Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : September 16, 2016
Accepted : October 26, 2016
Published online : December 31, 2016

Review article

Glycative stress and anti-aging: 2. The Evaluation of Glycative Stress:
Measurement of blood glucose, glycated proteins and intermediates.
Masayuki Yagi, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center,
Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan

Abstract

Various substances produced in the course of glycation reaction can be used as markers for the evaluation of glycativestress. The glycative stress markers in the stage before the production and accumulation of advanced glycation end products(AGEs) include blood glucose, glycated protein and intermediates for AGEs. Self-monitoring of blood glucose (SMBG) is a
simple method for the measurement of blood glucose. Glycated proteins include hemoglobin A1c (HbA1c) and glycoalbumin(GA), and can be measured by a clinical test organization. The intermediates for AGEs include 3-deoxyglucosone (3DG),glyoxal (GO), methylglyoxal (MG), glyceraldehyde and glycolaldehyde. 3DG can be measured by certain test organizations
that can be assigned with special clinical test items. However, for the measurement of other intermediates for AGEs, ameasuring system should be structured based on literatures as reference.

KEY WORDS: Glycative stress marker, measurement method, blood glucose, glycated protein, intermediates for AGEs

1. Glycative stress markers

Glycative stress is enhanced by elevated blood glucose
and the formations of carbonyl compounds and aldehyde
compounds, and consequently leads to the production andaccumulation of advanced glycation end products (AGEs) inthe body. This series of reactions is accelerated by ultraviolet
rays and oxidization. Various substances produced in thecourse of glycation reaction are used as markers for theevaluation of glycative stress. The measuring methods forblood glucose, glycated protein and intermediates for AGEs
are summarized, which are used as glycative stress markers
in the previous stage before the production and accumulationof AGEs, as follows.

2. Measurement of blood glucose

Blood glucose can be used as a glycative stress marker
because it evaluates elevated after-meal blood glucose
condition. Self-monitoring of blood glucose (SMBG) is thesimplest method to measure blood glucose. There are two
measuring principles for blood glucose, enzymatic electrodemethod and colorimetry method 1).

Enzymatic electrode method includes two methods,glucose oxidase (GOD) method and glucose dehydrogenase(GDH) method, depending on the difference of enzyme used
for dissolving glucose 2). In either measuring method, theelectrons generated when an enzyme dissolves blood glucose
are given to ferricyanide ions, i.e. electron mediators, andchanges them to ferricynide ions ( Fig. 1 ). After that, because
the electric current generated when a fixed voltage is applied
to electrode responds to the concentration of blood glucose,the current value can be measured as glucose concentration.
The enzyme colorimetric method includes two methods,
the hexokinase (HX) method and glucose oxidase/peroxidase(GOD/POD) method. In the HX method, after blood glucose
is converted to glucose-6-phosphate (G6P), nicotinamide
adenine dinucleotide (NADH) and tetrazolium are generated
by glucose - 6 -phosphate dehydrogenase (G6PDH), areactivated by diaphorase, and the hormazan dye colored fromblue to red or others is colorimetrically determined. In theGOD/POD method, quinone pigment, which is generated
when gluconic acid and hydrogen peroxide generated fromglucose by the action of GOD react to peroxidase (POD),
and the primary color substances of the reaction agent iscolorimetrically determined.

Contact Address: Professor Masayuki Yagi, PhD
Glycative Stress Research Center, Faculty of Life and Medical Sciences, Doshisha University
1-3, Tataramiyakodani, Kyotanabe-shi, Kyoto, 610-0394 Japan
Phone/Fax: +81-774-65-6394 E-mail: myagi@mail.doshisha.ac.jp
Co-author: Yonei Y, yyonei@mail.doshisha.ac.jp_ 205 _

Glycative Stress Research 2016; 3 (4): 205- 209(c) Society for Glycative Stress Research

The Evaluation of Glycative Stress
In SMBG, when the hematocrit level is near the reference
range (20-60 %), accurate measuring is possible. However,if the blood hematocrit level is less than 20%, as in the casesof anemia patients and dialysis patients, it shows a higher
level. On the other hand, if the hematocrit level is more than55 %, as in the cases of newborn babies and women before amenstrual period, it shows a lower level. In the GOD method,
the greater the dissolved oxygen partial pressure in blood is,the lower the level of blood glucose is measured 3) . In Japan,
SMBG instruments with various characteristics are sold by
a number of makers. Therefore, it is possible to select an
instrument according to necessity 4). Measuring time differs
by each instrument, but the difference is only 5-30 seconds.
SMBG is classified into specially controlled medical devices,class 3 by the Pharmaceutical Affairs Act. Therefore, theadvertisement of SMBG is not permitted, so general consumers
rarely have a chance to know the convenience of bloodmeasurement.

3. Measurement of glycated protein

Glycation reactions can be divided into “early stagereaction,” “intermediate stage reaction” and “ late stagreaction.” Amadori rearrangement products produced in earlystage reaction include glycated proteins, such as hemoglobin
A1c (HbA1c) and glycoalbumin (GA). They are used as theindexes reflecting the past condition of blood glucose for theevaluation of diabetes, and they are useful glycative stressmarkers, as are blood glucose levels 5) .
Valine, i.e. an amino acid of β-chain N-terminal ofhemoglobin A, became HbA1c, i.e. a glycated protein, throughSchiff base caused by a glycation reaction with glucose. As
glycation reaction depends on the concentration of bloodglucose, the glycation rate of hemoglobin increases, depending
upon the level of elevated blood glucose (concentrationand time). Therefore, the HbA1c thus formed, exits for120 days, which is the longevity of red blood cells. As aresult, the percentage of HbA1c reflects the blood glucose
condition for the past one to two months. There are threemethods for measuring HbA1c, the high performance liquid
chromatography (HPLC) method, immunization method
and enzyme method 6) . In the HPLC method, hemoglobin is
separated and analyzed by ion-exchange chromatography,
and the percentage of HbA1c is calculated from the peakarea of HbA1c and hemoglobin. HPLC method has themerit that it can find fetal hemoglobin (HbF) and abnormal
hemoglobin, other than HbA1c. The immunization method
is a measuring method using antibodies produced using
HbA1c as an antigen, and it can selectively measure only
HbA1c. Because it uses a highly specific antibody, it isimmune to the influence of hemoglobins other than HbA1c.
In the enzyme method, after glycated peptide of β-chain
N-terminal of hemoglobin is digested by protease, glycatedvaline peptide is measured by enzymatic assay using
frutosylpeptide oxidase. The enzyme method is suitable forprocessing a large number of specimens by an automatic
analyzer. However, for the immunization method andenzyme method, the total amount of hemoglobin shouldbe measured separately for the purpose of calculating
HbA1c. International standardization is applied for themeasurement of HbA1c, and the level cited in the National
Glycohemoglobin Standardization Program has been

introduced 7) . As a result, the level by the Japan Diabetes
Society (JDS), which has been used in routine medicaltreatment, is required to be adjusted approximately 0.4%
higher.
GA is a glycated protein of serum albumin. As the halflife period of serum albumin is about 20 days, GA reflects
the average blood glucose level for a period from two weeks
to one month in the past. GA can be applied for diabetes
patients with a wide fluctuating range of blood glucose leveland receiving drug therapy (insulin administration). Fourglycated moiety (Lys-199, 281, 439 and 525) have been
confirmed in an albumin molecule 8) , and albumin has thecharacteristic to comparatively respond more swiftly andmore largely to the fluctuation of blood glucose level than
HbA1c, and continue to transit. The HPLC method andenzyme method are used for the measurement of GA, andthe enzyme method is mainly used in clinical examinationsat present. In this method, after blood albumin is digested
by protease and glycated lysine is released, albumin ismeasured by enzymatic assay using ketoamine oxidase. The
total amount of albumin is specifically measured by thebromocresol purple method (BCP), and the percentage of
GA included in albumin is calculated.
HbA1c and GA are among the measurement items ofblood test for the examination of lifestyle related diseases
and screening test for blood donation, and they can be tested
by a clinical test organization.

4. Measurement of intermediates for AGEs

There are 3-deoxyglucosone (3DG) glyoxal (GO),methylglyoxal (MG), glyceraldehyde and glycolaldehyde
among the intermediates for AGEs ( Fig. 2 ) 9) . 3DG is anα-dicarbonyl compound formed from glycated protein, hasa reactivity 10,000 times higher than glucose, and is involvedin the formation of AGEs. If the concentration of 3DG in theblood plasma increases by 100 nmol/L, the risks of diabetic
retinopathy and nephropathy increase by about twice as much( Fig. 3 ) 10) . GO is formed by the auto-oxidation of glucose
and the peroxidation of lipids. MGO is also formed in theglycolytic pathway, and polyol pathway in cells. Because theglycation pathway of intermediates for AGEs is different
depending on each compound, the kinds of AGEs ultimately
formed are different. AGEs having glyceraldehydes asintermediate, in particular, are called toxic-AGEs (TAGE)
and are deeply involved in the onset and development ofcomplications of diabetes 11) .
3DG, GO and MG can be measured by the HPLC
assay with 2, 3 - diaminonaphthalene (DAN) pre-columnderivatization 12). For these measurements, after the blood
samples are deproteinated, they are added with DAN,
and the intermediates for AGEs are derivatized withaminonaphthalene and purified, then are analyzed by thereverse HPLC method. For the detection of intermediates for
AGEs converted to aminonaphthalene, ultraviolet absorption(wavelength of 268 nm) or fluorescence (excitation wavelengthof 271 nm and fluorescence wavelength of 503 nm) canbe applied ( Fig. 4 ). 3DG can be measured by certain test
organizations that can be assigned with special items.
However, for other intermediates for AGEs, a measuringsystem should be structured based on literatures as areference.

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Acknowledgement

This work was partially supported by the JapaneseCouncil for Science, Technology and Innovation, SIP(Project ID 14533567), “Technologies for creating nextgeneration agriculture, forestry and fisheries” (fundingagency: Bio-oriented Technology Research Advancement
Institution, NARO

Conflict of interest statement

There are no items deemed to be conflicts of interest in
this research.

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Fig. 1. Reaction scheme of the glucose sensor by an enzymatic electrode method.

GOD, glucose oxidase; GDH, glucose dehydrogenase; FAD, flavin adenine dinucleotide; PQQ, pyrroloquinoline quinone;
2[Fe(CN) 6]4-, ferrocynide ion; 2[Fe(CN) 6]3-, ferricyanide ion. (adapted from Ref. 2)_ 207 _The Evaluation of Glycative StressFig. 2. Alternative routes for the formation of various distinct AGEs in vivo.

AGEs, advanced glycation end products; Glc-AGE (AGE-1), glucose-derived AGEs; Glycer-AGE (AGE-2), glyceraldehyde-derived AGEs;
Glycol-AGE (AGE-3), glycolaldehyde-derived AGEs; MGO-AGE (AGE-4), methylglyoxal (MGO)-derived AGEs; GO-AGE (AGE-5),
glyoxal (GO)-derived AGEs; 3-DG-AGE (AGE-6), 3-deoxyglucosone (3-DG)-derived AGEs; P-NH 2, free amino residue of protein. GOLD,GO-lysine dimmer; GA-pyridine, 3-hydroxy-4-hydroxymethyl-1-(5-carboxypentyl) pyridinium cation; CML, N ε -(carboxymethyl)lysine;
DOLD, 3-DG-lysine dimmer; CEL, N ε -(carboxyethyl) lysine; MOLD, MGO-lysine dimmer; GLAP, glyceraldehyde derived pyridinium
compound. (adapted from Ref. 8)_ 208 _Glycative Stress Research

P < 0.001

1000

P < 0.001

P = 0.027

P = 0.008

Serum 3-DG (nmol/L)

800

600

400

200

0alr ia u r ia u r ia
ive
ive
rm
atatnnnu
roiiireeeNlifu m r otumlifroroPal b o al bpPonrmic rNoMNoControlNephropathyRetinopathy

Fig. 3. Serum 3DG concentrations in diabetic patients with various grades of nephropathy and retinopathy.

Control, nondiabetic patients and healthy volunteers; Normal, retinopathy was assessed by ophthalmologists using fundus
photography; Benferroni / Dunn multiple comparison test. 3DG, 3-deoxyglucosone. (adapted from Ref. 10)

Normoalbuminuria
Microalbuminuria
Proteinuria
NormalNonproliferative
Proliferative
3DG

120000100000peak intensity

80000

60000

40000

20000

GOMGO

0-20000

024681012141618

retention time (min)Fig. 4. HPLC chromatogram of intermediate for AGEs.

Human serum albumin (8 mg/mL) and glucose (0.05 mol/L) added into the phosphate buffer (pH 7.4), and incubated in60°C at 40 hours. HPLC measurement intermediate for AGEs was carried out after pre-label derivatization reaction by
2,3-diaminonaphthalene (DAN). HPLC condition; column, Unison UK-Phenyl (Imtakt), 75 x 3 mm ID; eluent, 0.05 mmol/
L phosphoric acid / ACN = 89 / 11; flow rate, 1.0 mL/min; detection, UV 268 nm. AGEs, advanced glycation end products;
3DG, 3-deoxyglucosone; GO, glyoxal; MGO, methylglyoxal; HPLC, high performance liquid chromatography.

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