Journal of Chromatographic Science 2015;53:537– 541 doi:10.1093/chromsci/bmu081 Advance Access publication July 17, 2014
Article
Determination of Free Fatty Acids in Human Serum by HPLC with Fluorescence Detection Minami Nishikiori1, Hideaki Iizuka1, Hideaki Ichiba1, Kiyomi Sadamoto2 and Takeshi Fukushima1* 1
Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510, Japan, and 2Faculty of Pharmaceutical Sciences, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama-shi, Kanagawa 245-0066, Japan
*Author to whom correspondence should be addressed. Email: [email protected] Received 17 January 2014; revised 26 May 2014
It has been suggested that serum concentrations of polyunsaturated essential fatty acids correlate with the symptoms or severity of various diseases, including depression and Alzheimer-type dementia, and that determination of serum fatty acids might be important for disease diagnosis. Thus, we developed to analyze serum fatty acids in healthy individuals by using a high-performance liquid chromatography method with fluorescence detection, because free fatty acids have a carboxyl group that can be derivatized with a fluorescent reagent, 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3benzoxadiazole. This approach could quantify five types of free fatty acids [a-linolenic acid (ALA), palmitoleic acid (PLA), arachidonic acid (AA), linoleic acid (LA) and oleic acid (OA)] in human serum. The detection limits of the method were in the range of 2.29 – 4.75 fmol (signal-to-noise ratio 3), and absolute concentrations of ALA, PLA, AA, LA and OA were 8.27 + 1.04, 18.8 + 2.95, 49.9 + 4.03, 230 + 18.1 and 201 + 22.1 mM, respectively.
Introduction Free fatty acids can function as an energy source, a constituent of the cell membrane and a physiologically active substance. In nature, the three types of fatty acid are saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs). Arachidonic acid (AA) and a-linolenic acid (ALA) are representatives of v-6 and v-3 PUFAs, respectively. The concentrations of fatty acids in serum are altered in patients with several diseases, including diabetes (1) and depressive disorder (2). In particular, PUFA levels appear to be correlated with symptoms or severity of neurological disorders such as Alzheimer-type dementia (3) and schizophrenia (4), and therefore, determination of serum fatty acids might be important for disease diagnosis. To date, although gas-chromatography provides superior resolution, high-performance liquid chromatography (HPLC) has been the method of choice for quantification of fatty acids, because the use of conventional HPLC has also been possible to analyze fatty acids without heating at high temperature. In particular, PUFAs possess olefin moieties in their structures, and cis/trans or positional isomerization may occur upon exposure to high temperatures of 200–2308C during the gas-chromatographic process (3). Many HPLC-based methods have been reported, and most utilize pre-column derivatization with fluorescence moieties; notable examples include 3-bromomethyl-6,7-methylenedioxy1-methyl-2(1H)-quinoxalinone (5), 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (6), 4-(N,N-dimethylaminosulfonyl)-7(1-piperazinyl)-2,1,3-benzoxadiazole, 4-nitro-7-(1-piperazinyl)-2, 1,3-benzoxadiazole (7), 2-(4-hydrazinocarbonylphenyl)-4,5diphenylimidazole (8), 9-anthryldiazomethane (9) and 1-pyrenyldiazomethane (10). We previously reported an HPLC-
fluorescence detection method based on labeling carboxyl groups with a fluorescent 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED, Figure 1) molecule; the method was validated using a standard of AA (11) (Figure 1). DBD-ED is a convenient reagent for its usage including high solubility in HPLC-grade CH3CN, used for the mobile phase. In addition, fluorescence DBD-ED adducts are relatively stable and have also been used as a pre-column derivatization reagent for quantification of N-acetylaspartic acid (12, 13), N-acetylneuraminic acid and 4(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (14). Here, we examined whether the method was suitable for the detection and quantification of five types of free fatty acid in human serum from healthy subjects. Experimental Human serum isolation Human serum was obtained from 34 healthy Japanese subjects. This study was approved by the Ethics Committee located in the Faculty of Pharmaceutical Sciences, Toho University (No. 24-6). Healthy volunteers (17 men: 26.6 + 4.30 years, and 17 women: 24.8 + 6.11 years) (mean + SD) were recruited with their informed consent. Approximately 5.0 mL blood was drawn from the arm vein into VENOJECT II tubes (VP-AS109K; Termo, Tokyo, Japan) between 11 : 00 and 12 : 00. Blood was transferred to a shaded container and allowed to stand for 30 min at room temperature. Samples were then centrifuged at 1,200 g for 15 min, and sera were stored at 2808C until analysis. Instrumentation and reagents a-linolenic acid (ALA), palmitoleic acid (PLA), arachidonic acid (AA), linoleic acid (LA), oleic acid (OA) and heptadecanoic acid used as internal standards (IS) were purchased from SigmaAldrich (St. Louis, MO). Dimethylformamide (DMF) and perchloric acid (60%) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Chloroform, n-heptane and trifluoroacetic acid (TFA, amino acid analysis grade) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DBD-ED, triphenylphosphine (TPP) and 2,20 -dipyridyl disulfide (DPDS) were purchased from Tokyo Chemical Industries, Ltd. (Tokyo, Japan). HPLC-grade acetonitrile (CH3CN) and methanol (MeOH) were purchased from Kanto Kagaku Kogyo (Tokyo, Japan). All chemicals were analytical-grade reagents. Methods Fatty acid extraction An extraction procedure of serum fatty acids was performed according to the previous paper (15) with minor modifications.
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Chemical structures of DBD-ED and the derivatives of fatty acid (AA).
Ten microliters of human serum was mixed with 10 mL of 50 mM heptadecanoic acid as an IS and 10 mL of CH3CN. Then, 30 mL perchloric acid was added for deproteinization, and 800 mL chloroform, 100 mL methanol, 100 mL n-heptane and 100 mL H2O were added to extract the fatty acids. After the solution was centrifuged at 3,000 g for 5 min, the organic layer (400 mL) was transferred to a brown tube and the solvents were evaporated to residue under reduced pressure by using a centrifugal concentrator, VC-36N (TAITEC Co., Ltd., Saitama, Japan) without heating for 20 min. The dried residue was dissolved in 50 mL DMF and mixed with 50 mL of 2 mM DBD-ED in CH3CN, 50 mL of 140 mM TPP in CH3CN and 50 mL of 140 mM DPDS in CH3CN to derivatize the fatty acids. After standing at room temperature for 120 min, an aliquot of the solution was diluted 50-fold with CH3CN – H2O (7:3). The diluted solution was filtered and 10 mL was subjected to HPLC analysis.
HPLC The HPLC system used consisted of a pump (LC-20AD; Shimadzu Corporation, Kyoto, Japan), a column oven (CTO10A VP; Shimadzu Corporation), a degasser (GASTOOR 704; FLOM, Tokyo, Japan) and a fluorescence detector (RF-20A XS; Shimadzu Corporation). A Cadenza CD-C18 column (4.6 250 mm, 3 mm; Imtakt Corporation, Kyoto, Japan) was used in a column oven setting at 408C, and the flow rate was constantly maintained at 1.0 mL/min. The excitation and emission wavelengths of the fluorescence detector were set at 450 and 560 nm, respectively. The mobile phases A, B and C used in the study were 0.1% TFA/CH 3 CN, 0.1% TFA/H 2 O and 0.1% TFA/MeOH, respectively. The time programs for the mobile phase were as follows: 0 – 25.00 min A% ¼ 60 – 70, B% ¼ 30 – 20 (linear gradient) and C% ¼ 10; 25.01 – 40.00 min A% ¼ 70, B% ¼ 20 and C% ¼ 10 (isocratic); 40.01 – 55.00 min A% ¼ 70 – 77, B% ¼ 20 – 13 (linear gradient) and C% ¼ 10; 55.01 – 65.00 min A% ¼ 77, B% ¼ 13 and C% ¼ 10 (isocratic) and 65.01 – 84.50 min A% ¼ 60, B% ¼ 30 and C% ¼ 10 (initialization). The resulting chromatogram was analyzed using CDS plus 5.0 (LAsoft, Chiba, Japan). Calibration curves for ALA, PLA, AA, LA and OA were constructed at the concentrations of 1 – 50, 1 – 200, 10 – 200, 25 – 800 and 25 – 800 mM,
538 Nishikiori et al.
respectively. The peak area ratio of the IS was then plotted against each concentration (n ¼ 4).
Results Figure 2a shows representative chromatograms of standard fatty acids, ALA, PLA, AA, LA and OA, and Figure 2b shows those present in human serum. A comparison of the panels reveals that fluorescence peaks corresponding to ALA, PLA, AA, LA and OA were observed in human serum. As depicted in Figure 3, the production of fluorescence adducts at room temperature reached a plateau after 120 min for AA. The time-course profile of the derivatization was very similar in both standard and serum samples (Figure 3a and b). The HPLC method presented here yielded detection limits between 2.29 and 4.75 fmol on the column (signal-to-noise ratio 3), and the recoveries were 108 – 113% (Table I). Table II indicates the concentrations of ALA, PLA, AA, LA and OA in human serum (17 male and 17 female patients) determined using the current HPLC method. As mentioned above, HPLC has the advantage of a low potential for the cis/trans isomerization of PUFAs caused by high temperature. Therefore, the data obtained using the present HPLC method are considered to be reliable. There was no correlation between fatty acid concentration and body mass index or age. Furthermore, none of the fatty acid concentrations was significantly different between male and female patients. Discussion In this study, we first derivatized fatty acids in human serum with DBD-ED, and serum ALA, PLA, AA, LA and OA were separated from each other on an octadecylsilyl column (Cadenza CD-C18) and analyzed using a fluorescence-based HPLC method. As shown in Figure 2a and b, fluorescence peaks derived from ALA, PLA, AA, LA and OA were observed in human serum. Validation data shown in Table I are satisfactory and consistent with those of previous reports (16). We note, however, that our method allows a lower serum volume (10 mL) to be used for fatty acid determination, in contrast to an earlier method that required .50 mL (17).
Figure 3. Time-course profile of the derivatization reaction of AA with DBD-ED. (a) Standard sample and (b) serum sample.
Table I Validations of the HPLC Method
LOQ (fmol) LOD (fmol) Precision (n ¼ 3) (%) Recovery (%)
Figure 2. Representative chromatograms of (a) blank, (b) standard fatty acids and (c) human serum samples. Peak: 1 ¼ ALA, 2 ¼ PLA, 3 ¼ AA, 4 ¼ LA, 5 ¼ OA.
With respect to the concentrations of ALA, PLA, AA, LA and OA in human serum (17 male and 17 female patients), no gender difference was observed. Furthermore, although AA is derived from LA by desaturase and elongase in vivo, we found no correlation between serum AA and LA concentration. Considering that all participants were healthy subjects, we infer that this lack of correlation is due to individual variations in the activity of pathways that metabolize both LA and AA. As shown in Table II, large individual differences on the serum concentrations of each fatty acid were observed in this study. On comparison with previous reports, we found that the concentrations of free fatty acids in serum varied between the reported values (3, 15, 17 –21) (Supplementary data, Table S1).
ALA
PLA
AA
LA
OA
10.5 3.14 1.56 111.7
7.63 2.29 2.68 111.8
11.5 3.44 3.21 108.0
8.43 2.53 3.00 108.9
15.8 4.75 2.43 113.4
We suggest that factors such as diet or the metabolic rate may account for such differences. Fatty acids, and in particular ALA and LA, are derived almost exclusively from the daily diet (22). Thus, there may be inter-subject differences in the daily intake of LA and ALA. This would be consistent with an earlier report of major differences in ALA and LA concentrations between individuals (18). In addition, only Japanese subjects were enrolled in this study, and therefore, their cultural diet and lifestyle are different from those of other nations. Moreover, the rate of fatty acid turnover may be influenced by variations in the levels of enzymes responsible for their metabolism. Thus, it is likely that these factors cause to racial differences contributed to the varied serum fatty acids concentrations. In this study, relatively young subjects (aged ,37 years) were recruited, and a broader range of subjects will be enrolled in the next study. Nevertheless, the present data may be used as a high quality baseline for the diagnosis of schizophrenia, because the onset of schizophrenia frequently occurs during adolescence.
Free Fatty Acids in Human Serum by HPLC with Fluorescence Detection 539
Table II Serum Concentrations of Fatty Acids in Healthy Subjects (mean + SE)
Total subjects (mM, n ¼ 34) (minimum –maximum) Male subjects (mM, n ¼ 17) (minimum –maximum) Female subjects (mM, n ¼ 17) (minimum –maximum)
ALA
PLA
AA
LA
OA
8.27 + 1.04 (2.30 –27.5) 6.72 + 0.90 (2.30 –14.2) 9.82 + 1.82 (3.26 –27.5)
18.8 + 2.95 (2.90 –83.3) 14.0 + 2.16 (2.90 –35.9) 23.5 + 5.34 (5.71 –83.3)
49.9 + 4.03 (20.5 –123) 42.4 + 4.63 (20.5 –107) 57.4 + 6.22 (27.6 –123)
230 + 18.1 (65.1–484) 209 + 25.0 (65.1–443) 251 + 25.7 (81.1–484)
201 + 22.1 (45.6 – 599) 164 + 20.9 (45.6 – 354) 237 + 37.6 (73.5 – 599)
Conclusion Our HPLC method enabled us to determine serum fatty acids using a low volume of human serum. This approach renders the HPLC method feasible for applications such as clinical diagnosis of diseases related to altered fatty acid metabolism.
Supplementary data Supplementary data are available at Journal of Chromatographic Science online.
Acknowledgments We would like to thank all the volunteers for providing serum specimens. We are grateful to all participants for their cooperation during the blood collection stage. We would also like to thank Drs H. Yamada and S. Iwasa for their helpful assistance with the study.
References 1. Reaven, G.M., Hollenbeck, C., Jeng, C.Y., Wu, M.S., Chen, Y.D.; Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM; Diabetes, (1988); 37: 1020 – 1024. 2. Horrobin, D.F., Bennett, C.N.; Depression and bipolar disorder: relationships to impaired fatty acid and phospholipid metabolism and to diabetes, cardiovascular disease, immunological abnormalities, cancer, ageing and osteoporosis; Prostaglandins, Leukotrienes Essential Fatty Acids, (1999); 60: 217– 234. 3. Wang, D.-C., Sun, C.-H., Liu, L.-Y., Sun, X.-H., Jin, X.-W., Song, W.-L., et al.; Serum fatty acid profiles using GC-MS and multivariate statistical analysis: potential biomarkers of Alzheimer’s disease; Neurobiology of Aging, (2012); 33: 1057– 1066. 4. Maekawa, M., Owada, Y., Yoshikawa, T.; Role of polyunsaturated fatty acids and fatty acid binding protein in the pathogenesis of schizophrenia; Current Pharmaceutical Design, (2011); 17: 168–175. 5. Yamaguchi, M., Hara, S., Takemori, Y., Nakamura, M.; High-performance liquid chromatography of monosialogangliosides in human plasma with fluorescence detection; Analytical Sciences, (1989); 5: 35–38. 6. Yasaka, Y., Tanaka, M., Shono, T., Tetsumi, T., Katakawa, J.; 2-(2,3Naphthalimino)ethyl trifluoromethanesulfonate as a highly reactive ultraviolet and fluorescent labelling agent for the liquid chromatographic determination of carboxylic acids; Journal of Chromatography, (1990); 508: 133–140. 7. Toyo’oka, T., Ishibashi, M., Takeda, Y., Nakashima, K., Akiyama, S., Uzu, S., et al.; Precolumn fluorescence tagging reagent for carboxylic acids in high-performance liquid chromatography: 4-substituted-7aminoalkylamino-2,1,3-benzoxadiazoles; Journal of Chromatography, (1991); 588: 61–71.
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8. Nakashima, K., Taguchi, Y., Kuroda, N., Akiyama, S., Duan, G.; 2-(4-Hydrazinocarbonylphenyl)-4,5-diphenylimidazole as a versatile fluorescent derivatization reagent for the highperformance liquid chromatographic analysis of free fatty acids; Journal of Chromatography Biomedical Applications, (1993); 619: 1 – 8. 9. Yoshida, T., Uetake, A., Yamaguchi, H., Nimura, N., Kinoshita, T.; New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives; Analytical Biochemistry, (1988); 173: 70– 74. 10. Nimura, N., Kinoshita, T., Yoshida, T., Uetake, A., Nakai, C.; 1Pyrenyldiazomethane as a fluorescent labeling reagent for liquid chromatographic determination of carboxylic acids; Analytical Chemistry, (1988); 60: 2067–2070. 11. Prados, P., Fukushima, T., Santa, T., Homma, H., Tsunoda, M., Al-Kindy, S., et al.; 4-N, N-Dimethylaminosulfonyl-7-N-(2-aminoethyl)aminobenzofurazan as a new precolumn fluorescence derivatization reagent for carboxylic acids (fatty acids and drugs containing a carboxyl moiety) in liquid chromatography; Analytica Chimica Acta, (1997); 344: 227–232. 12. Fukushima, T., Arai, K., Tomiya, M., Mitsuhashi, S., Sasaki, T., Santa, T., et al.; Fluorescence determination of N-acetylaspartic acid in the rat cerebrum homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization; Biomedical Chromatography, (2008); 22: 100– 105. 13. Song, Z., Ge, D., Ishii, K., Yamada, H., Toriumi, K., Watanabe, H., et al.; Determination of N-acetylaspartic acid concentration in the mouse brain using HPLC with fluorescence detection; Biomedical Chromatography, (2012); 26: 147– 151. 14. Ota, T., Yasuda, M., Iijima, R., Yui, S., Fukuuchi, T., Yamaoka, N., et al.; Development of a fluorescence analysis method for N-acetylneuraminic acid and its oxidized product ADOA; Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences, (2013); 932: 152–157. 15. Toyo’oka, T., Takahashi, M., Suzuki, A., Ishii, Y.; Determination of free fatty-acids in blood tagged with 4-(2-carbazoylpyroolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, by high-performance liquid-chromatography with fluorescence detection; Biomedical Chromatography, (1995); 9: 162–170. 16. Saito, M., Ushijima, T., Sasamoto, K., Ohkura, Y., Ueno, K.; 2-(5Hydrazinocarbonyl-2-furyl)-5,6-dimethoxybenzothiazole as a precolumn fluorescence derivatization reagent for carboxylic acids in highperformance liquid chromatography and its application to the assay of fatty acids in human serum; Analytical Sciences, (1995); 11: 103–107. 17. Sanchez-Avila, N., Mata-Granados, J.M., Ruiz-Jimenez, J., Luque de Castro, M.D.; Fast, sensitive and highly discriminant gas chromatography-mass spectrometry method for profiling analysis of fatty acids in serum; Journal of Chromatography A, (2009); 1216: 6864– 6872. 18. Yamaguchi, M., Matsunaga, R., Hara, S., Nakamura, M.; Highly sensitive determination of free fatty acids in human serum by highperformance liquid chromatography with fluorescence detection; Journal of Chromatography B, Biomedical Applications, (1986); 375: 27 – 35.
19. Puttmann, M., Krug, H., Vonochsenstein, E., Kattermann, R.; Fast HPLC determination of serum-free fatty-acids in the picomole range; Clinical Chemistry, (1993); 39: 825– 832. 20. Li, G.-L., Chen, G., Liu, Y.-Q., Jing, N.-H., You, J.-M.; A sensitive and selective HPLC-FLD method with fluorescent labeling for simultaneous detection of bile acid and free fatty acid in human serum; Journal of Chromatography B Analytical Technologies in the Biomedical and Life Sciences, (2012); 895– 896: 191– 195.
21. Kotani, A., Kusu, F., Takamura, K.; New electrochemical detection method in high-performance liquid chromatography for determining free fatty acids; Analytica Chimica Acta, (2002); 465: 199–206. 22. Griffiths, A.J., Humphreys, S.M., Clark, M.L., Fielding, B.A., Frayn, K.N.; Immediate metabolic availability of dietary fat in combination with carbohydrate; American Journal of Clinical Nutrition, (1994); 59: 53– 59.
Free Fatty Acids in Human Serum by HPLC with Fluorescence Detection 541
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Article
Determination of Free Fatty Acids in Human Serum by HPLC with Fluorescence Detection Minami Nishikiori1, Hideaki Iizuka1, Hideaki Ichiba1, Kiyomi Sadamoto2 and Takeshi Fukushima1* 1
Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510, Japan, and 2Faculty of Pharmaceutical Sciences, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama-shi, Kanagawa 245-0066, Japan
*Author to whom correspondence should be addressed. Email: [email protected] Received 17 January 2014; revised 26 May 2014
It has been suggested that serum concentrations of polyunsaturated essential fatty acids correlate with the symptoms or severity of various diseases, including depression and Alzheimer-type dementia, and that determination of serum fatty acids might be important for disease diagnosis. Thus, we developed to analyze serum fatty acids in healthy individuals by using a high-performance liquid chromatography method with fluorescence detection, because free fatty acids have a carboxyl group that can be derivatized with a fluorescent reagent, 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3benzoxadiazole. This approach could quantify five types of free fatty acids [a-linolenic acid (ALA), palmitoleic acid (PLA), arachidonic acid (AA), linoleic acid (LA) and oleic acid (OA)] in human serum. The detection limits of the method were in the range of 2.29 – 4.75 fmol (signal-to-noise ratio 3), and absolute concentrations of ALA, PLA, AA, LA and OA were 8.27 + 1.04, 18.8 + 2.95, 49.9 + 4.03, 230 + 18.1 and 201 + 22.1 mM, respectively.
Introduction Free fatty acids can function as an energy source, a constituent of the cell membrane and a physiologically active substance. In nature, the three types of fatty acid are saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs). Arachidonic acid (AA) and a-linolenic acid (ALA) are representatives of v-6 and v-3 PUFAs, respectively. The concentrations of fatty acids in serum are altered in patients with several diseases, including diabetes (1) and depressive disorder (2). In particular, PUFA levels appear to be correlated with symptoms or severity of neurological disorders such as Alzheimer-type dementia (3) and schizophrenia (4), and therefore, determination of serum fatty acids might be important for disease diagnosis. To date, although gas-chromatography provides superior resolution, high-performance liquid chromatography (HPLC) has been the method of choice for quantification of fatty acids, because the use of conventional HPLC has also been possible to analyze fatty acids without heating at high temperature. In particular, PUFAs possess olefin moieties in their structures, and cis/trans or positional isomerization may occur upon exposure to high temperatures of 200–2308C during the gas-chromatographic process (3). Many HPLC-based methods have been reported, and most utilize pre-column derivatization with fluorescence moieties; notable examples include 3-bromomethyl-6,7-methylenedioxy1-methyl-2(1H)-quinoxalinone (5), 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (6), 4-(N,N-dimethylaminosulfonyl)-7(1-piperazinyl)-2,1,3-benzoxadiazole, 4-nitro-7-(1-piperazinyl)-2, 1,3-benzoxadiazole (7), 2-(4-hydrazinocarbonylphenyl)-4,5diphenylimidazole (8), 9-anthryldiazomethane (9) and 1-pyrenyldiazomethane (10). We previously reported an HPLC-
fluorescence detection method based on labeling carboxyl groups with a fluorescent 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED, Figure 1) molecule; the method was validated using a standard of AA (11) (Figure 1). DBD-ED is a convenient reagent for its usage including high solubility in HPLC-grade CH3CN, used for the mobile phase. In addition, fluorescence DBD-ED adducts are relatively stable and have also been used as a pre-column derivatization reagent for quantification of N-acetylaspartic acid (12, 13), N-acetylneuraminic acid and 4(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (14). Here, we examined whether the method was suitable for the detection and quantification of five types of free fatty acid in human serum from healthy subjects. Experimental Human serum isolation Human serum was obtained from 34 healthy Japanese subjects. This study was approved by the Ethics Committee located in the Faculty of Pharmaceutical Sciences, Toho University (No. 24-6). Healthy volunteers (17 men: 26.6 + 4.30 years, and 17 women: 24.8 + 6.11 years) (mean + SD) were recruited with their informed consent. Approximately 5.0 mL blood was drawn from the arm vein into VENOJECT II tubes (VP-AS109K; Termo, Tokyo, Japan) between 11 : 00 and 12 : 00. Blood was transferred to a shaded container and allowed to stand for 30 min at room temperature. Samples were then centrifuged at 1,200 g for 15 min, and sera were stored at 2808C until analysis. Instrumentation and reagents a-linolenic acid (ALA), palmitoleic acid (PLA), arachidonic acid (AA), linoleic acid (LA), oleic acid (OA) and heptadecanoic acid used as internal standards (IS) were purchased from SigmaAldrich (St. Louis, MO). Dimethylformamide (DMF) and perchloric acid (60%) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Chloroform, n-heptane and trifluoroacetic acid (TFA, amino acid analysis grade) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DBD-ED, triphenylphosphine (TPP) and 2,20 -dipyridyl disulfide (DPDS) were purchased from Tokyo Chemical Industries, Ltd. (Tokyo, Japan). HPLC-grade acetonitrile (CH3CN) and methanol (MeOH) were purchased from Kanto Kagaku Kogyo (Tokyo, Japan). All chemicals were analytical-grade reagents. Methods Fatty acid extraction An extraction procedure of serum fatty acids was performed according to the previous paper (15) with minor modifications.
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Chemical structures of DBD-ED and the derivatives of fatty acid (AA).
Ten microliters of human serum was mixed with 10 mL of 50 mM heptadecanoic acid as an IS and 10 mL of CH3CN. Then, 30 mL perchloric acid was added for deproteinization, and 800 mL chloroform, 100 mL methanol, 100 mL n-heptane and 100 mL H2O were added to extract the fatty acids. After the solution was centrifuged at 3,000 g for 5 min, the organic layer (400 mL) was transferred to a brown tube and the solvents were evaporated to residue under reduced pressure by using a centrifugal concentrator, VC-36N (TAITEC Co., Ltd., Saitama, Japan) without heating for 20 min. The dried residue was dissolved in 50 mL DMF and mixed with 50 mL of 2 mM DBD-ED in CH3CN, 50 mL of 140 mM TPP in CH3CN and 50 mL of 140 mM DPDS in CH3CN to derivatize the fatty acids. After standing at room temperature for 120 min, an aliquot of the solution was diluted 50-fold with CH3CN – H2O (7:3). The diluted solution was filtered and 10 mL was subjected to HPLC analysis.
HPLC The HPLC system used consisted of a pump (LC-20AD; Shimadzu Corporation, Kyoto, Japan), a column oven (CTO10A VP; Shimadzu Corporation), a degasser (GASTOOR 704; FLOM, Tokyo, Japan) and a fluorescence detector (RF-20A XS; Shimadzu Corporation). A Cadenza CD-C18 column (4.6 250 mm, 3 mm; Imtakt Corporation, Kyoto, Japan) was used in a column oven setting at 408C, and the flow rate was constantly maintained at 1.0 mL/min. The excitation and emission wavelengths of the fluorescence detector were set at 450 and 560 nm, respectively. The mobile phases A, B and C used in the study were 0.1% TFA/CH 3 CN, 0.1% TFA/H 2 O and 0.1% TFA/MeOH, respectively. The time programs for the mobile phase were as follows: 0 – 25.00 min A% ¼ 60 – 70, B% ¼ 30 – 20 (linear gradient) and C% ¼ 10; 25.01 – 40.00 min A% ¼ 70, B% ¼ 20 and C% ¼ 10 (isocratic); 40.01 – 55.00 min A% ¼ 70 – 77, B% ¼ 20 – 13 (linear gradient) and C% ¼ 10; 55.01 – 65.00 min A% ¼ 77, B% ¼ 13 and C% ¼ 10 (isocratic) and 65.01 – 84.50 min A% ¼ 60, B% ¼ 30 and C% ¼ 10 (initialization). The resulting chromatogram was analyzed using CDS plus 5.0 (LAsoft, Chiba, Japan). Calibration curves for ALA, PLA, AA, LA and OA were constructed at the concentrations of 1 – 50, 1 – 200, 10 – 200, 25 – 800 and 25 – 800 mM,
538 Nishikiori et al.
respectively. The peak area ratio of the IS was then plotted against each concentration (n ¼ 4).
Results Figure 2a shows representative chromatograms of standard fatty acids, ALA, PLA, AA, LA and OA, and Figure 2b shows those present in human serum. A comparison of the panels reveals that fluorescence peaks corresponding to ALA, PLA, AA, LA and OA were observed in human serum. As depicted in Figure 3, the production of fluorescence adducts at room temperature reached a plateau after 120 min for AA. The time-course profile of the derivatization was very similar in both standard and serum samples (Figure 3a and b). The HPLC method presented here yielded detection limits between 2.29 and 4.75 fmol on the column (signal-to-noise ratio 3), and the recoveries were 108 – 113% (Table I). Table II indicates the concentrations of ALA, PLA, AA, LA and OA in human serum (17 male and 17 female patients) determined using the current HPLC method. As mentioned above, HPLC has the advantage of a low potential for the cis/trans isomerization of PUFAs caused by high temperature. Therefore, the data obtained using the present HPLC method are considered to be reliable. There was no correlation between fatty acid concentration and body mass index or age. Furthermore, none of the fatty acid concentrations was significantly different between male and female patients. Discussion In this study, we first derivatized fatty acids in human serum with DBD-ED, and serum ALA, PLA, AA, LA and OA were separated from each other on an octadecylsilyl column (Cadenza CD-C18) and analyzed using a fluorescence-based HPLC method. As shown in Figure 2a and b, fluorescence peaks derived from ALA, PLA, AA, LA and OA were observed in human serum. Validation data shown in Table I are satisfactory and consistent with those of previous reports (16). We note, however, that our method allows a lower serum volume (10 mL) to be used for fatty acid determination, in contrast to an earlier method that required .50 mL (17).
Figure 3. Time-course profile of the derivatization reaction of AA with DBD-ED. (a) Standard sample and (b) serum sample.
Table I Validations of the HPLC Method
LOQ (fmol) LOD (fmol) Precision (n ¼ 3) (%) Recovery (%)
Figure 2. Representative chromatograms of (a) blank, (b) standard fatty acids and (c) human serum samples. Peak: 1 ¼ ALA, 2 ¼ PLA, 3 ¼ AA, 4 ¼ LA, 5 ¼ OA.
With respect to the concentrations of ALA, PLA, AA, LA and OA in human serum (17 male and 17 female patients), no gender difference was observed. Furthermore, although AA is derived from LA by desaturase and elongase in vivo, we found no correlation between serum AA and LA concentration. Considering that all participants were healthy subjects, we infer that this lack of correlation is due to individual variations in the activity of pathways that metabolize both LA and AA. As shown in Table II, large individual differences on the serum concentrations of each fatty acid were observed in this study. On comparison with previous reports, we found that the concentrations of free fatty acids in serum varied between the reported values (3, 15, 17 –21) (Supplementary data, Table S1).
ALA
PLA
AA
LA
OA
10.5 3.14 1.56 111.7
7.63 2.29 2.68 111.8
11.5 3.44 3.21 108.0
8.43 2.53 3.00 108.9
15.8 4.75 2.43 113.4
We suggest that factors such as diet or the metabolic rate may account for such differences. Fatty acids, and in particular ALA and LA, are derived almost exclusively from the daily diet (22). Thus, there may be inter-subject differences in the daily intake of LA and ALA. This would be consistent with an earlier report of major differences in ALA and LA concentrations between individuals (18). In addition, only Japanese subjects were enrolled in this study, and therefore, their cultural diet and lifestyle are different from those of other nations. Moreover, the rate of fatty acid turnover may be influenced by variations in the levels of enzymes responsible for their metabolism. Thus, it is likely that these factors cause to racial differences contributed to the varied serum fatty acids concentrations. In this study, relatively young subjects (aged ,37 years) were recruited, and a broader range of subjects will be enrolled in the next study. Nevertheless, the present data may be used as a high quality baseline for the diagnosis of schizophrenia, because the onset of schizophrenia frequently occurs during adolescence.
Free Fatty Acids in Human Serum by HPLC with Fluorescence Detection 539
Table II Serum Concentrations of Fatty Acids in Healthy Subjects (mean + SE)
Total subjects (mM, n ¼ 34) (minimum –maximum) Male subjects (mM, n ¼ 17) (minimum –maximum) Female subjects (mM, n ¼ 17) (minimum –maximum)
ALA
PLA
AA
LA
OA
8.27 + 1.04 (2.30 –27.5) 6.72 + 0.90 (2.30 –14.2) 9.82 + 1.82 (3.26 –27.5)
18.8 + 2.95 (2.90 –83.3) 14.0 + 2.16 (2.90 –35.9) 23.5 + 5.34 (5.71 –83.3)
49.9 + 4.03 (20.5 –123) 42.4 + 4.63 (20.5 –107) 57.4 + 6.22 (27.6 –123)
230 + 18.1 (65.1–484) 209 + 25.0 (65.1–443) 251 + 25.7 (81.1–484)
201 + 22.1 (45.6 – 599) 164 + 20.9 (45.6 – 354) 237 + 37.6 (73.5 – 599)
Conclusion Our HPLC method enabled us to determine serum fatty acids using a low volume of human serum. This approach renders the HPLC method feasible for applications such as clinical diagnosis of diseases related to altered fatty acid metabolism.
Supplementary data Supplementary data are available at Journal of Chromatographic Science online.
Acknowledgments We would like to thank all the volunteers for providing serum specimens. We are grateful to all participants for their cooperation during the blood collection stage. We would also like to thank Drs H. Yamada and S. Iwasa for their helpful assistance with the study.
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