C and remained suitable for analysis for

up to 30 days. Each specimen was accompanied by a

postmortem ophthalmologic assessment of the fundus

condition.

Confocal fluorescence microscopy of RPE

monolayer. RPE monolayer samples were examined

using a Leica TCS SP5 confocal fluorescence micro-

scope (Germany) at the excitation wavelength of

488  nm. For comparative analysis of fluorescence in-

tensities across the RPE surface, fluorescence signals

were registered at 510  nm and 540  nm.

Image analysis was performed using the ImageJ

software (freely distributed by the National Institutes

of Health, USA). The software was used to delineate

the boundaries of selected cells within each sample

and to calculate the mean relative total fluorescence

intensity  (Σ) at the selected wavelengths within the

defined regions of interest. To ensure statistical re-

liability, measurements were obtained from at least

10-20cells per sample. Forcomparative assessment of

age-related changes in the content of oxidized bis-ret-

inoid forms in LGs, we calculated the ratio of total

fluorescence intensity at 510 nm to that at 540  nm.

All data were categorized into three age groups

for comparative analysis: group 1, donors aged 20-

39 years (n  =  34); group 2, donors aged 40-59 years

(n  =  36); and group 3, donors aged 60-70 years

(n  =  30).

Fluorescence lifetime measurements. The fluo-

rescence lifetime of LG suspensions was measured by

recording fluorescence decay kinetics by the TCSPC

method using a FluoTime  300 fluorimeter (PicoQuant,

Germany) at the New Materials and Technologies

AGE-RELATED CHANGES IN BIS-RETINOIDS OF LIPOFUSCIN GRANULES 1421

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Shared Use Center of the Institute of Biochemical

Physics. Sample fluorescence was excited using a

PicoQuant Solea laser (490  nm; 40-ps pulses), and the

fluorescence signal was recorded at 540  nm. Fluores-

cence decay kinetics were analyzed using a three-ex-

ponential decay model. Characteristic fluorescence

lifetimes and their contributions to the detected flu-

orescence decay curves were calculated according to

Equation  (1), taking into account the instrument re-

sponse function (IRF):

I(t) = IRF(t′)

exp[(− t−t′) / τ

]dt′

−∞

(1)

wherei is the component number, Ais the amplitude,

and τ is the fluorescence lifetime. The average fluo-

rescence lifetime was calculated using Equation  (2):

 = (A

× τ

+ A

× τ

+ A

× τ

)/(A

+ A

). (2)

Three samples (LG suspensions) were studied for

each donor age group: group 1, 17-32 years (n  =  64);

group 2, 33-63 years (n  =  56); group 3, 65-79 years

(n  =  43). Data analysis was performed using the

FluoFit software (PicoQuant GmbH).

RESULTS AND DISCUSSION

Fluorescent analysis of the relative content of

oxidized bis-retinoids in LGs from RPE of cadav-

eric eyes as a function of donor age. Fluorescence

spectroscopy of LG chloroform extracts and HPLC

analysis of bis-retinoids and their oxidized forms.

In our previous study, fluorescence spectra of chlo-

roform extracts obtained from LGs isolated from the

RPE of cadaveric eyes without visible pathology were

found to be largely identical across donors of differ-

ent ages. However, HPLC analysis revealed a small

but statistically significant age-dependent increase in

the relative content of oxidized bis-retinoids [36].

In the present study, we conducted a more

comprehensive and statistically robust analysis of

the content of oxidized bis-retinoid as a function of

donor age. All spectral data were grouped into three

age categories. Figure  2a presents the averaged fluo-

rescence spectra of LG chloroform extracts for these

groups. Thespectral differences between group1 (17-

30 years) and group 2 (34-61 years) were minimal

and within the limits of statistical error. In contrast,

comparison of group 1 with group 3 (65-78 years)

revealed a statistically significant increase in fluo-

rescence intensity in the short-wavelength region of

the spectrum (<592  nm). Because oxidation of bis-ret-

inoids is known to induce a blue shift of the fluores-

cence maximum [43], the observed increase in the

short-wavelength fluorescence was interpreted as

evidence for the age-related increase in the relative

content of oxidized bis-retinoids in LGs.

To quantitatively assess the relative content of

oxidized bis-retinoids in LGs in different age groups,

LG chloroform extracts from each individual RPE

sample were analyzed by HPLC. For consistency, the

HPLC data were grouped into the same three age

categories used for the averaged fluorescence spec-

tra (Fig.  2a). Arepresentative chromatogram is shown

in Fig.  2b. Previous studies have demonstrated that

oxidation and degradation products of bis-retinoids

are predominantly detected at shorter retention times

in HPLC analysis (peak groups 1, 2, and 3) [36, 43].

Age-related changes in the relative abundance of in-

dividual retinoid groups are summarized in Fig.  2c

and Table  1.

The data presented in Fig.  2c demonstrate an

age-related increase in the content of most detect-

ed compounds, except peaks 5, 6, and 8. The most

pronounced decrease was observed for peak 6, cor-

responding to iso-A2E. This phenomenon was pre-

viously reported in our studies [36, 43], where we

suggested that iso-A2E is more susceptible to oxida-

tion. To evaluate whether the relative abundance of

non-oxidized and oxidized bis-retinoids changes with

age, we calculated the ratio of the total content of ox-

idized bis-retinoids (Σ for peaks 1,2, and3) to that of

non-oxidized compounds (Σ for peaks 4, 5, 6, and 9)

for the three age groups (Fig.  2d, Table  1). As shown

inTable  1, this ratio increased with age (p  <  0.05), in-

dicating a slight but statistically significant increase

in the relative proportion of oxidized bis-retinoids.

However, it should be noted that HPLC analysis did

not account for the insoluble LG fraction, which also

exhibits fluorescent properties [14]. Consequently,

conclusions drawn solely from these results may be

incomplete. To address this limitation, we performed

comparative analysis of age-related changes in the

fluorescent properties of native LGs.

Determination of the ratio of oxidized and

non-oxidized bis-retinoids by measuring the flu-

orescence intensity of model lipofuscins and LGs

from human RPE cells using orange and yellow

filters. In this series of experiments, we applied

the spectral separation of the short-wavelength flu-

orescence of oxidized products from the long-wave-

length fluorescence of non-oxidized bis-retinoids as

described in [20] (see Materials and Methods). To ac-

curately evaluate the content of oxidized and non-oxi-

dized bis-retinoids in LGs from RPE cells, preliminary

experiments were carried out using a model system

(model lipofuscin) containing the synthetic LG fluo-

rophore A2E.

Model lipofuscin. Methylglyoxal-modified POSs,

which did not contain bis-retinoid fluorophores,

exhibited a fluorescence maximum in the region of

YAKOVLEVA et al.1422

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Fig. 2. Age-related changes in the fluorescence and bis-retinoid composition of LGs isolated from human RPE of cadaveric

eyes. a) Averaged fluorescence spectra of LG chloroform extracts: group 1, donor age 17-30 years (n = 15); group 2, donor

age 34-61 years (n = 30); group 3, donor age 65-78 years (n = 15). Fluorescence excitation wavelength, 488 nm; spectra

are normalized at 592 nm. b) Representative chromatogram of LG chloroform extract: peak groups 1-3 correspond to A2E

oxidation products [36]; peaks 4 and 6 are A2E and iso-A2E, respectively [36, 44]; peak group 9 is represented by other

bis-retinoids and their oxidation products [45-47] (detection by absorption at 430 nm). c) Distribution of individual groups

of compounds presented in the chromatogram in panel (b). d) Changes in the relative content of oxidized bis-retinoids

compared to non-oxidized ones in age groups 1-3. The y-axis represents the ratio of the summed contributions of oxidized

bis-retinoids to that of non-oxidized bis-retinoids; * p < 0.05.

Table 1. Relative content of non-oxidized and oxidized bis-retinoids in chloroform extracts from LGs from

donors of different age

Peak/peak group number

in chromatogram (Fig. 2b)

Age groups (Fig. 2c)*

Group 1 (17-30 years), % Group 2 (34-61 years), % Group 3 (65-78 years), %

1 11.52  ±  0.49 13.74  ±  0.24 15.17  ±  0.42

2 15.09  ±  0.39 17.35  ±  0.27 18.81  ±  0.39

3 3.54  ±  0.29 5.11  ±  0.10 5.30  ±  0.03

4 (A2E) 21.41  ±  0.52 24.36  ±  0.56 26.12  ±  0.31

5 2.07  ±  0.32 0.94  ±  0.06 0.42  ±  0.13

6 (iso-A2E) 16.49  ±  1.11 8.38  ±  0.95 4.59  ±  0.17

7 1.65  ±  0.12 2.71  ±  0.22 3.18  ±  0.20

8 3.47  ±  0.41 1.40  ±  0.27 0.17  ±  0.08

9 24.76  ±  0.18 26.01  ±  0.26 26.99  ±  0.19

Σ  (1, 2, 3)/Σ  (4, 5, 6, 9)* 0.48  ±  0.02 0.62  ±  0.01 0.68  ±  0.01

Note. * Data are presented as mean  ±  standard deviation (SD) for three independent HPLC measurements of the studied

sample; p < 0.05.

AGE-RELATED CHANGES IN BIS-RETINOIDS OF LIPOFUSCIN GRANULES 1423

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Fig.  3. a) Formation of model lipofuscin via the reaction of modified POSs with the fluorophore A2E. Fluorescence spectra

of modified POSs before (1) and after (2) addition of A2E. b) Fluorescence spectra of model lipofuscin (1, solid line) and

LGs from human RPE cells (2, dashed line). Excitation wavelength, 365 nm. c) Comparison of fluorescence of model lipo-

fuscin containing non-oxidized A2E(1) or predominantly oxidized A2E(2) as a fluorophore. Excitation wavelength, 488nm.

d) Dependence of the R value (R = I

ZhS

  −  1) on the percentage of oxidized A2E in model lipofuscin.

440-460  nm (excitation wavelength, 365  nm), char-

acteristic of Schiff bases (Fig.  3a, curve  1). Addition

of A2E was to such POSs resulted in fluorescence

quenching at 440-460  nm and a concomitant increase

in fluorescence intensity at 530-560  nm (Fig.  3a,

curve  2). The resulting model lipofuscin was almost

identical to lipofuscin extracted from RPE cells of

donors without pronounced visual pathologies in

terms of its fluorescence properties (Fig.  3b, curves  1

and  2, respectively).

The fluorescence spectra of model lipofuscin con-

taining the non-oxidized fluorophore A2E differed

markedly from those enriched with its oxidized forms

(Fig.  3c).

To quantify these spectral changes, we deter-

mined the parameter  R, defined as the ratio of the

total yellow-green fluorescence intensity (510-575  nm)

to the total orange-red fluorescence intensity (575-

715  nm). The value of R increased progressively

with the proportion of oxidized bis-retinoids in the

model lipofuscin (Fig.  3d). Although this relationship

was established based on changes in the fluores-

cence properties of a single lipofuscin fluorophore,

A2E, the resulting calibration curve can be used

to estimate the overall content of oxidized fluoro-

phores in LGs. This is possible because, despite the

variability in the excitation maxima of different

bis-retinoids, their emission maxima are relative-

ly similar [48]. In our previous studies [43, 49], we

demonstrated that irradiation of native LGs con-

taining a mixture of bis-retinoids produces effects

closely resembling those observed in irradiated A2E

solutions and A2E-containing liposomes. Specifically,

irradiation induces a blue shift of the fluorescence

maximum, reflecting a decrease in the concentration

of native fluorophores due to oxidation and concomi-

tant accumulation of oxidized products that fluoresce

at shorter wavelengths. This characteristic spectral

shift supports the use of A2E as a suitable mod-

el fluorophore for simulating RPE autofluorescence

in  vivo [50].

LGs from human RPE cells. Asmentioned earlier,

irradiation of LGs with visible light leads to changes

in their fluorescence spectrum due to the oxidation

of bis-retinoid fluorophores [51]. Consequently, irra-

diated LGs are expected to contain higher levels of

oxidized fluorophores, leading to an increase in the

ratio (R) of total yellow-green fluorescence intensity

to total orange-red fluorescence intensity. To test this

hypothesis, we determined the R values of LGs iso-

lated from cadaveric human RPE cells. LG samples

were collected separately from donors belonging

to three age groups: group 1, 20-39 years (n  =  18);

group 2, 40-59 years (n  =  21); and group 3, 60-68

years (n  =  19). Each LG sample was additionally ex-

posed to visible light (390-700  nm) for 1, 2, or 3 h.

Table  2 summarizes the R values obtained for LGs

from the three donor age groups as a function of

both donor age and duration of visible-light irradia-

tion.

YAKOVLEVA et al.1424

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Table 2. Comparison of R values for non-oxidized and irradiated LGs from RPE of three age groups of donors

LG sample R = I

ZhS

 − 1*

Age group

Group 1 (20-39 years) Group 2 (40-59 years) Group 3 (60-68 years)

Native (dark) LGs 1.58  ±  0.02 1.64  ±  0.03 1.66  ±  0.03

1-h irradiation 2.44  ±  0.04 2.44  ±  0.03 2.25  ±  0.04

2-h irradiation 2.77  ±  0.05 2.72  ±  0.05 2.88  ±  0.05

3-h irradiation 2.90  ±  0.05 2.88  ±  0.05 2.72  ±  0.05

Note. Fluorescence excitation wavelength, 488nm. *Data are presented as mean±SD for three independent measurements;

p < 0.05.

As shown in Table  2, the R value for native

(non-irradiated) LGs increased slightly with donor

age, which is in good agreement with the HPLC data

presented in Table  1. These findings indicate that the

content of oxidized fluorophores in native (non-irra-

diated) LGs increased with age. Moreover, based on

the calibration curve describing the dependence of R

on the percentage of oxidized A2E in model lipofus-

cin (Fig.  3d), it can be assumed that the amount of

oxidized fluorophores in native LGs isolated from

young donors (group 1, 20-39 years) was negligible.

However, after only 1  h of irradiation, more than

50% of fluorophore (A2E) became oxidized, and af-

ter 3  h of irradiation, the degree of oxidation ap-

proached 100%.

However, it should be noted that, in donors from

the older age group, the range of changes in the

Rvalue during irradiation of native LGs was marked-

ly smaller than in the young and middle-aged groups

(Table  2). Specifically, the increase in R was 84% for

group 1 (from 1.58 to 2.90), 76% for group 2 (from

1.64 to 2.88), and only 64% for group 3 (from 1.66

to 2.72).

This phenomenon is most likely associated with

the age-related accumulation of oxidized fluorophores

in LGs, which alters their susceptibility to further

photooxidation. In other words, LGs from older do-

nors likely contain a higher proportion of already ox-

idized products and, consequently, a lower proportion

of non-oxidized bis-retinoids available for oxidation.

As a result, the light-induced increase in the oxida-

tion indicator R becomes less pronounced with age.

This interpretation is consistent with the findings re-

ported in [52], where the aerobic photoreactivity of

RPE lipofuscin was shown to increase significantly

with age, potentially leading to a higher accumulation

of oxidized bis-retinoids.

Thus, comparative analysis of LG fluorescence

intensity using the ZhS-18 and OS-14 filters provides

a reliable approach for assessing age-related chang-

es in the content of oxidized fluorophores in native

LGs isolated from the RPE of cadaveric donor eyes.

The results obtained with this method demonstrate

an age-dependent increase in the relative content of

oxidized bis-retinoids.

Analysis of the content of oxidized bis-ret-

inoids in LGs from RPE as a function of donor

age using confocal fluorescence microscopy. In this

series of experiments, we investigated isolated RPE

monolayers obtained from cadaveric eyes of different

age donors. Because the fluorescence emission spec-

trum of oxidized bis-retinoids in LGs is shifted toward

shorter wavelengths relative to that of non-oxidized

forms [36], the relative content of oxidized bis-reti-

noids can be estimated by comparing fluorescence in-

tensities measured at two wavelengths. Foreach sam-

ple, fluorescence intensity was recorded at 510  nm

and 540  nm using confocal fluorescence microscopy

(Fig.  4,  a  and  b), and the ratio of these values was

calculated (see Materials and Methods). For compar-

ative analysis, data from all samples were grouped

into three age categories (Fig.  4c, Table  3).

It can be concluded from Fig.  4c that the relative

content of oxidized forms of bis-retinoids increased

with age. Although the observed changes were mod-

est, they were statistically significant.

In addition, full fluorescence spectra were re-

corded at individual points on the RPE monolayer

surface (Fig.  4d). These spectra revealed the same

trend observed in the steady-state fluorescence spec-

tra of LG chloroform extracts, namely, a slight blue

shift of the fluorescence maximum with increasing

donor age (Fig.  2a).

The obtained values of the fluorescence intensi-

ty ratio at 510 and 540  nm partially agree with the

data reported in [53]. In that study, the authors an-

alyzed the fluorescence intensity ratio (excitation at

488  nm) between two spectral ranges, 515-575  nm

AGE-RELATED CHANGES IN BIS-RETINOIDS OF LIPOFUSCIN GRANULES 1425

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Fig. 4. Confocal fluorescence microscopy of RPE monolayers from cadaveric donor eyes. Representative fluorescence intensi-

ty maps of individual RPE monolayers acquired at×20 magnification with excitation at488 nm and emission at 510nm(a)

and540nm(b). c)The ratio of the total fluorescence intensity recorded at a wavelength of510nm to the total fluorescence

intensity recorded at a wavelength of 540 nm. Data are presented as mean ± standard error of mean (SEM); * p < 0.05.

d)Averaged fluorescence emission spectra of the RPE monolayer; excitation at488nm: 1,donors aged 20-30years (n=10);

2, donors aged 60-65 years (n = 7). The spectra were normalized to the fluorescence intensity at 592 nm.

and 575-650  nm, across different age groups. Al-

though the reported values (0.64-0.75) were close to

those obtained in our study, the published data repre-

sented only the extreme values corresponding to RPE

donors aged 10 and 99 years. Furthermore, approxi-

mately half of the age-dependent data were derived

from the analysis of only a single sample for each

age group. Importantly, the authors did not observe a

consistent age-related increase in the fluorescence in-

tensity ratio across the selected spectral ranges (0.64,

0.68, 0.71, 0.67, 0.67, 0.60, 0.65, and 0.75 for donors

aged 10-99 years). In contrast, our study included a

substantially larger number of samples, providing

greater statistical reliability.

Thus, confocal fluorescence microscopy analysis

of oxidized bis-retinoids in LGs isolated from the RPE

of cadaveric donor eyes demonstrated a significant

age-related increase in the relative content of these

compounds.

Measurement of fluorescence lifetime of LGs

from RPE cells as a function of donor age using

fluorescence decay kinetics recording. In this ex-

periment, we used the TCSPC technique to evaluate

fluorescence lifetimes of LGs isolated from RPE cells.

The LG suspension samples of three age groups were

analyzed. Figure  5 presents the fluorescence decay

kinetics recorded for LG suspensions from group 1

(17-32 years, n  =  64), group 2 (33-63 years, n  =  56),

and group 3 (65-79 years, n  =  43). The fluorescence

decay curves were fitted using a three-exponential

decay model, allowing determination of the charac-

teristic fluorescence lifetimes and their relative con-

tributions (Table  4).

Table 3. The ratio of the total fluorescence intensity

of RPE monolayers recorded at a wavelength of 510nm

to the total fluorescence intensity recorded at a wave-

length of 540 nm (confocal fluorescence microscopy)

Age group Ratio of total

fluorescence intensity:

Σ  (510  nm)/Σ  (540  nm)*

(Fig.  4c)

Group 1, 20-39 years

(n = 34)

0.639  ±  0.008

Group 2, 40-59 years

(n = 36)

0.656  ±  0.006

Group 3, 60-70 years

(n = 30)

0.671  ±  0.005

Note. Fluorescence excitation wavelength, 488 nm. * Data

are presented as mean  ±  SD for three independent measure-

ments; p  <  0.05.

YAKOVLEVA et al.1426

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

Fig. 5. a)Fluorescence decay kinetics of LG suspensions: 1,donor age, 17-32years (n=64); 2,donor age, 33-63years (n=56);

3,donor age, 65-79years (n=43). IRF, instrument response function; fluorescence excitation wavelength, 490 nm; emission

wavelength, 540 nm. b) Distribution of fluorescence lifetimes (τ

) by age groups specified in panel (a). c) Distribution of

fluorescence intensity by age groups specified in panel (a); * p < 0.05.

Table 4. Characteristic fluorescence lifetimes (τ

) and their contributions (I

) for LGs from three age groups

Age group τ

, ns τ

, ns I

, % I

, % t

 + I

)/I

Group 1

17-32 years, n=64

0.18  ±  0.007 0.88  ±  0.01 3.4  ±  0.05 50  ±  0.6 39  ±  0.7 11.8  ±  0.2 0.31 1.02

Group 2

33-63 years, n=56

0.3  ±  0.01 1.0  ±  0.03 4.4  ±  0.07 32  ±  1.8 48  ±  1.1 19.6  ±  0.5 0.63 2.11

Group 3

65-79 years, n=43

0.4  ±  0.01 1.2  ±  0.07 4.9  ±  0.30 32  ±  2.1 49  ±  2.2 19.0  ±  1.1 0.80 2.12

Note. Fluorescence excitation, 490 nm; fluorescence emission, 540 nm. Samples represented total LG suspensions. Data are

presented as mean ± SD for three independent measurements; p < 0.05.

Analysis of the kinetic curves shows that fluo-

rescence component with a characteristic lifetime

in the range of 0.18-0.40 ns can be attributed to

non-oxidized bis-retinoids, including A2E. The τ

lifetime component (0.88-1.2 ns) is characteristic of

weakly oxidized bis-retinoids, whereas the long-

lived component τ

(3.4-4.9 ns) corresponds to highly

oxidized bis-retinoids and their degradation prod-

ucts [28, 29].

The data obtained (Table  4) demonstrate a clear

age dependence of the relative contributions (I

) as-

sociated with the characteristic fluorescence life-

times (τ

). Specifically, the contribution of the short-

lived component τ

decreases with age, indicating

a reduction in the relative content of non-oxidized

bis-retinoids. Incontrast, the contributions of the lon-

ger-lived components, τ

and τ

, increase with age,

reflecting the accumulation of oxidized bis-retinoid

species. These findings provide direct evidence of

the age-related shift toward a higher proportion of

oxidized bis-retinoids.

In addition, both the mean fluorescence life-

time (t

) and the ratio representing the relative

contribution of oxidized forms, (I

 + I

)/I

, increased

with age, further supporting the conclusion that

the content of oxidized bis-retinoids rises over time

(Table  4). Previous studies have reported that patho-

logical conditions are associated with elevated mean

fluorescence lifetimes (t

) compared with normal

tissue [26]. Our results similarly demonstrate an

age-dependent increase int

, suggesting that aging is

accompanied by progressive accumulation of oxidized

bis-retinoids.

CONCLUSION

This study reports age-related changes in the

composition of fluorophores (bis-retinoids and their

oxidation and degradation products) in LGs from RPE

cells of cadaveric eyes from different age donors. The

investigated materials included chloroform extracts

AGE-RELATED CHANGES IN BIS-RETINOIDS OF LIPOFUSCIN GRANULES 1427

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

of LGs, suspensions of native LGs, and LG-containing

RPE cell monolayers derived from human cadaveric

eyes. Fluorescence spectroscopy, HPLC, confocal fluo-

rescence microscopy, and fluorescence lifetime mea-

surements (TCSPC) were employed.

HPLC analysis demonstrated an age-associated

increase in the relative abundance of oxidized bis-ret-

inoids compared with non-oxidized forms, as reflect-

ed by the increase in the ratio of total oxidized to

non-oxidized species (0.48  →  0.68; Table  1). Fluores-

cence spectroscopy and confocal microscopy revealed

an increased contribution of the short-wavelength

emission relative to long-wavelength emission with

age (Tables  2 and  3). Fluorescence lifetime measure-

ments further showed an age-dependent increase in

mean lifetime from 0.31 to 0.80  ns (Table  4). Collec-

tively, these spectral and temporal changes indicate

an increasing contribution of oxidized bis-retinoids to

the overall fluorescence signal in aging LGs.

However, the data obtained using different meth-

odologies do not allow for a reliable quantitative com-

parison of age-dependent changes in the content of

oxidized bis-retinoids. Several factors contribute to

this limitation. First, sample collection required an ex-

tended period of time (over five years) to obtain suffi-

cient material, making it impossible to generate fully

matched age cohorts across all experimental modal-

ities due to logistical and social constraints. Second,

differences in sample preparation and varying de-

grees of sample preservation may have influenced the

measured parameters. Third, the non-invasive nature

of FAF spectral analysis (an approach aligned with

personalized medicine) may further contribute to the

inter-sample heterogeneity. Therefore, the variability

of measured parameters can be significant. These fac-

tors complicate the establishment of robust diagnostic

thresholds distinguishing normal aging from patholo-

gy and explain why no standardized, widely accepted

diagnostic method based on FAF spectral analysis is

currently available. Nevertheless, active efforts in this

direction are underway in multiple research laborato-

ries [54], given its potential for preclinical detection

of degenerative processes in the retina and RPE.

In summary, our work provides the first evidence

that aging is associated with an increased relative

contribution of bis-retinoid oxidation products in LGs

compared with non-oxidized forms. These findings

suggest that the development of preclinical diagnostic

approaches in ophthalmology based on FAF spectral

analysis must account for age-related alterations in

the qualitative and quantitative composition of LG

fluorophores in RPE cells. Aging in RPE cells is ac-

companied, among other processes, by the accumula-

tion of LGs containing photo-oxidizable bis-retinoids,

leading to the increased formation of cytotoxic deg-

radation products, including aldehydes and ketones.

Abbreviations

AMD age-related macular degeneration

A2E N-retinylidene-N-retinylethanolamine

FAF fundus autofluorescence

FLIO fluorescence lifetime imaging

ophthalmoscopy

HPLC high-performance liquid

chromatography

LG lipofuscin granule

RPE retinal pigment epithelium

TCSPC time-correlated single-photon

counting

Acknowledgments

The work used equipment (FluoTime 300 fluorime-

ter) of the New Materials and Technologies Center of

Collective Equipment Use of the Emanuel Institute of

Biochemical Physics.

Contributions

M.A.O., A.E.D., M.A.Ya., and T.B.F. developed the con-

cept and supervised the study; M.A.Ya., A.A.K., N.L.A.,

P.M.Sh., S.A.B., and A.E.D. conducted experiments;

T.B.F. wrote the text of the article; M.A.O., A.E.D.,

M.A.Ya., V.A.K., and T.B.F. edited the manuscript.

Funding

The work was supported by the Ministry of Science

and Higher Education of the Russian Federation (proj-

ect no. 122041400102-9) and State Assignment to the

Lomonosov Moscow State University. Spectral and

kinetic studies were carried out using equipment of

the New Materials and Technologies Shared Research

Center of the Institute of Biochemical Physics (project

no. 125020501478-5).

Ethics approval and consent to participate

Experiments using cadaveric donor material (eyes)

were conducted in compliance with the Law of the

Russian Federation no.  4180-I of December 22, 1992

“On Transplantation of Human Organs and/or Tis-

sues” (with the latest amendments and additions of

December 8, 2020); Section II “Removal of Organs

and/or Tissues from Cadavers”; Clause 8 “Presumption

of Consent for the Removal of Organs and/or Tissues”;

Clause 10 “Permission for the Removal of Organs and/

or Tissues from Cadavers.”

According to the license L041-00110-47100574034

issued by the Federal Service for Surveillance of

Healthcare of the Russian Federation on April7, 2020,

and Permit no. 2010.243 issued by the Federal Service

for Surveillance in Healthcare and Social Development

on June 24, 2010, the Eye Tissue Bank at the Svya-

toslav Fyodorov Eye Microsurgery Complex receives

cadaveric eyes from the thanatology departments of

the Moscow Bureau of Forensic Medical Examination.

YAKOVLEVA et al.1428

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026

These authorizations permit the use of ocular tis-

sues obtained postmortem for both transplantation

and scientific research purposes. In accordance with

the Scientific Cooperation Agreement no.  137 dated

June8, 2011 between the Center for Fundamental and

Applied Biomedical Problems of the Eye Microsurgery

Complex and the Emanuel Institute of Biochemical

Physics, approval was granted by the Chief Physician

of the Eye Microsurgery Complex to conduct exper-

imental studies in the Laboratory of Physicochem-

ical Foundations of Receptor Processes at Institute

of Biochemical Physics using RPE derived from ca-

daveric eyes. Prior to tissue use, donor material was

subjected to mandatory serological screening for HIV

types I and II, hepatitis B and C viruses, and syphilis.

Following corneal retrieval for transplantation pur-

poses, the remaining ocular tissues were transferred

by the Eye Tissue Bank to Institute of Biochemical

Physics in a certified container designed for the trans-

port of human biological materials. After isolation of

the RPE, the remaining cadaveric ocular material was

returned to the Eye Tissue Bank for disposal in ac-

cordance with established bioethical and regulatory

procedures.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

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