Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : December 18, 2023
Accepted : January 16, 2024
Published online : March 31, 2024
doi:10.24659/gsr.11.1_1

Original article

Rapid measurement of fumaric acid in culture medium as an evaluation of
metabolic abnormality of adipocytes
Himeno Takahashi 1), Yuki Tominaga 2), Ryoji Nagai 1, 2)
1) Laboratory of Food and Regulation Biology, Graduate School of Agriculture, Tokai University, Kumamoto, Japan
2) Graduate School of Bioscience, Tokai University, Kumamoto, Japan

Abstract

It has been reported that adipocytes accumulate triglycerides and concurrently produce adiponectin, a beneficial
adipocytokine, to assist insulin function, and conversely produce unbeneficial adipokines, which have negative effects on
the organism. We previously reported that the production of fumaric acid increases under hyperglycemic conditions due tomitochondrial stress and that fumaric acid reacts with the thiol group of cysteine residue to form S-(2-succinyl)cysteine (2SC),
which inhibits adiponectin secretion and modifies a variety of proteins, resulting in the dysfunction of adipocytes. Therefore,
we considered that a simple measurement of fumaric acid produced by adipocytes would enable rapid evaluation of metabolic
abnormalities in adipocytes, and investigated a measurement system for this purpose. After culturing adipocytes for 4 days, adecrease in pH was observed due to the production of organic acids. Hydroxylamine hydrochloride and iron (III) chloride, areagent for carboxylic acid detection, reacted with fumaric acid, malic acid, and citric acid. Similarly, fumaric acid levels by
the enzymatic method and organic acids were found to be elevated by the carboxylic acid qualitative reagent in the medium
in which adipocytes were cultured. Therefore, it was confirmed that functional abnormalities of adipocytes can be easily
evaluated by the carboxylic acid qualitative reagent.

KEY WORDS: fumaric acid, adipocytes, S-(2-succinyl)cysteine (2SC), mitochondrial function

Introduction

Previously, we analyzed non-enzymatic post-translational
modifications in adipocytes, which play an important role inmetabolic regulation. As a result, we found that fumaric acid,
which increases due to abnormal mitochondrial function,
reacts with the thiol group of cysteine, resulting in S-(2discovered a phenomenon produced by succinyl)cysteine(2SC) 2). 2SC subsequently causes abnormalities in thefunction of adiponectin, a beneficial cytokine 3), and GAPDH,
which is essential for glucose metabolism 4), leading toabnormal glucose metabolism. Furthermore, 2SC conversionof glutathione 5) causes a decrease in antioxidant capacity,which in turn increases the production of AGEs, advanced
glycation end products, through the production of carbonylcompounds 6). Recently, it has been reported that fumaricacid itself, a 2SC precursor, is involved in the development ofinflammatory reactions 7), for example, fumaric acid has beenreported to promote DNA methylation in human monocytes
and cause them to secrete inflammatory cytokines 8). In
addition, α-ketoglutarate, succinate, fumarate, and malate are
increased in macrophages under inflammatory conditions,and glutamate, pyruvate, and 2-oxoglutaric acid, succinate,

Japan's aging population is increasing rapidly, and withit, advances in medical care for the elderly have made thecountry one of the longest-living countries in the world interms of average life expectancy. However, according to a
2019 survey by the Ministry of Health, Labour and Welfare,there is a gap of 8.7 years between average life expectancy
and healthy life expectancy for men and 12 years for women,
which means that elderly people are forced to live with adeteriorated quality of life for approximately 10 years before
their death 1). Diabetes mellitus is one of the most common
lifestyle-related diseases that cause poor quality of life, andthe number of people suffering from this disease, including
those in the pre-diabetic group, has rapidly increased to20.5 million in Japan. Since 95 % of diabetic patients aretype 2, which is mainly caused by lifestyle-related disorders,the disease can be treated with early intervention. However,
due to poor glycemic control, various complications, mainly
vascular disorders such as atherosclerosis, often develop
within 5 to 10 years.

Correspondence to: Ryoji Nagai, Ph.D.,
Laboratory of Food and Regulation Biology, Graduate School of Agriculture, Tokai University
Sugidoh 871-12, Mashiki-machi, Kamimashiki-Gun, Kumamoto 861-2205
TEL : + 81- 96 -386 -2692 e-mail: nagai-883@umin.ac.jp
Co-authors: Takahashi H, 2cnfm012@mail.u-tokai.ac.jp;
Tominaga Y, 1CTLD 001@mail.u-tokai.ac.jp_ 1 _Glycative Stress Research 2024; 11 (1): 1- 6(c) Society for Glycative Stress ResearchRapid Measurement of Fumaric Acid

fumarate, and malate are increased in obese adipose tissue.
Therefore, chronic inflammation in adipose tissue contributes
to increased organic acids in the TCA cycle 7, 9). Therefore,
suppressing the excessive production of fumaric acidassociated with metabolic abnormalities may inhibit fumaricacid-mediated inflammatory reactions in living organisms.
In this study, we investigated a simple method to evaluate
functional abnormalities of adipocytes by measuring fumaricacid produced when adipocytes are cultured under a highglucose condition.

order to specifically measure fumarate with this method, it isnecessary to establish a measurement system using HPLC.

Measurement of fumaric acid

Fumaric acid was determined using the Fumarate assay
kit (MAK060, Sigma-Aldrich, St. Louis, MO, USA). Ten μLof the assay sample was added to a 96-well plate. Master
reaction mix (100 μl) was added to each well and pipetted.
The absorbance at 450 nm was then measured in a microplate
reader. Fumaric acid standard was added to each 0 (blank),
2, 4, 6, 8, and 10 nmol/well to generate a calibration curve forquantifying fumaric acid and calculate the concentration of
fumaric acid in the samples.

Materials and methods
Cell culture

The mouse preadipocyte cell line (3T3-L1 cells) was
purchased from the JCRB cell bank. Cells were passaged in
Dulbecco's Modified Eagle Medium (DMEM) (low glucose
medium) containing phenol red with 100 μg/mL streptomycin,
100 μg/mL penicillin, and 10 % calf serum (CS; Fujifilm
Wako Ltd., Osaka, Japan) was used for passaging.
3T3-L1 was differentiated using DMEM medium
containing 10 % fetal bovine serum (FBS), 10 μg/mL
insulin, 0.3 μM dexamethasone, and 0. 5 μM 3- isobutyl-1methylxanthine (IBMX) 2). Subsequently, the medium waschanged every 2 days using a maturation medium of DMEM
supplemented with 10 % FBS and 5 μg/mL of insulin. On the
fourth day after differentiation, the medium was collected,
treated with a 0.45 μm filter, and stored at -20 °C.

Statistical analysis

All data were expressed as mean ± standard deviation± standard deviation (SD). One-way analysis of varianceand Bonferroni's post test were used to examine statisticalsignificance. Statistical analysis was performed using the
EZR software package 11).

Results

Color changes in adipocyte culture media

To confirm the variation in organic acid production by
3T3-L1 as a function of glucose concentration, we evaluatedthe color changes in the culture medium of 3T3-L1 adipocytes
cultured in DMEM medium for 4 days at different glucose
concentrations. No change in color tone was observed in themedium alone, while the supernatant cultured in low glucose
became slightly lighter in color and changed to yellow inhigh glucose (Fig. 1-a). Since phenol red may have changed
the color tone, the pH of the culture medium was next
measured. The results showed that when 3T3-L1 adipocytes
were cultured in high glucose, the pH was significantly lower
than in other conditions (Fig. 1-b).

Measurement of pH

The measurements of pH were performed using abench-top pH meter (F-51, Horiba, Kyoto, Japan) and a pHelectrode (9615S). Measurements were calibrated prior to
analysis with a neutral phosphate standard (pH 6.86), aphthalate standard (pH 4.01), and a borate standard (pH 9.18).

Qualitative analysis for carboxylic acid

All organic acids in the TCA cycle have a carboxy
group (R-COOH). Determination of organic acids was
performed by a modification of the method of Murata et al. 10).
Specifically, to 50 μL of each sample, 50 μL of a saturated
ethanol solution of hydroxylamine hydrochloride (Fujifilm
Wako Ltd., Osaka, Japan) and 50 μL of a 1 % ethanol
solution of dicyclohexylcarbodiimide (DCC; Fujifilm Wako
Ltd., Osaka, Japan) were added, mixed, and left for 2 minutes.
The iron (Ⅲ) chloride reagent was prepared by dissolving
10 mg of iron (Ⅲ) chloride hexahydrate (Nacalai TesqueCo., Ltd., Kyoto, Japan) in 1 mL of ethanol, adding 10 μL
of hydrochloric acid, and 50 μL was added to the above
reaction solution. Then, 200 μL was added to each 96-wellplate, and the absorbance was measured at 550 nm witha microplate reader (Infinite 200 PRO, Tecan). The color
tone of the iron chelate complex is not changed by organic
acids. Therefore, with a carboxylic acid qualitative reagent,
it is not possible to detect each organic acid individually,
and only the total amount is detected. Present experiment
was conducted to measure the total amount of organic acidsin order to confirm the correlation between the increase infumaric acid and the increase in organic acids. Therefore, in

Determination of organic acids by qualitative
reagents

The color reaction of carboxylic acids was used tomeasure organic acids in the culture medium. In this study,
DCC was used as a condensing agent for carboxylic acids,
which were converted to hydroxamic acid by condensing
with hydroxylamine via DCC, and then reacted with trivalentiron chloride to form a colorful iron chelate complex thatcan be detected (Fig. 2-a). As a result of the measurementof carboxylic acids contained in organic acids using thisproperty, a concentration-dependent increase in the amountof carboxylic acid was confirmed in the standards of fumaricacid, citric acid, and malic acid, which are representative
organic acids (Fig. 2-b).

Comparison of accuracy of fumaric aciddetermination

To confirm the accuracy of the detection of carboxylic
acid content by qualitative reagents, a comparison wasmade using the Fumarate assay kit. Concentration increased_ 2 _Glycative Stress Research

approximately 4-fold under high glucose conditions ( Fig.
3-a). Similarly, carboxylic acid levels were significantly
increased in the supernatant of 3T3-L1 cultures grown underhigh glucose conditions (Fig. 3-b).
A strong correlation of fumarate levels was obtained
between the enzymatic assay and the colorimetric method(Fig. 3-c).

transplantation showed that blood 2SC significantly decreased
with transplantation, indicating that some fumaric acid is
involved in the decrease in renal function 6).
Although the interaction between macrophages andadipocytes via free fatty acids is generally known in normal
chronic inflammation 13), the involvement of fumaric acid withrespect to inflammation has been reported in recent years.
For example, it is known that inflammatory stimuli withlipopolysaccharide induce mitochondrial remodeling andincreased fumaric acid production in macrophages 14) andthat the fumaric acid produced induces interferon-β (IFN-β)
production by macrophages 7). Therefore, fumaric acid has beenreported to be closely related to inflammatory reactions, butsince fumaric acid itself is metabolized, it is not a marker offumaric acid-derived inflammatory reactions and it is difficult
to explain the relationship with inflammatory reactions. The
measurement of 2SC, a derivative measurement of fumarate,is expected to become an important marker to explain fumaricacid-derived biological phenomena in vivo.

Discussion

Previously, we measured 2SC by GC-MS, but due to thedifficulty of pretreatment, we developed a system in which
2SC was measured by LC-MS/MS without pretreatment. As
a result, 2SC accumulated in the brain of mice with aging, but
no age-related changes were observed in the liver 12), indicatingthat 2SC may also be involved in age-related changes in brain
function. In addition, blood 2SC increases with decreasing
renal function, and a longitudinal study before and after renal     
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Fig. 1. Color changes in adipocyte culture media.

Culture medium and culture supernatant from a 4-day culture of 3T3-L1 adipocytes were collected and evaluated for color
and pH changes in the medium. (a ) Photographs of culture medium and culture supernatant. A; DMEM medium (Glucose
1,000 mg/L + FBS), B; DMEM medium (Glucose 4,500 mg/L + FBS) C; 3T3-L1 culture supernatant (Glucose 1,000 mg/L
+ FBS), D; 3T3-L1 culture supernatant (Glucose 4,500 mg/L + FBS). ( b ) pH value of each medium by pH meter. Data areshown as mean ± SD, *p < 0.05; **p < 0.001, n = 3. FBS, fetal bovine serum; SD, standard deviation.

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b)Fig. 2. Determination of organic acids by qualitative reagents.
( a ) Structural changes in the color reaction of carboxylic acids with qualitative reagents. ( b) Fumaric acid,citric acid, and malic acid were prepared at concentrations of 0.2, 0.4, 0.8, and 1.0 mM, treated (n = 1) withqualitative reagents, and measured at 550 nm absorbance. DCC, dicyclohexylcarbodiimide.

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Glycative Stress Research

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Fig. 3. Comparison of accuracy of fumaric acid determination.

Fumarate levels were determined in culture supernatants of 3T3-L1 cells grown in normal medium and 3T3-L1 cells differentiated andcultured for 4 days. ( a ) Fumarate determination by Fumarate assay kit. ( b ) Absorbance of color reaction using a qualitative reagent.
Data are shown as mean ± SD, *p < 0.001, n = 3. SD, standard deviation. ( c ) Correlation of fumarate levels between the enzymatic
assay kit and the colorimetric method.

Conclusion

between the color change of the medium and the change
in fumaric acid produced by the enzymatic method. In the
future, this measurement system will facilitate the searchfor compounds that inhibit the production of fumaric acid.

The present study revealed that carboxylic acids in themedium are qualitatively measured by a colorimetric method.
In order to explore the inhibition of fumaric acid production,
it is necessary to culture adipocytes with high glucose andevaluate the changes in the concentration of fumaric acid in
the culture medium. Previously, we derivatized fumaric acidand quantified it by GC-MS, but pretreatment and GC-MS
measurements were difficult for multi-sample analysis 2). In
addition, the measurement of fumaric acid by the enzymatic
method is expensive, so development of a simpler method
is required. However, the evaluation based on the color
change of the culture medium alone is unreliable, since thecolor change is also affected by bacterial infection and otherfactors. Therefore, we used an organic acid coloring reagent
to evaluate fumaric acid production, and found a correlation

Conflict of interest statement
None.

Research grantThis work was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science and Technology (JSPS KAKENHI #20K05895).

_ 5 _Rapid Measurement of Fumaric Acid

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