Article pubs.acs.org/jpr
High-Throughput LC−MS/MS Method for Direct Quantification of Glucuronidated, Sulfated, and Free Enterolactone in Human Plasma Natalja P. Nørskov,*,† Cecilie Kyrø,‡ Anja Olsen,‡ Anne Tjønneland,‡ and Knud Erik Bach Knudsen† †
Aarhus University, Department of Animal Science, AU-Foulum, Blichers Alle 20, P.O. Box 50, DK-8830 Tjele, Denmark Danish Cancer Society Research Center, Strandboulevarden 49, DK-2100 Copenhagen, Denmark
‡
S Supporting Information *
ABSTRACT: Sulfation and glucuronidation constitute a major pathway in humans and may play an important role in biological activity of metabolites including the enterolignan, enterolactone. Because the aromatic structure of enterolactone has similarities to steroid metabolites, it was hypothesized that enterolactone may protect against hormone-dependent cancers. This led to numerous epidemiological studies. In this context, there has been a demand for rapid, sensitive, high-throughput methods to measure enterolactone in biofluids. Different methods have been developed using GC−MS, HPLC, LC−MS/MS and a fluoroimmunoassay; however, most of these methods measure the total concentration of enterolactone, without any specification of its conjugation pattern. Here for the first time we present a high-throughput LC−MS/MS method to quantify enterolactone in its intact form as glucuronide, sulfate, and free enterolactone. The method has shown good accuracy and precision at low concentration and very high sensitivity, with LLOQ for enterolactone sulfate at 16 pM, enterolactone glucuronide at 26 pM, and free enterolactone at 86 pM. The short run time of 2.6 min combined with simple sample clean up and high sensitivity make this method attractive for the high-throughput of samples needed for epidemiological studies. Finally, we have adapted the new method to quantify enterolactone and its conjugates in 3956 plasma samples from an epidemiological study. We found enterolactone glucuronide to be the major conjugation form and that conjugation pattern was similar between men and women. KEYWORDS: enterolactone glucuronide, enterolactone sulfate, free enterolactone, LC−MS/MS, human plasma, method
1. INTRODUCTION Enterolactone was discovered more than 30 years ago1 and since then scientific research has indicated that this compound might be of particular interest for human health due to its antioxidative, estrogenic/antiestrogenic, and antiproliferative properties.2 Already in the early 1980s it was hypothesized that lignans and especially enterolactone may protect against breast cancer, and this has led to a number of case-control and prospective studies;3,4 however, results from these studies have been controversial because the effect of enterolactone as a weak estrogen has been shown to be both agonistic and antagonistic.2 These results have been difficult to explain, but one suggestion has been that the effect of enterolactone may depend on the level of endogenous estrogens, as has been seen in the case of coumestrol.2 Estrogens are the most important hormones involved in breast cancer.5,6 The main circulating form of estrogen is the estrogen sulfates, which have been measured in high concentrations in the breast tissue of patients with mammary carcinoma.7,8 Estrogen glucuronides have also been detected in urine and bile, but they are less abundant and more readily cleared from the body.5 Similarly to the estrogens, the main circulating forms of enterolactone are enterolactone glucuronide and enterolactone sulfate;9 however, so far little attention has been paid to the conjugation of lignans, as it has been complicated to measure enterolactone in its intact and inactive © XXXX American Chemical Society
form as glucuronide and sulfate. Nevertheless, the information regarding the ratio and the mode of conjugation may be important for our understanding of the biological role and possible health effects of enterolactone. To our knowledge, only two quantitate studies have been reported on the conjugation pattern of enterolactone in plasma and urine. One study measured enterolactone in urine and found enterolactone glucuronide to be the major form (94% in women (n = 4) and 85.5% in men (n = 2)), followed by sulfate and further by free enterolactone, sulfoglucuronide, disulfate, and diglucuronide.10 The second study, found that free enterolactone and enterolactone sulfate formed the major proportion of plasma enterolactone.11 These studies were performed more than 20 years ago, and since then no simple and rapid method has been developed to quantify conjugated enterolactone with a highthroughput of samples. Most methods developed so far are based on hydrolysis of enterolactone with the enzymes βglucuronidase/sulfatese prior to the measurements. All epidemiological studies published to date base their results on the analyses of the total concentration of enterolactone in plasma without any specification of its conjugation pattern. The purpose of this study was to develop a quantitative method for direct determination of glucuronidated, sulfated, Received: December 10, 2015
A
DOI: 10.1021/acs.jproteome.5b01117 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research
Figure 1. (A) (±)-enterolactone-mono-β-D-glucuronide. (B) (±)-enterolactone monosulfate ammonium salt.
ng/mL) concentrations to be able to calculate batch-to-batch variation.
and free enterolactone. Moreover the major purpose of our approach was to simplify the sample preparation so that incubation with enzymes could be avoided and at the same time to shorten the analytical time. Here we present a sensitive and simple method for quantification of enterolactone glucuronide, sulfate, and free enterolactone using LC−MS/MS. Finally, the method was applied to a large number of human plasma samples from an epidemiological study.
Samples from Epidemiological Study
We used plasma samples from the Danish Diet, Cancer and Health cohort.13 The cohort consisted of 53 057 men and women aged 50−64 years at recruitment (baseline) in 1993− 1997. At baseline, none were diagnosed with cancer registered in the Danish Cancer Registry. 14 At recruitment, the participants visited the study center in Copenhagen or Aarhus. Prior to this visit, they were asked to fill in a food frequency questionnaire (FFQ). At the study center, the participants further completed a lifestyle questionnaire with information on, for example, smoking status and physical activity. Both the FFQ and the lifestyle questionnaires were checked for errors and missing information at the study center. Furthermore, lab technicians performed anthropometric measurements of the participants, which included, for example, height, weight, and waist circumference.15 For each participant, 30 mL of blood was drawn and spun and divided into plasma, serum, erythrocytes, and buffy coat. The samples were thereafter stored in liquid nitrogen (max −150°). The participants were not asked to be fasting, but time since last meal was recorded.15 Between baseline and end of 2009, 4038 incident breast cancer, prostate cancer, and colorectal cancer cases were registered in the Danish Cancer Registry.14 Of these, plasma samples of 3921 of them were available from the biobank. In addition, in 46 of the breast cancer cases, plasma drawn at time of diagnosis was available. Thus, in total 3956 plasma samples from 3913 participants were measured in our laboratory.
2. EXPERIMENTAL SECTION Chemicals and Reagents
The standards used for the method development were: (±)-enterolactone-mono-β-D-glucuronide and (±)-enterolactone monosulfate ammonium salt custom-made for this project by ReseaChem (Burdorf, Switzerland) (Figure 1) and enterolactone from Plantech (Berkshire, U.K.). Enterolactone glucuronide and enterolactone sulfate were the mixtures of regioisomers and had the purity of >95%; see the detailed information in Tables S1 and S2 of the Supporting Information. The internal standard was glycocholic acid (glycine-1 13C) from Sigma-Aldrich (St. Louis, MO). Acetonitrile (ACN) and methanol (MeOH) were purchased from Fluka/Sigma-Aldrich and were of HPLC grade. Working Solutions
Enterolactone glucuronide and sulfate were dissolved in 50/40/ 10% ACN/MeOH/H2O, whereas enterolactone was dissolved in 100% ACN and kept at −80 °C. Then, the working solution containing all lignan standards in the concentration of 200 ng/ mL was prepared and used for preparation of standard curves and spiking of test plasma. The working solution and the standard curves were kept at −80 °C at all times. Stability of the internal standard (IS) was previously tested in water.12 We used standard curves to investigate freeze and thaw and long-term stability over 3 months.
Solid-Phase Extraction Clean up of Plasma
Plasma samples were centrifuged, and 100 μL of the sample was transferred to another tube and diluted with 300 μL of water. The sample volume may be increased to 200 μL to increase the sensitivity of free enterolactone and enterolactone sulfate, if necessary. Test of 10 samples with 100 and 200 μL showed the same results; however, no systematic validation has been performed with 200 μL of sample volume. Samples were cleaned up using Solid-Phase Extraction (SPE) Oasis HLB 96well plates 30 μm (10 mg) and a vacuum manifold with constant flow rate from Waters (Torrance, CA). The SPE method included conditioning, washing, and elution of enterolactone and enterolactone conjugates. The binding plate (96-well plate) (Figure 2) was conditioned with 500 μL of MeOH and then 500 μL of water. Afterward the samples
Test Plasma
To obtain the lowest possible concentration of enterolactone and its conjugates in human plasma, we pooled plasma samples with a known concentration of enterolactone from a previously conducted study. This pooled low enterolactone concentration human plasma was used for development and validation of the method and for preparation of QC samples. Standards of enterolactone, enterolactone glucuronide and sulfate were added to QC samples at low (0.0977 ng/mL) and high (6.25 B
DOI: 10.1021/acs.jproteome.5b01117 J. Proteome Res. XXXX, XXX, XXX−XXX
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ionization (ESI) source and operated in negative ionization mode. The flow injection analysis (FIA) was performed to optimize the turbo V source of the instrument. The ESI source settings for the ionization of enterolactone and enterolactone glucuronide and sulfate were as follows: curtain gas 20 psig, Gas1 50 psig, Gas2 20 psig, temperature 300 °C, and ionization spray operated at −4500 eV. Deprotonated molecules were detected in multiple reaction monitoring (MRM) mode. The compound-dependent parameters were optimized for each lignan by syringe infusion of pure standard and are listed in Table 1. Data analysis was performed in Analyst software 1.6 from AB Sciex. Table 1. Compound-Dependent LC−MS/MS Parameters, Declustering Potential (DP), Entrance Potential (EP), Collision Energy (CE), and Cell Exit Potential (CEP)
enterolactone enterolactone glucuronide enterolactone sulfate
Q1 mass
Q3 mass
DP
EP
CE
CEP
297.1 473.1
253.1 297.1
−140 −25
−10 −10
−26 −34
−21 −25
377.1
297.1
−55
−10
−32
−13
Calibration Curves
Calibration curves were prepared from the working solution in the range of 0.0061−12.5 ng/mL in 50/40/10% ACN/MeOH/ H2O. The IS was added to the calibration curves in the final concentration of 10 ng/mL. The analyte/internal standard concentration ratio was plotted against the analyte/internal standard peak area ratio as a linear regression curve with 1/x weighting.
Figure 2. Analytical scheme of the sample SPE clean-up procedure.
were loaded and allowed to elute slowly through the HLB material. Vacuum was applied in the end of elution to be sure that all of the samples eluted through the sorbent. The sorbent was then washed with 0.5 mL of 5% MeOH. Again, the vacuum was applied to dry the sorbent. After the washing step, the elution plate was used to collect the eluting enterolactone, enterolactone glucuronide, and sulfate from the binding plate. The elution of the analytes was performed with 300 μL of 50/ 40/10% ACN/MeOH/H2O. Vacuum was applied in the end of elution to be sure that all of the solvent eluted through the sorbent. Samples were then diluted directly in the elution plate with 900 μL of water containing glycocholic acid (glycine-1 13 C) as the internal standard with a final concentration of 10 ng/mL. This sample clean-up procedure takes ∼1 h. Samples were spun down at 20 °C for 5 min prior to LC−MS/MS measurements.
Method Validation
This method was validated according to the guidelines of the U.S. Food and Drug Administration (FDA)16 and European Medicines Agency of Science Medicines Health.17 Validation of the method was performed to demonstrate the accuracy, precision, and recovery of the measurements. Intrabatch accuracy, precision, and recovery of enterolactone and its conjugates were tested at three concentrations: low (0.0977 ng/mL), medium (1.56 ng/mL), and high (6.25 ng/ mL) using five measurements per concentration. Accuracy was determined by spiking test plasma with standards post-SPE clean up and calculating the relative error (RE) between true and measured values. Precision was calculated as relative standard deviation (RSD), with acceptance criteria for both accuracy and precision that they should not exceed by >15%. Recoveries of the enterolactone and its conjugates were determined by spiking test plasma with standards pre-SPE clean up. The recovery of the analyte represents the amount of the analyte recovered after the sample preparation procedure and is expressed in percentage. Blank plasma was used to subtract the background during the calculations. Solvent-based blank samples were used to investigate the carry-over effect. Interbatch precision and recovery were determined using QC samples, which can provide the basis for accepting or rejecting the batch. Three low and three high QC samples were used for each batch/plate. A similar acceptance criterion was used for interbatch precision as for intrabatch precision of 15%. The matrix effect was evaluated for all three analytes and the IS. For this, we have calculated the matrix factor (MF) for each analyte and IS as the ratio between the peak area in the
LC−MS/MS Equipment and Method
Samples were measured on MicroLC 200 Series from Eksigent/ AB Sciex (Redwood City, CA) and QTrap 5500 mass spectrometer from AB Sciex (Framingham, MA). The MicroLC was equipped with a Phenyl Column, 300 mm × 0.5 mm with 3.0 μm particle size from Eksigent/AB Sciex. The LC conditions were the following: Eluent A was water with 0.1% formic acid and eluent B was acetonitrile with 0.1% formic acid. The column was equilibrated for 2 min. The gradient started at 15% of eluent B, held constant for 0.5 min, and increased to 75% of eluent B over 1.9 min and kept constant for 0.2 min. The total run time was 4.6 min. The autosampler racks and the column were maintained at 20 and 23 °C, respectively. The mass spectrometer was equipped with an electrospray C
DOI: 10.1021/acs.jproteome.5b01117 J. Proteome Res. XXXX, XXX, XXX−XXX
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Figure 3. Typical MRM chromatogram of authentic standards in concentration 1.56 ng/mL. Enterolactone glucuronide RT (1.33), enterolactone sulfate RT (1.38), internal standard (IS) RT (1.51), and enterolactone RT (1.65).
performed directly on conjugates of enterolactone. The developed method is also very applicable for automatization. The authentic standards of enterolacrone, enterolactone glucuronide, and sulfate were further used to optimize LC− MS/MS conditions. The developed chromatographic separation was of only 2.6 min per samples without any coeluting peaks, Figure 3. It was our intention not to separate the isomers of enterolactone glucuronide and sulfate. The column containing phenyl sorbent gave the best resolution, symmetrical peak-shape and no isomer separation compared to the usually used for lignans C18 column (data not shown). Moreover, the tandem mass spectrometry was the method of choice due to its superior selectivity and sensitivity. The negative ion mode provided the best ionization efficiency of deprotonated molecules. The MS/MS transitions were optimized by manual tuning and the compound-dependent parameters are listed in Table 1. The most abundant fragments, which offered the best sensitivity and signal-to-noise characteristics, were chosen for quantification. In the case of enterolactone glucuronide and sulfate the most abundant fragments were represented by the loss of the glucuronic acid and sulfate moieties, 473.1/297.1 and 377.1/297.1, respectively. The combination of rapid sample clean up and short chromatographic run time with high sensitivity and selectivity of the MS/MS technology makes this method well suited for high-throughput of samples.
presence and absence of matrix. If the ratio was close to 100% no ion suppression has occurred.
3. RESULTS AND DISCUSSION Method Development
This method was developed to measure high-throughput of samples and therefore can be used for example in epidemiological studies. With this method it was possible to measure 3956 samples from an epidemiological study in less than 2 months. Large epidemiological studies need analytical methods that are not time-consuming and have minimal losses of the analytes due to sample transfer. So far, no simple and rapid method has been developed for quantification of enterolactone glucuronide, sulfate, and free enterolactone in human plasma. The GC−MS method that was developed many years ago was very labor-intensive involving some 20 SPE steps and separate GC−MS injections per sample.10,11 Later one LC−MS method was reported on enterolactone conjugates in serum.18 Knust et al. developed a method to quantify enterolactone glucuronide in serum and urine; however, many extraction steps were included in the clean-up procedure of serum samples. Other methods on the quantification of enterolactone include the time-consuming and crucial step of hydrolysis, in which only total concentration of enterolactone can be measured. Compared with the previously reported methods, our method consists of only one purification step using SPE (Figure 2). SPE was chosen due to its high selectivity, speed of extraction, and good potential for removing interferences from the samples. Furthermore, much lower volumes of organic solvents are required for SPE than in the liquid−liquid extraction (LLE) used by Knust et al.18 One of the main advantages of the current method is that no hydrolysis of the samples is necessary because the measurements are
Method Validation
Calibration curves were constructed using authentic standards of enterolactone, enterolactone glucuronide, and enterolactone sulfate in a pure solvent. The test of calibration curves in both pure solvent and test plasma showed that the different matrices had no discernible effect on the calibration slope or intercept. Moreover, the calculations of the MF of each analyte and IS in the test plasma showed that the ratio was close to 100%, indicating that no ion suppression had occurred. The linear D
DOI: 10.1021/acs.jproteome.5b01117 J. Proteome Res. XXXX, XXX, XXX−XXX
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determined for each analyte and are listed in Table 3. Even though test plasma contained a low concentration (25
0−10
10−25
>25
1846 (94.1%)
113 (5.8%)
2 (0.1%)
1815 (93.0%)
128 (6.6%)
9 (0.4%)
Calculated as enterolactone sulfate/(enterolactone sulfate + enterolactone glucuronide).
have been performed only with unconjugated enterolactone and not with conjugation/deconjugation in the target cells. For estrogen, it has been proven that estrogen sulfate does not bind to the estrogen receptor (ER) and that it has no direct biological activity; however, estrogen sulfate acts as a precursor to the active estrogen and therefore has an indirect effect on the target tissue.7 Moreover, it has been suggested that conjugation and deconjugation of estrogens can be compared with an activation/deactivation mechanism that plays an important role in the prognosis of breast cancer risk because only the free form of estrogen can bind to the ER receptor.25,26 Conjugation of the molecules is therefore most likely a very important mechanism by which the body can control the level of active versus inactive metabolites. When lignan studies began in the 1980s, the main problem was the lack of rapid and sensitive techniques to measure enterolactone and its conjugates in biofluids. Over the past 35 years many good methods have been developed using GC−MS, HPLC, LC−MS/MS, and a fluoroimmunoassay used by many epidemiological studies; however, the quantification of enterolactone has always been based on its total concentration and not on its intact forms. Here we have presented a rapid, reproducible, and sensitive LC−MS/MS method developed to quantify three intact forms of enterolactone in plasma: enterolactone glucuronide, enterolactone sulfate, and free enterolactone. The prospect of directly measuring the enterolactone and its conjugates in plasma may therefore offer a new perspective on the role of lignans in human health.
glucuronidated, with a wide concentration range among the human samples, from 0.2 to 650 nM and mean concentration of 28.5 nM. The concentration of enterolactone sulfate varied from 0 to 30 nM with mean concentration of 1.3 nM, whereas the concentration of free enterolactone was quantifiable in only a few samples. Our results are therefore in good agreement with the first studies performed on enterolactone in the 1980s, in which it was concluded that enterolactone was almost exclusively conjugated with glucuronic acid.1,9,10 Our further calculations on the mean percentage distribution of enterolactone glucuronide and sulfate in plasma for men and women (Table 4) have revealed that percentage distribution for men and women had similar patterns. Moreover, the calculations on percentage distribution of enterolactone sulfate have shown that very few people have enterolactone sulfate higher than 25%, only two men (0.1%) and four women (0.4%) (Table 5). The majority of both men and women had enterolactone sulfate not higher that 10%. Because our results are the first in which enterolactone glucuronide and sulfate were quantified in such a high number of samples, they are difficult to compare with the previous results of Axelson and Setchell9 and Adlercreutz et al.,10,11 in which only a few urine and plasma samples were measured; however, it is important to point out that the method used for quantification of lignan conjugates at that time was adapted from the method on quantification of steroid conjugates.20,21 The authors have also noted that the mode of conjugation of enterolactone resembled that of steroid hormones and that similarly to steroids the conjugation of lignans probably occurred in the liver.9,10,22 Adlercreutz et al.10 also discussed the importance of relating the conjugation of enterolactone to its biological effects. They found that enterolactone is sulfated by human breast and liver cancer cells and that enterolactone sulfate is the dominant form of enterolactone in such cell cultures.11 There are also many similarities in the enterohepatic circulation of lignans and estrogen, and it was established that the same UDPGT-dependent conjugation system participates in the conjugation of polyphenols and estrogens and that polyphenols may have an influence on their level in the body.8,23,24 All of this suggests that the conjugation of enterolactone may play a far more important role in relation to its biological activity and thus have different effects on, for example, cancer than what is currently expected. The divergent results on whether enterolactone has estrogenic or antiestrogenic effects may also be related to the fact that experiments
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b01117. Table S1. Certificate of analysis for enterolactone glucuronide. Table S2. Certificate of analysis for enterolactone sulfate. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: +4587157724. Fax: +4587154249. E-mail: Natalja.
[email protected]. G
DOI: 10.1021/acs.jproteome.5b01117 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research Notes
(17) Guideline on bioanalytical method validation. Eurpean Medicines Agency of Science Medicine Health. Committee for Medicinal Products for Human Use (CHMP), February 2012. Available on the internet at http://www.ema.europa.eu/docs/en_ GB/document_library/Scientific_guideline/2011/08/WC500109686. pdf. (18) Knust, U.; Hull, W. E.; Spiegelhalder, B.; Bartsch, H.; Strowitzki, T.; Owen, R. W. Analysis of enterolignan glucuronides in serum and urine by HPLGESI-MS. Food Chem. Toxicol. 2006, 44 (7), 1038− 1049. (19) Clarke, D. B.; Lloyd, A. S.; Botting, N. P.; Oldfield, M. F.; Needs, P. W.; Wiseman, H. Measurement of intact sulfate and glucuronide phytoestrogen conjugates in human urine using isotope dilution liquid chromatography-tandem mass spectrometry with C13(3) isoflavone internal standards. Anal. Biochem. 2002, 309 (1), 158−172. (20) Fotsis, T.; Adlercreutz, H. the Multicomponent Analysis of Estrogens in Urine by Ion-Exchange Chromatography and GC-MS 0.1. Quantitation of Estrogens after Initial Hydrolysis of Conjugates. J. Steroid Biochem. 1987, 28 (2), 203−213. (21) Axelson, M.; Sahlberg, B. L.; Sjövall, J. Analysis of profiles of conjugated steroids in urine by ion-exchange separation and gas chromatographymass spectrometry. J. Chromatogr., Biomed. Appl. 1981, 224 (3), 355−370. (22) Dean, B.; Chang, S.; Doss, G. A.; King, C.; Thomas, P. E. Glucuronidation, oxidative metabolism, and bioactivation of enterolactone in rhesus monkeys. Arch. Biochem. Biophys. 2004, 429 (2), 244−251. (23) Adlercreutz, H.; Hockerstedt, K.; Bannwart, C.; Bloigu, S.; Hamalainen, E.; Fotsis, T.; Ollus, A. Effect of Dietary-Components, Including Lignans and Phytoestrogens, On Enterohepatic Circulation and Liver-Metabolism of Estrogens and on Sex-Hormone Binding Globulin (SHBG). J. Steroid Biochem. 1987, 27 (4−6), 1135−1144. (24) Zhu, B. T.; Taneja, N.; Loder, D. P.; Balentine, D. A.; Conney, A. H. Effects of tea polyphenols and flavonoids on liver microsomal glucuronidation of estradiol and estrone. J. Steroid Biochem. Mol. Biol. 1998, 64 (3−4), 207−215. (25) Raftogianis, R.; Creveling, C.; Weinshilboum, R.; Weisz, J. Estrogen Metabolism by Conjugation. J. Natl. Cancer Inst. Monogr. 2000, 27, 113−124. (26) Pettersson, K.; Gustafsson, J. A. Role of estrogen receptor beta in estrogen action. Annu. Rev. Physiol. 2001, 63, 165−192.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Innovation Fund Denmark for financing the project “The effects of enterolignans in chronic diseases - ELIN” (0603-00580B).
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REFERENCES
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