A spectrophotometric assay for monoamine oxidase activity with 2, 4-dinitrophenylhydrazine as a derivatized reagent

Article abstract of DOI:10.1016/j.ab.2016.06.020

A simple, rapid and reliable spectrophotometry was developed to determine monoamine oxidase (MAO). In this study, 2,4-dinitrophenylhydrazine (DNPH), a classic derivatizing reagent, was used to detect MAO-dependent aldehyde production; and traditional DNPH

Full text of DOI:10.1016/j.ab.2016.06.020

Analytical Biochemistry 512 (2016) 18e25
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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
A spectrophotometric assay for monoamine oxidase activity with 2,
4-dinitrophenylhydrazine as a derivatized reagent
Guili Huang 1, Fei Zhu 2, Yuhang Chen 3, Shiqiang Chen 4, Zhonghong Liu 5, Xin Li 6,
Linlin Gan 7, Li Zhang 8, Yu Yu*
Department of Medicinal Chemistry, College of Pharmacy, Chongqing Medical University, 1 Yi Xue Yuan Rd, Chongqing 400016, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple, rapid and reliable spectrophotometry was developed to determine monoamine oxidase (MAO).
In this study, 2,4-dinitrophenylhydrazine (DNPH), a classic derivatizing reagent, was used to detect MAO-
dependent aldehyde production; and traditional DNPH spectrophotometry was simplified. Benzylamine
and serotonin oxidation were catalyzed by MAO-B and MAO-A, respectively, to aldehydes. These were
derivatized with DNPH, and the corresponding quinones were further formed by adding NaOH. These
DNPH derivatives with large conjugated structures were directly measured spectrophotometrically at
465 nm and 425 nm, without the need for precipitating, washing and suspending procedures. The
addition of NaOH caused a red shift of the maximum absorption wavelength of these derivatives, which
Received 25 March 2016
Received in revised form
8 June 2016
Accepted 20 June 2016
Available online 5 August 2016
Keywords:
2,4-dinitrophenylhydrazine
Monoamine oxidase
DNPH spectrophotometry
reduced the interference of free DNPH. MAO-B protein was as low as 47.5 mg in rat liver with correlation
coefficients ranging within 0.995e0.999. This method is 2e3 times more sensitive than direct spectro-
photometry. The detection of MAO inhibition through this method showed that IC50 values of rasagiline
are 8.00 ꢀ 10ꢁ9 M for MAO-B and 2.59 ꢀ 10ꢁ7 M for MAO-A. These results are similar to the values
obtained by direct spectrophotometry. Our study suggests that DNPH spectrophotometry is suitable to
detect MAO activity, and has the potential for MAO inhibitor screening in the treatment of MAO-
mediated diseases.
© 2016 Published by Elsevier Inc.
1. Introduction
Monoamine oxidase (MAO) [1,2] (MAO; EC 1.4.3.4), a fla-
voenzyme of the mitochondrial outer membrane, plays an impor-
tant role in the regulation of the metabolism of exogenous amines,
Abbreviations: 5-HT, 5-hydroxytryptamine; DNPH, 2, 4-dinitrophenylhydrazine;
MAO, monoamine oxidase; MAO-A, monoamine oxidase-A; MAO-B, monoamine
oxidase-B; MAOI, monoamine oxidase inhibitor.
* Corresponding author. Department of Medicinal Chemistry, College of Phar-
macy, Chongqing Research Center for Pharmaceutical Engineering, Chongqing
Medical University, 1 Yi Xue Yuan Road, Chongqing 400016, China. Tel.: þ86 023
68485983.
E-mail addresses: 15215135399@163.com (G. Huang), 1612763995@qq.com
(F. Zhu), 316970293@qq.com (Y. Chen), 634638141@qq.com (S. Chen),
781522794@qq.com (Z. Liu), 704921339@qq.com (X. Li), 365620247@qq.com
(L. Gan), 474941459@qq.com (L. Zhang), yuyu3519@163.com (Y. Yu).
the level of neurotransmitters (such as dopamine, norepinephrine
and serotonin) and intracellular amine stores. MAO exists as two
isoforms (MAO-A and MAO-B), according to their substrate speci-
ficity and sensitivity to specific inhibitors [3]. MAO-A preferentially
oxidizes norepinephrine (NE) and serotonin (5-hydroxytryptamine,
5-HT). In contrast, MAO-B mainly oxidizes phenethylamine (PEA)
and benzylamine [4]. MAO-A and MAO-B are widely expressed in
most tissues at different concentrations, as shown in Table 1 [1];
and abnormal MAO activities are linked to various diseases [5,6].
Since amine neurotransmitters are metabolized extensively by
MAO in the CNS system [7], an increase in MAO expression or ac-
tivity may be related to depression, Parkinson's disease and Alz-
heimer's disease. At present, selective MAO-A and MAO-B
inhibitors have been used to treat the aforementioned and other
diseases [6,8]. Thus the exploration of proper methods to
1
Tel.: þ86 15215135399.
2
Tel.: þ86 15123828591.
3
Tel.: þ86 13996337514.
4
Tel.: þ86 18723484454.
5
Tel.: þ86 18680745865.
6
Tel.: þ86 18716318927.
7
Tel.: þ86 15922562348.
8
Tel.: þ86 15310097251.
http://dx.doi.org/10.1016/j.ab.2016.06.020
0003-2697/© 2016 Published by Elsevier Inc.

G. Huang et al. / Analytical Biochemistry 512 (2016) 18e25
19
Table 1
was used to measure MAO activity in this study; and MAO-
dependent aldehyde products were derivatized to form DNPH de-
rivatives with large conjugated structures and strong UV absorp-
tion. In addition, we modified the traditional DNPH
spectrophotometry, simplified the procedure, and reduced inter-
ference from free DNPH, as reported by Mesquita et al. A reaction
scheme (Fig. 1) illustrated the mechanism of this proposed method.
The sensitivity of this proposed method for the measurement of
MAO activity was compared with direct spectrophotometry, and
the validation of the proposed method was carried out by deter-
mining MAO inhibition by monoamine oxidase inhibitors (MAOI).
Distribution of MAO-A and MAO-B in humans and in the brains of selected species.
Tissue
Total activity (%)
MAO-A
MAO-B
Brain
Human (striatum:all brain regions) and Guinea pig
Monkey and Cat
Pig
20
25
40
55
50
100
80
75
60
45
50
0
Rat
Mice, Glia and astrocytes
Aminergic neurons
Other tissues
Liver(human, rat, rabbit) and Lings (rat)
Small intestine
Kidney (rat)
Human platelet and Chromaffin cell
Pheochromocytoma and PC-12 cells
55
~75
25
5
45
25
75
95
5
2. Material and methods
2.1. Materials
95
MAO-B inhibitor (rasagiline, [N-propargyl-1R(þ)-aminoindan]),
and 5-HT creatinine sulfate monohydrate (1064561, TCL) were used
in this study. Benzylamine, DNPH and Triton X-100 were purchased
from Adamas Reagent Co., Ltd. All other reagents were of analytical
grade.
determine MAO activity is crucial for disease diagnosis, and in
screening more potent MAO inhibitors.
MAO activity has been determined by measurement of oxygen
consumption and the concentration of hydrogen peroxide (H2O2) or
an oxidized monoamine product based on their catalytic oxidative
deamination of monoamines [9]:
2.2. MAO sample preparation
Male Sprague-Dawley rats (13e15 months old) were obtained
from the Experimental Animal Center of Chongqing Medical Uni-
versity. These rats were maintained on a 12-h lightedark cycle, and
fed standard rat chow and tap water ad libitum. Rats were sacri-
ficed by decapitation, and livers and brains tissues were dissected
out, washed with sucrose (0.3 M) and frozen at ꢁ80 ꢂC until
analyzed. Rat liver mitochondrial MAO was isolated by the method
reported by Castillo [16] with a slight modification. The liver tissue
(5.0 g) or brain tissue (2.5 g) was homogenized at 1:40 (w/v) in
0.3 M of ice-cold sucrose, and centrifuged at 1824 g for 10 min. The
supernatants were collected and further centrifuged at 12,768 g for
35 min to obtain crude MAO protein precipitations. These pre-
RCH2NR1R2 þ H2O þ O2/RCHO þ NR1R2 þ H2O2
The measurement of oxygen consumption by an oxygen-
sensitive electrode requires specialized equipment, and is not
appropriate for the rapid measurement of a large numbers of
samples [10]. The H2O2 method is insensitive and not specific for
MAO, because most biological and synthetic compounds also
absorb at 230 nm. Additionally, complicated cellular processes
could influence the concentration of H2O2 [10,11]. Aldehydes,
which are oxidized monoamine products, are produced by the in-
cubation of amine substrates with MAO; and these can be detected
by spectrophotometry. This method is widely used without
requiring expensive or specialized equipment and reagents. How-
ever, this method is limited for the measurement of MAO with low
activity or concentration. Thus, improving the sensitivity of the
spectrophotometric assay is essential to detect MAO activity, and
this has the potential to screen MAO inhibitors in MAO-mediated
disease treatment.
It has been reported that carbonyls can be measured through
the direct or indirect reaction of carbonyl moieties with reagents
[12] such as DNPH, tritiated sodium borohydride, fluorescein thi-
osemicarbazide and fluorescein amine, since these derivatized
compounds possess the characteristic of absorbance, radioactivity,
or fluorescence, respectively. DNPH has been widely used to
determine carbonyl groups at approximately 370 nm [14]. How-
ever, the use of traditional DNPH spectrophotometry is time-
consuming and labor intensive due to complicated process,
including precipitating, washing and re-suspending with organic
solvents (ethanol/ethyl acetate and benzene). Moreover, free DNPH
also has absorption at approximately 370 nm; hence, the extraction
and washing steps of these derivatives are necessary to remove the
interference of unreacted DNPH.
cipitates were resuspended in 500 ml (for liver) or 250 ml (for brain)
of 0.3 M of sucrose, and were layered onto 40 ml (for liver) or 20 ml
(for brain) of 1.2 M of sucrose. The precipitates were centrifuged in
1.2 M of sucrose at 12,687 g for 40 min, in order to obtain MAO
protein precipitates. Following a single wash in potassium phos-
phate buffer (pH 7.60, 100 mM), the pure liver and brain MAO
protein precipitates were suspended in 40 ml (for liver) and 10 ml
(for brain) of potassium phosphate buffer; and stored in aliquots of
1 ml at ꢁ80 ꢂC for subsequent analysis. All steps were performed at
0e4 ꢂC conditions.
The protein concentrations of MAO protein precipitates were
determined using the Bicinchoninic acid (BCA) method [17], with
bovine serum albumin (BSA) as the standard. MAO protein con-
centration was expressed as microgram per microliter (
mg/ml).
3. Assay procedure
3.1. DNPH spectrophotometry
The DNPH spectrophotometry reported by Basha [18] was
Mesquita et al. reported a simplified DNPH spectrophotometric
assay to quantitatively determine carbonyls. The key procedure is
that the addition of NaOH makes the reaction system alkalizate
after adding DNPH [15]. This simplified method reduced the
interference of DNPH by shifting the maximum absorbance wave-
length of the derivatives. This allowed for the direct measurement
in the sample solution without the aforementioned process
described in the traditional DNPH method.
modified to detect MAO activity. The assay mixtures (1.5 ml) con-
tained potassium phosphate buffer (pH 7.60, 25 mM), 200
ml of
MAO protein homogenates and benzylamine (for detecting MAO-
B), or 5-HT (for detecting MAO-A). The solution was incubated for
20 min at 37 ꢂC before adding 200
m
l of 0.016 M benzylamine in
l of 0.02 M 5-HT and the incubation was continued
for 60 min. Next, 400 l of 2 M DNPH in 1 M HCl was added. After
buffer or 150
m
m
incubation for 40 min at room temperature, 2 ml of 1.25 M NaOH
containing 5 g/l of Triton X-100 was added; and the reaction
In order to improve the sensitivity of spectrophotometry, DNPH

20
G. Huang et al. / Analytical Biochemistry 512 (2016) 18e25
Fig. 1. The reaction scheme for catalytic oxidative deamination of monoamines and DNPH derivatives of target aldehydes (benzaldehyde or 5-hydroxyindole acetaldehyde).
mixture was kept for an additional 30 min at room temperature.
Finally, absorption was measured by a spectrophotometer at
465 nm and 425 nm for MAO-B and MAO-A, respectively.
and Service Solutions (SPSS) program.
4. Results and discussion
3.2. Direct spectrophotometry
4.1. UV spectroscopic characterization
The activities of MAO-A and MAO-B were determined by
The absorption spectra of reaction substrates and products were
measured at room temperature using a spectrometer of 1 nm
spectral resolution from 800 nm to 200 nm. The concentrations of
benzylamine, 5-HT and DNPH were 0.0008 M, 0.0002 M and
0.0125 M, respectively; and the absorption spectra are shown in
Fig. 2. The results indicate that the absorption peak of DNPH-
derivatives for MAO-B and MAO-A are at 465 nm and 425 nm,
respectively, without interference from 5-HT (275 nm), benzyl-
amine (256 nm) and DNPH (365 nm). The alkalized reaction system
with the addition of NaOH and the transformation from DNPHy-
drazones to quinone anion not only simplifies the processes of
protein precipitating, washing and redissolving, but also avoids the
use of organic solvents such as benzene, ethanol/ethyl acetate, and
guanidine hydrochloride. Compared to the method reported by
Basha [18], our method shortened experimental time from
approximately 200 mine100 min. Moreover, the alkaline condition
shifted the maximum wavelength of the DNPH derivatives from
375 nm [19] to 465 nm (for determining MAO-B) and 425 nm (for
determining MAO-A), in which interference from the absorption of
free DNPH or other substances were modest.
UVeVis spectrophotometry. Briefly, 800
potassium phosphate buffer (pH 7.60, 100 mM), 200
m
l of a solution containing
l of MAO
m
protein homogenates and benzylamine (for detecting MAO-B) or 5-
HT (for detecting MAO-A) was incubated for 20 min at 37 ꢂC. The
reaction was started by adding 150
100 mM buffer or 200 l of 0.02 M 5-HT, and incubated for 60 min.
The reaction was stopped by adding 200 l of 10% perchloric acid or
200 l of 2 M HCl. Then, 3 ml of hexamethylene (for detecting MAO-
ml of 0.016 M benzylamine in
m
m
m
B) or n-butyl acetate (for detecting MAO-A) was added to the re-
action solution; and the tubes were vortexed for 3 min. Following
centrifugation at 10,625 g for 6 min, absorbance was determined
using a spectrophotometer at 242 nm and 280 nm for MAO-B and
MAO-A, respectively.
3.3. Determination of MAO inhibitory activity by MAO inhibitors
in vitro
The stock solution of rasagiline was prepared with ultra-pure
water, and the preincubation was carried out in the presence of
increasing concentrations of rasagiline. In order to compare this
assay with direct spectrophotometry, MAO-B and -A activities were
determined by DNPH spectrophotometry and direct spectropho-
tometry, as described above. Finally, IC50 values of rasagiline for
MAO-B and MAO-A were calculated using the Statistical Product
4.2. Optimization of DNPH spectrophotometry
In order to improve the sensitivity further, experimental con-
ditions containing the concentration of substrates, pH and the

G. Huang et al. / Analytical Biochemistry 512 (2016) 18e25
21
Fig. 2. Absorption spectra (800 nme200 nm) of 5-HT (0.0002 M), benzylamine (0.0008 M), DNPH (0.0125 M), DNPH-derivatives for MAO-B and MAO-A, and the MAO protein were
234.3 and 468.5 g, respectively, to obtain DNPH-derivatives for MAO-B and MAO-A. The graph shows that the absorption peaks are at 275 nm, 256 nm, 365 nm, 465 nm and
m
425 nm, respectively.
concentration of potassium phosphate buffer, as well as reaction
time and DNPH derivatizing time were optimized using four liver
(Fig. 4) and calibration curves (Fig. 5) were constructed using the
origin 8 program. The spectra showed that the absorption of
derivatized compounds increased with the increase in MAO protein
concentration. Fig. 5 shows a linear relationship between MAO
protein concentrations and absorbance with correlation co-
efficients ranging from 0.995 to 0.999. Regression equations for
MAO-B and MAO-A were A ¼ 0.00421x and A ¼ 0.00258x by DNPH
spectrophometry, respectively; and A ¼ 0.00151x and A ¼ 0.00074x
by direct spectrophotometry, respectively.
There is an excellent linear relationship between DNPH spec-
trophotometry and direct spectrophotometry for the measurement
of MAO activity, whereas the slopes of DNPH spectrophotometry
were greater than that obtained by direct spectrophotometry based
on regression equations. This indicates that the DNPH spectro-
photometric assay is more sensitive than is direct spectropho-
tometry. Compared to direct spectrophotometry, less MAO protein
is needed when using the DNPH spectrophotometric assay to attain
equivalent absorbance. Based on regression equations, the sensi-
tivity of DNPH spectrophotometry was found to be approximately
2.8 and 3.5-fold greater than that obtained with the direct spec-
trophotometric method for measuring MAO-B and MAO-A activ-
ities, respectively. The possible reason could be that DNPH
derivatives for MAO-A and MAO-B possess different conjugated
structures, and usually have different UV absorption coefficients;
which might lead to difference in sensitivity.
MAO protein homogenates (4.69
mg/ml) for each experimental
condition. The effects of pH and the concentration of the potassium
phosphate buffer were evaluated in various concentrations (25, 50,
75 and 100 mM) and pHs (7.20, 7.40, 7.60 and 7.80). These results
indicated that the increase in potassium phosphate buffer con-
centration was associated with a significant reduction in MAO-B
activity, but this had minor effects on MAO-A activity. Further-
more, these maximum absorbances for MAO-A and MAO-B were
obtained with the potassium phosphate buffer (pH 7.60, 25 mM)
(Fig. 3a and b). In order to measure the substrate content required
to react with MAO proteins, the influence of substrate concentra-
tion was investigated between 0.8 and 4 mM. It was found that
benzylamine (3.2 mM) and 5-HT (4 mM) produced the maximum
absorbance (Fig. 3c). In order to determine the time required for the
amine substrate (benzylamine or 5-HT) to react with the MAOs, the
influence of reaction time was also investigated. The results indi-
cated that the increase in time was associated with a corresponding
increase in absorbance up to 60 min, as shown in Fig. 3d. Further-
more, in order to determine the time required for DNPH to react
with all produced aldehydes, the influence of DNPH derivatizing
time was investigated. Maximum absorbance was obtained after
for 40 min of DNPH derivatization. Thus, 40 min was used as the
optimum DNPH derivatizing time (Fig. 3e).
Sensitivity and precision were determined by measuring the
activity of equal amounts of MAO proteins (4.69
mg/ml in the liver,
4.3. Validation of DNPH spectrophotometry
3.00 g/ l in the brain) from rat liver and brain. In DNPH spectro-
m
m
photometry, it was shown that the relative standard deviation
(RSD) values for precision (n ¼ 5) and repetition (n ¼ 3) were
0.69e3.34% and 3.92e7.44%, respectively. In direct spectropho-
tometry, it was found that the RSD values for precision (n ¼ 5) and
repetition (n ¼ 3) were 2.54e3.29% and 1.26e8.09%, respectively.
Table 2 summarizes the results for precision and repetition, and
indicates that both DNPH spectrophotometry and direct spectro-
photometry povide good precision and repetition in the measure-
ment of MAO activity. However, Fig. 6 and Table 2 show that the
The validity of the proposed method was evaluated following
Bio-analytical Method Validation. MAO protein concentration was
measured by BCA assay, and average total protein concentrations of
the liver and brain MAO protein precipitations were found to be
approximately 4.69 and 3.00
mg/ml, respectively. The calibration
curves were constructed in rat liver MAO proteins under optimum
conditions. Absorbance for various concentrations of MAO (from
23.4 to 468.5
mg) was determined by DNPH spectrophotometry and
compared to direct spectrophotometry. The absorption spectra

22
G. Huang et al. / Analytical Biochemistry 512 (2016) 18e25
Fig. 3. Effects of reaction conditions of DNPH spectrophotometry on MAO activity. a, concentration of potassium phosphate buffer; b, pH of potassium phosphate buffer; c,
concentration of substrate (benzylamine or 5-HT); d, reaction time and e DNPH derivatizing time. The absorbance are the mean of four samples and the results are shown as
mean SD (n ¼ 4).
absorbance by DNPH spectrophometric assay is approximately 2e3
times more sensitive by direct spectrophotometric assay, because
benzaldehyde and 5-hydroxyindole acetaldehyde are derivatized
by DNPH to form the corresponding DNPHydrazones and quinone
anion that have large conjugated structures with the transitions
bioluminescent assay could increase sensitivity and reduce inter-
ference with external fluorescence; but expensive luciferin detec-
tion reagents (esterase and luciferase) are needed. Moreover, MAO-
B and MAO-A activities were constructed in the presence of specific
inhibitors or specific MAO-A/-B proteins. In addition, HPLC could
improve sensitivity and specificity, but it is more expensive and
laborious [24e26]; and the HPLC column has a limited life time if
guanidine hydrochloride is used to dissolve the DNPhydrazone
compound [14,27]. More than 205 and 90 min are needed in the
traditional DNPH spectrophotometry reported by Basha [18] and
direct spectrophotometry, respectively; and our method only
required 105 min. Therefore, this proposed method has advantages
in time and experimental operation, and is more sensitive, when
compared to direct spectrophotometry.
p/p* (Fig. 1). This results in stronger UV absorptions compared to
the underivatized products. This behavior is similar to reported
calibration curves. Fig. 5 shows that lower MAO concentrations are
required for DNPH spectrophotometry to achieve the same absor-
bance in the two assays. In order to achieve an absorbance of
0.2e0.8, the MAO protein concentration required was approxi-
mately 47.5 and 77.5
DNPH spectrophotometry; while MAO protein concentration was
132 and 270 g for MAO-B and MAO-A, respectively, by direct
mg for MAO-B and MAO-A, respectively, by
m
spectrophotometry. Although the sensitivity of DNPH spectropho-
tometry is lower than ELISA [20,21], fluorophotometry [22,23] and
radioisotope assay [7], the radioisotope assay is time-consuming
and expensive, while flurophotometry is more likely to be
affected by the fluorescent interference of test compounds. Based
on luminescent signals, the bioluminescent assay has been devel-
oped for assaying MAO activity; and is well-suited for high-
throughput screening inhibitors [11]. Furthermore, the
4.4. Analysis of the inhibition of rasagiline for MAO in vitro
This proposed DNPH spectrophotometric method was used to
determine the inhibition of rasagiline, in order to reflect MAO ac-
tivity in rat livers and brains in vitro. The change on maximum
absorbance was associated with the interaction between rasagiline
and enzymes (Fig. 7). IC50 values were calculated by SPSS, and are

G. Huang et al. / Analytical Biochemistry 512 (2016) 18e25
23
Fig. 4. The increasing concentration of MAO protein results in a corresponding increasing in absorbance by DNPH spectrophotometric method (a,b) and direct spectrophotometric
method (c,d).
Fig. 5. Comparison of calibration curves for DNPH spectrophotometry (square) and direct spectrophotometry (circle). From left to right, the loads were 23.4, 46.9, 117, 234, 469 mg of
MAO protein. The graph shows that the regression equation for MAO-B and MAO-A are A ¼ 0.00421x, R2 ¼ 0.995 and A ¼ 0.00258x, R2 ¼ 0.997 by DNPH spectrophometry and
A ¼ 0.00151x, R2 ¼ 0.996 and A ¼ 0.00074x, R2 ¼ 0.999 by direct spectrophotometry.
Table 2
Repetition and precision analysis of DNPH spectrophotometry.
Methods
Tissues
Repetition
Mean SD
Precision
RSD
Mean SD
RSD
DNPH spectrophotometry
Liver MAO-B
Liver MAO-A
Brain MAO-B
Brain MAO-A
Liver MAO-B
Liver MAO-A
Brain MAO-B
Brain MAO-A
1.53 0.06
0.840 0.06
1.21 0.09
1.07 0.05
0.792 0.01
0.307 0.01
0.681 0.05
0.371 0.03
3.92%
7.14%
7.44%
4.67%
1.26%
3.26%
7.34%
8.09%
1.45 0.01
0.897 0.03
1.24 0.01
1.02 0.01
0.788 0.02
0.304 0.01
0.723 0.02
0.392 0.01
0.69%
3.34%
0.81%
0.98%
2.54%
3.29%
2.77%
2.55%
Direct spectrophotometry
The equal volume tissue homogenates were incubated for 20 min at 37 ꢂC before addition of benzylamine (for MAO-B) or 5-HT (for MAO-A) and continuously incubated 60min,
MAO activities were determined by DNPH spectrophotometry and direct spectrophotometry, and the absorbances are given as mean SD (n ¼ 5). The RSD is presented as the
following equation: RSD (%) ¼ (SD ꢀ 100)/mean.
presented in Table 3. In order to compare the reliability of this
proposed assay, the inhibition of rasagiline for MAO activity was
also measured by direct spectrophotometry. There were no signif-
icant differences in IC50 (P > 0.05) between the DNPH and direct
spectrophotometry methods. Rasagiline was approximately 60
times more potent for the inhibition of MAO-B compared with
MAO-A in both DNPH and direct spectrophotometry. In addition,
IC50 values of rasagiline by DNPH spectrophotometry were
8.00 ꢀ 10ꢁ9 M for MAO-B and 2.59 ꢀ 10ꢁ7 M for MAO-A; while IC50
values of rasagiline were 4.43
ꢀ
10ꢁ9
M for MAO-B and

24
G. Huang et al. / Analytical Biochemistry 512 (2016) 18e25
Table 4
Published IC50 (M) values by different methods.
Tissue
MAO-B
MAO-A
Reference
Rat brain
Rat brain
4.43 0.92 ꢀ 10ꢁ9 4.12 1.23 ꢀ 10ꢁ7 MBH. Youdim [28]
4
0.001 ꢀ 10ꢁ9
0.41 0.12 ꢀ 10ꢁ6 H Zheng [7]
Human brain 1.4 0.35 ꢀ 10ꢁ8
7.1 0.93 ꢀ 10ꢁ7
7.10 ꢀ 10ꢁ7
7.3 ꢀ 10ꢁ8
MB.H. Youdim [28]
JJ. Chen [29]
JJ. Chen
Human brain 1.4 ꢀ 10ꢁ8
Rat brain
Rat brain
Rat brain
F344/N rats
2.5 ꢀ 10ꢁ9
2.3 ꢀ 10ꢁ9
1.49 ꢀ 10ꢁ7
4.12 ꢀ 10ꢁ7
JJ. Chen
4.43 ꢀ 10ꢁ9
JJ. Chen
MK Hu [23]
2.1 0.6 ꢀ 10ꢁ6
MAO activity inhibitions by rasagiline were published and taken from reference.
5. Conclusion
In this study, we developed a sensitive and simple spectropho-
tometry to detect MAO activity using DNPH as a derivatizing agent.
The DNPH spectrophotometry is suitable for measuring MAO with
low activity or at a low concentration. This method could be po-
tentiality used to evaluate IC50 values of inhibitors with the following
advantages: (1) compared to direct spectrophotometry, this method
is more sensitive for measuring MAO with low activity or concen-
tration, since DNPhydrazones have a larger conjugated structure and
stronger UV absorption; (2) by adding NaOH, the alkalization of this
reaction system shifts the absorption wavelength from 370 nm to
425/465 nm, which eliminates interference from free DNPH; (3) this
method shortens the experimental time, since the application of an
alkaline solution simplifies the tedious procedures of precipitating,
washing and redissolving processes; (4) this method avoids the use
of organic solvents and expensive or sophisticated equipment,
showing that this method has certain advantages in experimental
Fig. 6. An equal volume of liver or brain homogenate was incubated with substrate
37 ꢂC and the MAO-B and MAO-A activities were determined with DNPH spectro-
photometry and direct spectrophotometry. The absorbance is presented as mean SD
(n ¼ 5) and the ratio (r) shows the increased sensitivity by DNPH spectrophotometry
compared to direct spectrophotometry.
4.12 ꢀ 10ꢁ7 M for MAO-A by a radioisotope method, as reported in
published literature [28,29]. These are shown in Table 4. These
indicate that the IC50 values and selectivity were in good agreement
with the range of the reported and obtained values using the
radioisotope assay. It is worth noting that DNPH spectrophotom-
etry has the potential to be widely used, since it does not need a
radioisotope and expensive instruments [12,13]. Thus, our method
has the potential to measure MAO activity even in large samples,
and could be widely applied for screening MAOIs.
Fig. 7. In vitro inhibition of rasagiline against Sprague-Dawley (SD) rat liver and brain MAO. Rasagiline was added at concentration of 10ꢁ7 M to 10ꢁ9 M. Then the solution was
incubated with liver and brain homogenates for 20 min at 37 ꢂC before adding benzylamine or 5-HT and incubated continuously 60 min. Then absorbance was determined by DNPH
spectrophotometry (a) and direct spectrophotometry (b). The inhibition was calculated as the equation: inhibitionð%Þ ¼ ð1 ꢁ A=BÞ ꢀ 100%.
Table 3
IC50 values (M) for inhibition of MAO-B and MAO-A by rasagiline in SD rat liver and brain in vitro.
Methods
Liver
Brain
MAO-B
MAO-A
MAO-B
MAO-A
DNPH spectrophotometry
Direct spectrophotometry
5.00 ꢀ 10ꢁ9*
3.38 ꢀ 10ꢁ7*
8.00 ꢀ 10ꢁ9*
2.59 ꢀ 10ꢁ7*
5.80 ꢀ 10ꢁ9
3.97 ꢀ 10ꢁ7
8.00 ꢀ 10ꢁ9
2.11 ꢀ 10ꢁ7
The inhibition by rasagiline at various concentrations was measured by DNPH spectrophotometry and direct spectrophotometry. IC50 values were calculated using 5e7
concentrations of rasagiline by SPSS program, *P > 0.05: no significant difference between DNPH spectrophotometry and direct spectrophotometry.

G. Huang et al. / Analytical Biochemistry 512 (2016) 18e25
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studies when compared with other methods.
In conclusion, this proposed method possesses properties of
simplicity, sensitivity, safety and rapidity. It is suitable for
measuring MAO activity, and has the potential to be used for
screening MAOIs in many laboratories.
Acknowledgments
The authors gratefully thank Pharmacy college of Chongqing
Medical University for assistance with instruments. I thanks Hon-
gmei Guo for guidance in my experiment, and my companions for
their care. Lastly, this paper is supported by The National Natural
Science Foundation of China (81172097, 30772595 and 30371632).
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