MS Based Simultaneous Determination of

Article pubs.acs.org/ac

High-Throughput LC−MS/MS Based Simultaneous Determination of Polyamines Including N‑Acetylated Forms in Human Saliva and the Diagnostic Approach to Breast Cancer Patients Haruhito Tsutsui,† Toshiki Mochizuki,† Koichi Inoue,† Tatsuya Toyama,‡ Nobuyasu Yoshimoto,‡ Yumi Endo,‡ Kenichiro Todoroki,† Jun Zhe Min,† and Toshimasa Toyo’oka*,† †

Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan ‡ Department of Oncology, Immunology & Surgery, Graduate School of Medical Sciences, Nagoya City University, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan ABSTRACT: The determination of polyamines and their N-acetylated forms was performed by ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC−MS/MS). The polyamines efficiently reacted with 4-(N,N-dimethylaminosulfonyl)7-fluoro-2,1,3-benzoxadiazole (DBD-F) in 0.1 M borax (pH 9.3) at 60 °C for 30 min. The resulting derivatives were analyzed by electrospray ionization (ESI)-MS and sensitively detected by selected reaction monitoring (SRM). Furthermore, a rapid separation of the polyamine derivatives within 10 min was performed by UPLC using an antipressurized column packed with 1.7-μm octadecylsilyl (ODS) silica gel. The limits of detection (S/N = 3) on the SRM chromatograms were at the attomole level (9−43 amol). This procedure was used to successfully determine 11 polyamines, including their N-acetylated forms, in the saliva of patients with primary and relapsed breast cancer and healthy volunteers. The level of several polyamines (Ac-PUT, Ac-SPD, Ac-SPM, DAc-SPD, and DAc-SPM) increases in breast cancer patients. Furthermore, the levels of three polyamines (Ac-SPM, DAc-SPD, and DAc-SPM) were significantly higher only in the relapsed patients. The present method proved highly sensitive and is characterized by specificity and feasibility for sample analysis. Consequently, the proposed method is useful for the noninvasive salivary diagnosis of cancer patients and could be applied to determine polyamines in several specimens of biological nature.

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are high-molecular-mass substances such as glycoprotein and enzyme. On the contrary, biogenic amines such as histamine and polyamine are known as markers of low-molecular-mass compounds for various diseases. Naturally occurring polyamines are components of all cell types that exert multiple functions, such as RNA and DNA stabilizers, growth factors, second messengers, antioxidants, nutrients, and metabolic regulators.3−5 Furthermore, it is widely accepted that the levels of polyamines have correlations with several diseases. For instance, the marked increase of the biosynthesis of polyamines has been associated with rapid tumor growth,6 resulting in the increase of their levels in urine and plasma.7−9 Since L-ornithine (ORN) is the main precursor of polyamines, a method capable of simultaneously determining ORN and its derivatives in complex matrixes is important to unveiling the biological functions of these molecules in biochemical and physiological processes. The concentration of N,N-diacetyl-spermine (DAc-SPM) in urine increases in breast cancer patients.10 However, the

reast cancer cases are increasing worldwide, not only in European and American countries, but also in Asian countries.1 Although noninvasive nonmelanoma skin cancer is the most frequent, breast cancer comprises more than 20% of the invasive cancers in women, and its incidence has significantly increased in the past 40 years, probably due to lifestyle. Breast cancer incidence is linked to the patients’ ages, with only 5% of all cases occurring in women younger than 40 years old. Many breast cancers can be easily diagnosed by analyzing the biopsy with a microscope. While screening techniques aid in the determination of cancer chances, additional analyses are needed to verify whether a lump is a real cancer. Among the screening tests, mammography, clinical examination, genetic screening, magnetic resonance imaging, and ultrasound have been employed. However, women struggle against these tests because of the embarrassment, waste of time, and high expenses. Consequently, another screening method, which is a simple, not embarrassing, and low cost procedure, is highly desired. Many tumor markers correspond to specific diseases, and they are used in oncology to help detect the presence of cancer. Several tumor markers such as CA15-3 are known to increase in breast cancer patients.2 However, elevated levels of tumor markers could be induced by different causes. Tumor markers © XXXX American Chemical Society

Received: August 9, 2013 Accepted: November 14, 2013

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dx.doi.org/10.1021/ac402526c | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), was used for the determination of polyamines in the saliva of breast cancer patients and healthy volunteers.

Table 1. Detection of Polyamines Labeled with DBD-F by UPLC−ESI-MS/MS

a

polyamine

precursor ion [M + H]+

product ion (m/z)

cone voltage (V)

collision energy (eV)

LODa (S/N = 3) (amol)

Ac-PUT DAc-SPD DAP PUT CAD DAH (IS) ORN Ac-SPD DAc-SPM SPD Ac-SPM SPM

356.13 455.19 525.13 539.14 553.16 567.17 583.13 638.21 737.27 821.22 920.29 1103.3

311.08 100.08 437.03 451.10 465.06 479.07 495.03 550.11 100.08 733.12 834.20 1015.2

30 30 30 30 30 30 30 40 40 50 50 50

10 22 20 20 20 20 22 25 33 26 38 38

34 9.0 12 6.0 18 n.d. 23 43 21 10 10 24

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EXPERIMENTAL SECTION Materials and Chemicals. Eleven polyamines were used: ornithine (ORN), putrescine (PUT), cadaverine (CAD), spermine (SPM), N1N8-diacetyl-spermidine (DAc-SPD), and N1N12-diacetyl-spermine (DAc-SPD) were from Wako (Osaka, Japan); diaminopropane (DAP) and 1,6-diaminohexane (DAH) were from Tokyo Kasei (Tokyo, Japan); N8-acetylspermidine (Ac-SPD), N1-acetyl-putrescine (Ac-PUT), and N1acetyl-spermine (Ac-SPM) were from Sigma-Aldrich (St. Louis, MO); and spermidine (SPD) was from Kanto Chemicals (Tokyo, Japan). DAH was used as the internal standard (IS). DBD-F was obtained from Tokyo Kasei. LC−MS-grade formic acid (FA) and acetonitrile were purchased from Kanto Chemicals. The water used throughout the study was either deionized or distilled by using the Aquarius PWU-200 automatic distillation apparatus (Advantec, Tokyo, Japan). All other reagents and solvents were of analytical grade and used without further purification. UPLC−ESI-MS/MS. The high-throughput liquid chromatography system was an ACQUITY ultraperformance liquid chromatograph (UPLC-I class, Waters). The reversed-phase (RP)

LOD, limit of detection; n.d., not determined.

concentration change of other polyamines is not clearly understood. Therefore, we tried to determine 11 polyamines, including their acetylated forms, simultaneously by highthroughput liquid chromatography−electrospray ionization tandem mass spectrometry (LC−ESI-MS/MS). The proposed method, which included the derivatization with

Figure 1. Structures of the tested polyamines. B



Article

Table 2. Calibration Curves of Polyamines Labeled with DBD-F polyamine

Figure 2. General reaction scheme between polyamines and DBD-F.

calibration range (fmol)

SPM

1−1000

Ac-SPM

1−1000

SPD

1−1000

DAc-SPM

1−1000

Ac-SPD

1−1000

ORN

1−1000

CAD

1−1000

PUT

1−1000

DAP

1−1000

DAc-SPD

1−1000

Ac-PUT

1−1000

linear equation y = 0.00102x − 0.00346 y = 0.0193x − 0.0678 y = 0.0235x − 0.104 y = 0.0361x − 0.0187 y = 0.0213x − 0.0659 y = 0.0225x + 0.484 y = 0.0365x − 0.406 y = 0.0482x − 0.0344 y = 0.0259x − 0.112 y = 0.0910x + 0.0610 y = 0.0357x − 0.106

linearity (R2)

RSD (%) (N = 5)

0.9947

3.0−5.3

0.9997

4.3−5.3

0.9991

1.3−6.5

1.0000

2.6−4.8

0.9996

5.1−9.9

0.9985

6.9−9.2

0.9965

2.4−5.9

0.9998

3.4−7.7

0.9991

0.7−5.6

1.0000

3.5−8.8

0.9995

2.9−6.7

DBD-F in acetonitrile at 60 °C for 30 min. Thereafter, the reaction mixtures were filtered through a Millex-LG membrane with 0.2-μm pores and 4 mm in inner diameter. An aliquot (5 μL) of the filtrate was analyzed by UPLC−ESI-MS/MS. Collection and Pretreatment of Human Saliva. Saliva (∼1 mL) was directly collected into a collecting tube (without a collection device) from breast cancer patients (primary, n = 8; relapse, n = 22) and healthy volunteers (n = 14) and stored below −20 °C until used. The subjects fasted and did not brush their teeth 1 h before the samples were collected. All the subjects provided written informed consent. The experimental procedures were conducted in accordance with the ethical standards of the Helsinki Declaration and were approved by the Ethics Committee of the University of Shizuoka and Nagoya City University. After thawing, the saliva sample was centrifuged at 3000g for 10 min to precipitate the denatured mucins. The supernatant (30 μL) was mixed with acetonitrile (120 μL) containing IS (3 pmol), vortexed for 30 s, and centrifuged again at 3000g for 5 min. The solvent of supernatant was then removed. The residue was redissolved in 0.1 M borax, reacted with DBD-F, and subjected to UPLC−ESI-MS/MS as described in the section Derivatization of Polyamines with DBD-F. Analytical Validation. Calibration Curves. Fixed concentrations of the polyamines (2−2000 nM) were prepared by sequential dilutions of the stock solutions. The working solutions (30 μL) were pretreated and reacted as indicated in the section Derivatization of Polyamines with DBD-F. The derivatization solutions (5 μL) were then analyzed by UPLC−ESIMS/MS. The calibration curves were constructed by plotting the peak area ratio of the polyamines to IS (y) versus the concentration of polyamines (x, ng/mL) using linear regression weighted by 1/x (n = 5). Intraday and Interday Assays. The precisions of the intraand interday assays were determined by repeating the measurement (n = 5) on the saliva samples obtained from two volunteers in 1 day and over a 5-day period. The precision was reported as the relative standard deviation (RSD, %).

Figure 3. UPLC−ESI-MS/MS spectra of SPD, Ac-SPD, and DAc-SPD derivatives.

analysis was performed using an ACQUITY UPLC BEH C18 column (1.7 μm, 100 mm × 2.1 mm i.d.; Waters) at 40 °C. The mobile phase consisting of solvent A (0.1% FA in water) and solvent B (0.1% FA in acetonitrile) was delivered at the flow rate of 0.4 mL/min. The gradient elution was as follows: B% = 20, 60, 90, 98, 98, 20, and 20 (0, 8, 10, 11, 12, 13, and 20 min). The separated compounds were detected by a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA). Unless otherwise stated, the polyamines derivatized with DBD-F were analyzed by UPLC−ESI-MS/MS using the positive-ion mode. The detection conditions were as follows: capillary voltage, 3.00 kV; cone voltage, 30−50 V; desolvation gas flow, 1000 L/h; cone gas flow, 150 L/h; collision energy, 20−25 eV; nebulizer gas flow, 7.0 L/h; collision gas flow, 0.15 mL/min; desolvation temperature, 500 °C; collision cell exit potential, 5 V. MassLynx, version 4.1 analytical software was used for the system control and data processing. The precursor and product ions of each molecule are summarized in Table 1. Derivatization of Polyamines with DBD-F. The polyamines and DAH (IS) were dissolved in acetonitrile each at 0.1 μM concentration. The solutions (30 μL each) were vigorously mixed and the solvent was removed. The resulting residue was dissolved in 150 μL of 0.1 M sodium tetraborate (borax, pH 9.3) and then reacted with an equal volume of 40 mM C



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Table 3. Recovery of Polyamines Spiked in the Saliva of Healthy Volunteers intact amt (fmol)

spiked amt (fmol)

SPM Ac-SPM SPD DAc-SPM Ac-SPD ORN CAD PUT DAP DAc-SPD Ac-PUT

10.9 2.80 6.70 67.6 8.50 109 55.6 230 151 74.5 242

1.70 1.70 1.70 16.7 1.70 16.7 16.7 16.7 16.7 16.7 16.7


7.80 1.20 6.60 85.2 4.10 50.4 58.8 257 68.6 56.4 324

1.70 1.70 1.70 16.7 1.70 16.7 16.7 16.7 16.7 16.7 16.7

recovery (%) Saliva A 99.2 99.5 98.8 92.8 99.9 99.5 96.4 102 86.3 108 96.3 Saliva B 100 99.5 100 101 94.8 99.3 93.2 97.0 87.9 93.4 102

Table 4. Intraday and Interday Assays of Polyamines in Human Saliva by Proposed Procedure saliva A (fmol) SPM Ac-SPM SPD DAc-SPM Ac-SPD ORN CAD PUT DAP DAc-SPD Ac-PUT

9.76 2.87 6.69 58.2 8.00 131 51.1 185 173 66.6 218


9.52 2.81 6.23 55.2 7.90 125 50.5 180 166 60.6 208

RSD (%) Intraday 4.89 1.04 7.25 3.95 0.560 4.28 5.97 3.23 4.56 7.30 9.85 Interday 5.15 5.61 2.28 8.24 3.90 2.91 9.74 4.12 6.55 4.81 5.37

saliva B (fmol)

RSD (%)

7.76 1.23 6.72 86.4 4.27 50.7 59.6 264 67.0 59.4 331

6.53 3.10 4.56 5.81 1.65 8.30 5.32 5.21 5.71 9.22 9.08

7.65 1.17 6.56 84.4 4.05 51.2 56.3 259 66.2 55.1 329

4.68 8.09 2.51 5.26 3.79 5.74 8.93 2.28 7.11 5.09 2.40

RSD (%)

spiked amt (fmol)

recovery (%)

RSD (%)

1.6 2.2 1.2 3.5 5.0 1.5 3.1 2.9 1.9 7.0 3.4

3.30 3.30 3.30 33.4 3.30 33.4 33.4 33.4 33.4 33.4 33.4

99.3 93.1 106 103 94.1 97.0 101 99.0 93.7 96.5 96.7

0.70 1.7 2.9 1.3 0.90 2.3 5.9 1.5 1.4 1.3 4.7

3.6 8.0 3.6 13 11 2.4 5.0 3.1 8.9 8.3 3.7

3.30 3.30 3.30 33.4 3.30 33.4 33.4 33.4 33.4 33.4 33.4

96.4 93.1 103 95.8 104 97.6 99.1 96.6 89.4 93.7 101

7.3 2.7 2.9 9.9 1.3 6.7 7.2 8.0 11 5.2 3.1

Table 5. Freeze/Thaw Stability of Polyamines in Human Saliva saliva A freeze/thaw cycle

saliva B freeze/thaw cycle

polyamine

initial (%)

1

2

1

2


100 100 100 100 100 100 100 100 100 100 100

95.2 98.8 102 98.2 95.9 88.8 100 103 99.9 92.2 94.5

101 98.5 98.8 89.5 96.7 92.2 97.6 98.2 103 89.1 102

106 93.4 102 98.8 101 92.2 98.9 99.2 98.8 89.9 95.5

99.8 95.5 101 93.4 101 93.0 97.7 97.7 99.2 87.7 102

polyamines were determined from respective calibration curves. The recovery was defined as F/(F0 + A)·100 (%), where F and F0 are the concentrations of the polyamines in the spiked and unspiked samples, respectively, and A is the spiked concentration. Freeze/Thaw Stability. The stability of the polyamines in the saliva during freeze/thaw was examined by determining two saliva samples with/without additional freeze/thaw cycles.

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RESULTS AND DISCUSSION Derivatization of Polyamines with DBD-F. Polyamines possess hydrophilic and strong basic properties in virtue of their primary and secondary amino functional groups (Figure 1). The simultaneous separation of polyamines by RP chromatography is challenging because they adsorb on the column resins. Furthermore, sensitive detection could not be achieved due to their low absorption in the ultraviolet (UV) wavelength region.

Recovery Tests. The polyamines (30 μL; SPM, Ac-SPM, SPD, and Ac-SPD: 1.7 and 3.3 fmol; ORN, CAD, PUT, DAP, Ac-PUT, DAc-SPD, and DAc-SPM: 16.7 and 33.4 fmol) were spiked in 30-μL saliva samples (n = 5). The spiked and unspiked solutions were then pretreated and subjected to UPLC− ESI-MS/MS, as described in the section Collection and Pretreatment of Human Saliva. The concentrations of the D



Article

Figure 4. SRM chromatograms of polyamine derivatives obtained from the human saliva of a healthy volunteer. The UPLC−ESI-MS/MS conditions are described in the Experimental Section and Table 1

Although several methods based on gas chromatography (GC),11,12 LC,13−19 and capillary electrophoresis (CE)20,21 have been established, in virtue of its high sensitivity and selectivity, the coupling of high performance liquid chromatography (HPLC) separation and fluorescence detection seems to be a reliable strategy to determine polyamines in real matrixes. Several fluorescent probes, such as dansyl chloride, 9-fluorenylmethyl chloroformate, and o-phthaladehyde/thiol, were used to derivatize polyamines before and after HPLC separation.22 By using this method, polyamines were successfully determined in plasma and urine. However, in complex matrixes, the simultaneous determination of polyamines at very low concentrations remains challenging. We also used the fluorescent labeling technique to determine polyamines in hair samples.23 However, endogenous materials in the samples interfered with the polyamines that eluted fast. Thus, a more sensitive method for the specific and simultaneous determination of polyamines in trace amounts in real samples is needed.

Recently, MS became the major technique to determine trace amounts of chemicals in real matrixes such as urine and blood. Therefore, we attempted the simultaneous determination of polyamines by combining derivatization and MS detection.23,24 In this study, we chose DBD-F to label the polyamines contained in the saliva, because of its high sensitivity and reactivity toward amino groups.25 Figure 2 shows the general scheme for the labeling reaction of polyamines with DBD-F. The reaction efficiently proceeds in alkaline medium. On the basis of previous reports,25,26 we reacted the polyamines with DBD-F at 60 °C for 30 min in 0.1 M borax (pH 9.3). Under this condition, primary and secondary amines seem to be completely labeled in the presence of excess amounts of DBD-F, although the absolute yields of the derivatization reaction are not obvious. Separation and Detection of DBD-Labeled Polyamines. Instead of using a conventional HPLC system, to rapidly separate the DBD-labeled polyamines by UPLC, we E



Article

Figure 5. Average concentration of polyamines in the saliva of breast cancer patients and healthy volunteers (mean ± standard deviation). N, healthy volunteer (n = 14); B, primary patient (n = 12); R, relapsed patient (n = 24).

adopted an antipressurized column packed with a small, porous resin. A tandem quadrupole mass spectrometer was used to achieve highly sensitive detection. DBD-labeled polyamines maintain their basic properties because they contain secondary and tertiary amines. Therefore, we used the acidic mobile phase consisting of water−acetonitrile containing 0.1% FA to achieve simultaneous separation. The acidic mobile phase also favors the highly sensitive MS detection due to the high protonation efficiency by FA. Figure 3 shows the MS/MS spectra of SPD, Ac-SPD, and DAc-SPD as the representative polyamines tested. The derivatives of 11 authentic polyamines, including their acetylated forms, were perfectly separated in the selected reaction monitoring (SRM) chromatogram within 10 min. By using the optimized conditions for MS detection (Table 1), the limit of detection (S/N = 3) of the molecules under study was in the range 9−43 amol. The sensitivity of the detection against the various derivatized polyamines was slightly different, probably because of the different number of DBD groups; however, the intensity of the peaks did not correlate with the number of amino groups. The MS/MS detection conditions, such as the collision energy, might be another reason for the difference. However, the reasons for the intensity difference are not straightforward. Since the MS/MS detection proved highly sensitive at the attomole level, we adopted the UPLC−ESIMS/MS strategy to simultaneously determine the polyamines in saliva samples after DBD-F labeling. Validation of the Present Method. The calibration curves of the derivatized polyamines at five different concentrations (1−1000 fmol) were first determined (n = 5), and their parameters are shown in Table 2. A good linearity (R2 > 0.995) between the MS intensity and the injected amount of each polyamine was obtained. To validate the present method, we determined the recovery (%) and precision (RSD) of the measurements. The RSD values of the different injection amounts fell in the range of 0.7−9.9%. As shown in Table 3, the recovery of the polyamines that were spiked in the saliva samples, were in the range of 86.3−108%. Furthermore, as

Figure 6. Percentage (%) of polyamines in healthy volunteers, primary patients, and relapsed patients.

shown in Table 4, the intraday and interday precisions (RSD) were within 9.9 and 9.7%, respectively. The freeze/thaw stability was also good enough for real sample analysis (more than 87.7% after two freeze/thaw cycles) (Table 5). According to the good stability, linearity, precision, and recovery values obtained, the method proposed here can be used to analyze human saliva. Determination of Polyamines in Human Saliva. In general, the determination of polyamines such as DAc-SPM and DAc-SPD in cancer patients is carried out using urine samples, because urine is the best studied biological sample among other types of specimens. However, the fluctuations in the content of the various components during the day should be considered. In addition, hygiene during collection and handling are other important factors. The pretreatment of urine is a very complicated and time-consuming step. Furthermore, due to the high polarity, low molecular mass, and high hydrophobicity of polyamines, their determination at the trace amount level is complicated because it is impaired by the interference with F



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CONCLUSION Here, a simple and feasible method for the determination of polyamines and their acetylated forms by using LC−ESI-MS/ MS was developed. The polyamines were labeled with DBD-F and sensitively detected by ESI-MS/MS. Furthermore, a highthroughput separation within 10 min was carried out by using UPLC equipped with an antipressurized column packed with 1.7-μm octadecylsilyl (ODS) silica gel particles. The proposed method is the first reported test for the simultaneous determination of 11 polyamines in saliva. The levels of several N-acetylpolyamines, including Ac-PUT, Ac-SPD, Ac-SPM, DAc-SPD, and DAc-SPM, were higher in breast cancer patients than in healthy volunteers. Since our proposed method is highly sensitive, specific, and feasible, it is a useful, noninvasive diagnosis tool for cancer patients through the determination of polyamines in various biological matrixes. At present, further study of the polyamine analysis associated with various types of cancers is in progress in our laboratory.

other endogenous substances. Thus, the use of urine as a diagnostic sample is not recommended. Recently, saliva gained much attention for clinical examination and therapeutic drug monitoring,27−30 because it ensures quick, noninvasive, easy, repeatable, and stress-free sampling. Furthermore, saliva samples are reasonably clean and can be easily stored. For these reasons, the use of saliva as a diagnostic sample is increasing. However, the determination of various polyamines at the same time in the saliva has not yet been performed. Here, the separation of polyamines contained in the saliva of breast cancer patients and healthy volunteers has been performed. To identify the endogenous polyamines, 30 μL of saliva collected from a healthy volunteer was pretreated and determined as described in the Experimental Section. Figure 4 displays the SRM chromatograms obtained from the saliva of a healthy person after derivatization with DBD-F. The peaks corresponding to the polyamines including IS were clearly separated within 10 min without interference of endogenous materials. However, an unknown peak appeared on the SRM chromatogram of Figure 4e. Based on the chromatogram, the derivative has the same molecular mass and very similar structure of N8-Ac-SPD. Indeed, the MS and MS/MS spectra of both derivatives were almost superimposable (data not shown). Furthermore, the peak appears on the SRM chromatogram derived from the transition of m/z 638.2 → 550.1. Therefore, the unknown peak, eluted after the peak corresponding to N8-Ac-SPD, seems to be the derivative of N1-Ac-SPD (an isomer of N8-Ac-SPD). Since the peak corresponding to N8-Ac-SPD did not interfere with that of N1-Ac-SPD, the present method seems to be applicable for the simultaneous determination of polyamines in saliva. Concentration of Polyamines in the Saliva of Breast Cancer Patients and Healthy Volunteers. A total of 44 saliva samples from breast cancer patients (8 primary and 22 relapsed patients) and 14 healthy volunteers have been analyzed. Figure 5 shows the average concentrations of the polyamines in the saliva. The concentrations of several N-acetylpolyamines (i.e., Ac-PUT, Ac-SPD, Ac-SPM, DAc-SPD, and DAc-SPM) were significantly higher in the cancer patients than in the healthy subjects. Because a large variance of each polyamine was observed (Figure 5), the concentration difference of the polyamines in each patient is significant. Therefore, the percentage ratios of the 11 polyamines in each group were calculated based on the total polyamine concentrations in each person (Figure 6). In spite of the large concentration difference, several polyamines (Ac-PUT, Ac-SPD, Ac-SPM, DAc-SPD, and DAc-SPM) in the patients tended to be higher than those in the healthy subjects, and three polyamines (Ac-SPM, DAc-SPD, and DAc-SPM) were extremely high only in the relapsed patients. Because N-acetylpolyamines are known as diagnostic marker molecules, the present results are not in conflict with previous reports.31,32 It is unclear whether the large concentration difference is due to the difference in the cancer stage and/or relapsed state of the patients. The individual sample difference seems to be another factor affecting the concentration difference. However, the results suggest that the diagnosis of breast cancer might be possible by simultaneously determining the various polyamines. A detailed study including large sample numbers is planned with the cooperation of the hospital.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-54-264-5656. Fax: +81-54-264-5593. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science.

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REFERENCES

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