Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : December 14, 2022
Accepted : January 10, 2023
Published online : March 31, 2023
doi:10.24659/gsr.10.1_1

Original article

Determination of AGEs in mice femur by LC-MS/MS
Yoshitaka Hiraoka 1), Hikari Sugawa 2), Ryoji Nagai 1, 2)
1) Laboratory of Food and Regulation Biology, Graduate School of Agriculture,
Tokai University, Kumamoto, Japan
2) Laboratory of Food and Regulation Biology, Department of Food and Life Sciences,
School of Agriculture, Tokai University, Kumamoto, Japan

AbstractLiquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used in the research on advanced glycation endproducts (AGEs), enabling the measurement of several types of AGEs. However, there still remain many unknowns regarding
how AGE accumulate in tissues during aging and age-related diseases, and the relationship between each biological tissue andAGE accumulation. We have established a method for the determination of AGEs in various tissues using mice as an animalmodel and have quantified AGEs in various soft tissues, turning out to be difficult to quantify AGEs in bone tissue, which isa hard tissue, unlike soft tissue processed easily. In this study, we attempted to quantify AGEs in mouse femur using LC-MS/MS. As a result, N ε-(carboxymethyl)lysine (CML) and N δ-(5-hydro-5-methyl-4-imidazolon-2-yl)- ornithine (MG-H1) wereclearly detected in mouse femur, and the amount of CML in femur was significantly higher than that in the crystalline lens.
These results confirm that AGE content is high in tissues such as bone, where metabolism and inflammatory reactions occur
actively through the action of osteoblasts and osteoclasts. The fact that AGEs in hard tissues as well as soft tissues of mice
can now be quantified using LC-MS/MS is expected to make it more effortless to study AGEs in rodents in the future.

KEY WORDS: glycation, N ε-(carboxymethyl) lysine (CML),		

N δ-(5-hydro-5-methyl- 4 -imidazolon-2-yl)-ornithine (MG-H1), femur, lensIntroduction

body. Although it has been difficult to quantify AGEs in vivo,
recent advances in AGE research have made it possible tomeasure several AGEs using liquid chromatography-tandem
mass spectrometry (LC-MS/MS). It is now recognized that
AGE accumulation in the crystalline lens changes withdifferences in blood glucose levels in rats and humans, andpreventive effects and therapeutic strategies for cataracts
targeting inhibition of AGE accumulation in the lens areexpected. However, it is still unclear what tissues produce oraccumulate AGEs, which increase with aging and diseases,
and the relationship between each tissue and AGE accumulation.
Therefore, we have established a method to quantify AGEsin various tissues using mice as an animal model. We havereported that, using mice, CML accumulation in the lensincreases with the onset of diabetes 6), and that AGE accumulate
in the liver, kidney, and brain, and that their accumulationin the brain and kidney increases with aging 7). However,

It has been shown that advanced glycation end-products(AGEs), generated from non-enzymatic reactions between
proteins and carbonyl compounds, accumulate in livingorganisms as well as in foods, and that their accumulation rateis correlated with aging and the progression of lifestyle-relateddiseases 1, 2). Among AGEs, N ε-(carboxymethyl)lysine (CML)
is an AGE produced in an oxidation-dependent manner 3, 4)
and has been shown to be an indicator of oxidative stress.
Whereas, among Arg-derived AGEs, N δ-(5-hydro-5-methyl4-imidazolon-2-yl)-ornithine (MG-H1), a major AGE of Argderived AGEs, is produced via glyceraldehyde-3-phosphatein the glycolysis system, and evaluated as an indicator ofabnormal glucose metabolism since it is increased in diabeticpatients 5). Thus, multiple concurrent measurements of AGEstructures are important for assessing abnormalities in theAddress correspondence to: Ryoji Nagai, Ph.D.
Laboratory of Food and Regulation Biology, Department of Bioscience,
School of Agriculture, Tokai University
Toroku 9-1-1, Higashi-ku, Kumamoto, Kumamoto 862-8652, Japan.
TEL &FAX: +81-96 -386-2692 e-mail: nagai-883@umin.ac.jp
Co-authors; Hiraoka Y, zisg64mul721@gmail.com;
Sugawara H, sh855655@tsc.u-tokai.ac.jp

Glycative Stress Research 2023; 10 (1): 1- 5(c) Society for Glycative Stress Research_ 1 _Determination of AGEs in Mice Femur by LC-MS/MSbone is a hard tissue, the processing for crushing andmeasurement is different from that for soft tissues, and theamount of sample obtained from mice is small due to theirsize, which is a drawback of AGE measurement in bone. In
this study, we attempted to quantify AGEs in mouse femur,
the largest bone in size, from mice.

HILIC column (150 × 2.1 mm, 5 μm; Merck Millipore,Billerica, MA, USA) was used for the LC section 6). Targets
were separated by a gradient mode of mobile phase A (0.1 %formic acid) and mobile phase B (acetonitrile in 0.1 %formic acid). The flow rate was 0.2 mL/min and the column
oven temperature was set at 40 °C.

Statistical analysis

Methods

Results are expressed as mean ± standard deviation (SD).
Significant difference tests were calculated using the MannWhitney U test with p < 0.05 being considered a significant
difference.

Animal Experiments

Animal experiments were performed with the approvalof the Tokai University Ethics Committee (approval number:#191036). Male Sea:ddY mice (5-weeks-old) were purchased
and housed in a pathogen-free barrier-free facility (12-hourlight/dark cycle) for 3 months. CE-2 (Clea, Tokyo, Japan)
and water were fed ad libitum. After 3 months, lenses andfemurs were sampled from mice under 30 % isoflurane (v/v)
anesthesia.

Results

Mice were housed for one week after purchase and kept
for 3 months. The fasting blood glucose level of mice before
autopsy was 111 ± 18 mg/mL. AGEs in mouse lens and femur
bone were quantified by LC-MS/MS; the predicted sites offragment ions used for CML and MG-H1 measurements arelisted in Fig. 1 9). As a result, CML and MG-H1 were clearly
detected in mouse femur, and the intensity of detection wasapproximately 20 times higher than that of CML and MGH1 in lens (Fig. 2). The CML content in mouse femur was
significantly higher than that in lens ( Fig. 3 ), even whencorrected for the content of Lys in each tissue.

Measurement of CML and MG-H1 in each tissueLenses were homogenized in 1 mM diethylenetriamine
pentaacetic acid (DTPA) solution and crushed, and protein
quantification was performed by bicinchoninic acid (BCA)
method (Pierce, Rockford, IL, USA). The blood was removed
from femurs. Then, femur samples ware lyophilized, andcrushed using liquid nitrogen. The samples were thenremoved lipid and minerals to be used for the assay 8). For
measurements, 0. 3 mg of lens protein and 1 mg of crushed
femur were used. Sodium borate buffer (0.2 M boric acid, 2mM DTPA, pH 9.1) was then added in equal volume to thesample, and NaBH4 solution (2 mM NaBH4, 0.1 M NaOH)
was added at one-tenth the volume of the sample and allowed
to stand for 4 hours. Then, 20 % trichloroacetic acid (TCA)
solution was added, stirred, and the proteins were precipitatedby centrifugation (12,000 rpm, 5 minutes, 4 °C ) and thesupernatant was discarded. Subsequently, pretreatment for
LC-MS/MS measurements was performed according to theprevious studies 6). Specifically, 0.01 nmol of CML and MGH1 internal standard (PolyPeptide Laboratories, Strasbourg,France) and 5 nmol of Lysine internal standard (Cambridge
Isotope Laboratories, Inc, Tewksbury, MA, USA) was added
to samples and hydrolyzed using 1 mL of 6 M HCl at 100 °C
for 18 h. The mixture was then centrifuged and concentrated,followed by the removal of hydrochloric acid using acentrifugal concentrator. To the dried sample, 1.0 mL ofultrapure water was added to dissolve the sample in thesolvent. The sample was then placed on a Strata-X-C column(Phenomenex, Torrance, CA, USA) to remove unnecessarycomponents, washed with 2 % formic acid, eluted with 7%
ammonia, and allowed to dry. The dried sample wasdissolved in 20 % acetonitrile 0.1% formic acid solution
and used for LC-MS/MS measurements.

Discussion

Although the Maillard reaction was first reported in 1912,
more than 100 years ago, it remains difficult to accurately
assess its progression. Browning, changes in fluorescenceintensity, and immunological techniques using anti-AGE
antibodies have been used, however the quantification of
biological AGEs is more difficult and therefore remains at theresearch level, limiting the clinical use of AGE measurements.
We have previously reported that AGEs are artificiallygenerated by heating 10) and alkali treatment 11). However,
the measurement of AGEs by gas chromatography-mass
spectrometer (GC-MS) requires several steps of derivatization,
which is disadvantageous for the analysis of multiple samples.
In addition, as a biological sample, the lens of the eye
has been frequently measured for the evaluation of biological
AGEs because it is easy to crush and briefly yields soluble
protein. However, it is technically more difficult to quantify
AGEs in bone tissue than in lens due to the hardness of thebone. In this study, we attempted to quantify AGEs in mouse
femur using LC-MS/MS, which does not require derivatization.
As a result, we were able to measure AGEs in bone protein
by delipidating, demineralization, and hydrochloric acidhydrolysis of femur bone.
Lens proteins in the eye have been considered to be
one of the tissues with high AGE content due to their low
turnover rate. However, CML levels in the femur wereunexpectedly higher than in the lens, and MG-H1 levels
were similar to those in the lens. Production of carbonylcompounds and reactive oxygen species is increased inmetabolically active organs 5) and inflammatory responses 12).

LC-MS/MS conditions

For LC-MS/MS measurements, a TSQ Vantage triple
stage quadrupole mass spectrometer (Thermo Fisher Scientific,Waltham, MA, USA) was used for the MS section. A ZIC ®-_ 2 _Glycative Stress Researcha)b)

ParentParention ion
Fragment
Fragment
ion ionOm/z 130m/z 130OC
H2N HCH
2N CCH OHOParentParention ionFragment
Fragment
ion ionOH

H2N HCHC
2N CCH OH
CH 2 CH 2

CH 2 CH 2

CH 2 CH 2

CH 2 CH 2
CH 2 CH 2NH

CH 2 *CH 2 *HOHO O

OH

CH 2 CH 2CH 2 CH 2NHONHNHCNCNHNHNO

O

CH3* CH3*O(IS:m/z(IS:117)
m/z 117 )m/z 114m/z
114m/z 232)
(IS:232)
229m/zm/z 229m/z (IS:m/z 205m/z (IS:205m/z(IS:207m/z) 207 )Fig. 1. Structures of CML (a) and MG-H1 (b).

The straight lines indicate the parent ion and the dotted lines the predicted sites of the measured fragment ions. CML,N ε-(carboxymethyl)lysine; MG-H1, N δ -(5-hydro-5 -methyl- 4 -imidazolon-2-yl)-ornithine.

a)

b)

c)

Fig. 2. Chromatograms of AGEs in mouse tissue detected by LC-MS/MS.

d)

The upper peak represents AGEs in tissue, and the lower peak represents each AGE internal standard in tissue. CMLpeaks femur (a ) and lens ( b ). MG-H1 peaks femur (c ) and lens (d ). AGEs, advanced glycayion endproducts; LC-MS/MS,liquid chromatography-tandem mass spectrometry; CML, N ε-(carboxymethyl)lysine; MG-H1, N δ-(5-hydro-5-methyl- 4 imidazolon-2 -yl)- ornithine.

_ 3 _Determination of AGEs in Mice Femur by LC-MS/MSa)

b)

N.S.

0.5

*MG-H1 (mmol/mol Lys.)

CML (mmol/mol Lys.)

0.25
0.20.150.10.05
00.4
0.3
0.2
0.10

Femur

Lens

Femur

Lens

Fig. 3. Comparison of CML and MG-H1 content between tissues.
CML (a ) and MG-H1 ( b ) content in mouse tissues in bone and lens, n = 3, significance level *p < 0.05. N.S., notsignificant; CML, N ε-(carboxymethyl) lysine; MG-H1, N δ-(5-hydro-5-methyl- 4-imidazolon-2 -yl)-ornithine.

Since bone homeostasis is maintained by the balance between
osteoblasts and osteoclasts, metabolism of bone is relatively
active and generates AGEs 8, 13).
Suzuki et al. 13) reported that accumulation of CML in
osteoblasts induces apoptosis, resulting in an increased riskof osteoporosis. In the present study, bone CML was higher
than that in the lens, suggesting that AGE accumulation
may affect bone homeostasis more than we had previouslyexpected. The fact that AGEs can be quantified in hardtissues as well as soft tissues of mice by LC-MS/MS without
derivatization is expected to make it more effortlessly tostudy AGEs in rodents.

Conflict of interest declaration
There are no relevant matters concerning this study.

Funding or Acknowledgement
We received a scientific research grant from JSPS
KAKENHI (grant no. 20K05895) provided to Ryoji Nagai.

_ 4 _Glycative Stress Research

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