Glucoselysine is derived from fructose and accumulates in the eye lens of diabetic rats

Article abstract of DOI:10.1074/jbc.RA119.010744

Prolonged hyperglycemia generates advanced glycation endproducts (AGEs), which are believed to be involved in the pathogenesis of diabetic complications. In the present study, we developed a polyclonal antibody against fructose-modified proteins (Fru-P antibody) and identified its epitope as glucoselysine (GL) by NMR and LC-electrospray ionization (ESI)- quadrupole TOF (QTOF) analyses and evaluated its potential role in diabetes sequelae. Although the molecular weight of GL was identical to that of fructoselysine (FL), GL was distinguishable from FL because GL was resistant to acid hydrolysis, which converted all of the FLs to furosine. We also detected GL in vitro when reduced BSA was incubated with fructose for 1 day. However, when we incubated reduced BSA with glucose, galactose, or mannose for 14 days, we did not detect GL, suggesting that GL is dominantly generated from fructose. LC-ESI-MS/MS experiments with synthesized [¹³C₆]GL indicated that the GL levels in the rat eye lens time-dependently increase after streptozotocininduced diabetes. We observed a 31.3-fold increase in GL 8 weeks after the induction compared with nondiabetic rats, and N_∈-(carboxymethyl)lysine and furosine increased by 1.7- and 21.5-fold, respectively, under the same condition. In contrast, sorbitol in the lens levelled off at 2 weeks after diabetes induction. We conclude that GL may be a useful biological marker to monitor and elucidate the mechanism of protein degeneration during progression of diabetes.

Full text of DOI:10.1074/jbc.RA119.010744

cro
ARTICLE
Glucoselysine is derived from fructose and accumulates in
the eye lens of diabetic rats
Received for publication, August 21, 2019, and in revised form, October 4, 2019 Published, Papers in Press, October 8, 2019, DOI 10.1074/jbc.RA119.010744
Rei-ichi Ohno‡, Kenta Ichimaru§, Seitaro Tanaka§, Hikari Sugawa‡, Nana Katsuta§, Shiori Sakake§, Yu-ki Tominaga§,
Ikuho Ban¶, Jun-ichi Shirakawa¶, X Yoshiki Yamaguchiʈ, Emi Ito**, Naoyuki Taniguchi‡‡, and X Ryoji Nagai‡§¶1
From the ‡Laboratory of Food and Regulation Biology, Graduate School of Bioscience, the §Laboratory of Food and Regulation Biology,
Graduate School of Agriculture, and the ¶Laboratory of Food and Regulation Biology, Department of Bioscience, School of Agriculture,
Tokai University, 9-1-1 Toroku, Kumamoto, Higashi-ku, Kumamoto 862-0970, Japan, the ʈLaboratory of Pharmaceutical Physical
Chemistry, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan, the
**Department of Diabetic Complications, Diabetes Research Center, Research Institute National Center for Global Health and Medicine,
1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan, and the ‡‡Department of Glyco-Oncology and Medical Biochemistry, Osaka
International Cancer Institute, 3-1-69 Otemae, Chuoku, Osaka 541-8567, Japan
Edited by Gerald W. Hart
Prolonged hyperglycemia generates advanced glycation end-
products (AGEs), which are believed to be involved in the patho-
genesis ofdiabeticcomplications. Inthepresentstudy, we devel-
oped a polyclonal antibody against fructose-modified proteins
(Fru-P antibody) and identified its epitope as glucoselysine (GL)
by NMR and LC-electrospray ionization (ESI)- quadrupole TOF
(QTOF) analyses and evaluated its potential role in diabetes
sequelae. Although the molecular weight of GL was identical to
that of fructoselysine (FL), GL was distinguishable from FL
because GL was resistant to acid hydrolysis, which converted all
of the FLs to furosine. We also detected GL in vitro when
reduced BSA was incubated with fructose for 1 day. However,
when we incubated reduced BSA with glucose, galactose, or
mannose for 14 days, we did not detect GL, suggesting that GL is
dominantly generated from fructose. LC-ESI-MS/MS experi-
ments with synthesized [13C6]GL indicated that the GL levels in
the rat eye lens time-dependently increase after streptozotocin-
induced diabetes. We observed a 31.3-fold increase in GL 8
weeks after the induction compared with nondiabetic rats, and
N⑀-(carboxymethyl)lysine and furosine increased by 1.7- and
21.5-fold, respectively, under the same condition. In contrast,
sorbitol in the lens levelled off at 2 weeks after diabetes induc-
tion. We conclude that GL may be a useful biological marker to
monitor and elucidate the mechanism of protein degeneration
during progression of diabetes.
amino and thiol groups of amino acids to form advanced glyca-
tion end-products (AGEs) through the Schiff base and Amadori
products of the Maillard reaction (1). Accordingly, AGEs are
used as markers for carbohydrate metabolism in vivo. For
instance, hemoglobin A1c (HbA1c) (2), glycoalbumin (3, 4),
and fructoselysine (FL) (5) are well-known glycated proteins
and lysed by glucose, serving as clinical markers for the diagno-
sis of diabetes. The modification of proteins by glycation and
AGEs denatures the protein structures, thereby reducing enzy-
matic activity (6–9). Recent studies have demonstrated that
AGEs accumulate in tissues during aging (10–12) as well as
under pathological conditions such as diabetic complications,
including kidney failure (13, 14), retinopathy (15–17), neurop-
athy (18), and atherosclerosis (19, 20).
We previously reported that N⑀-(carboxymethyl)lysine
(CML), a major antigenic AGE, was generated by oxidation
with hydroxyl radicals (21), hypochloric acid (22), and per-
oxynitrite (23), demonstrating its role as an oxidative marker.
Thornalley et al. (14) demonstrated that the level of the MG-
␦
derived AGE, N -(5-hydro-5-methyl-4-imidazolon-2-yl)-orni-
thine is increased in the plasma of patients with kidney failure.
We also previously reported that a glycolaldehyde (GA)-de-
rived AGE, GA-pyridine accumulates in human atherosclerosis
⑀
lesions (19). CML and N -(carboxyethyl)lysine (CEL) are
known to accumulate in the human lens with aging (10–11).
These reports demonstrated the variation in the AGEs gen-
erated in different tissues and under different pathological con-
ditions. Therefore, we hypothesized that levels of AGEs or
glycated products could be a valuable marker for metabolic
abnormalities.
Reducing sugars and carbonyl compounds, such as glucose
and methylglyoxal (MG),2 nonenzymatically react with the
Although extensively researched, we focused on fructose-
derived AGEs that accumulate in the lens. We previously devel-
oped a polyclonal antibody against fructose-modified protein
and showed that the epitope of the antibody accumulates in the
lens of diabetic rats and the content correlated with the amount
of sorbitol in the lens (24). Because activation of the polyol
The authors declare that they have no conflicts of interest with the contents
of this article.
1 To whom correspondence should be addressed: Laboratory of Food and
RegulationBiology, DepartmentofBioscience, School ofAgriculture, Tokai
University Toroku 9-1-1, Higashi-ku, Kumamoto City, Kumamoto 862–
8652, Japan. Tel./Fax: 81-96-386-2692; E-mail: nagai-883@umin.ac.jp.
2 The abbreviations used are: MG, methylglyoxal; ACN, acetonitrile; AGE,
advanced glycation end-product; RdBSA, reduced bovine serum albumin;
LC-ESI, liquid chromatography-electrospray ionization; LC-ESI-QTOF, LC-
ESI-quadrupole-timeofflight;BCA, bicinchoninicacid;Cbz, carboxybenzyl;
CML, N⑀-(carboxymethyl)lysine; FA, formic acid; FL, fructoselysine; Fru-P,
fructose-modified protein; GL, glucoselysine; HbA1c, hemoglobin A1c;
KLH, keyhole limpet hemocyanin; MRM, multiple reaction monitoring; TBS,
Tris-buffered saline; GA, glycolaldehyde; STZ, streptozotocin; HSQC, het-
eronuclear single quantum coherence; HMBC, heteronuclear multiple
bond correlation.
17326 J. Biol. Chem. (2019) 294(46) 17326–17338
© 2019 Ohno et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of glucoselysine in diabetic lens
Table 1
Biochemical parameters of rats after the induction of diabetes
Weeks after STZ injection
0
1
2
3
4
6
8
Body weight
Normal (n ϭ 6)
DMa (n ϭ 6)
Blood glucose
Normal (n ϭ 6)
DM (n ϭ 6)
HbA1c
157.2 Ϯ 5.2
157.1 Ϯ 5.8
201.7 Ϯ 7.4
231.7 Ϯ 9.3
277.5 Ϯ 12.3
235.7 Ϯ 19.0
307.7 Ϯ 15.0
254.0 Ϯ 21.2
362.2 Ϯ 20.9
257.0 Ϯ 44.5
401.0 Ϯ 24.9
281.0 Ϯ 46.9
183.3 Ϯ 13.3
201.9 Ϯ 19.2
87.3 Ϯ 17.0
99.1 Ϯ 35.3
63.1 Ϯ 27.4
98.3 Ϯ 7.6
89.7 Ϯ 20.3
105.7 Ϯ 9.1
105.8 Ϯ 8.8
211.0 Ϯ 38.4
537.1 Ϯ 68.6
321.3 Ϯ 18.7
384.1 Ϯ 39.7
380.8 Ϯ 84.6
461.5 Ϯ 38.5
496.4 Ϯ 38.2
Normal (n ϭ 6)
DM (n ϭ 6)
NDb
ND
3.1 Ϯ 0.1
4.5 Ϯ 0.2
ND
ND
ND
ND
ND
3.7 Ϯ 0.1
9.2 Ϯ 0.7
5.8 Ϯ 0.3
7.5 Ϯ 0.2
8.6 Ϯ 0.8
a DM, diabetic group.
b ND, not determined.
pathway is enhanced under a hyperglycemic condition and is CML in the rat lens increased by 5.9- (Fig. 1G) and 1.1-fold (Fig.
known to be associated with several diabetic complications, 1H), respectively, following the induction of diabetes.
such as retinopathy, neuropathy, and kidney failure (25), the
Identification of the epitope structure of Fru-P antibody
accurate measurement of fructose-derived AGEs may clarify
the mechanism of the development and progression of such
complications. However, the epitope structure of the poly-
clonal antibody has not been identified since this first report
more than 20 years ago (24).
To analyze the epitope structure of the Fru-P antibody, the
antibody reactivity was measured by noncompetitive ELISA. As
shown in Fig. 2A, Fru-P antibody reacted with fructose-modi-
fied BSA (Fru-BSA) and keyhole limpet hemocyanin (KLH) in a
dose-dependent manner, whereas the reactivity to BSA modi-
fied with other aldehydes and native BSA was negligible. After
incubation of fructose along with the basic amino acids, lysine
and arginine, which are considered to be preferentially modi-
fied by aldehydes (26), the antibody was found to react only with
fructose-modified acetyl-lysine in a dose-dependent manner
(Fig. 2B), suggesting that its epitope structure was generated
from lysine.
In the present study, we prepared a new antibody by a
method different from a previously reported one designated as
Fru-P antibody. Then we evaluated reactivity of its antibody
with lens proteins of streptozotocin (STZ)-induced diabetic
rats. We also compared reactivity of CML antibody in the same
rats. Furthermore, the epitope structure of Fru-P antibody was
identified and a system was developed for its quantification
with LC-ESI-QTOF and LC-ESI-MS/MS to investigate its rela-
tionship to diabetes.
Carboxybenzyl (Cbz)-lysine was used to produce the epitope
structure and the eluent was monitored with a UV detector at
270 nm, which is a characteristic of the Cbz group (first-step
purification). Cbz-lysine was eluted at a retention time of
26–27 min, and the reaction mixture of Cbz-lysine and fructose
generated several peaks, four of which were isolated (Fig. 2C).
By competitive ELISAs, the reactivity of Fru-P antibody to
coated Fru-BSA showed significant competition with Fru-BSA
(as a positive control) and fraction 2 (Fig. 2D). Because fraction
2 contained at least two peaks, the reaction mixture was ana-
lyzed with a different elution program to improve the separa-
tion efficiency. As shown in Fig. 2E, Cbz-lysine was eluted at a
retention time of 40–42 min. Fractions 2-1 and 2-2 were iso-
lated and their reactivities were measured against the antibody.
As a result, the reaction of Fru-P antibody to coated Fru-BSA
was competed only by fraction 2-1 and fraction 2 (as a positive
control) (Fig. 2F), demonstrating that fraction 2-1 is the epitope
of Fru-P antibody.
Results
Reactivity of Fru-P antibody with rat lens proteins
Type 1 diabetes was induced by STZ injection in Wistar rats,
and the change in GL in the lens proteins was compared with
other biological parameters. As shown in Table 1, the body
weight of normal rats showed a steep increase in a time-depen-
dent manner, whereas body weight of diabetic rats increased
more gradually, with ϳ70.1% lower body weight than normal
rats after 8 weeks. The fasting blood glucose levels of diabetic
rats dramatically increased after the induction of diabetes, and
gradually increased during the feeding period. In contrast, the
blood glucose level of the normal rats did not change during
the feeding period except at 8 weeks (Table 1). Furthermore, the
levels of HbA1c increased during the feeding period in the dia-
betic rats; however, it did not change even at 8 weeks (Table 1)
in normal rats. Western blotting showed that the levels of the
epitope of the Fru-P antibody in the rat lens dramatically
increased by the induction of diabetes (Fig. 1A), whereas the
CML level was hardly affected by diabetes onset (Fig. 1D). The
NMR structural analysis
NMR analysis was conducted to determine the structure of
results of densitometric analysis of the bands detected by West- fraction 2-1, formed from the condensation of Cbz-lysine and
ern blotting demonstrated that the epitope of Fru-P antibody fructose. The one-dimensional 1H NMR spectrum showed the
and CML increased 4.3- (Fig. 1B) and 1.1-fold (Fig. 1E), respec- presence of two anomeric signals at 5.53 and 5.03 ppm (Fig. 3A),
tively, following the induction of diabetes. The residual proteins suggesting that the sugar is in an equilibrium between ␣ and ␤
in the polyacrylamide gel, stained by Coomassie Brilliant Blue hemiacetal forms. The coupling constants of each anomeric
as a loading control, showed no difference (Fig. 1, C and F). signal were 3.3 and 8.4 Hz, respectively, suggesting that the
Furthermore, the levels of AGEs measured by ELISA also dem- sugar is a glucose-like and not a mannose-type configuration.
onstrated that the epitope structure of the Fru-P antibody and The NMR signals were assigned by a series of one- and two-
J. Biol. Chem. (2019) 294(46) 17326–17338 17327

Identification of glucoselysine in diabetic lens
Figure 1. Reactivity of Fru-P antibody or CML antibody to the rat lens proteins after the induction of diabetes. Lens proteins (10 ␮g/lane) were applied
to 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The proteins bound to the membrane were detected by Western blot using Fru-P
antibody (A) or CML antibody (D) and were quantified by densitometric analysis (B and E). Residual proteins on the polyacrylamide gel after blotting were
stained by Coomassie Brilliant Blue as a loading control (C and F). Each well of a 96-well immunoplate was coated with 10 ␮g/ml of lens protein, and the
reactivity of 0.5 ␮g/ml of Fru-P antibody (G) or CML antibody (H) were visualized by horseradish peroxidase-conjugated anti-rabbit IgG antibody and 1,2-
phenylenediamine dihydrochloride as described under “Experimental procedures.” Nor, normal at 8 weeks (n ϭ 3); DM, diabetes at 8 weeks (n ϭ 3). The data
are presented as mean Ϯ S.D. #, p Ͻ 0.001 versus normal at 8 weeks (Student’s t test).
dimensional experiments and the assignments are shown in composition, the chemical properties of these compounds were
Fig. 3B. To identify the linkage between the sugar and lysine,
1H-13C HMBC and 1H-15N HMBC spectra were collected (Fig.
3, C and D). Several HMBC peaks were successfully observed
between sugar and the lysine side chain. Taken together, it was
concluded that the ⑀-amino group of the lysine side chain is
connected to the C2 position of glucose in the Fru-P antibody
epitope (Fig. 3E). Therefore, the epitope of Fru-P antibody is
also referred to hereafter as glucoselysine (GL).
compared. As shown in Fig. 5A, GL was stable under acid hy-
drolysis against 6 M HCl at 100 °C for 48 h, whereas all of the FL
was degraded within 18 h under the same condition. Further-
more, FL was degraded by acid hydrolysis in a time-dependent
manner up to 4 h, and furosine was generated by 1 h incubation
with HCl and a complete conversion was seen as early as 4 h
(Fig. 5B). To compare the yields of FL and GL, they were incu-
bated with BSA in the presence of fructose, glucose, galactose,
or mannose; reduced BSA (RdBSA) was used to exclude the GL
present in native BSA. The level of GL increased in a time-de-
pendent manner under incubation with fructose, but not with
glucose, galactose, or mannose, demonstrating that GL was
preferentially generated from fructose (Fig. 5, C and E). In con-
trast, the level of furosine, the hydrolysate of FL, increased in a
time-dependent manner when RdBSA was incubated with glu-
cose, whereas a small amount of furosine was generated from
fructose (Fig. 5D), suggesting that FL was predominantly gen-
erated from glucose, galactose, and mannose (Fig. 5F). To clar-
ify the stability of GL under oxidative stress, GL was exposed to
hydroxyl radicals because CML is generated by the oxidation of
FL. As shown in Fig. 5G, CML was generated by exposure of FL
Structural analysis by LC-ESI-QTOF
To confirm the structure analysis by NMR, the epitope struc-
ture was also analyzed by ESI-Q-TOF-MS/MS. An ion peak at
ϩ
m/z 443.2025 [M ϩ H] for fraction 2-1 was estimated as
C20H30N2O9 (m/z 443.2024) (Fig. 4A). After the Cbz group was
deprotected by catalytic reduction using palladium carbon, a
peak at m/z 309.1656 (C12H25N2O7) (Fig. 4B) corresponded to a
similar mass-charge ratio of FL and GL. The fragment ions
detected at m/z 309.1656 by MS/MS are shown in Fig. 4C and
Table 2.
Chemical properties of GL and FL
Because GL and FL, generated from fructose and glucose,
respectively, have the same m/z (C12H25N2O7) and elemental to hydroxyl radicals from iron (II) chloride with hydrogen per-
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Identification of glucoselysine in diabetic lens
Figure2. IsolationoftheepitopestructureoftheFru-Pantibody. Aminoacidsproducingtheepitopewereanalyzedandtheepitopestructurewasisolated
by HPLC. A, each well of a 96-well immunoplate was coated with modified proteins, and the reactivity of Fru-P antibody (0.5 ␮g/ml) was visualized by
horseradish peroxidase-conjugated anti-rabbit IgG antibody and 1,2-phenylenediamine dihydrochloride as described under “Experimental procedures.” B,
Fru-BSA (0.01 ␮g/ml) was coated on the immunoplate and then blocked with gelatin. Fifty microliters of each competitor was added in the presence of the
same volume of Fru-P antibody (0.5 ␮g/ml). The antibody bound to the well was visualized as described above. C, 100 ␮l of fructose-modified Cbz-lysine (50
mM) was applied to first-step HPLC and separated into four fractions (Fr. 1–4). D, the reactivity of Fru-P antibody with the four isolated fractions was measured
by competitive ELISA. E, Fr. 2 obtained from first-step HPLC was further applied to second-step HPLC. F, the reactivity of Fru-P antibody with Fr. 2-1 and 2-2
isolated from the second-step HPLC system was measured by competitive ELISA.
oxide. In contrast, the CML level was below the detectable limit increased by 31.3-fold after 8 weeks (Figs. 6, B and C, and 7, B
when GL was exposed to hydroxyl radicals under the same con- and C). Although GL was detectable in rat kidneys, there was no
dition. These data demonstrated that GL does not generate statistical significance between the normal and diabetic groups
CML under oxidative conditions.
(0.341 Ϯ 0.203 mmol/mol of lysine versus 0.402 Ϯ 0.182 mmol/
mol of lysine, mean Ϯ S.D., n ϭ 6 each).
Quantification of sorbitol, furosine, CML, and GL in the rat lens
by LC-ESI-QTOF or LC-ESI-MS/MS
Discussion
The levels of sorbitol, furosine, CML, and GL in lens of dia-
The detailed pathogenic mechanisms and factors related to
betic and normal rats were analyzed by LC-ESI-QTOF or LC- diabetic complications remain elusive; however, the AGEs have
ESI-MS/MS with an internal standard. Measurement of the recently emerged as a strong candidate mediating these effects
fructose level in lens was not very stable probably because it (25). Although several AGEs structures have been identified in
rapidly reacts with proteins to form a protein-adduct. There- biological samples, CML is the most measured AGE in the
fore, we measured sorbitol, upstream of the formation of fruc- world. For instance, CML accumulates with progression of dia-
tose in the polyol pathway, by LC-ESI-QTOF. The level of sor- betes (27), atherosclerosis (19), and diabetic complications,
bitol reached a maximum and levelled off at 2 weeks after the such as nephropathy (28) and retinopathy (17). The result of
induction of diabetes (Fig. 6A). As shown in Fig. 7A, the quan- Western blotting and ELISA revealed that the Fru-P antibody
titative ions of GL and [13C6]GL were detected by LC-ESI-MS/ showed higher reactivity with diabetic rat lens than anti-CML
MS. The levels of GL significantly increased in a time-depen- antibody. It is likely, therefore, that the epitope of the Fru-P
dent manner (Fig. 7B). Specifically, the levels of furosine and antibody contributed to the modification of lens proteins under
CML increased by 21.5- and 1.7-fold, respectively, at 8 weeks hyperglycemia. Although an antibody against fructose-modi-
after the induction of diabetes, whereas the level of GL fied proteins was first reported by one of our groups in 1998 (24,
J. Biol. Chem. (2019) 294(46) 17326–17338 17329

Identification of glucoselysine in diabetic lens
Figure 3. NMR analysis of the epitope structure recognized by the Fru-P antibody. NMR spectra of fraction 2-1 shown for (A) 1D 1H, (B) 2D 1H-13C HSQC, (C)
2D 1H-13C HMBC, and (D) 2D 1H-15N HMBC. The assignments are labeled for each peak. E, chemical structure of glucoselysine.
29), the epitope structure had not been identified until now. genic AGE (33), or the proteins modified by MG and GA,
Furthermore, although the level of AGEs is a common clinical demonstrating that the epitope structure of the antibody differs
biomarker of diabetes and related complications, the detection from that of AGEs identified to date. Detection of GL by Fru-P
of AGEs in physiological samples with immunological methods antibody may be affected by amino acid sequence and the 3-D
is affected by pretreatment of heating (30), alkaline treatment structure around the GL because the molecular mass of the side
(31), and the presence of autoantibodies against AGEs (32). residue on GL is merely 162 Da (whole molecular mass of GL
Therefore, identification of each AGE structure and precise including lysine is 308 Da).
quantification technology are required to evaluate the biologi-
Heyns et al. (34) proposed that fructose forms lysine-adduct
cal significance of AGEs, which is a limitation for clinical and named it as GL, but did not confirm the structure by instru-
laboratories.
mental analysis. Furthermore, Kawasaki et al. (24) demon-
Here, we demonstrated that Fru-P antibody recognizes fruc- strated that the reactivity of Fru-P antibody to the diabetic rat
tose-modified KLH and BSA, but not their native proteins. Fur- lens is positively correlated with sorbitol levels. Taken together,
thermore, this antibody did not recognize CML, a major anti- these reports suggested that fructose-modified proteins accu-
17330 J. Biol. Chem. (2019) 294(46) 17326–17338

Identification of glucoselysine in diabetic lens
Figure 4. Structural analysis by LC-ESI-QTOF. A, ESI-QTOF analysis of fraction 2-1 showing an ion peak at m/z 443.2024 [M ϩ H]ϩ calculated as C20H30N2O9.
B, ESI-QTOF analysis of de-protected fraction 2-1 showing an ion peak at m/z 309.1656 [M ϩ H]ϩ calculated as C12H25N2O7. C, MS/MS analysis of the de-pro-
tected fraction 2-1 of m/z 309.1656 [M ϩ H]ϩ detected fragment ions are indicated in Table 2.
Table 2
generated from fructose, whereas FL was generated from man-
Identification of glucoselysine fragment ion formulas by LC-ESI-QTOF
nitol, galactose, and glucose. Therefore, GL has potential to
Expected m/z and formula
Detected m/z
Difference of m/z
become a useful marker for protein modifications by fructose.
FL is an Amadori rearrangement product, which is oxidized to
form CML (21). In contrast, GL was not generated from CML
after exposure to hydroxyl radicals, demonstrating its suitabil-
ity for the clinical detection of AGEs. Because blood glucose
levels sometimes transiently rise when animals get excited, lev-
els of FL, measured as furosine in lens (Fig. 6B), and hemoglobin
A1c (Table 1) were also determined to evaluate the variation of
glucose level in the present study.
84.0808 (C5H10N)
84.0799
Ϫ0.0009
Ϫ0.0019
Ϫ0.0044
Ϫ0.0043
Ϫ0.0068
Ϫ0.0030
130.0863 (C6H12NO2)
210.1125 (C11H16NO3)
225.1234 (C11H17N2O3)
255.1339 (C12H19N2O4)
273.1445 (C12H21N2O5)
130.0844
210.1081
225.1191
255.1271
273.1415
mulate in the lens in accordance with progression of the polyol
pathway.
Nakayama et al. (35) and Turk et al. (36) demonstrated that
the level of AGEs measured by the fluorescence intensity of lens
proteins in normal rats did not change between 6 and 14 weeks
of age. In contrast, the level of GL determined by LC-ESI-
MS/MS showed a significant increase in the lens of normal rats
by 8 weeks, suggesting that GL may increase as part of the aging
process. Because the present study focused on identification of
the structure generated from fructose, the presence of the
structure in vivo, and its increase by the pathogenesis of diabe-
tes in rat lens proteins, further study is required to clarify
whether GL increases in accordance with normal aging.
Kawasaki et al. (24) demonstrated that the reactivity of Fru-P
antibody to rat lens proteins increased after the induction of
diabetes for 8 weeks. Surprisingly, in the present study, the
Here, we showed that FL is converted into furosine by acid
hydrolysis, whereas GL is stable under the same conditions.
Although the FL content is typically estimated based on
furosine levels after acid hydrolysis (data not shown), our pre-
vious experiment showed that the parent ion of FL remained
detectable in biological samples even after acid hydrolysis for
18 h. However, because all of the purified FL was converted into
furosine after acid hydrolysis for 4 h, it is likely that the level
detected at m/z 309.1656, corresponding to the parent ion of
FL, in physiological samples after acid hydrolysis reflected the
presence of GL.
The parent ion of GL was also detected in commercial BSA
after acid hydrolysis (data not shown), suggesting that GL may
be generated in bovine serum. To account for the potential
presence of endogenous GL and FL in biological samples, BSA reactivity of Fru-P antibody to the rat lens and the GL content
was reduced with sodium tetrahydroborate before incubation measured by LC-ESI-MS/MS both increased 1 week after the
with several kinds of carbohydrates to clarify the precursors for induction of diabetes. Furthermore, the levels of GL, FL, and
GL and FL. The results showed that GL was predominantly CML increased by 31.3-, 21.5-, and 1.7-fold, respectively, 8
J. Biol. Chem. (2019) 294(46) 17326–17338 17331

Identification of glucoselysine in diabetic lens
Figure5. Chemicalpropertiesofglucoselysine(GL)andfructoselysine(FL). ThestabilityofGL(A)andFL(B)andformationoffurosineunderacidhydrolysis
under 6 M HCl at 100 °C for 48 h were measured by LC-ESI-QTOF. The formation of GL (C) and furosine (D) during incubation of reduced-BSA (RdBSA) with
fructose (Fru) or glucose (Glu) at 37 °C for up to 14 days. Comparison of GL (E) and furosine (F) formation during incubation of RdBSA with Fru, Glu, galactose
(Gal), and mannose (Man) at 37 °C for 14 days. G, GL and FL were incubated in 50 mM sodium phosphate buffer (pH 7.2) in the presence or absence of FeCl2 (0.4
mM) and H2O2 (0.1 mM) at 37 °C for 1 h, and CML formation was determined by LC-ESI-QTOF. The data are presented as mean Ϯ S.D. (n ϭ 3). #, p Ͻ 0.001 versus
control or Fru (Bonferroni test).
weeks after the induction of diabetes. There has been no previ- predictive markers of cataract. In this study, we focused on the
ous report of an AGE structure increasing by ϳ20-fold within 8
weeks after the onset of diabetes. These results demonstrated
that GL in the lens accumulates in accordance with the progres-
sion of the polyol pathway; the increasing rate was higher than
FL and CML. Therefore, GL accumulation reflects the degen-
eration of lens proteins by fructose at an early stage. However,
the level of GL in the rat kidney did not increase by the induc-
tion of diabetes. We have previously reported that the total 24-h
urinary protein and albumin concentrations at 27 weeks were
approximately five and six times higher, respectively, in dia-
betic rats compared with nondiabetic rats (27). Because rats
were maintained for only 8 weeks after the induction of diabetes
in the present study, kidney function may have been still
uncompromised and GL levels in the kidneys did not alter
significantly.
association between GL accumulated in the rat lens and pro-
gression of diabetes, so the association with diabetic complica-
tions was not evaluated. Because GL shows a distinctly higher
rate of increase than other AGEs, it may increase with the onset
and progression of diabetic complications such as cataract. To
clarify the possibility of using GL as a marker for polyol-related
diabetic complications, further study is required to investigate
its association with various durations of diabetes and related
complications.
Experimental procedures
Sample preparation
GA-modified BSA and MG-modified BSA were prepared by
incubating 2 mg/ml of BSA (1.5 mM lysine residues; initial frac-
tion by heat shock, minimum 98%; Sigma) with 30 mM GA or
MG (Sigma), respectively, at 37 °C for 7 days in PBS (26). To
prepare the Fru-BSA, 10 mg/ml of BSA was mixed with 0.37 M
D-fructose (Kanto Chemical Co., Inc., Tokyo, Japan), and the
solution was lyophilized, followed by incubation at 90 °C for 30
min (39). CML-BSA was prepared as described previously (26).
In the previous report, crystallin protein in the lens was
shown to be denatured by MG, ribose, galactose, or fructose
inducing aggregation, which is one of the causes of cataract. It
has been reported that these aldehyde or hexoses form MG-H1,
CEL, or CML (37, 38) suggesting that AGEs reflect the progres-
sion of cataract. However, these AGEs in the lens did not cor-
relate with the development of cataract and they cannot be In brief, 10 mg/ml of BSA was incubated at 37 °C for 24 h with
17332 J. Biol. Chem. (2019) 294(46) 17326–17338

Identification of glucoselysine in diabetic lens
Figure 6. Quantification of sorbitol, furosine, and CML in the rat lens by LC-ESI-QTOF or LC-ESI-MS/MS. Changes in sorbitol (A), furosine (B), and CML (C)
in rat lens proteins were measured by LC-ESI-MS/MS. Nor, normal group (n ϭ 6); DM, diabetic group (n ϭ 6). The data are presented as mean Ϯ S.D. #, p Ͻ 0.001,
versus normal at 1 week (Bonferroni test). In addition, A, *, p Ͻ 0.05, DM at 1 week versus DM 2 weeks. B, #, p Ͻ0.001, DM at 1 week versus DM at 2 weeks; DM at
2 weeks versus 4 weeks. C, #, p Ͻ 0.001, DM at 2 weeks versus DM at 8 weeks (Bonferroni test).
75 mM glyoxylic acid monohydrate (FUJIFILM Wako Pure in a pathogen-free barrier facility (12-h light/dark cycle) and
Chemical., Inc., Osaka, Japan) and 0.45 M NaCNBH3 in 0.2 M were fed a normal rodent chow diet (Clea, Tokyo, Japan). The
sodium phosphate buffer (pH 7.8), followed by dialysis against rats were randomly divided into six groups (n ϭ 6 per cage).
PBS. To prepare the fructose-modified amino acids (Fru- Diabetes was induced in 6-week-old male rats (body weight
␣
acetyl-Lys, Fru-acetyl-Arg), 50 mM N -acetyl-lysine (Tokyo ϳ150 g) by a single intravenous (tail vein) injection of STZ (50
␣
Chemical Industry Co., Ltd.) or N -acetyl-Arg (Sigma) were mg/kg body weight) in 0.2 ml of 0.05 M saline-citrate buffer (pH
mixed with 0.37 M fructose, and the solution was lyophilized. 4.5). Diabetic rats (at 1, 2, 4, and 8 weeks after STZ injection)
The lyophilized sample was incubated at 90 °C for 30 min. and nondiabetic rats (at 1 and 8 weeks after breeding start) were
These modified BSA or amino acids were then subjected to killed by decapitation under anesthesia with isoflurane. Serum
noncompetitive or competitive ELISA using Fru-P antibody.
samples were collected and analyzed to determine the blood
glucose levels by Glucose CII-testwako (FUJIFILM Wako Pure
Chemical) and HbA1c levels were determined by DCA vantage
(SIEMENS Healthineers, Erlangen, Germany). The tissue spec-
imens were immediately frozen and stored at Ϫ80 °C until
analysis.
Preparation of Fru-P antibody
To prepare a polyclonal antibody against Fru-P, 0.1 mg of
Fru-BSA in 50% Freund’s complete adjuvant was injected intra-
dermally on a rabbit, followed by nine booster injections of 0.1
mg of Fru-BSA in 50% Freund’s incomplete adjuvant. Serum
obtained 10 days after the final immunization was precipitated
with ammonium sulfate. The precipitated antiserum protein
was subjected to further affinity column. BSA or Fru-BSA were
coupled to HiTrap NHS-activated HP Column (HiTrap-NHS).
Anti-BSA antiserum was removed from the antiserum by the
BSA-coupled HiTrap-NHS. Anti-Fru-BSA antiserum was puri-
fied by Fru-BSA-coupled HiTrap NHS, Fru-P antibody, which
reacted to Fru-BSA was obtained (40, 41). The reactivity of the
Fru-P antibody against Fru-BSA was measured by ELISA.
Detection of fructose-modified protein by Western blotting
using Fru-P antibody
For the detection of Fru-BSA by Western blot, whole lens
proteins (10 ␮g) were subjected to 12% SDS-PAGE and trans-
ferred onto polyvinylidene fluoride membranes under semi-dry
conditions using a Trans-blot (Bio-Rad). The membrane was
blocked by incubation with 5% BSA in Tris-buffered saline
(TBS; 1 mM Tris-HCl, pH 7.4, and 0.15 M NaCl) at room tem-
perature for 1 h with gentle agitation, and washed three times
with TBS containing 0.05% Tween 20 for 5 min each time. The
membrane was incubated with 0.5 ␮g/ml of Fru-P antibody at
Animal experiments
All animal experiments were approved by Tokai University 4 °C overnight. After washing, the membrane was incubated
(approval number: 181089) and complied with Guidelines for with horseradish peroxidase-conjugated streptavidin diluted to
the Care and Use of Animals for Scientific Purposes at Tokai 1:5000 for 1 h at room temperature and then washed three
University (established April 1, 2007). Wistar rats were pur- times with TBS containing 0.05% Tween 20 for 5 min each time.
chased from Kyudo (Kumamoto, Japan). The rats were housed The chemiluminescence method was employed to amplify the
J. Biol. Chem. (2019) 294(46) 17326–17338 17333

Identification of glucoselysine in diabetic lens
Figure 7. Quantification of GL in the rat lens by LC-ESI-MS/MS. A, typical chromatogram of GL and [13C6]GL in the rat lens obtained by LC-ESI-MS/MS.
Changes in GL (B) in rat lens proteins were measured by LC-ESI-MS/MS. The variability in the rate of CML, furosine, and GL are indicated (C). Nor, normal group
(n ϭ 6); DM, diabetic group (n ϭ 6). The data are presented as mean Ϯ S.D. †, p Ͻ 0.01, versus normal 1 week (Student’s t test); #, p Ͻ 0.001, versus normal at 1
week (Bonferroni test). In addition, B, #, p Ͻ 0.001, DM at 1 week versus DM at 2 weeks; DM at 2 weeks versus DM at 4 weeks; DM at 4 weeks versus DM at 8 weeks
(Bonferroni test).
signal using an Amersham Biosciences ECL Prime system (GE Purification of the epitope structure of Fru-P antibody
␣
Healthcare) (24). The density of detected bands in each lane
was measured using Image Gauge software in LAS-3000 mini
(FUJIFILM, Tokyo, Japan). The part on the gel outside the lanes
was utilized as control, where no protein was applied.
To prepare fructose-modified N -Cbz-lysine (Fru-Cbz-Lys),
␣
70 mM N -Cbz-lysine (Sigma) was mixed with 0.37 M fructose,
and the solution was lyophilized. The lyophilized sample
was incubated at 90 °C for 30 min. For preparative HPLC
(SHIMAZDU, Kyoto: system controller, CBM-20A; pump,
LC-20AD; UV detector, SPD-20A; autosampler, SIL-20A; col-
umn oven, CTO-20AC), 30 ␮l of the Fru-Cbz-Lys was injected
into the Cosmosil Packed column 5C18-AR-II (20 ϫ 250 mm;
Nacalai Tesque Inc., Kyoto, Japan). The column was main-
tained at 40 °C. The mobile phase was 0.1% TFA (FUJIFILM
Wako Pure Chemical, Inc.), with a two-step gradient of ace-
tonitrile (ACN) (0–2.5 min, 10–25% ACN; 2.5–20 min,
25–47.5% ACN; 20–22.25 min, 47.5–60% ACN; 22.25–40
min, 60% ACN) with a flow rate of 3 ml/min. Effluents were
monitored for UV of 270 nm and separated into four fractions
(fraction 1, 21–22.5 min; fraction 2, 24.5–25.5 min; fraction 3,
27–28.5 min; fraction 4, 37.5–39 min). These fractionations
were repeated 20 times, and each fraction was lyophilized. The
lyophilized samples were reconstituted in water, and aliquots of
each fraction were subjected to competitive ELISA using Fru-P
ELISA
ELISAs were performed as described previously (20, 42).
In brief, for noncompetitive ELISA, each well of a 96-well
immunoplate (Thermo Fisher Scientific) was coated with 0.1
ml of the sample in PBS and blocked with 0.5% gelatin
hydrolysate in PBS. The wells were incubated for 1 h with 0.1
ml of the 0.5 ␮g/ml of the Fru-P antibody or CML antibody.
The antibody bound to wells was detected by horseradish
peroxidase-conjugated anti-mouse IgG antibody (Thermo
Fisher Scientific). For competitive ELISAs, each well of the
96-well immunoplate was coated with 0.1 ml of 0.01 ␮g/ml of
Fru-BSA in PBS and blocked with 0.5% gelatin hydrolysate in
PBS. After addition of 50 ␮l of the sample, the same volume
of the Fru-P antibody solution was added to each well. The
antibody bound to wells was detected by horseradish perox-
idase-conjugated anti-mouse IgG antibody.
17334 J. Biol. Chem. (2019) 294(46) 17326–17338

Identification of glucoselysine in diabetic lens
Table 3
antibody as described above. Fraction 2 was confirmed to con-
tain the Fru-P antibody epitope by competitive ELISA. To fur-
ther separate fraction 2, Fru-Cbz-Lys was applied to an HPLC
system with a different gradient condition. The mobile phase
was 0.1% TFA with a two-step gradient of ACN (0–3 min, 10%
ACN; 3–5 min, 10–25% ACN; 5–25 min, 25–27.5% ACN;
25–35 min, 27.5–40% ACN; 35–40 min, 40–60% ACN). Elu-
ents were monitored for UV of 270 nm and separated into two
fractions (fraction 2–1, 34–36 min; fraction 2–2, 36–38 min).
These fractionations were repeated 20 times, and each fraction
was subjected to competitive ELISA using Fru-P antibody.
Fraction 2-1 was then subjected to NMR and LC-ESI-QTOF.
The Cbz group of fraction 2-1 was deprotected by catalytic
reduction using 1 ml of methanol containing 5% palladium car-
bon (Nacalai Tesque Inc., Kyoto, Japan) under a hydrogen
atmosphere (43).
Retention time and m/z values of AGEs, Amadori product, and amino
acids determined by LC-ESI-QTOF
Analyte
Retention time
Parent ion
min
m/z
CML
14.1
205.1183
207.1308
309.1656
315.1858
255.1339
261.1541
147.1128
153.1329
[2H2]CML
GL
14.1
15.7
[
13C6]GL
15.7
Furosine
13C6]Furosine
Lysine
13C6]Lysine
13.5
[
13.5
15.8
[
15.8
40 °C. The mobile phase was 0.1% FA (pH 3.8), with a two-step
gradient of ACN (0–2 min, 90% ACN; 2–16 min, 90–10%
ACN; 16–19 min, 10% ACN). The flow rate was set to 0.2
ml/min and the injection volume was 5 ␮l. GL, CML, furosine,
lysine, and their internal standards were detected by ESI-posi-
tive MS. The [2H2]CML standard was purchased from Poly-
Peptide Laboratories (Strasbourg, France), and the [13C6]lysine
standard was purchased from Cambridge Isotope Laboratories
Inc. (Tewksbury, MA). The retention time and quantitative
ions are indicated in Table 3.
NMR structural analysis
Fraction 2-1 was characterized by NMR using a DRX-500
spectrometer equipped with a cryogenic TXI probe (Bruker
BioSpin, Billerica, MA). The probe temperature was set to 298
K. The sample (fraction 2-1, 5 mg) was dissolved in 600 ␮l of
D2O (99.99 atom% D), and the 1H chemical shifts were reported
relative to the external standard of 4,4-dimethyl-4-silapentane-
1-sulfonic acid. The 13C- and 15N-chemical shifts were cali-
brated using an indirect reference based on the X/1H resonance
ratio of 0.251449530 (13C/1H) and 0.101329118 (15N/1H). NMR
Measurement of sorbitol in the rat lens by LC-ESI-QTOF
LC was conducted on a ZIC௡-HILIC column maintained at
40 °C. The mobile phase was 10 mM ammonium acetate, with
an isocratic flow of 80% ACN. The flow rate was set to 0.2
ml/min and the injection volume was 5 ␮l. Sorbitol and its
internal standard were detected by ESI-negative MS. The
D-[13C6]sorbitol standard was purchased from Cambridge Iso-
tope Laboratories Inc. (Tewksbury, MA). The retention times
of sorbitol and [13C6]sorbitol were 5.9 min, respectively. The
quantitative ions of sorbitol and [13C6]sorbitol were m/z
181.0707 and 189.0908, respectively.
1
signals were assigned by 1D H, 1D-selective TOCSY, 1D-se-
lective NOESY, 1D 13C, 2D 1H-1H DQF-COSY, 1H-13C HSQC,
1H-13C HSQC-TOCSY, 1H-13C HSQC-NOESY, 1H-13C
HSQC-ROESY, 1H-13C HMBC, and 1H-15N HMBC. Data pro-
cessing and analysis were performed using XWIN-NMR (ver-
sion 3.5, Bruker BioSpin). NMR spectra were displayed with
XWIN-PLOT (version 3.5, Bruker BioSpin).
Structural analysis by LC-ESI-QTOF
Measurement of GL and furosine in the rat lens and kidney by
LC-ESI-MS/MS
The pooled elution of fraction 2-1 was dried and resuspended
in 20% ACN containing 0.1% formic acid (FA). The accurate
mass of fraction 2-1 was determined by LC-ESI-QTOF using a
compact mass spectrometer (Bruker Daltonics, Bremen, Ger-
many). The mobile phase was an isocratic flow of 80% ACN
containing 0.1% FA. The flow rate was set to 0.2 ml/min and the
injection volume was 5 ␮l. Instrument calibration was per-
formed externally for each assessment with 50% 2-propanol
containing 5 mM sodium formate. The samples were analyzed
at flow injection by ESI-positive MS multiple reaction monitor-
ing (MRM). The ionization source temperature was 200 °C, and
the capillary voltage was 4.5 kV. Collision-induced dissociation
was performed using nitrogen with the collision energy set to 20
eV and pressure of 1.6 bar. Data were acquired with a stored
mass range of m/z 50–1000. The composition formula of
detected ions was analyzed by SmartFormula manually (Bruker
Daltonics, Bremen, Germany) (14).
LC was conducted using the same conditions as described
above for the measurement of AGEs or Amadori product by
LC-ESI-QTOF. The accurate masses of GL and furosine were
determined by LC-ESI-MS/MS using a TSQ Quantiva system
(Thermo Fisher Scientific) with ESI-positive MRM. The flow
rate was set to 0.2 ml/min and the injection volume was 10 ␮l. A
traditional ESI probe was used to quantify GL and furosine in
the rat lens, and the parameters were optimized as follows:
nitrogen sheath gas, 35 Arb; auxiliary gas, 15 Arb; spray voltage,
3.5 kV; ion transfer tube temperature, 270 °C; and vaporizer
temperature, 250 °C. Argon gas was used as the collision gas
and the pressure was set at 2.0 mTorr. GL content in lens
and kidney was measured by quantitative ion, m/z 210.1
(C11H16NO3), which is one of the fragment ions of GL as shown
in Table 2. The retention time and quantitative ions are indi-
cated in Table 4 (44).
Measurement of CML, GL, furosine in vitro, and CML in the rat
lens by LC-ESI-QTOF
Chemical properties of GL and FL
For evaluation of the stability of GL or FL under acid hydro-
LC was conducted on a ZIC௡-HILIC column (2.1 ϫ 150 mm, lysis, 10 pmol of GL or FL standard was hydrolyzed with 1 ml of
5 ␮m; Merck Millipore, Billerica, MA, USA) maintained at 6 M HCl at 100 °C for 0–48 h. The pooled samples were dried
J. Biol. Chem. (2019) 294(46) 17326–17338 17335

Identification of glucoselysine in diabetic lens
Table 4
Preparation for measurement of CML in the rat lens
Retention time and m/z values of AGEs, Amadori products, and amino
Rat lens samples (0.3 mg each) were used for measurement of
CML. The samples were reduced with NaBH4 (2 ␮l of 1 M
NaBH4 in 0.1 N NaOH) in 20 ␮l of 200 mM sodium borate buffer
(pH 9.1) at room temperature for 4 h. CML and lysine internal
standards were added to the pellets, which were hydrolyzed
with 1 ml of 6 M HCl at 100 °C for 18 h. The dried sample was
resuspended in 1 ml of 0.1% TFA and passed over a Strata-X-C
column, which had been pre-washed with 1 ml of methanol and
equilibrated with 1 ml of the solution, similar to the sample
solvent, and loaded to the column. The column was then
washed with 2% FA and eluted with 20% methanol containing
7% ammonia. The pooled elution fractions were dried and
resuspended in 0.2 ml of 20% ACN containing 0.1% FA. The
samples were then subjected to LC-ESI-QTOF.
acids by LC-ESI-MS/MS
Retention
Collision
energy
Parent
ion
Fragment
ion
Analyte
time
min
15.7
15.7
13.5
13.5
15.8
15.8
eV
18.8
19.0
15.1
15.7
15.0
15.0
m/z
GL
13C6]GL
Furosine
13C6]Furosine
Lysine
13C6]Lysine
309.2
315.2
255.1
261.2
147.1
153.1
210.1
216.1
192.1
198.1
84.0
[
[
[
89.0
and resuspended in 100 ␮l of 20% ACN containing 0.1% FA.
The samples were then subjected to LC-ESI-QTOF.
For formation of GL and FL under physiological condition,
BSA was reduced with 0.1 M NaBH4 and 0.2 M sodium borate
buffer (pH 9.1) at room temperature for 4 h. The RdBSA was
then dialyzed against 0.2 M sodium phosphate buffer (pH 7.4) at
4 °C for 18 h, and the protein concentration was measured by
the bicinchoninic acid (BCA) protein assay (Pierce, Rockford,
IL). RdBSA (1.5 mg/ml) was incubated with 30 mM fructose
(Fru-RdBSA), glucose (Kanto Chemical Co., Inc.; Glu-RdBSA),
galactose (FUJIFILM Wako Pure Chemical., Inc., Osaka, Japan;
Gal-RdBSA), or mannose (Kanto Chemical Company Inc.;
Man-RdBSA) at 37 °C for 0–14 days in 0.2 M sodium phosphate
buffer (pH 7.4). The samples were dialyzed against diluted
water at 4 °C for 18 h, and the protein concentrations were
measured by the BCA assay. Ten micrograms of modified BSA
samples were hydrolyzed with 1 ml of 6 M HCl at 100 °C for 18 h.
The pooled samples were dried and resuspended in 50 ␮l of 20%
ACN containing 0.1% FA. The samples were then subjected to
LC-ESI-QTOF.
Preparation for measurement of GL and furosine in the rat
lens and kidney
Rat lens or kidney samples (0.3 or 0.2 mg each) were used for
measurement of GL and furosine. GL, furosine, lysine, and their
internal standards were added to the samples, which were
hydrolyzed with 1 ml of 6 M HCl at 100 °C for 18 h. The dried
sample was resuspended in 1 ml of 0.1% TFA and filtered
through a Sep-Pak C18 column, which had been pre-washed
with 1 ml of methanol and equilibrated with 1 ml of the solution
similar to the sample solvent. The pooled flow-through frac-
tions and 1 ml of the same solution fractions were dried and
resuspended in 1 ml of 20% ACN containing 0.1% FA. The
samples were then subjected to LC-ESI-MS/MS (44, 45).
Preparation for measurement of sorbitol in the rat lens
One milligram of rat lens samples were used for measure-
ment of sorbitol. The solution volume was adjusted to 0.2 ml
with 1% TFA. The sorbitol internal standard was added to the
solutions, which were filtered by a 10,000 molecular weight
cut-off filter. The filtered solutions (0.1 ml each) were added to
0.9 ml of 1% TFA and passed over a Sep-Pak C18 column, which
had been pre-washed with 1 ml of methanol and equilibrated
with 1 ml of 1% TFA, and then loaded to the column. The
pooled flow-through fractions and 1 ml of the same solution
fractions were dried and resuspended in 0.1 ml of 20% ACN.
The samples were then subjected to LC-ESI-QTOF.
Conversion of GL or FL to CML under oxidative stress
GL or FL (20 pmol) was incubated 37 °C for 1 h with 0.4 mM
FeCl2 in the absence or presence of 0.1 mM H2O2 in 50 mM
sodium phosphate buffer (pH 7.2). The sample solutions were
added to 1 ml of 0.1% TFA and passed over a Strata-X-C col-
umn (Phenomenex, Torrance, CA), which had been pre-
washed with 1 ml of methanol and equilibrated with 1 ml of
0.1% TFA. The samples were loaded to the column and the
column was washed with 3 ml of 2% FA and eluted with 1.5 ml
of 20% methanol containing 7% ammonia. The pooled elution
fractions were dried and resuspended in 0.2 ml of 20% ACN
containing 0.1% FA (21). The samples were then subjected to
LC-ESI-QTOF.
Statistical analysis
Data were expressed as mean Ϯ S.D. (Table 1, Figs. 1 and
5–7). The differences in the levels of GL, CML, sorbitol, and
furosine between the groups were examined for statistical sig-
nificance using the one-way analysis of variance (normal, 1
week versus normal 8 weeks; DM, 1, 2, 4, and 8 weeks). Student’s
t test and subsequent post hoc analysis (Bonferroni correction
method) were used to correct for multiple comparisons.
Preparation for measurement of AGEs and Amadori product in
the rat lens and kidney
The rat lens was homogenized with 0.2 ml of 1 mM diethyl-
enetriamine-N,N,NЈ,NЉ,NЉ-pentaacetic acid (Dojindo Labora-
tories, Kumamoto, Japan) by Shake master auto version 2 (Bio-
medical Science, Tokyo, Japan). The kidneys, cut into several
pieces, removed blood by gentle agitation at 4 °C for 72 h in
PBS. The rat kidney was homogenized with 5 ml of 0.5%
CHAPS/PBS (Dojindo Laboratories, Kumamoto, Japan) by
Polytron PT 10–35 GT (Kinematica, Luzern, Swiss). The pro-
tein concentrations of homogenized samples were measured by
the BCA method.
Author contributions—R.-i. O., K. I., S. T., H. S., N. K., S. S., Y.-k. T.,
I. B., J.-i. S., Y. Y., and E. I. formal analysis; R.-i. O., K. I., S. T., H. S.,
N. K., S. S., Y.-k. T., I. B., J.-i. S., E. I., and R. N. investigation; R.-i. O.
writing-original draft; N. T. conceptualization; N. T. supervision;
N. T. and R. N. writing-review and editing; R. N. funding acquisition;
R. N. methodology; R. N. project administration.
17336 J. Biol. Chem. (2019) 294(46) 17326–17338

Identification of glucoselysine in diabetic lens
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Acknowledgments—We are grateful to Hikari Sato and Mime Nagai
(Department of Bioscience, School of Agriculture, Tokai University)
for their collaborative endeavors.
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17338 J. Biol. Chem. (2019) 294(46) 17326–17338

Glucoselysine is derived from fructose and accumulates in the eye lens of diabetic
rats
Rei-ichi Ohno, Kenta Ichimaru, Seitaro Tanaka, Hikari Sugawa, Nana Katsuta, Shiori
Sakake, Yu-ki Tominaga, Ikuho Ban, Jun-ichi Shirakawa, Yoshiki Yamaguchi, Emi Ito,
Naoyuki Taniguchi and Ryoji Nagai
J. Biol. Chem. 2019, 294:17326-17338.
doi: 10.1074/jbc.RA119.010744 originally published online October 8, 2019
Access the most updated version of this article at doi: 10.1074/jbc.RA119.010744
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